The Quest for Height: Grow Taller | Increase Height | Bone Size

In Bone Marrow Chondrogenesis of Mesenchymal Stem Cells

2012-01-26T12:08:00.000-08:00

The goal of Lateral Synovial Joint Loading is to use lateral loads to induce hydrostatic pressure in the epiphyseal bone marrow. This hydrostatic pressure results in chondrogenic differentiation. Unlike with osteoblasts, chondrocytes are both hydrophillic(water loving) and have their hypertrophy and cellular proliferation coupled in columns. It is likely some combination of these two properties that allow chondrocytes to be much more effective at lengthening bone than osteoblasts. Thus, studies of in vivo(within the body) studies of chondrogenic differentiation would be very useful in providing insights to the LSJL method. As they could both tell us how best to induce chondrogenic differentiation and whether the chondrocytes generated by LSJL could increase height.

In vivo chondrogenesis of adult bone-marrow-derived autologous mesenchymal stem cells.

"The purpose of this study has been to investigate the possible effects of the normal joint cavity environment on chondrocytic differentiation of bone-marrow-derived mesenchymal stem cells (MSCs)[not the epiphyseal bone marrow which is our target area for inducing MSC chondrogenic differentiation]. Autologous bone marrow was aspirated from the iliac crest of male sheep. MSCs were purified, expanded, and labeled with the fluorescent dye PKH26. Labeled MSCs were then grown on a three-dimensional porous scaffold of poly (L-lactic-co-glycolic acid) in vitro and implanted into the joint cavity by a surgical procedure. At 4 or 8 weeks after implantation, the implants were removed for histochemical and immunohistochemical analysis. The cells labeled with red fluorescent PKH26 in the implants expressed type II collagen and synthesized sulfated proteoglycans. However, the osteoblast-specific marker, osteocalcin, was not detected by immunohistochemistry indicating that the implanted MSCs had not differentiated into osteoblasts by being directly exposed to the normal joint cavity. To investigate the possible factors involved in chondrocytic differentiation of MSCs further, we co-cultured sheep MSCs with the main components of the normal joint cavity, viz., synovial fluid or synovial cells, in vitro. After 1 or 2 weeks of co-culture, the MSCs in both co-culture systems expressed markers of chondrogenesis. These results suggest that synovial fluid and synovium from normal joint cavity are important for the chondrocytic differentiation of adult bone-marrow-derived MSCs[the interstitial fluid flow from LSJL may help synovial fluid get to the epiphysis and synovial tissue might be able to be accessed via the periosteum]."

"in vivo experiments have demonstrated that MSCs implanted into surgically created, osteochondral defects in
rabbits are able to differentiate into chondrocytes and improve the quality of cartilaginous repair"<-But not yet whether MSCs in intact epiphyseal bone marrow can differentiate into chondrocytes. Osteochondral defects give more access to various fluids and nutrients. However, bone is already well vascularized.

Since LSJL involves synovial joint loading it seems likely that loading the synovial joint plays some role in inducing the chondrogenesis and just increasing the hydrostatic pressure in the bone marrow. To study this, you'd have to perform LSJL on rats, after removing their joint capsule, and then compare the results to the rats in the other study.

The LSJL scientists studied the effects of surgical holes on bone formation but not chondrogenesis. But it's likely that any chondrogenesis in the bone would've increased the bone measurements like cortical area and cortical thickness. Thus, the increase in intramedullary pressure may be the main determinant for both chondrogenic and osteogenic differentiation but synovial fluid transport from the joint cavity may enhance chondrogenesis.

Here's a study about what happens to extra MSCs that come into the body:

The dynamic in vivo distribution of bone marrow-derived mesenchymal stem cells after infusion.

"Bone marrow-derived mesenchymal stem cells (MSCs) have the potential to differentiate along different mesenchymal lineages including those forming bone, cartilage, tendon, fat, muscle and marrow stroma that supports hematopoiesis. This differentiation potential makes MSCs candidates for cell-based therapeutic strategies for mesenchymal tissue injuries and for hematopoietic disorders by both local and systemic application. In the present study, rat marrow-derived MSCs were ex vivo culture-expanded, labeled with (111)In-oxine, and infused into syngeneic rats via intra-artery (i.a.), intravenous (i.v.) and intraperitoneal cavity (i.p.) infusions[so put directly into the blood stream basically]. In addition, for i.a. and i.v. infusions, a vasodilator, sodium nitroprusside, was administered prior to the cell infusion and examined for its effect on MSC circulation. The dynamic distribution of infused MSCs was monitored by real-time imaging using a gamma camera immediately after infusion and at 48 h postinfusion. After 48 h, radioactivity in excised organs, including liver, lungs, kidneys, spleen and long bones, was measured in a gamma well counter and expressed as a percentage of injected doses. After both i.a. and i.v. infusion, radioactivity associated with MSCs was detected primarily in the lungs and then secondarily in the liver and other organs. When sodium nitroprusside was used, more labeled MSCs cleared the lungs resulting in a larger proportion detected in the liver. Most importantly, the homing of labeled MSCs to the marrow of long bones was significantly increased by the pretreatment with vasodilator[Vasodilators widen the blood vessels like Viagra]. These results indicate multiple homing sites for injected MSCs and that the distribution of MSCs can be influenced by administration of vasodilator."

"Vascular cell adhesion molecule-1 (VCAM-1) is expressed by the marrow stromal cells and may play an important role in the interchange of cells between bone marrow and blood."<-So areas with bone marrow already like the long bones express VCAM-1. The more vascularized the blood the more likely it is for MSCs in the blood to bind to the bone marrow.

So to get more MSCs to the epiphysis you'd want more epiphyseal bone marrow to be there and to take a vasodilator.

Induction of chondrogenesis in muscle, skin, bone marrow, and periodontal ligament bydemineralized dentin and bone matrix in vivo and in vitro.

"Induction of chondrogenesis in vivo by rolls of demineralized dentin[dentin is a part of your tooth] implanted in muscle, subcutaneous connective tissue of skin, medullary cavity of femur[not the epiphysis though], and periodontal ligament of rat was investigated. Specimens were examined at various times up to 21 days after implantation, using light microscopy and morphometric analysis. Induction of cartilage occurred most quickly in muscle, followed by subcutaneous connective tissue of skin and medullary cavity of femur, and most slowly in periodontal ligament. Significantly more cartilage was found in muscle than in subcutaneous connective tissue of skin and medullary cavity of femur at the times examined, and least of all in periodontal ligament. Outgrowth of cells from rat muscle, dermis and subcutaneous tissue, bone marrow and periodontal ligament cultured in vitro on demineralized bone matrix for up to 35 days produced similar results."

"Implant in medullary cavity of femur. Wounds were made. A skin incision, approximately 3.0 cm in length,
was made along the lateral aspect of the right thigh, and the femoral muscles were retracted to expose the lateral aspect of the femur. A defect approximately 1.4 mm in diameter was cut in the mid-region of the lateral aspect of the femoral diaphysis, by means of a #3 round bur mounted in a slow-running dental handpiece cooled with phosphate-buffered saline, pH 7.0. The underlying bone marrow was excised with a dental
excavator, and debris was removed by being washed with phosphate-buffered saline. One dentin roll was implanted in the medullary cavity proximal to the wound, and parallel to the long axis of the bone. The muscle was sutured with 3.0 silk and the skin with 4.0 silk."

It took 10 days for cartilage to form in the bone. The cartilage formed directly next to the dentin roll. No chondrogenesis was evident after day 21.

"demineralized bone matrix is mitogenic for mesenchymal cells; and second, that demineralized bone matrix binds fibronectin, an event that is necessary for induction of cartilage"<-It's possible that hydrostatic pressure demineralizes bone matrix which allows for new cartilage growth to occur. Acid is also a way to dimeralize bone matrix.

In this study cartilage only formed directly next to the dimineralized dentin roll. Thus dimineralized matrix is likely necessary for cartilage growth. Thus for LSJL to be more effective we may need to dimineralize the bone matrix. Hydrostatic pressure may already contribute to demineralization of the bone as bone tissue is hydrophobic.

Lactic Acid is naturally produced in response to exercise. Ascorbic acid(Vitamin C), which can induce chondrogenesis, is naturally occurring. The main minerals that give bone it's stiffness are Vitamin D, phosphorus, and Vitamin C. However, people with osteomalacia are not growing taller. In fact people with osteomalacia have stunted height. So, we want only partial bone demineralization only on the surface area of the bone to allow for chondrogenesis to occur on it. Hydrostatic pressure may demineralize the surface area of the bone without affecting the mineralization of the interior of the bone providing a safe way to increase height.

The longitudinal ends of the bones may provide a demineralized bone portion for mesenchymal stem cells to differentiate into chondrocytes in the joint capsule. Osteoarthritis in fact involves changes in subchondral bone mineralization which may affect the stem cells ability to undergo chondrogenesis.

Osteoclasts operate by using an electron transport pump to acidify the surface of the bone. So osteoclasts would also be a way to dimineralize a bone surface to allow for chondrogenesis.

Surgically, you could insert a roll of demineralized dentin into your growth plate and then take stem cells followed by a vasodilator.

Growing Taller with natriuretic peptides?

2012-01-25T15:42:00.000-08:00

There's a patent pending on free patents online for a new height increase product called a composition for increasing body height:

"This invention provides a composition for increasing a body height of a patient with short stature or an individual other than patients with short stature. More specifically, the invention provides: a composition for increasing the body height of an individual comprising a guanyl cyclase B (GC-B) activator as an active ingredient, the composition being to be administered to an individual free from FGFR3 abnormality; a method for increasing the body height of an individual free from FGFR3 abnormality which comprises activating GC-B; a method for screening an agent for increasing the body height of an individual which comprises selecting an agent for increasing the body height using GC-B activity as an indication; and a method for extending a cartilage bone free from FGFR3 abnormality which comprises activating GC-B in an individual."

"Guanyl cyclase (GC) is a membrane protein belonging to the enzyme family that catalyzes the synthesis of the second messenger cGMP from GTP, and its examples include GC-A, GC-B, . . . , and GC-F. GC-B is found mainly in vascular endothelial cells, and thought to be involved in relaxation of the smooth muscle."

"Natriuretic peptides (NPs) are divided into ANP (atrial sodium peptide), BNP (brain natriuretic peptide) and CNP (type c natriuretic peptide), and they are thought to elevate an intracellular cGMP level through two guanyl cyclase conjugated receptors (NPR-A for ANP and BNP, and NPR-B for CNP) and to perform intracellular signal transduction mediated by a plurality of cGMP effecter molecules (Ann Rev Biochem 1991; 60: 229-255). NPs have been reported to play an important role in the control of humoral homeostasis and blood pressure (J Clin Invest 1987; 93:1911-1921, J Clin Invest 1994; 87: 1402-1412), and their expression and biological activity in various tissues other than the cardiovascular system are known (Endocrinol 1991; 129:1104-1106, Ann Rev Biochem 1991; 60: 553-575). Concerning cartilage bones, effectiveness of overexpression of BNP (Proc. Natl. Acad. Sci., U.S.A., 1998, 95: 2337-2342) or CNP in the joints on the treatment of achondrogenesis resulting from mutation of a fibroblast growth factor receptor 3 (FGFR3) gene has been reported (Nat. Med., 2004, 10 (1): 80-86; Japanese Patent Publication No. 2003-113116 A)."

Mesenchymal stem cells have the ability to develop into blood cells and not natriuretic peptides so if this method works on the growth plate it must be by a method different than LSJL.

"We have prepared a C-type natriuretic peptide (CNP) transgenic mouse, which expresses CNP, a guanyl cyclase B (GC-B) activator, systemically with elevated blood level of CNP, and then studied the effect of CNP on body height or on growth cartilage. As a result, we have now found that in the CNP transgenic mouse the increase in body height is accelerated, that the femoral growth plate cartilage becomes significantly thickened, and that, through the property analyses of such CNP transgenic mice, the increase in body height is accelerated by the effect of CNP on hematogenously in the absence of an abnormality in FGFR3."

Now remember an acceleration of height gain does not necessarily mean an increase in final adult height.

"The thickness of the growth cartilage of CNP Tgm(CNP transgenic mice) was histologically analyzed using the mean thickness of the resting layer, proliferating layer and hypertrophy layer of the growth cartilage on the patellar surface femur, and the total of the three layers (as the thickness of growth cartilage). As a result, it was confirmed that each thickness of the resting layer, proliferating layer and hypertrophy layer, and the total thickness thereof for CNP Tgm were greater with statistical significance than those of the wild type. It was also demonstrated that CNP accelerates the increase in body height in animals by increasing each thickness of the resting layer, proliferating layers and hypertrophy layer of other cartilage bones, such as the tibiae, radiuses or ulnae, in addition to those of the cartilage bone of femora."

Now C-type natriuretic peptide affects the resting layer which might be the limiting factor in terms of height growth. Therefore, this supplement does have potential to increase height since it does act locally on the growth plate and affects the starting stage of growth.

"In the present invention, the term “FGFR3 abnormality” refers to achondrogenesis or achondroplasia, which is caused by growth inhibition of cartilage bones resulting from mutations in the fibroblast growth factor receptor 3 (FGFR3) gene, or achondrogenesis or achondroplasia caused by function control failure of FGFR3 or overexpression of FGFR3 gene resulting from mutations in the FGFR3 gene."

I don't get what a cartilage bone is. Maybe it refers to a long bone which has cartilage in the growth plate.

There are lots of studies that state that C-type natruiretic peptides are essential in growth and can account for growth variation like this one:

A genome-wide association study of northwestern Europeans involves the C-type natriuretic peptide signaling pathway in the etiology of human height variation.

"Northwestern Europeans are among the tallest of human populations. The increase in body height in these people appears to have reached a plateau, suggesting the ubiquitous presence of an optimal environment in which genetic factors may have exerted a particularly strong influence on human growth. Therefore, we performed a genome-wide association study (GWAS) of body height using 2.2 million markers in 10 074 individuals from three Dutch and one German population-based cohorts. Upon genotyping, the 12 most significantly height-associated single nucleotide polymorphisms (SNPs) from this GWAS in 6912 additional individuals of Dutch and Swedish origin, a genetic variant (rs6717918) on chromosome 2q37.1 was found to be associated with height at a genome-wide significance level (P(combined) = 3.4 x 10(-9)). Notably, a second SNP (rs6718438) located approximately 450 bp away and in strong LD (r(2) = 0.77) with rs6717918 was previously found to be suggestive of a height association in 29 820 individuals of mainly northwestern European ancestry, and the over-expression of a nearby natriuretic peptide precursor type C (NPPC) gene, has been associated with overgrowth and skeletal anomalies. We also found a SNP (rs10472828) located on 5p14 near the natriuretic peptide receptor 3 (NPR3) gene, encoding a receptor of the NPPC ligand, to be associated with body height (P(combined) = 2.1 x 10(-7)). Taken together, these results suggest that variation in the C-type natriuretic peptide signaling pathway, involving the NPPC and NPR3 genes, plays an important role in determining human body height."

So yes maybe guanyl cyclase activator could increase height. In adults it's unclear if manipulation of the C-type natriuretic peptide signaling pathway could "re-awaken the growth plates". Someone like alkoclar claims that enhancing CNP expression has the ability to increase adult height.

Here's a study that shows that CNP causes skeletal overgrowth during development but I don't have access to the full study:

C-Type Natriuretic Peptide and Overgrowth

"Natriuretic peptides are a family of structurally related peptides with different distinct biological effects. C-type natriuretic peptide (CNP)-mediated signaling is important for endochondral ossifica-tion and intervenes in the control of chondrocyte maturation by regulating the balance between proliferation and terminal differentiation[there's no proliferating chondrocytes in adults so how would CNP increase adult height? Unless you cause the differentiation of stem cells into chondrocytes with LSJL]. CNP is encoded by the NPPC gene on human chromosome 2 for which, so far, no mutations have been described in humans. Recently, two independent articles reported the description of 3 patients with a similar clinical phenotype characterized by the pres-ence of skeletal anomalies and overgrowth. In all 3 cases, the clinical picture was associated with the presence of a balanced translocation involving chromosome 2 and causing overexpression of the NPPC gene and an increased plasma concentration of its product, CNP[to cause skeletal overgrowth in development increase plasma concentration of CNP]. Transcriptional dysregulation of NPPC has been ascribed to the separation of the gene unit from the long-range regulatory element with a transcriptional silencing effect on its expression and CNP overproduction has been correlated to the skeletal overgrowth phenotype observed."
In the study "Overexpression of the C-type natriuretic peptide (CNP) is associated with overgrowth and boneanomalies in an individual with balanced t(2;7) translocation" overexpression of CNP in osteoblastic cells in mice resulted in skeletal overgrowth this supports the theory that increasing CNP plasma levels can increase skeletal size as adults definitely have osteoblastic cells.

Here's an article about achondroplasia(dwarfism) and how CNP may be a possible cure:

"[CNP acts by inducing intracellular cGMP through Guanylyl Cyclase B]"

"genetically engineered mice have short bones when null for CNP and long bones when CNP is overexpressed"<-is this dose dependent however? Would more CNP increase height indefinitely at higher and higher levels?

"growth plates in these mice are shortened and widened [in CNP over- and -under expression] in a manner similar to that detected in mice with loss- and gain-of-function mutations for FGFR3"<-FGFR3 gain-of-function causes dwarfism.

"transgenic mice [were generated] in which CNP was overexpressed in the growth plate; expression of the gene encoding CNP, designated Nppc, was driven by the type II collagen cartilage-specific promoter (Col2). The Col2-Nppc transgenic mice displayed excessive skeletal growth that was mainly postnatal[after birth]."<-So more Type II Collagen more CNP?

"Offspring of the mating that carried both the Col2-Nppc and Col2-FGFR3 transgenes had near normal body lengths when measured over 10 weeks"

"the over-expression of CNP did not appear to rescue the reduced proliferation of growth plate chondrocytes detected in the Col2-FGFR3"<-meaning that possibly the amount of proliferation of growth plate chondrocytes does not have much affect on height. Or, it's possible that the positive benefits of CNP overexpression are greater than the effects of FGFR3 overexpression.

"[cultured tibias were treated] from Col2-FGFR3 ach mice with different doses of CNP. Bone length showed a dose response to the CNP"<-But we can't be sure that this dose response occurs forever.

"The dose that restored bone length to normal also restored synthesis of 2 markers of cartilage matrix biosynthesis(glycosaminoglycan and collagen),which were reduced in the Col2-FGFR3 mice, to near normal"<-so cartilage matrix is likely the best determinant of height and not chondrocyte proliferation. That means that something like High Molecular Weight Hyaluronic Acid could possibly increase height during puberty.

"FGFR3 signals through STAT1 to down regulate chondrocyte proliferation and differentiation and through the MAP kinase-ERK pathway to negatively control matrix synthesis in the growth plate. They propose that CNP blocks the MAP kinase inhibitory signals of FGFR3 to increase matrix synthesis and thereby counters the restraining consequences of FGFR3 on bone growth"<-So other possible target proteins for height increase are STAT1 and MAPK inhibitory signals.

Still, CNP should only help during development. After development, you would need to induce a new cartilagenous matrix followed by endochondral ossification.

Why does hydrostatic pressure induce chondrocyte growth?

2012-01-23T12:25:00.000-08:00

We hear a lot in biology that structure equals function. The structure of a compound correlates to the purpose. The induction of stimulus to stem cells that a chondrocyte is better to counteract than the other cell types that a stem cell can differentiate into is likely to induce stem cells to differentiate into chondrocytes. Stem Cells have been found to differentiate into chondrocytes as a result of hydrostatic pressure and microgravity. Note that going into a pool actually reduces hydrostatic pressure by counteracting the internal hydrostatic pressure generated from the blood in the body.

The fate of mechanically induced cartilage in an unloaded environment.

"According to mechanobiologic theories, persistent intermittent mechanical stimulation is required to maintain differentiated cartilage. In a rat model for bone repair, we studied the fate of mechanically induced cartilage after unloading. In three groups of rats, regenerating mesenchymal tissue was submitted to different loading conditions in bone chambers[epiphyseal bone marrow is like a bone chamber and also contains mesenchymal tissue so it is similar to the terms of this study]. Two groups were immediately killed after loading periods of 3 or 6 weeks (the 3-group and the 6-group). The third group was loaded for 3 weeks and then kept unloaded for another 3 weeks (the (3 + 3)-group). Cartilage was found in all loaded groups[so loading mesenchymal tissue resulted in chondrogenic differentiation of MSCs]. Without loading, cartilage does not appear in this model[remember that in a random position machine(in which stem cells differentiated into chondrocytes in response to microgravity), the cartilage does not undergo complete unloading but only constant stimulation to gravity from different directions while getting a rest in others]. In the 3-group there was no clear ongoing endochondral ossification, the 6-group showed ossification in 2 out of 5 cartilage containing specimens, and in the (3 + 3)-group all cartilage was undergoing ossification. These results suggest a tendency of the cartilage to be maintained also under unloaded conditions until it is reached by bone that can replace it through endochondral ossification[once the stem cells have been differentiated they stay chondrocytes upon unloading].Additional measurements showed less amount of new bone in the loaded specimens. In most of the loaded specimens in the 3-group, necrotic bone fragments were seen embedded in the fibrous tissue layer close to the loading piston, indicating that bone tissue had been resorbed due to the hydrostatic compressive load[hydrostatic pressure can resorb bone helping to induce a more youthful state]. In some specimens, a continuous cartilage layer covered the end of the specimen and seemed to protect the underlying bone from pressure-induced resorption[cartilage protects the bone from hydrostatic pressure-induced resorption which is why stem cells under hydrostatic pressure differentiate into chondrocytes to protect bone from resorption, these new chondrocytes can new form growth plates which will result in you growing taller!]. We suggest that one of the functions of the cartilage forming in the compressive loaded parts of a bone callus is to protect the surrounding bone callus from pressure-induced fluid flow leading to resorption."

"The bone conduction chamber consists of a threaded titanium cylinder, formed out of two half cylinders held together by a hexagonal closed screw cap. One end of the implant is screwed into the bone. The interior of the chamber has a diameter of 2 mm, and is 7 mm long. There are two bone ingrowth openings at one end where tissue can grow in from the cortical and the subcortical cancellous bone but not from extracortical soft tissue. At 3 weeks after the implantation, the specimens within the chamber usually contain 3 different zones of ingrown tissue as described previously. At the bottom there is a zone with cancellous bone with a marrow cavity, followed higher up by more immature woven bone formed by membranous ossification occurring as an advancing ossification frontier. Above this frontier there is vascularized fibrous tissue. At the fourth and sixth week the marrow cavity has expanded, the cancellous bone is found higher up, and the fibrous zone is thinner."

You can see in b that it is very similar to an apparatus used to generate hydrostatic pressure.

"The load chamber consists of the same two half cylinders with its two ingrowth openings. The hexagonal screw cap is replaced by a cap equipped with a 1.8 mm diameter piston protruding into the chamber, from the subcutaneous end towards the intraosseous end. By applying a known force on the top of the piston, a mechanical load can be transmitted to the tissue within the chamber. When loading is interrupted, the piston returns to its original position by means of a spring and no further mechanical stimuli act upon the tissue within the chamber. The inside diameter of the LC is the same as in the BCC (2 mm), the distance between the chamber bottom with its ingrowth openings and the piston is 5 mm when the chamber is unloaded, and 1.5 mm when the piston is in its most downward position. The top, with its mobile parts, is covered with a rubber coat to prevent overlying tissues from interfering with the moving parts. Originally we aimed at evaluating the ingrowth distance of new bone and used an unloaded control. In the first 3-group, bilateral chambers were thus used, a BCC on one side and an LC on the other side. Since it became more interesting to merely show the presence of cartilage, the unloaded control BCC was left out in the later six-series and the (3+3)-series."
"The loading device consisted of a metal rod, a spring and a metal cylinder that could glide over the rod, at the same time compressing the spring"<-this is very similar to a clamp except the clamp is applied laterally rather than axially.

After 6 weeks of loading the mean cartilage area in millimeters squared was 0.017 with the highest being 0.065. There were non-responders so there definitely could be a genetic component in not responding to hydrostatic pressure.

The scientists reported tissue ingrowth but no mention of tissue outgrowth is made(which is what causes height growth). The study is definitely indicative however of the ability for hydrostatic pressure to form new cartilagenous regions in bone marrow.

"It has been suggested that intermittent hydrostatic compressive stress and/or low oxygen tension will stimulate mesenchymal tissue to form cartilage"<-still don't know what purpose low oxygen tension stimulating chondrocyte differentiation does yet.

"High shear stresses were hypothesized to be associated with formation of fibrous tissue, whereas high hydrostatic compressive stresses seemed to guide the cellular differentiation into the chondrogenic pathway"<-which is why you have to load the epiphysis in LSJL. The area that is clamped will experience hydrostatic pressure but the other areas of the bone will mostly experience shear strain.

The scientists in the study used a bone conduction chamber to generate hydrostatic pressure. Then there's a piston used to push the fluid in the bone.

"As cortical bone is stiffer than the newly formed membranous trabecular bone within the bone chamber, the deformations of cortical bone are less than the strain threshold for cartilage to form. This indicates that bone resorption can be initiated by hydrostatic stresses, as low as the lowest stresses that induce cartilage.In conclusion, mechanically induced cartilage can be maintained in unloaded conditions."<-So hydrostatic pressure is possible to cause resorption of the cortical bone allowing for more stem cell growth and thus a more youthful organ.

"In a few specimens, a continuous cartilaginous layer had developed adjacent to the bone frontier, which had reached far into the chamber. No signs of bone resorption or necrotic trabeculae were found in these cases. It seems that this cartilage, which covered the whole end of the specimen, protected the underlying bone from resorption"<-cartilage is the best adaptive mechanism to hydrostatic pressure so the body responds to hydrostatic pressure by encouraging chondrogenesis. "Cartilage could play a protective role against hydrostatic stress-induced resorption of the underlying bone, possibly by hindering massive fluid flow through the bone"
The formation of chondrocytes may be an adaptive mechanism in response to hydrostatic pressure. In the study, 40% of the cartilage containing groups underwent endochondral ossification. However, it's possible that more of the groups could undergo endochondral ossification after the 6 week period.

So like muscle adapts to microdamage by growing bigger(over simplified), bone responds to hydrostatic pressure by forming cartilagenous possible growth plate like regions.

Hydrostatic pressure and the actomyosin cortex drive mitotic cell rounding.

"During mitosis, adherent animal cells undergo a drastic shape change, from essentially flat to round. Mitotic cell rounding is thought to facilitate organization within the mitotic cell and be necessary for the geometric requirements of division. However, the forces that drive this shape change remain poorly understood in the presence of external impediments, such as a tissue environment. Here we use cantilevers to track cell rounding force and volume. We show that cells have an outward rounding force, which increases as cells enter mitosis. We find that this mitotic rounding force depends both on the actomyosin cytoskeleton and the cells' ability to regulate osmolarity[so LSJL requires both a sensitive actin cytoskeleton and sufficient hydrostatic pressure]. The rounding force itself is generated by an osmotic pressure. However, the actomyosin cortex is required to maintain this rounding force against external impediments[the actin cytoskeleton is need to prevent chondrogenic differentiation]. Instantaneous disruption of the actomyosin cortex leads to volume increase, and stimulation of actomyosin contraction leads to volume decrease. These results show that in cells, osmotic pressure is balanced by inwardly directed actomyosin cortex contraction[osmotic pressure is required to generate the stimulus to a chondrogenic phenotype but the actin cytoskeleton is required to respond to the stimulus]. Thus, by locally modulating actomyosin-cortex-dependent surface tension and globally regulating osmotic pressure, cells can control their volume, shape and mechanical properties."

"If the osmolarity is higher inside the cell than outside, water will flow into the cell and generate a hydrostatic pressure"<-LSJL alters solute concentration by means of compression.

"Introduction of hypotonic medium (−Δ100 mosM l⁻¹) led to an immediate increase in the volume of metaphase cells (40 ± 6%; n = 9), indicating that water entered the cells. This was accompanied by a concurrent increase in the measured rounding pressure (76 ± 20%; n = 9), presumably because the intracellular pressure increased. Within 3 min of the osmolarity changing, the cell volume and rounding pressure returned to close to their original values. This is probably because, in response to increased osmotic pressure, regulatory volume decrease causes cells to release ions"<-so pressure may have to be sustained longer for LSJL to be active to prevent dedifferentiation or perhaps LSJL needs to be applied in 3 minute intervals.

"The exchange of a proton with a Na⁺ ion increases the intracellular osmolarity because pH is strongly buffered in the cytoplasm; thus, a Na⁺ ion has a greater effect on osmolarity than a proton"<-High Sodium intake may help with LSJL?

Confirmation that the LSJL scientists believed it can cause height growth

2012-01-10T09:54:00.000-08:00

Here's a grant submitted by Ping Zhang that was proposed to do research on whether LSJL could cause adult height growth!

Load-Driven Bone Lengthening

"The long-term objective of the proposed project is to understand the mechanism of load-driven bone lengthening. Although distraction osteogenesis is effective to treat the patients with limb length discrepancy, this invasive procedure occasionally generates problems such as premature consolidation, delayed union, and infection. In order to investigate a possibility of load-driven non-invasive therapy[like LSJL], we will focus on knee loading a form of joint loading modalities. In this R03 project, we address a set of questions (1) Does knee loading enhance proliferation of chondrocytes in the growth plate and lengthen the proliferative and hypertrophic zones in the distal femur and the proximal tibia[if knee loading can cause differentiation of stem cells into chondrocytes it can cause new height growth]? (2) Is the lengthening effect dependent on ages[will it work on adults?]? (3) Does injection of insulin growth factor-2 (IGF-2) into the growth plate enhance the loading effects? We hypothesize (a) Lateral loads, applied to the knee with appropriate loading conditions, can lengthen both the femur and the tibia through stimulating proliferation of chondrocytes in the growth plate; (b) Efficacy of load-driven bone lengthening is stronger in the youth than the elderly; and (c) The lengthening effect can be augmented in the elderly by injecting IGF-1 in the growth plate[so LSJL will work even in aged individuals!!!! However, aged individuals do not have growth plates so maybe he means injecting IGF-1 into the bone marrow?]. In order to examine the above hypotheses, three specific aims are proposed using a hindlimb of C57BL/6 mice as a model system. " Evaluation of load-driven bone lengthening under varying mechanical conditions " Comparison of the loading effects among mice with varying ages " Effects of local administration of IGF-2 into the growth plate with and without knee loading. We will use a custom-made piezoelectric mechanical loader, and conduct bone histomorphometric and gene expression analyses using varying imaging modalities. The proposed study is expected to contribute to developing a non-invasive physical therapy for treatment of patients with limb length discrepancy. PROJECT NARRATIVE Currently the invasive surgical procedure (distraction osteogenesis) is commonly employed to lengthen long bones. However, this procedure presents a series of potential risks such as premature consolidation, delayed union, bone infection, joint stiffness, and fracture of newly formed bone. The proposed R03 project will contribute to examining a possibility of non-invasive physical therapy for treatment of patients with limb length discrepancy"

Here's the grant name to track it: Grant 1R03AR055322-01A1.

Here's a link with more info on the project.

How do the growth plates make us taller?

2012-01-04T11:15:00.000-08:00

We know that water plays a key role in the endochondral ossification process and with LSJL induced hydrostatic pressure causing chondrogenic differentiation of stem cells. We're not sure exactly on how the growth plate physically makes us taller. In understanding that, we can understand more about how the growth plate works or we can mimic the methods that the growth plate uses to induce bone deformation to perform a new height increasing method that doesn't involve the growth plate.

A Review of the Actual Knowledge of the Processes Governing Growth and Development of Long Bones

"Before the appearance of the ossification centers epiphyseal growth rests exclusively on chondrocytes proliferation (interstitial growth), without any detectable differentiated cellular organization."<-this bodes well for a method like seeks to induce chondrogenic differentiation out of nowhere like LSJL.

"When endochondral ossification starts a defined spatial disposition of chondrocytes and a corresponding organization of the intracellular matrix is set up."<-Since another phase of growth occurs before endochondral ossification it would be best to understand that in order to understand how LSJL and the growth plates increase height.

"In the late epiphyseal growth another mechanism is active in addition to endochondral ossification, namely, articular cartilage interstitial growth and subchondral remodeling."<-Being in this stage could affects gains from a height increase routine like LSJL.

"in the early gestational age the first ossification centers in long bones cartilage model appear at level of the diaphysis when the physes were still completely formed by cartilage, but already conforming to the final shape of the proximal or distal end of the bone."<-Location of ossification centers could possibly be influenced by shear strain. The change from chondrocytes to bone doesn't change the shape of the epiphysis'.

"Chondrocytes of physes are small and evenly distributed inside the matrix; their proliferation and the matrix synthesis accounts for the size increments during the pre-ossification phases but without any appreciable change of shape."<-So what increases limb size is the proliferation of chondrocytes and matrix synthesis. Maybe something like Growth Hormone is more effective in this phase and chondrocytes don't have limited proliferative capacity in this stage. If this is true then embryonic stem cells could be of some use in height growth and not just mesenchymal.

Stages of endochondral ossification:

"1. Proliferation, enlargement, and lining up of chondrocytes."
"2. Hypertrophy of chondrocytes."
"3. Calcium Phosphate deposition on the matrix interposed between hypertrophic chondrocytes."
"4. Apoptosis of hypertrophic chondrocytes and vessels penetration between calcified cartilage septa."
"5. Differentiation of osteoblasts and deposition of bone matrix on the calcified cartilage matrix(primary bone trabeculae).
"6. Remodeling of trabeculae from primary to secondary(only lamellar without a core of calcified cartilage)."

Stages 1, 2, and 4 are likely to result in a change of bone shape(increase in size). Apoptosis likely releases force that can cause bone deformation. Calcium Phosphate deposition likely marks the point where the matrix has reached it's peak size.

"In the structured bone epiphysis, the peripheral(side to side) expansion of the ossification center ceases when the proliferative cells layer chondrocytes have reached the periosteum or, on the joint front, the level corresponding to the subchondral bone."<-This likely points to the fact the periosteum is key. Maybe the starting width of the bone plays a role in height growth as that determines how much proliferation chondrocytes undergo. The wider the growth plate the more force that can be exerted when the chondrocytes undergo apoptosis. Any means of expansion of width including exercise could play a role in height growth until the proliferative later chondrocytes have reached the periosteum. However, this likely occurs very early in development likely before 4 years of age.

"IF the density of cells remain evenly distributed, the shape of the model cannot change but only its volume increases."<-If the shape of your bone is changing but you still have actively differentiating growth plates that means LSJL is working because otherwise the shape of your bone should remain the same.

Conclusion: Starting bone width could influence height growth. The wider the base the more force that can be exerted by apoptotic chondrocytes resulting in taller bone. Chondrocytes may not have a defined proliferative capacity at this moment so this could explain GH-induced Gigantism. High levels of GH before year 4 could result in a wider base resulting in more final growth. Measurement method to gauge effectiveness of LSJL before puberty: If bone is changing in shape that indicates influence of LSJL and speaks to it's effectiveness.

Here's some info about how growth plate chondrocytes specifically influence the growth plate. The more influential the chondrocytes are the more promising that proves LSJL could be. If chondrocytes can control a great deal then that means that causing stem cells to differentiate into chondrocytes could form new growth plates.

The growth plate chondrocyte and endochondral ossification.

"Endochondral ossification is the process that results in both the replacement of the embryonic cartilaginous skeleton during organogenesis and the growth of long bones until adult height is achieved. Chondrocytes play a central role in this process, contributing to longitudinal growth through a combination of proliferation, extracellular matrix secretion and hypertrophy[apoptosis likely plays a key role as well]. Terminally differentiated hypertrophic chondrocytes then die, allowing the invasion of a mixture of cells that collectively replace the cartilage tissue with bone tissue. The behaviour of growth plate chondrocytes is tightly regulated at all stages of endochondral ossification by a complex network of interactions between circulating hormones (including growth hormone and thyroid hormone), locally produced growth factors (including Indian hedgehog, WNTs, bone morphogenetic proteins and fibroblast growth factors) and the components of the extracellular matrix secreted by the chondrocytes (including collagens, proteoglycans, thrombospondins and matrilins)[So Hyaluronic Acid and Chondroitin could affect height growth]. In turn, chondrocytes secrete factors that regulate the behaviour of the invading bone cells, including vascular endothelial growth factor and receptor activator of NFκB ligand."

"Intramembranous ossification, which occurs in the flat bones of the skull, involves direct differentiation of embryonic mesenchymal cells into the bone-forming osteoblasts."<-the flat bone of the skull remember is covered by periosteum at the top so it's possible to grow taller there by thickening the periosteum. Also intramembranous ossification shows that it's possible for osteoblasts to increase height

"Ossification of the cartilage model is preceded by hypertrophy of the chondrocytes in the prospective mid-shaft of the bone, and deposition of a periosteal bone collar by recently differentiated osteoblasts surrounding the mid-shaft"<-osteoblasts form the periosteum. How we can use this concept to increase height remains to be seen.

"Blood vessels, osteoclasts (cartilage- and bone-resorbing cells), as well as bone marrow and osteoblast precursors then invade the model from the bone collar and proceed to form the primary centre of ossification."<-so the growth plate originates from the periosteum. Finding out how the osteoblasts form the periosteum and how the periosteum forms the growth plate will be key in finding new ways to grow taller.

"Skeletal maturity occurs when the expanding primary centre of ossification meets the secondary centre of ossification, thus obliterating the growth plate"<-this is an interesting theory. If this is the case then if you just remove the primary center of ossification you would grow forever.

"Following hypertrophy, chondrocytes undergo physiological death, and the transverse septa of the cartilage ECM surrounding them are removed, allowing entry of the mixture of cells responsible for the expansion of the ossification centre."<-this is interesting as it does not mention apoptosis which is likely responsible for physically increasing the size of bone. Perhaps the entry of the mixture of cells is what drives the bone expansion in this model.

"The growth plate chondrocyte constructs the transient growth plate tissue, which has the necessary capacity to move in space through continued self-renewal and localized degradation, but simultaneously maintain the mechanical stability of the growing bone."<-So in this model the growth plate stays the same while the bone grows because of "continued self-renewal and localized degradation" of growth plate tissue. The growth plate continuously remodels to stay the same size while the hypertrophic zone is eventually replaced by bone.

"Accumulating evidence indicates that the growth plate chondrocyte orchestrates the invasion of its own domain by the ossification front not only through preparation of the cartilage tissue, but also by secreting soluble molecules that regulate the behaviour of the invading cells."<-chondrocytes have the ability to form their own growth plates which means that a method that results in new chondrocytes in bone like LSJL could result in new growth plates.

"The growth plate chondrocyte contributes to bone elongation through a combination of proliferation, ECM secretion and hypertrophy. The relative contributions of these parameters vary with growth rate, which varies with anatomical location, age and species; the higher the growth rate the greater the contribution from cellular hypertrophy and the smaller the contribution from matrix synthesis"<-thus you may want less hypertrophy and more matrix synthesis.

"IHH stimulates chondrocyte proliferation through inactivation of the repressor form of Gli3, in particular, as demonstrated by the phenotype of double IHH-null/Gli3-null mice[you may need IHH for LSJL to work or another deactivator of Gli3]. IHH binds to aggrecan through its chondroitin sulphate side-chains, and in the mouse growth plate normal sulphation of chondroitin sulphate is required for normal IHH protein distribution and signaling, and for chondrocyte proliferation. IHH signaling, and thus proliferation of growth plate chondrocytes, is also dependent on the presence of an intact primary cilium[If the freshly differentiated chondrocytes differentiated from stem cells as a result of hydrostatic pressure don't have an intact primary cilium then LSJL will likely not work], a structure consisting of a basal body and a ciliary axoneme that extends several micrometres from the surface of the chondrocyte and most other cells"

"Fibroblast growth factors (FGFs) acting through FGF receptor-3 (FGFR3) are important regulators of chondrocyte proliferation, but activation of this receptor represses proliferation rather than promoting it"<-CNP inhibits FGFR3 which is why CNP stimulates height growth. FGFR3 mutations cause CNP stimulators to not work because CNP must interact with FGFR3 to exert it's height increasing effects.

"Cartilage ECM consists primarily of large aggregates of aggrecan and the glycosaminoglycan, hyaluronan, packed in amongst fibrils of collagen type II"<-Hyaluronic Acid supplementation does increase serum levels of hyaluronan. HA may help increase height due to the contribution of ECM to height growth.

"The collagen fibrils provide the framework for the tissue and the strongly hydrophilic hyaluronan aggrecan aggregates allow the tissue to withstand compression."<-hydrophilic means "water-loving" this is why hydrostatic pressure causes stem cells to differentiate into chondrocytes so they can form hydrophilic tissues to absorb water.

"In addition to the protein components of cartilage ECM, hyaluronan plays an important role in the contribution of ECM secretion to growth. Mice in which the gene for hyaluronan synthase-2 (Has2) is inactivated in tissues derived from limb bud mesoderm possess abnormally short limbs"<-thus taking HA during puberty may help you grow taller. But, only some HA is absorbed and that is divided amongst several tissues -> NOW Foods Hyaluronic Acid 100mg 2X Plus, 60 Vcaps.

"Expression and secretion of components of cartilage ECM, including collagen type II and aggrecan are stimulated by a variety of soluble factors present in the growth plate, including IGF1, BMPs and other members 1 of the TGFβ superfamily, and are absolutely dependent on the transcription factor SOX9"<-inactivation of SOX9 may be a reason for LSJL failure in some cases.

"SOX9-activated transcription appears to be modulated by epigenetic mechanisms[epigentic mechanisms usually involve methylation, telomere length, or possibly phosphorylation], since it occurs predominantly in hyperacetylated chromatin[hyperacetylated chromatin may be a supplement that could induce SOX9 transcription]; the histone acetyltransferase p300 associates with SOX9 and enhances SOX9-dependent transcription. Moreover, inhibition of histone deacetylases (HDACs) stimulates expression of SOX9-activated cartilage ECM genes and induces histone acetylation in the region of the Col2a1 enhancer in primary chondrocyte cultures[another possibility for supplementation would be inhibitors of histone deacetylases]. Over-expression of HDAC1 or 2 in chondrocytes results in down-regulation of expression of Aggrecan and Col2a1, providing further evidence for epigenetic control of this aspect of chondrocyte function"<-Acetyltransferase may also be something worth looking into to grow taller.

"As post-proliferative chondrocytes undergo hypertrophy, they experience changes in gene expression that allow them to modify the structure and composition of the surrounding ECM[the change in the structure of the chondrocytes changes the function of the chondrocyte on the surrounding ECM]. The synthesis of collagen type II is down-regulated and the synthesis of the non-fibrillar collagen type X, expression of which is specific to hypertrophic chondrocytes, is initiated"

"The chondrocytes of MMP-13-null mice undergo apparently normal hypertrophy, indicating that collagen degradation is not necessary. Similarly, aggrecan degradation does not appear to be required."<-thus MMP-13 inhibitors may help you grow taller. Enhanced MMP-13 expression was a key factor behind F-spondin indicating that MMP-13 likely has height reducing effects.

"Results of studies in which growth plate organ cultures were treated with hyaluronidase have provided evidence for a role for hyaluronan in the enlargement of the lacunae of hypertrophic chondrocytes"<-Hyaluronic Acid makes the lacunae of hypertrophic chondrocytes bigger which likely plays a role in the change of overall bone size that's part of height growth.

"Recently proliferated chondrocytes are flattened in the longitudinal axis of the growing bone, and the subsequent increase in cell volume is manifest as a disproportionate increase in height, relative to width of the cell, that is, in the direction of bone growth"<-So the direction of the height growth is based on the shape of the chondrocyte. This could explain why people are growing mainly in width rather than height. The hydrostatic pressure is molding the chondrocytes in a way as to increase width more than height.

"Degradation of the cartilage matrix surrounding growth plate chondrocytes does not appear to be required for hypertrophy, as noted in the section on chondrocyte hypertrophy above, but it is required for invasion of the growth plate by the cells of the centre of ossification."<-thus slowing down degradation of the cartilage matrix will slow down ossification which will allow the growth plates to be active longer. The growth plate chondrocytes will still have the problem of limited proliferative capacity though.

Chondrocytes play a heavy role in the process of endochondral ossification. Their shape, secretion of molecules, all drive the process. Thus a process that can create new chondrocytes like LSJL with it's ability to induce stem cell differentiation into chondrocytes by means of hydrostatic pressure must receive serious consideration.

The growth plate chondrocyte and endochondral ossification.

"Endochondral ossification is the process that results in both the replacement of the embryonic cartilaginous skeleton during organogenesis and the growth of long bones until adult height is achieved. Chondrocytes play a central role in this process, contributing to longitudinal growth through a combination of proliferation, extracellular matrix secretion and hypertrophy. Terminally differentiated hypertrophic chondrocytes then die, allowing the invasion of a mixture of cells that collectively replace the cartilage tissue with bone tissue. The behaviour of growth plate chondrocytes is tightly regulated at all stages of endochondral ossification by a complex network of interactions between circulating hormones (including growth hormone and thyroid hormone), locally produced growth factors (including Indian hedgehog, WNTs, bone morphogenetic proteins and fibroblast growth factors) and the components of the extracellular matrix secreted by the chondrocytes (including collagens, proteoglycans, thrombospondins and matrilins). In turn, chondrocytes secrete factors that regulate the behaviour of the invading bone cells, including vascular endothelial growth factor and receptor activator of NFκB ligand. This review discusses how the growth plate chondrocyte contributes to endochondral ossification, with some emphasis on recent advances."

"chondrocytes undergo proliferation, which is observed as the presence of pairs of chondrocytes in a single lacuna within the cartilage ECM, prior to their separation from each other by secretion of ECM"<-The secretion of ECM separates the chondrocytes. This secretion of ECM may exert a deforming force on the entire bone.

"Following proliferation, the chondrocytes undergo a period of high secretory activity, as they deposit the typical cartilage ECM components around themselves, while remaining in multicellular clusters, often arranged in columns parallel to the long axis of the bone. These cells gradually undergo hypertrophy, modeling their surrounding ECM as they expand, then mineralizing it. Following hypertrophy, chondrocytes undergo physiological death, and the transverse septa of the cartilage ECM surrounding them are removed, allowing entry of the mixture of cells responsible for the expansion of the ossification centre. Thus, the growth plate chondrocyte plays multiple important roles during its lifespan. It constructs the transient growth plate tissue, which has the necessary"<-the type of physiological death is not explained here whether it involves the expulsion of water. Under this model the expansion of the ECM and the expansion of the ossificiation centre could be the factor responsible for the increase in bone size rather than water force.

"Secretion of ECM by growth plate chondrocytes makes an important contribution to growth. Cartilage ECM consists primarily of large aggregates of aggrecan and the glycosaminoglycan, hyaluronan, packed in amongst fibrils of collagen type II. These three components of cartilage ECM confer on the growth plate the mechanical stability required by this integral component of the growing skeleton. The collagen fibrils provide the framework for the tissue and the strongly hydrophilic hyaluronan-aggrecan aggregates allow the tissue to withstand compression. Both collagen type II and aggrecan are almost exclusively expressed in cartilage[Meaning these are good markers of chondrogenic differentiation]."

"The chondrocytes of MMP-13-null mice undergo apparently normal hypertrophy, indicating that collagen degradation is not necessary"<-thus MMP-13 inhibition may be a way to grow taller.

"the role of hyaluronan may be to initiate hypertrophy-inducing intracellular signaling in chondrocytes emerging from the proliferative phase"<-Thus Hyaluronic Acid may help you grow taller.

"Given the importance of hyaluronan-aggrecan complexes for swelling of the growth plate ECM, however, it is likely that this swelling per se is required for the concomitant expansion of the chondrocyte."<-Swelling indicates the importance of water.

"Hydroxyapatite crystals (comprised primarily of calcium and phosphate) are deposited in the ECM surrounding late hypertrophic chondrocytes. The matrix vesicles released by these cells contain a combination of proteins including phosphate transporters, phosphatases and annexins, and provide the nucleation site for mineralization"

"Most hypertrophic chondrocytes appear to undergo rapid death in the last row of lacunae before the ossification front. A number of publications have described these cells as dying by apoptosis, but the evidence for this conclusion is based on the detection of molecular features known to be associated with apoptosis, such as DNA strand breaks and caspase activation, rather than on the more definitive morphological changes observed on ultrastructural examination"<-so it can not be definitively said that chondrocytes undergo apoptosis.

"Cells undergoing apoptosis show intense condensation of chromatin into geometric shapes, and fragmentation of the nucleus and cytoplasm into membrane-bound apoptotic bodies. A number of careful ultrastructural studies have failed to identify chondrocytes undergoing apoptosis in growth plates of several species"<-failure to identify apoptosis does not mean it does occur.

"Each of these cell types undergoes a distinctive series of morphological changes following hypertrophy: light chondrocytes appear to disintegrate within their cell membrane and dark chondrocytes progressively extrude their cytoplasm into the extracellular space. Nuclear condensation occurs very late and is irregular."<-light chondrocytes disintegrating within their cell membrane would not exert a bone deforming force. However dark chondrocytes extude their cytoplasm could.

Here's how the growth plates relate to the cortical bone:

Cortical bone development under the growth plate is regulated by mechanical load transfer

"Longitudinal growth of long bones takes place at the growth plates. The growth plate produces new bone trabeculae, which are later resorbed or merged into the cortical shell. This process implies transition of trabecular metaphyseal sections into diaphyseal sections[so parts of the bone that were formerly part of the epiphysis can turn into part of the diaphysis]. We hypothesize that the development of cortical bone is governed by mechanical stimuli. We also hypothesize that trabecular and cortical bone share the same regulatory mechanisms for adaptation to mechanical loads. To test these hypotheses, we monitored the development of the tibial cortex in growing pigs, using micro-computer tomography and histology. We then tested the concept that regulatory mechanisms for trabecular bone adaptation can also explain cortical bone development using our mechanical stimulation theory, which could explain trabecular bone (re)modelling. The main results showed that, from the growth plate towards the diaphysis, the pores of the trabecular structure were gradually filled in with bone, which resulted in increased density and cortical bone. The computer model largely predicted this morphological development. We conclude that merging of metaphyseal trabeculae into cortex is likely to be governed by mechanical stimuli[Hiroki Yokota once mentioned that LSJL thickened cortex]. Furthermore, cortex development of growing long bones can be explained as a form of trabecular bone adaptation, without the need for different regulatory mechanisms for cortical and trabecular bone."

So trabecular bone is just cortical bone that hasn't experienced enough mechanical load. Remember that the epiphysis doesn't usually bear load unless loaded laterally like with LSJL. Growth plates form new trabeculae below the growth plate.

"both trabecular and cortical bone are formed and resorbed by the same cells, the osteoblasts and osteoclasts. Moreover, it is believed that osteocytes are mechanosensitive through extracellular fluid flow, which in theory can be applied to both trabecular and cortical bone"

"Growth plates cause longitudinal growth by producing new trabeculae that are subsequently resorbed or merged into the cortical shell[forming new growth plates like by LSJL can form new trabeculae]. Considered in a longitudinal spatial–temporal context, the process of cortical bone development implies transition of trabecular metaphyseal sections into cortical diaphyseal sections. At the metaphyseal level, the cortex consists of younger bone compared with the cortex at diaphyseal level as a result of new bone production from the growth plate. This means that the cumulative number of loading cycles to which bone is subjected over time increases from the growth plate towards the diaphysis."<-the reason that diaphyseal bone is different from epiphyseal bone is that it is subjected to more loading cycles.

"At 6 weeks, at growth plate level, the cortex consisted of trabecular bone. At the metaphyseal level, a cortex was present, but it could barely be distinguished from trabecular bone. At the diaphyseal level, a cortex was clearly present; the endosteal surface was irregular owing to trabeculae merging into the cortex and osteoclastic bone resorption. At the periosteal side, circumferential[meaning outside the bone] plexiform[meaning part of a network] plates were present, which were interconnected by radially orientated bone rods"

"The pores in, for example, the diaphysis at 6 weeks were mainly orientated in the longitudinal direction; their structure was very irregular with many branches that were not perpendicular to each other[meaning that growth from the growth plate is not perfectly structured either]. The pores, i.e. vascular canals being incorporated to form primary osteons, were mainly orientated parallel to the long bone axis"

"Mechanical loading is not considered to be the direct stimulus for bone remodelling but rather the stimulus that determines the expression of biochemical signalling molecules. The local concentration of this biochemical factor is subsequently compared with a set-point value at the bone surface. Upon exceeding a certain threshold, local bone apposition occurs."<-so there may be a conditioning effect that reduces the concentration of the biochemical factors in response to the same loads.

Skeletal dysplasias and the growth plate.

"The zone of polarizing activity plays a role that regulates anterior–posterior patterning, the apical ecdodermal ride regulates shape of the developing limb, and the dorsal ectoderm regulates dorsal–ventral patterning. The mesodermal cells differentiate to form the cartilaginous template of the long bones. Blood vessels invade the central portion of the cartilaginous template and a primary ossification center forms. Secondary ossification centers form on each end of the bone, leaving cartilage at the ends of the bones that ultimately becomes the articular cartilage, and cartilage between the ossification regions that become the growth plate"<-the zone of polarizing activity, the apical ecdodermal rode, and the dorsal ectoderm are things to investigate in terms of growing taller.

"One such factor is the transcription factor SOX9 that is expressed in mesenchymal cells as they transform to chondrocytes. Studies in knockout mice show that this transcription factor is necessary for pleuripotential mesenchymal cells to become chondrocytes precursors. SOX9 activates the expression of a variety of genes that important in chondrocyte function, such as type II collagen. Fibroblast growth factor signaling plays an important role in a variety of growth plate chondrocytes functions but has a major role regulating chondrocyte proliferation. There are a variety of fibroblast growth factor ligands and receptors expressed in the developing chondrocytes and the growth plate, suggesting that this signaling pathway is crucial to chondrocyte function. As the growth plate chondrocytes mature, parathyroid hormone-related protein (PTHrP) and Indian hedgehog act in a feedback loop regulating chondrocyte differentiation. Cells in the pre-hypertrophic zone of the growth plate express the secreted protein Indian hedgehog"<-Sox9 is not expressed during LSJL and this could be a problem.

Here's a study that reviews the process during endochondral ossification:

Histology of epiphyseal cartilage calcification and endochondral ossification

"Cartilage calcification is carried out by chondrocytes as they hypertrophy and begin to secrete matrix vesicles. Calcification initiates when calcium phosphates appear inside these matrix vesicles, forming hydroxyapatite crystals that eventually break through the membrane to form calcifying globules, as in bone calcification[this is the first term I've heard endochondral ossification describe this way where hypertrophy chondrocytes secrete calcium phosphate crystals]. However, the extracellular environment in cartilage is different from that in bone:cartilage is abundant in proteoglycans but contains a small amount of osteopontin. Hypertrophic chondrocytes secrete vesicles in the cartilaginous matrix of intercolumnar septae only, forming well-calcified longitudinal septae and poorly-calcified transverse partitions. Such pattern of vesicledeposition permits the invasion of endothelial cells, which infiltrate into cartilage and induce migration of osteogenic and osteoclastic cells. Osteoclasts resorb the excess of calcified globules in the partitions, shaping calcified cartilage cores paralleling the longitudinal axis of long bones. After the formation of these calcified cartilage cores, endochondral ossification involves a series of well-defined events in which osteogenic cells deposit new bone onto the cartilage core and form primary trabecules. This review presents the histology of epiphyseal cartilage calcification and endochondral ossification."

"It is generally accepted that the first clue of the start of a center of ossification in a developing cartilage is hypertrophy of chondrocytes residing in the middle portion of the hyaline cartilage shaft[it's interesting that it forms in the center whereas at the epiphyseal growth plates the hypertrophic chondrocytes are at the end]. This finding is consistent with the enlargement of their lacunae at the expense of the intervening cartilaginous matrix. The matrix remaining in the region of the hypertrophic zone becomes calcified. The osteogenic capability of cells from the perichondrium covering the mid-portion of the shaft is subsequently activated. These cells then launch the process of intramembranous ossification. Osteoclasts invade the calcified intramembranous bone inside the mid-shaft cartilaginous anlage, accompanied by migrating blood vessels and osteogenic cells. After that, osteogenic cells differentiate into osteoblasts and start the deposition of new bone in the former hypertrophic zone. The cartilage anlage will then depict cartilaginous extremities sandwiching newly-formed bone, and will be termed epiphyseal cartilage."<-Maybe this initial stage of forming a middle hypertrophic cartilage center and then separating into two epiphyseal cartilages is necessary to form an epiphyseal growth plate. Of course, the center of the circle when the diameter of the center of the circle is stretched into a straight line is the end of the cricle. So, this circular form of endochondral ossification could be the same as epiphyseal ossification but just in a different shape. Or this different type of endochondral ossification could be some kind of regulatory mechanism so you don't form a bone within a bone or fuse two bones together.

"Chondrocyte precursors differentiate into chondrocytes and start secreting cartilaginous matrix abundantly, in a process known as appositional growth[<-increasing the number of chondrocyte precursors during development would be one way to grow taller]. Meanwhile, differentiated chondrocytes may undergo cell division and synthesize cartilaginous matrix simultaneously, in what is termed interstitial growth[appositional is growth from without so the chondrocytes expand the area outwardly, interistitial growth is growth from within the structure itself; it's possible that only appositional growth results in an increase in organism size whereas interstitial growth only increases bone quality; however sufficient interstitial growth should increase size(height). But appositional growth should increase height more(so stem cells differentiating into chondrocytes)]. The normal development and growth of endochondral bone is dependent on a balance between proliferation and differentiation of chondrocytes and the consequent appositional and interstitial growth; it also depends on effective vascular invasion allowing space and nutrition for bone matrix deposition. The spatial progression of chondrocytic differentiation is extremely important for enabling endochondral ossification[so we have to make sure that the genes that allow for this spatial expression are sufficiently expressed likely something like Sox9]. Chondrocytes scattered throughout the resting zone show enlarged cisterns ofrough endoplasmic reticulum (rER), accumulated glycogen granules and round cell shape. They will become proliferative chondrocytes, which align in the longitudinal columns known as "stacked coins". Then, proliferative chondrocytes differentiate into the hypertrophic phenotype, which features an enlarged and translucent cell body and express collagen type I and X, alkaline phosphatase (ALPase), proteoglycans and osteopontin."

"The extracellular matrices, as an ion reservoir, may constitute the adequate microenvironment for the initiation of calcification. Proteoglycans are complexes of various glycosaminoglycan (GAG) chains and core proteins. The GAG chains are highly negatively charged, which can attract free divalent cations such as Ca2+. It seems likely that proteoglycans significantly impact the dynamics of the extracellular fluid's mineral ionic content[this proteoglycan ability to manipulate extracellular fluid mineral ionic content is likely why chondrocytes can help you grow taller whereas bone cannot]. Since cartilage contains abundant proteoglycans,cartilage matrices may serve as a reservoir for free divalent cations. Especially, crystal ghosts appear to be rich in sulfated GAG chains, especially, chondroitin sulfate."

"In cartilage, small leucine-rich proteoglycans -- decorin, biglycan, fibromodulin, lumican, andepiphycan, also referred to as PG-Lb -- are present. In situ hybridization andimmunohistochemistry have verified that hypertrophic chondrocytes in developing epiphyseal growth plate cartilage express high levels of biglycan mRNA. Immunoelectron microscopy of the growth plate revealed that the prominent immunolabelling was confined to the Golgi apparatus and cisternae of rER of hypertrophic chondrocytes, and to the early calcified cartilage matrices of the longitudinal septum of the lower hypertrophic zone[the proteoglycans go away as ossification occurs]. Therefore, proteoglycans in the extracellular matrix of the lower hypertrophic zone may be degraded by proteases and removed before calcification, and this seems to be the mechanism by which a matrix that does not possess the ability to calcify is transformed into one that has that capability[this process could also change the osmotic balance in the growth plate being a source of energy with which to generate height growth]. Interestingly, however, the concentration of sulfate or cartilage proteoglycans was not shown to change before and after cartilage calcification. Decorin/biglycan-double knockout mice revealed osteopenia as a result of impaired GAG-linking to decorin- and biglycan-core proteins, whereas calcification was unaffected. Collagen calcification based on the proposed process of removal of small proteoglycans before calcification may deserve further investigation, at least in cartilage."<-so biglycan and decorin may be a key to growing taller. However, knockout mice only had reduce bone density and not reduced height growth.

"According to observations of the osteoid in bone derived from the quick frozen-freeze substitutiontechnique with electron energy loss spectroscopy (EELS), which enables elemental mapping at the molecular level, calcium was primarily localized to proteoglycans, whereas phosphate was predominantly localized to collagen fibrils. Therefore, even if the extracellular fluid as a whole is supersaturated with Ca2+ and PO43−, it seems feasible that, in non-calcified sites, the extracellular meshwork of organic substances limits the production of hydroxyapatite and inhibit sprecipitation of calcified crystals by controlling the spatial distribution of Ca2+ and PO43−. However, these findings were obtained from osteoid in bone, and localization of calcium and phosphate in the cartilage matrix using TEM-EELS is still in progress."<-so proteoglycans mainly serve to attract calcium thus why mice with knockouts of proteoglycans would suffer from reduced bone mineralization. So, maybe proteoglycans attracting water doesn't affect height growth.

"We postulate that osteoclasts/chondroclasts could resorb the excessive transverse accumulations of calcifying globules in order to align the calcified cartilage parallel to the longitudinal axis of the long bone, after hypertrophic chondrocytes calcify the longitudinal intercolumnar regions. This supposition is based on the observation that osteoclasts/chondroclasts do not resorb the calcified cartilage matrix completely, so that migrating osteoblasts can independently form mixed spicules of cartilage and bone."<-so the job of the osteoclasts is likely to help make sure that the calcified cartilage is aligned with the bone so the bone doesn't grow in a dysfunctional manner.

"vascular invasion rather than osteoclastic resorption is dominant for endochondral ossification"

"without osteoclasts, long bones do grow and elongate; however, there is a picture of mixed spicules with central cartilage cores and bone matrix in the periphery, forming a disorganized meshwork with intense trabecular connectivity."<-so osteoclasts organize the bone and vascular evasion by a compound such as VEGF(which is related to estrogen) mainly affects bone growth and elongation.

"chondrocytes in the terminal region of the hypertrophic zone undergo apoptosis as their lacunae are pierced by invading blood vessels "<-So that would mean that VEGF(and in turn estrogen) highly regulates growth plate apoptosis.

"MMP-9, a gelatinase that degrades components of the cartilaginous extracellular matrix with high specificity for degraded collagens, plays a key role in endochondral ossification, specifically capillary invasion into hypertrophic cartilage."<-if capillary invasion affects height growth by means of controlling apoptosis, then inhibiting MMP-9 may elongate height growth.

However, both MMP-9 and MMP-13 deletion showed no effect on height growth.

"MT1-MMP-/-mice revealed deficient vascularization during secondary ossification of epiphyseal cartilage and growth plate development."<-And dwarfism, so it's possible that enhancing MT1-MMP may enhance height growth. MT1-MMP has been shown to be upregulated in response to dynamic compression of chondrocytes. MT1-MMP also helps form cartilage canals.

"MT1-MMP-dependent dissolution of uncalcified cartilages, coupled with apoptosis of non-hypertrophicchondrocytes, mediates remodeling of these cartilages into other tissues. Therefore, MT1-MMP appears to be important for removal of uncalcified cartilage in individual growth."<-Alternatively, that could play a role in why MT1-MMP affects height growth.

Growth plate columns are often referred to as stacked coins. It's possible that the hypertrophic chondrocytes act as a base to provide force against the bone whereas the weaker small coins of the proliferating chondrocytes allow for division and growth. So the hypertrophic chondrocytes provide the force(secreting cartilagenous matrix) while the smaller proliferating chondrocytes provide the growth.

Imagine you have a bunch of quarters between two bricks. You want to increase the space between the two bricks. You wouldn't try to squeeze in more quarters, you'd try to put in more pennies. The quarters of course secreting matrix that lifts the bricks up allowing for the pennies to divide and the pennies eventually growing into quarters.

So this could be a possibility of why osteoblasts don't make us taller. Osteoblasts can proliferate and hypertrophy just like chondrocytes but they don't make us taller. This procedure is not as well coupled as chondrocyte proliferation and hypertrophy is. If osteoblasts were stacked into columns then perhaps they could make us taller.

According to this hypothesis, delaying apoptosis of the hypertrophic chondrocytes wouldn't make us grow taller forever and this is what has been confirmed by analysis of estrogen where too high and low estrogen are bad for growth. Hypertrophic chondrocytes likely can't provide enough force to push against two bones indefinitely and you'd need the hypertrophic chondrocyte layer to become bone(or brick) eventually to prevent "collapse" So to maximize height you'd want an equilibrium quantity of estrogen to ensure that the optimal amount of Force is generated. Extremely high levels of estrogen cause reduced growth likely because the hypertrophic chondrocytes undergo apoptosis is optimal. Too low levels of estrogen causes reduced growth by reducing chondrocyte proliferation. This goes with our hypothesis as without estrogen triggering VEGF the hypertrophic chondrocyte layer will become large and unwieldy thus the body uses negative feedback to inhibit chondrocyte proliferation.

The study "Epiphyseal plate transplantation between sites of different growth potential." reported "As growth progressed, we saw distraction of the metatarsal epiphyseal plate, similar to the clinical and experimental cases reported." This shows evidence that the growth plate is capable of exerting a distraction force on the bone environment. The study also reported that growth of the plate is dependent on the growth plate itself rather than where the growth plate is located. The cases mentioned showed that the growth plate itself distracted in response to a distractive force being place on it. Slightly different that then the study here where growth plates where transplanted, but it still shows the potential of the growth plate to exert force on the surrounding bone to create more room that is to distract.

The study "Free microvascular epiphyseal-plate transplantation. An experimental study in dogs." involved transplanting multiple growth plates into sites that did not normally have growth plates and found longitudinal growth in all cases. Therefore, it's likely that a method designed to create new growth plates via stem cell differentiation into chondrocytes like LSJL is likely to be effective if it can in fact induce chondrogenesis.

Grow Taller with Ascorbic Acid(Vitamin C)

2011-12-28T11:14:00.000-08:00

Previously, we talked about the potential of vitamins augmenting potential height growth methods. Now, we find a study that shows the ability of Ascorbic Acid to directly induce chondrogenic differentation.

The cell line used in the study is ATDC5 which is a rat cell line so human chondrocyte cells might have different results.

The mechanism of ascorbic acid-induced differentiation of ATDC5 chondrogenic cells.

"The ATDC5 cell line exhibits a multistep process of chondrogenic differentiation analogous to that observed during endochondral bone formation. Previous investigators have induced ATDC5 cells to differentiate by exposing them to insulin at high concentrations. We have observed spontaneous differentiation of ATDC5 cells maintained in ascorbic acid-containing α-MEM[so it has to be Vitamin C containing MEM alpha, MEM stands for Minimum Essentail Medium a listing of what the contains can be found here, none of the compounds would be anything particularly unusual to find in the body except for something like phenol red which is used more as a measurement tool]. A comparison of the differentiation events in response to high-dose insulin vs. ascorbic acid showed similar expression patterns of key genes, including collagen II, Runx2, Sox9, Indian hedgehog, and collagen X. We took advantage of the action of ascorbic acid to examine signaling events associated with differentiation. In contrast to high-dose insulin, which downregulates both IGF-I and insulin receptors, there were only minimal changes in the abundance of these receptors during ascorbic acid-induced differentiation[so ascorbic acid is better than Insulin as it doesn't impact IGF-1 and insulin receptors]. Furthermore, ascorbic acid exposure was associated with ERK activation[so this leads us to believe that activating ERK increases chondrogenic differentiation and may help us to grow taller], and ERK inhibition attenuated ascorbic acid-induced differentiation. This was in contrast to the inhibitory effect of ERK activation during IGF-I-induced differentiation. Inhibition of collagen formation with a proline analog markedly attenuated the differentiating effect of ascorbic acid on ATDC5 cells. When plates were conditioned with ATDC5 cells exposed to ascorbic acid, ATDC5 cells were able to differentiate in the absence of ascorbic acid. Our results indicate that matrix formation early in the differentiation process is essential for ascorbic acid-induced ATDC5 differentiation. We conclude that ascorbic acid can promote the differentiation of ATDC5 cells by promoting the formation of collagenous matrix and that matrix formation mediates activation of the ERK signaling pathway[so forming the cartilage matrix is key to early height growth], which promotes the differentiation program."

"At the growth plate, ascorbic acid deficiency can result in decreased chondrocyte proliferation, impaired matrix synthesis, and a reduction in osteoblast cell number"

"ERK activation can be mediated by the cross-linking of extracellular matrix with integrins. In human chondrocytes, the interaction of collagen II with the β1-integrin receptor has been shown to activate ERK" <-So injecting type II collagen into the bone and increasing serum levels of the Beta1-integrin receptor may be one way to grow taller.

"Given that ascorbic acid is required for the formation of collagen triple helices, we hypothesized that the ability of ascorbic acid to induce ATDC5 cell differentiation depends on its ability to promote synthesis of collagen matrix."<-So amounts of ascorbic acid greater than that required to form collagen triple helices would be useless.

"Ascorbic acid stimulates procollagen hydroxylation and processing and is required for collagen fibril assembly and collagen secretion"<-collagen hydroxylation is the addition of hydroxyl groups to the amino acids proline or lysine. Supranormal levels of ascorbic acid, proline, and lysine could stimulation supranormal collagen fibril assembly and collagen secretion.

"The rate-limiting step in this overall process is the hydroxylation and secretion of unprocessed procollagen chains that accumulate in the endoplasmic reticulum of ascorbic acid-deficient cells"<-so you have to have enough Vitamin C such that no cell is definition to maximize height growth.

"collagen II hydrogels could stimulate the differentiation of bone marrow mesenchymal stem cells in the absence of growth factors."<-Thus injecting type II collagen hydrogels into your bone marrow could make you taller.

Since the compounds of Vitamin C and the other compounds of the medium are so readily available in the body it's likely to be a problem with the stem cells themselves that's inhibiting chondrogenic differentiation.

Thus study was done on adult stem cells:

Effects of osteogenic differentiation inducers on in vitro expanded adult mesenchymal stromalcells.

"Aim of this study was to analyze the role of the biochemical osteogenic inducers, i.e. ascorbic acid, dexamethasone, and ß-glycerophosphate, employed in the current protocols for osteogenic differentiation of MSC in vitro, to address the requirements for reliable differentiation systems.?Methods: MSC were isolated from the bone marrow of donors (46-73 years of age) undergoing total hip replacement, and expanded in vitro. At confluence, MSC were cultured under four different conditions: a-MEM plus serum (basal medium or C1), basal medium plus ascorbate (C2), basal medium plus ascorbate and dexamethasone (C3), or basal medium plus ascorbate, dexamethasone and ß-glycerophosphate (C4). Morphology, proliferation, mineralization, alkaline phosphatase, collagen and expression of bone-related genes of MSC under the different media were analyzed at fixed time points.?Results: MSC proliferation and the number of colony forming units were increased by ascorbic acid, whereas dexamethasone enhanced the proportion of ALP-positive CFU and was critical for mineral deposition. Runx-2 and type I collagen gene expression decreased along with additive-induced MSC differentiation[we don't want Runx-2 and type I collagen as that is osteogenic while we want chondrogenic so we would not want Beta-glyerophosphate(dexamethasone has been found to have potential height decreasing effects)], i.e. from C1 to C4, while ALP and osteocalcin were differently regulated.?Conclusion: Our findings support the role of different inducers on the sequential stages of MSC expansion and osteogenic differentiation in vitro, suggesting the addition of DEX following proliferation to ensure mineralization, as an index of in vivo osteogenic potency of human mesenchymal cells."

Chondrogenic markers were not measured in this study. However, using a different cell line produced very different results in regards to Vitamin C.

Here's a study that shows that age doesn't affect stem cell differentiation however:

Human mesenchymal stem cell proliferation and osteogenic differentiation during long-term ex vivo cultivation is not age dependent.

"Mesenchymal stem cells (MSCs) are of major clinical interest for the development of cell-based strategies to treat musculoskeletal diseases including critical-size bone defects caused by trauma, degenerative disorders, or infections. Elderly people mainly suffer from critical-size bone defects from the rising incidence of trauma, osteoporosis, and arthroplasties. In this study we investigated the influence of donor age on proliferation and osteogenic differentiation in long-term ex vivo cultures of primary human MSCs from patients in different age groups. Fifteen patients (8 men/7 women) comprised three age groups: (I) <50 years, (II) 50-65 years, and (III) >65 years. MSCs harvested from bone marrow derived from routine surgical procedures were isolated and cultured in standard medium over eight passages. Osteogenic differentiation was induced by dexamethasone (10 nM), ascorbic acid (300 μM), and β-glycerophosphate (3.5 mM). Osteogenic differentiation capacity of MSCs was quantified by alkaline phosphatase (ALP) activity, fluorescence-activated cell sorting (FACS) analysis of the surface markers CD9, CD90, CD54, CD166, CD105, CD44, and CD73, and RT-PCR for Coll I and II, Cbfa 1, ALP, OC, BSP1, and GAPDH genes characterized the phenotypic changes during monolayer expansion. In vitro chondrogenic differentiation was analyzed by immunohistochemistry and RT-PCR[chondrogenic differentiation was studied]. Progenitor cells could be expanded in the long term from all bone marrow donations. FACS single staining analysis from MSCs showed no significant difference between the age groups. The surface antigen CD166 was predominantly found in all cell cultures independently of differentiation stage. Comparison of expanded and differentiated MSCs within a single age group showed that undifferentiated MSCs had higher CD44 levels. Osteogenic stimulation of MSCs was confirmed by measuring ALP activity. The highest ALP activity was found in probands of the age group >65 years. Additionally, we observed a tendency toward male-specific ALP increase during differentiation. Osteogenic marker gene expression in MSCs was detected by RT-PCR. No significant expression differences were detected between the three donor age groups. Micromass culture of MSCs resulted histologically and immunohistologically in a chondrogenic phenotype[This page lists the micromass protocol for MSCs to generate chondrocytes]. Elderly osteoprogenitor cell donors are a highly clinically relevant patient population. In summary, cultivation leads to a reduced osteogenic differentiation capacity regardless of age. Because donor age does not affect osteogenic differentiation potential, it should not be used as an exclusion criterion for autologous transplantation of human adult MSCs."

"Pellet cultures of MSCs resulted in the formation of dense nodules consistent with chondrogenic differentiation."<-So forming a pellet culture is most important in inducing chondrogenesis. Can hydrostatic pressure like that induced by LSJL result in the MSC formation of pellet cultures?

So Vitamin C enhances both chondrogenic and osteogenic differentiation. Age does not seem to affect whether differentiation is headed towards an osteogenic or chondrogenic path but rather how the stem cells are cultured. Hydrostatic pressure may change the culture within the bone marrow to be more chondrogenic.

Increasing serum levels of Vitamin C, proline, and lysine may increase height but there may be negative feed mechanisms and points of diminishing returns.

Twin Studies indicate the likelihood of environmental factors influencing height growth

2011-12-23T11:23:00.000-08:00

Even if height is 100% genetic, genes are still manipulable through DNA Methylation status, telomere length, chromatin folding, and histone acetylation. Things like mechanical stimulation, hydrostatic pressure, and shear strain like that induced by Lateral Synovial Joint Loading are capable of upregulating and downregulating genes. So too are chemicals capable of upregulating and downregulating genes. Leptin for instance stimulates the PI3K pathway.

Twin studies can provide us insight on environmental factors that can effect height. Even if height is determined solely by genetics, environmental factors can manipulate epigentic factors and genetic regulation. If height is found to be 20% environmental in twin studies where twins engage in normal activities that means that supranormal activities like LSJL may affect height much more. And remember bone is a substance and that means it shares the properties of all substances like being capable of being stretched.

An assessment of the individual and collective effects of variants on height using twins and a developmentally informative study design.

"In a sample of 3,187 twins and 3,294 of their parents, we sought to investigate association of both individual variants and a genotype-based heightscore involving 176 of the 180 common genetic variants with adult height identified recently by the GIANT consortium. First, longitudinal observations on height spanning pre-adolescence through adulthood in the twin sample allowed us to investigate the separate effects of the previously identified SNPs on pre-pubertal height and pubertal growth spurt. We show that the effect of SNPs identified by the GIANT consortium is primarily on prepubertal height[so genes mainly influence height prepuberty? intersting]. Only one SNP, rs7759938 in LIN28B, approached a significant association with pubertal growth. Second, we show how using the twin data to control statistically for environmental variance can provide insight into the ultimate magnitude of SNP effects and consequently the genetic architecture of a phenotype. Specifically, we computed a genetic score by weighting SNPs according to their effects as assessed via meta-analysis. This weighted score accounted for 9.2% of the phenotypic variance in height, but 14.3% of the corresponding genetic variance. Longitudinal samples will be needed to understand the developmental context of common genetic variants identified through GWAS, while genetically informative designs will be helpful in accurately characterizing the extent to which these variants account for genetic, and not just phenotypic, variance."

"Twin and adoption studies suggest that height is highly heritable (∼80%)"<-20% however is a very large amount of unheritable variance.

Interestingly, according to table 1 there's a .25 cm increase in height males between age 20 and 29(however there are so many other possibilities for this increase rather than a .25cm growth in adult males between 20 and 29, note for instance how females show an average height reduction between 25 and 29).

"Shared environmental effects accounted for 9% (0%, 27%) and 11% (0%, 31%) of the variance in the intercept and slope, respectively"<-environment accounts for around 10% of height in normal cases and in extraordinary cases that could be much more like with extreme loading or by LSJL.

Heritability of adult body height: a comparative study of twin cohorts in eight countries.

"A major component of variation in body height is due to genetic differences, but environmental factors have a substantial contributory effect. In this study we aimed to analyse whether the genetic architecture of body height varies between affluent western societies. We analysed twin data from eight countries comprising 30,111 complete twin pairs by using the univariate genetic model of the Mx statistical package. Body height and zygosity were self-reported in seven populations[this could be a problem as people with higher self esteem could report their height as higher] and measured directly in one population[we still have this data though]. We found that there was substantial variation in mean body height between countries; body height was least in Italy (177 cm in men and 163 cm in women) and greatest in the Netherlands (184 cm and 171 cm, respectively). In men there was no corresponding variation in heritability of body height, heritability estimates ranging from 0.87 to 0.93 in populations under an additive genes/unique environment (AE) model. Among women the heritability estimates were generally lower than among men with greater variation between countries, ranging from 0.68 to 0.84 when an additive genes/shared environment/unique environment (ACE) model was used. In four populations where an AE model fit equally well or better, heritability ranged from 0.89 to 0.93. This difference between the sexes was mainly due to the effect of the shared environmental component of variance, which appears to be more important among women than among men in our study populations. Our results indicate that, in general, there are only minor differences in the genetic architecture of height between affluent Caucasian populations, especially among men."

"Twin correlations for MZ male pairs were uniformly high in all countries, ranging from 0.87 to 0.94 in male
and from 0.84 to 0.94 in female MZ pairs"<-Still however a significant part that is not correlated between twins.

"Heritability of body height is lower among women than among men and there is also greater geographic variation in the heritability estimates among women"<-The study also found little effect of sex-linked characteristics on height so it may be an estrogen production factor. High levels of estrogen can cause apoptosis of growth plate chondrocytes and estrogen may be influenced by environmental factors.

This study found that genetic factors account for 90% when self-reporting bias is removed. 10% is still a large place for enivornmental factors however and gives the possibility for even more environmental influence when you have extraordinary stimulation like LSJL.

Bias, precision and heritability of self-reported and clinically measured height in Australian twins.

"Many studies of quantitative and disease traits in human genetics rely upon self-reported measures. Such measures are based on questionnaires or interviews and are often cheaper and more readily available than alternatives. However, the precision and potential bias cannot usually be assessed. Here we report a detailed quantitative genetic analysis of stature. We characterise the degree of measurement error by utilising a large sample of Australian twin pairs (857 MZ, 815 DZ) with both clinical and self-reported measures of height. Self-report height measurements are shown to be more variable than clinical measures. This has led to lowered estimates of heritability in many previous studies of stature. In our twin sample the heritability estimate for clinical height exceeded 90%. Repeated measures analysis shows that 2-3 times as many self-report measures are required to recover heritability estimates similar to those obtained from clinical measures. Bivariate genetic repeated measures analysis of self-report and clinical height measures showed an additive genetic correlation >0.98[well still 2% is environmental]. We show that the accuracy of self-report height is upwardly biased in older individuals and in individuals of short stature. By comparing clinical and self-report measures we also showed that there was a genetic component to females systematically reporting their height incorrectly; this phenomenon appeared to not be present in males. The results from the measurement error analysis were subsequently used to assess the effects of error on the power to detect linkage in a genome scan. Moderate reduction in error (through the use of accurate clinical or multiple self-report measures) increased the effective sample size by 22%; elimination of measurement error led to increases in effective sample size of 41%."

"This corresponds to a decline in height of 4.5 cm over a 40 year period"<-So posture correction and stimulating cartilage growth in the discs of the spine can restore height of at least 2 inches.

This study however found a higher percentage of environmental factors affected height. It used self reported height but it measured the accuracy of self-reported height against height measurements.

Genetic and environmental influences on growth from late childhood to adulthood: a longitudinal study of two Finnish twin cohorts.

"Two cohorts of monozygotic and dizygotic (same sex and opposite sex) Finnish twin pairs were studied longitudinally using self-reported height at 11-12, 14, and 17 years and adult age (FinnTwin12) and at 16, 17, and 18 years and adult age (FinnTwin16). Univariate and multivariate variance component models for twin data were used.
From childhood to adulthood, genetic differences explained 72-81% of the variation of height in boys and 65-86% in girls. Environmentalfactors common to co-twins explained 5-23% of the variation of height, with the residual variation explained by environmental factors unique to each twin individual. Common environmental factors affecting height were highly correlated between the analyzed ages (0.72-0.99 and 0.91-1.00 for boys and girls, respectively). Genetic (0.58-0.99 and 0.70-0.99, respectively) and unique environmental factors (0.32-0.78 and 0.54-0.82, respectively)[this is what we're looking more for as unique environmental factors we can alter] affecting height at different ages were more weakly, but still substantially, correlated.
The genetic contribution to height is strong during adolescence. The high genetic correlations detected across the ages encourage further efforts to identify genes affecting growth. Common and unique environmental factors affecting height during adolescence are also important, and further studies are necessary to identify their nature and test whether they interact with genetic factors."

"Reliability of self-reported height, analyzed in sub-samples of both twin cohorts after completion of the last wave of questionnaires, was found highly correlated with measured height in FinnTwin12 (r = 0.99, N = 797) and FinnTwin16 (r = 0.99, N = 566) "

"Nutrition is universally the most important environmental factor affecting growth. For example, milk [likely due to lactoferrin and IGF-1] consumption has been found to be positively associated with height in children and adults"

"growth before puberty is the main determinant of adult stature"<-Thus why some believe you should delay puberty to grow taller.

Here's one study that shows that the environmental factor muscle loading may affect foot length.

Association between foot growth and musculoskeletal loading in children with Prader-Willi syndrome before and during growth hormone treatment.

"In 37 children with PWS, foot length (FL) before and after 6 years of growth hormone therapy (GHT) was retrospectively evaluated with parental and sibling's FL, height, and factors reflecting musculoskeletal loading, such as weight for height (WfH), lean body mass (LBM; dual energy X-ray absorptiometry, deuterium labeled water), physical activity (accellerometry), and walk age. Because of the typically biphasic evolution of body mass and the late walk age in PWS, 2 age groups were separated (group 1, >2.5 years; group 2, < or =2.5 years).
Children with PWS normalized height, but not FL after 6 years of GHT. Parental FL correlation with PWS's FL was lower than with sibling's FL. In group 1, FL positively correlated with WfH, LBM, and physical activity[after 2.5 years of age foot length correlated with physical activity, body weight, and muscle mass]. In group 2, FL negatively correlated with age at onset of independent ambulation. Foot catch-up growth with GHT was slower in group 2 compared with group 1.
In PWS, FL is positively associated with musculoskeletal loading. Small feet in children with PWS before and during long-term GHT may be more than just another dysmorphic feature, but may possibly reflect decreased musculoskeletal loading influencing foot growth and genetic and endocrine factors[decreased musculoskeletal loading may affect foot growth in all individuals and not just children with PWS]."

"In patients with GH deficiency or GH insensitivity syndrome, the size of hands and feet is reduced in proportion to the patient's body height, feet being relatively longer than hands, with both normalizing on GH therapy (GHT)"<-height is composed larger of long bone length whereas feet length is mainly determined by short and irregular bone size. This may indicate that GH plays a larger role on short bone growth than long bone growth.

"GHT normalizing height and hand length, but not foot length (FL)"<-that feet would be influenced by different factors than hands is very interesting.

" In hemiplegic children, the inactive leg is shorter than the active one"

"Factors other than GH and the genetic background may have an impact on foot growth."

"the later the onset of musculoskeletal loading, the shorter the FL"<-this means that you may only need to pass a threshold of musculoskeletal loading to maximize foot length and that as long as you are passed that threshold your foot length growth is maximized. The lack of catch-up growth is not good news for the possibility of environmental musculoskeletal stimulation being a significant way to increase foot length(as long as you are above threshold).

"in the older group of children only, there was a significantly positive correlation between FL and WfH, indicating that body mass may be a stimulus for foot growth"<-this data however is very positive for environmental musculoskeletal stimulation stimulating foot length with body weight correlating positively with foot length.

" in addition to height, parental FL variables of musculoskeletal loading were significant predictors of FL measures."<-So even in parents who did not have PWS, musculoskeletal loading was correlated with foot length[weight and exercise]

So environmental factors accounted for around at most 20% of height and less than 2% of height in some studies. Musculoskeletal loading is one such environmental factor that was found to affect foot length. Another corollary environmental factor to that is nutrition which affects body weight which is positively correlated to foot length(referred to as weight for height in the study). Is it possible that foot length could be influenced by factors other than bone length? There's arch size which may decrease with increasing loads(thus increasing foot length). Fat mass and lean body mass can contribute to foot size(however, those things can contribute to hand size as well).

Less people load their hands than walk(less people provide mechanical stimulation to hands than feet). Thus, if mechanical stimulation stimulates foot growth thus foot length should increase greater than hand length in normal individuals(as not as many people do push-ups as walk). Thus, meaning PWS individuals are less far behind in hand length than foot length. This too means that studying hand size with exercise would be an interesting study. One twin does push-ups whereas another lives life normally.

Huge News: More LSJL studies on longitudinal growth!

2011-12-21T13:02:00.000-08:00

Here's a study that shows that LSJL increases height in adult rats!

Knee loading promotes longitudinal bone growth in both young and adult mice

"Treatment of limb length discrepancy requires differential bone lengthening. Joint loading, a recently developed loading modality, can enhance anabolic responses. The object of this study was to examine whether knee loading to one limb would be useful for correcting length discrepancy in young and adult mice.

Methods: 21 young (8 weeks of age) and 15 adult (16 weeks of age) C57/BL/6 female mice were used[rodents retain a cartilagenous template post growth plate cessation however if LSJL still involves differentiation of stem cells into chondrocytes then it can still work in human adult mice it'll just be harder as mice already have a cartilagenous matrix to work with]. The left hindlimb was given 5-min loading bouts (0.5 N at 5 Hz) for 10 days (young mice) and 20 days (adult mice), while the right hindlimb was used as sham loading control. Mice were sacrificed 2 weeks after the last loading. Tibia length was measured with a digital caliper, and its weight was determined with an electronic balance. BMD and BMC were determined with a PIXImus densitometer.

Results: First, knee loading lengthened tibiae in both young and adult mice. Compared to sham loading control, the longitudinal tibia length was elevated by 2.3% from 16.68 ± 0.06 mm (control) to 17.06 ± 0.05 mm (loading) (p < 0.001) in young mice and 1.5% from 17.82 ± 0.04 mm (control) to 18.09 ± 0.03 mm (loading) (p < 0.001) in adult mice[so the growth plates were not senescent in adult mice which means that it is not proof that LSJL induces chondrogenic differentiation]. Second, the tibia weight was increased by 10.4% in young mice (p < 0.001) and by 6.0% in adult mice (p < 0.01). Third, loads also elevated BMD and BMC. In young mice, for instance, BMD was increased from 0.0409 ± 0.0003 g/cm2 (control) to 0.0427 ± 0.0003 g/cm2 (loading) (p < 0.001) and BMC was elevated from 0.0136 ± 0.0002 g (control) to 0.0145 ± 0.0002 g (loading) (p < 0.01). Lastly, based on the normalized change (% of control), young mice increased both length (p < 0.05) and weight (p < 0.01) more than adult mice did.

The current study demonstrates that knee loading enhances longitudinal bone growth in both young and adult mice, and the effects are stronger in young mice than adult mice. Joint loading may have a potential usage in development of load-driven therapies for limb length discrepancy and short stature."

Here's a patent related to LSJL:

"Apparatus and methods enhance bone formation. The methods and apparatus provide a mechanical load applied to the epiphysis of a bone associated with a joint[just like we are trying to do with LSJL]. The magnitude of the mechanical load is oscillated and is applied periodically for a short durations of time. The applied mechanical force induces formation of trabecular and cortical bones. Embodiments of the apparatus include passively and actively actuated bands which are positioned around at least a portion of a joint and provide mechanical loading on the joint, for example, on the epiphysis of a bone and oriented traverse to the longitudinal axis of the bone[so essentially lateral loading]."<-We are not really oscillating the loads but I suppose we could with the clamp. Rotate the clamp tighter than loosen a little, then a little tighter than before and so on...

Some difficulties for an LSJL like advice:

"generating strain in the diaphysis in limb bones requires a loading device that is large enough to span the entire length of a long bone, for example, a femur or tibia[this device however was designed to stimulate bone formation in the entire bone whereas we are only looking for chondrogenic differentiation in the epiphysis]. Accordingly, the portability and ease of use of such devices are detrimentally affected. In addition, it may be difficult to determine the appropriate loading magnitude for particular individuals; therefore, operation of such a device may cause unwanted bone fractures[<-no reported bone fractures have been reported with LSJL however]. Furthermore, enhancing bone formation by generating a strain in the diaphysis may require that all bones be treated independently. Moreover, the use of such devices can require dedicated exercise time which may not be easily incorporated into daily activities[this is an issue people have been having with LSJL]."

Here's some methods described to perform LSJL. They are not specifically described to generate height increase but they are all designed to increase intramedullary pressure and fluid flow which is our goal as to generate chondrogenic differentiation:

"The method of enhancing bone formation in a mammal can include applying a mechanical load to a joint of the mammal so that viscoelastic joint tissues of the joint are deformed[this is what we do with the clamp or dumbell]. In one embodiment the method includes oscillating the amplitude of the mechanical load, for example at about 2 Hz, so that fluid flow is generated in the bones forming the joint. The fluid flow can be interstitial cellular fluid flow and include the diaphysis of a bone. The method can also include varying the amplitude of the mechanical load to generate a streaming potential measured on the periosteal surface of the diaphysis of the bones forming the joint, for example so that the peak-to-peak amplitude of the mechanical load is about 0.5 N. The joint can be, but is not limited to, one of the knee, ankle, hip, shoulder, elbow, and wrist, and the mechanical load in one embodiment is applied laterally to the joint to cause viscoelastic deformation of joint tissues. The method can also include applying a circumferential band around at least a portion of the joint and exercising the joint to vary the load applied to the joint by the band[basically like putting a ring on the finger or something like a ring on your leg]. The method can also include positioning a wall of a bladder in contact with the joint and advancing a fluid into the bladder[a bladder is a hydration system like the platypus, the wall would be one side of the bag that holds the fluid, so you would put the side of the bladder onto the joint and then put fluid into the bladder(something dense would work best)]. Alternatively, applying the mechanical load to the joint can include positioning a band around at least a portion of the joint and tightening the band.
In another embodiment the method of enhancing bone formation in a mammal having a joint connecting a first bone and a second bone includes applying a mechanical load to the epiphysis of at least one of the first bone and the second bone. The magnitude of the mechanical load can be oscillated and the mechanical load can be applied laterally to the joint such that fluid flow is generated in at least one of the first bone and the second bone. The orientation of the mechanical load can be in a direction that is about transverse to the longitudinal axis of the bone and can be applied to the joint tissue connecting the first bone and the second bone[so you would stack your joints one on top of each other and then load the top joint with a dumbell and hope that you get both joints]."

The hydration system would be interesting to try. A platypus hydration system is available for about 30$. We'd have to find the best liquid to put in the platypus. We'd want the most dense liquid but that's still save and affordable(not something like mercury). For the circumferential band, you could tie off two exercise bands at the end of two joints and then go running.

"In yet another embodiment, the apparatus for enhancing bone formation includes a band configured to fit around at least a portion of a joint of a mammal, a bladder coupled to the band, and a pump in fluid communication with the bladder and operable to advance a fluid, for example water or air, into the bladder. The pump can have a controller for oscillating the volume of fluid in the bladder. The band can form a loop having an adjustable circumference. The band can also be a belt that defines a loop having a circumference configurable to fit around a joint and an electric motor operatively coupled to the belt and capable of varying the circumference of the loop. The band can alternatively include an electro-chemical material, for example, a polymer that undergoes dimensional changes upon exposure to an electric field, such as polypyrrole or polythiophene. Oscillation of the electric field can be used to change the circumference of the loop, thereby varying the mechanical load on the joint.
In another exemplary embodiment, the apparatus for enhancing bone formation includes a band configured to be positioned around a joint and an element coupled to that band that is configured to apply lateral pressure to the joint. The band can include an elastic wrap. The element can also include a pad, for example a fluid filled bladder. In one embodiment two pads are positioned on opposite sides of the joint."

"that may be driven by air pressure approximately 40 kPa (5.8 psi) was needed to provide 0.5 N to murine elbows in the second exemplary study discussed below. Assuming that 100 N is used to press a lateral wall of a human knee joint, 51 kPa (7.4 psi) is required for a 50 mm in diameter bladder"<-So we can figure out that 40 kPa produces 0.5N is for mice. So humans need 11more kPa of pressure to generate 0.5N of force given a 50mm in diameter bladder. So for humans 51 kPa per 0.5N of force given a 50mm bladder.

We can combine clamping and a bladder hydration system. We could clamp the sides of the bone while putting the platypus hydration system on top using just water at this time. Anyone have ideas for substances more dense than water?

Here's a new study on elbow loading:

Elbow loading promotes longitudinal bone growth of the ulna and the humerus.

"Mechanical stimulation plays a critical role in bone development and growth. In view of recently recognized anabolic responses promoted by a joint-loading modality, we examined the effects of elbow loading on longitudinal growth of the ulna and the humerus. Using a custom-made piezoelectric loader, the left elbow of growing C57/BL/6 female mice was given daily 5-min bouts of dynamic loading for 10 days. The right forelimbs of those mice served as contralateral controls, and the limbs of non-treated mice were used as age-matched controls. The effects of elbow loading were evaluated through measurement of bone length, weight, bone mineral density (BMD), and bone mineral content (BMC), as well as mRNA expression levels of load-sensitive transcription factors such as c-fos, egr1, and atf3. The results revealed that the humerus was elongated by 1.2% compared to the contralateral and age-matched controls (both p < 0.001), while the ulna had become longer than the contralateral control (1.7%; p < 0.05) and the age-match control (3.4%; p < 0.001)[We can't be sure if these actually increased final height or merely accelerated height growth until we actually identify how lateral joint loading increases growth]. Bone lengthening was associated with increases in bone weight, BMD and BMC. Furthermore, the mRNA levels of the selected transcription factors were elevated in the loaded ulna and humerus. Interestingly, the increase was observed not only at the elbow but also at the wrist and shoulder in the loaded limb[loading at one area likely changes hydrostatic pressure throughout the bone and the periosteum of one bone is connected to another]. The present study demonstrates that joint loading is potentially useful for stimulating bone lengthening and treating limb length discrepancy."

" In the hypertrophic zone of the growth plate in the proximal tibia, the number of chondrocytes and their cellular height were elevated[increase in number of chondrocytes indicates possibility of new stem cells differentiating into chondrocytes thus bypassing proliferative capacity, also increase in cellular height means more height growth per chondrocyte thus also likely leading to a resultant final adult height rather than more acceleration]. Thus, joint loading is potentially useful to lengthen long bones in the hindlimb and the growth plate at the loading site exhibits morphological alterations; however, no studies have been conducted for the forelimb. Furthermore, little is known about potential alterations in gene expression not only in the loaded joint but also at the other end of long bones (e.g., the wrist and shoulder for elbow loading), which are not directly under mechanical loading."

"mice were mask-anesthetized using 1.5% isoflurane and received loads to the left elbow in the lateral-medial direction with the custom-made piezoelectric mechanical loader"<-the mice were anesthetized meaning no muscle contraction but that doesn't mean that muscle contraction can't increase hydrostatic pressure.

" Loads were 0.5 N at 5 Hz and given for 5 min per day for 10 days"<-5 bouts of 1/2 the force of gravity per second. Hard to convert this to our purposes but the load seems pretty low. I am now loading at 100 seconds. Perhaps lower loading is needed for LSJL and longer duration. This study uses electricity but a pizeoelectric current is generated by the bone in response to deformation due to loading thus the same effect is achieved due to loading.

"To avoid a local stress concentration between the elbow and the loader, both the loading surface and supporter were covered with silicon rubber"<-thus supporting the usage of socks and clothing to eliminate some of the irritation of LSJL.

"In response to lateral loads applied to the knee ex vivo, the maximum strain at the loading site was in the order of a few millistrains and the strain at the midshaft cortical bone was in the order of 10 microstrains"<-this is incredibly low

"The longitudinal strain along the bone length was positive, indicating that tensile force acts in the growth plate[so the growth plate was stretched but previous growth plate stretching methods have had a lack of success]. As a biophysical mechanism for induction of bone formation with joint loading, it is proposed that alterations in intramedullary pressure[Hydrostatic pressure is likely a subset of intramedullary pressure thus inducing chondrocyte differentiation] are induced and interstitial molecular transport is activated by a dynamic pressure gradient"

Unfortunately, there were no histological slides but it's good to know that LSJL is still being explored for longitudinal growth by two of the creators. P. Zhang and H. Yokota.

There's also been a review study recently that Hiroki Yokota is a part of that mentions LSJL and may provide some insight:

Mechanical intervention for maintenance of cartilage and bone.

"Mechanical loading provides indispensible stimuli for growth and development of the articular cartilage and bone. Interestingly, depending on loading conditions loads applied to the joint can be beneficial as well as harmful to skeletal maintenance and remodeling. Moderate loads to the synovial joint, for instance, suppress the expression levels of matrix metallproteinases (MMPs), while loads above a threshold tend to increase their destructive activities[although some catabolic effects of MMPs may be good for height growth, MMPs may degrade bone allowing for cartilage growth]. This report focuses on two recently developed loading modalities from animal studies, joint motion and joint loading. Their unique characteristics and potential usages for maintenance of the articular cartilage and stimulation of bone remodeling are reviewed. Also described are biophysical and molecular mechanisms which likely are responsible for the load-driven maintenance of cartilage and bone, and a possibility of developing load-mediated treatments of osteoporosis and osteoarthritis."

"Moderate shear stress(2–5 dyn/cm2) reduced MMP expression levels, while high shear stress (10–20 dyn/cm2) increased them. Similarly, moderate hydrostatic pressure (1–5 MPa) suppressed MMP-1 expression, while higher loads (10 MPa) elevated it."<-you're likely not to get above 5MPa of hydrostatic pressure even with very large loads so that is not really an issue with LSJL.

"The required magnitude of loads for joint loading is in general smaller than that for axial loading (e.g. 0.5 N for elbow loading and 2–3 N for ulna axial loading in mice). Bone is less stiff in a lateral direction than an axial direction."<-Note that more than 0.5N is likely required for humans. 0.5N is what was used in the mouse arm lengthening study.

"It has been proposed that joint loading periodically alters the pressure in the medullary cavity and activates molecular transport in a lacunocanalicular network in cortical bone."<-It is our hypothesis that this increase in pressure in the medullary cavity induces chondrogenic differentiation. The medullary cavity is continuous into the spaces of the spongy bone of the epiphysis. It is these spaces where we aim to induce chondrogenic differentiation and thus induce endochondral ossification to grow taller.

"That is, a pressure gradient in the medullary cavity generates oscillatory fluid flow in the porous bone cortex."<-and fluid flow into the spongy bone spaces of the epiphysis.

"Modulation of the intramedullary pressure with knee loading is exerted throughout the length of the tibia and the femur."<-the epiphysis is part of the entire length thus knee loading like by LSJL alters pressure in the epiphysis.

"Suppression of inflammatory responses—Proinflammatory cytokines such as IL-1β upregulate the expression and activity of MMP-1 and MMP-13. It has been shown using cultured chondrocytes that
mechanical stimulation, given in a form of fluid flow shear stress, can suppress the IL-1β-induced upregulation of MMP-1 and MMP-13. In accordance with those in vitro results, joint motion in vivo is able to reduce inflammatory responses in a murine collagen-induced arthritis model. Additionally, in an antigen-induced arthritis model in rabbits, continuous passive motion suppressed transcription of IL-1β and synthesis of inflammatory mediator COX-2 and MMP-1. These mechanical signals also induced IL-10 synthesis, suggesting that moderate joint loading can generate anti-inflammatory signals."<-there are good and bad MMPs for height growth. MMP-1 and MMP-13 seem to be bad for height growth.

"Bone lengthening—When knee loading was applied to one leg, the loaded tibia and femur were reported to be longer than the non-loaded contralateral bones. Histological analysis revealed that in response to knee loading, the number of cells in the growth plate of the proximal tibia increased and their cellular shape was altered. The result suggests a possibility of using knee loading for treating limb length discrepancies in children."<-If LSJL increases the number of cells in the growth plate by differentiation of stem cells into chondrocyte than LSJL will work in adults as well.

"Does mechanical loading of joints activate molecular interactions between the articular cartilage and
the subchondral bone? Homeostasis of the articular cartilage is affected through interactions with the subchondral bone underneath the cartilage. For instance, both MMPs and ADAMTS need to be post-translationally activated, and this activation process is regulated by many factors including MMPs themselves and many proteoglycans. It has not yet been investigated whether mechanical loading to joints regulates activities of MMPs and ADAMTS through interactions between the articular cartilage and the subchondral bone."<-Thus loading of the articular cartilage may itself play a role in the height gain by triggering a response in the subchondral bone in response to the stimulation of the articular cartilage.

Here's another study involving Hiroki Yokota that mentions that lateral joint loading modality:

Mechanical Loading: Bone Remodeling and Cartilage Maintenance

"Bone remodeling and cartilage maintenance are strongly influenced by biomechanical signals generated by mechanical loading. Although moderate loading is required to maintain bone mass and cartilage homeostasis, loading can cause deleterious effects such as bone fracture and cartilage degradation. Because a tight coupling exists between cartilage and bone, alterations in one tissue can affect the other[So an increase in TGF-Beta expression in bone can affect cartilage growth]. Bone marrow lesions are often associated with an increased risk of developing cartilage defects, and changes in the articular cartilage integrity are linked to remodeling responses in the underlying bone. Although mechanisms regulating the maintenance of these two tissues are different, compelling evidence indicates that the signal pathways crosstalk, particularly with the Wnt pathway[loading the cartilage may also have an impact on the bone enabling the stimulation of cartilage growth within bone]. A better understanding of the complex tempero-spatial interplay between bone remodeling and cartilage degeneration will help develop a therapeutic loading strategy that prevents bone loss and cartilage degeneration."

Here's the section that mentions LSJL:

"One of the loading modalities (i.e., joint loading) applies moderate lateral loads to bone as well as joint tissues including articular cartilage and synovium. In the knee loading modality, loads are transmitted not only to the distal femur and the proximal tibia but also to the articular cartilage of the femur and tibia. Recent data indicate that this joint loading modality is capable of stimulating bone remodeling throughout the lengths of the femur and the tibia, as well as suppressing the expression and activities of matrix metalloproteinases (MMPs) in the articular cartilage. According to a currently proposed mechanism, joint loading induces a periodic alteration in the pressure in the medullary cavity[it's this alteration in pressure in the epiphysis that we hope induces chondrogenic differentiation] and activates a Wnt signaling pathway in bone. In the articular cartilage, however, little is known about the joint loading–driven mechanism of MMP downregulation and the role of Wnt signaling."

"Moderate loading of the articular cartilage generates mechanical signals that increase the synthetic activities of the chondrocyte while suppressing its catabolic actions"<-moderate loading is prochondrogenic so it'll make it easier for stem cell in the marrow to differentiate into chondrocytes. So you should definitely do some form of moderate loading in addition to LSJL.

Note that joint rotation may increase VEGF which is important for the formation of cartilage canals. Joint rotation may help augment the effects of LSJL.

"Mechanical stimulation also suppresses cell death through signaling pathways including tumor necrosis factor-α (TNF-α) and Wnt"<-normal mechanical stimulation may help you grow taller through normal height development by suppressing TNF-alpha.

"CITED2 expression is increased by moderate flow shear (5 dyn/cm²), intermittent hydrostatic pressure (1–5 MPa)[thus CITED2 is stimulated by LSJL], and joint motion. The induction of CITED2 in vivo by joint motion loading was correlated with the downregulation of MMP-1 and the maintenance of cartilage matrix integrity"

"CITED2 suppresses MMP-1 expression by preventing MMP transactivator Ets-1 from recruiting limiting amounts of co-activator p300 to the MMP-1 promoter"

"Mechanical overloading also stimulates expression of vascular endothelial growth factor (VEGF), which appears to be involved in the induction of MMP-1, −3, and −13 expressions"<-Note that LSJL does upregulate MMP-3. So it's possible that LSJL does stimulate VEGF expression.

"Quickly after osteocyte stimulation, there is an increase in intracellular Ca²⁺ concentrations and the release of adenosine triphosphate. These events are followed by the participation of secondary messengers prostaglandin and nitric oxide. Further downstream are elements of the Wnt pathway, which regulate bone formation and bone remodeling by promoting osteoblast proliferation and differentiation"<-Note that this release in adenosine triphosphate(ATP) can stimulate chondrocytes as well.

Here's a study not directly written by H. Yokota but was cited as being highly influential on the previous study:

Mechanical loading, cartilage degradation, and arthritis

"Joint tissues are exquisitely sensitive to their mechanical environment, and mechanical loading may be the most important external factor regulating the development and long-term maintenance of joint tissues. Moderate mechanical loading maintains the integrity of articular cartilage; however, both disuse and overuse can result in cartilage degradation. The irreversible destruction of cartilage is the hallmark of osteoarthritis and rheumatoid arthritis. In these instances of cartilage breakdown, inflammatory cytokines such as interleukin-1 beta and tumor necrosis factor-alpha stimulate the production of matrix metalloproteinases (MMPs) and aggrecanases (ADAMTSs), enzymes that can degrade components of the cartilage extracellular matrix. In order to prevent cartilage destruction, tremendous effort has been expended to design inhibitors of MMP/ADAMTS activity and/or synthesis. To date, however, no effective clinical inhibitors exist. Accumulating evidence suggests that physiologic joint loading helps maintain cartilage integrity; however, the mechanisms by which these mechanical stimuli regulate joint homeostasis are still being elucidated. Identifying mechanosensitive chondroprotective pathways may reveal novel targets or therapeutic strategies in preventing cartilage destruction in joint disease."

"There appears to be a critical threshold of 15–20 megapascals (MPa) for cell death and collagen damage due to a single impact load in bovine cartilage explants"<-15 MPa is incredibly high. I think we'd be lucky to get 2 MPa at most with LSJL style loading. In one study they found that 25lbs of loading increased Hydrostatic pressure by 12-14mmHg. There are 7500mmHg in one MPa. The smallest MPa listed as damaging is 5 MPa in the article.

"the range of nonphysiological load intensities in vivo should be greater than those reported in these in vitro studies."<-Due to distribution of force in a living organism thus the threshold for a damaging amount of pressure should be higher than 5MPa.

"The MMP family consists of the collagenases, (MMPs 1, 8, and 13) which degrade collagens types I, II, and III, the gelatinases (MMPs 2 and 9), which target denatured collagen, the stromelysins (MMPs 3, 7, 10, and 11), which degrade several ECM proteins and are involved in proenzyme posttranslational activation, the membrane-type MMPs (MT-MMP 1–4), and a diverse subgroup including MMPs 12, 20, and 23."<-LSJL downregulates MMP-1 which is good as you don't want degradation of type II collagen. You may want degradation of type I collagen however to make room for chondrogenesis. LSJL upregulates MMP-3 although whether this is beneficial is unknown.

"LIPUS promotes synthesis of several matrix components in chondrocytes in vitro, including type II collagen, type X collagen, and aggrecan. LIPUS also increased production of type II collagen in an experimental OA rat model and ameliorated histological cartilage damage when compared to untreated groups."<-Since LIPUS stimulates Type II Collagen and Aggrecan it too can help stimulate chondrogenesis of mesenchymal stem cells.

"Mechanical overloading stimulates expression of VEGF, which appears to be necessary for mechanically induced MMP-1, -3, and -13 expressions"<-Since LSJL upregulates MMP-3 and VEGF is necessary to the mechanically induced upregulation of MMP-3, it is logical that LSJL increases VEGF fashion possibly in a non-estrogen mediated fashion.

"Epigenetic regulation includes the activation of normally silent genes through DNA hypomethylation, which allows for an open chromatin structure, or silencing of normally expressed genes through DNA hypermethylation, which prevents access of transcription factors to their promoter."<-DNA hypomethylation could be used to activate tall genes in normal individuals? It is hard to control specific gene methylation however.

"gene transcription can also be regulated by deacetylation through histone deacetylases (HDACs)."

"there is evidence in adult endothelial cells that shear stress regulates gene expression. Shear stress at 10 dyn/cm²/sec modifies core histones H3 and H4 in human umbilical vein endothelial cells."<-Shear stress modifies gene expression. It's not bone or cartilage cells but it shows that shear stress can modify gene acetylation. LSJL causes shear stress.

Here's a study involving Sun HB who seems to be working with Hiroki Yokota on LSJL related issues.

Mechanotransduction and cartilage integrity.

"Mechanotransduction is the process by which biomechanical signals regulate cell activity and behavior. Chondrocytes are able to sense and react to mechanically induced changes within the cartilage matrix[this is important for people who still have growth plates]. Chondrocyte mechanotransduction is initiated at the interface between the cell membrane and extracellular matrix, and the processing of these mechanical signals involves mechanoreceptors such as ion channels and integrins. For example, membrane stretch, a condition that chondrocytes experience during compression or during hypo-osmotic conditions that cause swelling, activates potassium channels. The function of ion channels in chondrocyte membranes is not clear, but they may be involved in chondrocyte functions such as cell proliferation and matrix secretion[so ion channels are involved in anabolic therefore height increasing functions]. Integrins are heterodimeric transmembrane receptors consisting of α and β subunits and interact with cytoskeletal proteins such as fibronectin, vitronectin, and osteopontin.Mechanical stimulation of human chondrocytes increases expression of aggrecan and decreases MMP-3 gene expression in a pathway involving the α5β1 integrin and IL-4 release. However, this response to mechanical stimulation is absent in chondrocytes derived from OA cartilage, suggesting abnormal chondrocyte signaling may be involved in OA disease progression[this may be one of the reasons why osteoarthritis doesn't help make you taller]"

So growth plates are capable of anabolic responses in response to mechanical load. OA however involves endochondral ossification so maybe mechanical stimulation doesn't effect chondrocytes undergoing endochondral ossification such as growth plate chondrocytes.

"One transcriptional regulator that appears to play a crucial role in cartilage homeostasis is CITED2 (CBP/p300-interacting transactivator with ED-rich tail 2). CITED2 is a transcriptional coregulator that does not bind DNA directly. It positively regulates transcription by recruiting CBP (cAMP-responsive element-binding protein) and p300 to interact with other DNA-binding transcription factors such as Lhx2, PPARα, PPARγ, Smad 2, and TFAP2. CITED2 also negatively regulates target genes by competing for CBP/p300 binding with transcription factors including Ets-1, NF-κB, HIF-1α, STAT2, and p53. Through these mechanisms, CITED2 is able to regulate many cellular processes such as embryonic development, cell proliferation, inflammation, and matrix turnover."

"With regard to cartilage integrity, CITED2 expression in chondrocytes in vitro is increased by moderate intensities of flow shear and intermittent hydrostatic pressure[LSJL induces both these things but does flow shear and hydrostatic pressure stimulate CITED2 release in stem cells?] (IHP) and in chondrocytes in vivo by joint motion. Increased CITED2 expression in vivo correlated with the maintenance of cartilage integrity and the suppression of collagenase MMP-1, suggesting the anticatabolic effects of physiologic joint loading were mediated by CITED2. As demonstrated by competitive binding and transcriptional activity assays, CITED2 suppresses MMP-1 transcription by competing with MMP transactivator Ets-1 for binding to its coactivator p300. In addition to MMP-1, Ets-1 binds to the promoter regions of other MMPs including MMP-2, -3, -8, -9, and -13. Therefore, it is likely CITED2 may regulate additional MMPs through a similar manner.

Upstream of CITED2, moderate IHP loading phosphorylated p38δ, which was required for the transactivation of CITED2[stem cells have p38 meaning that hydrostatic pressure likely can stimulate CITED2 activation in stem cells too]. p38 belongs to the MAP kinase family, which is activated in response to mechanical stresses. While moderate loading activated p38δ and CITED2, high levels of IHP phorphorylated p38α and MMP-1, but not CITED2. This may explain why CITED2 is specifically activated by moderate loading and also suggests that p38α is involved in the upregulation of MMP-1[MMP-1 is catabolic however very high levels of hydrostatic pressure has been shown to be anabolic in inducing chondrocyte differentiation]. The data indicate different members of the p38 family may act as a “mechanosensitive switch” in chondrocytes, which act to upregulate or downregulate MMP expression based on the mechanical loading regimes. The evidence that CITED2 is inducible by IL-4 and may interact with components of NF-κB, suggests a potential role of CITED2 as a central mediator in these mechanotransduction pathways involved in maintaining cartilage integrity"

So CITED2 competes with MMP-1 a catabolic protein. So moderate levels of hydrostatic pressure may be better with existing growth plates to activate CITED2 and not MMP-1 but high levels of hydrostatic pressure may be better to induce new chondrocyte differentiation where growth plates are absent.

Here's a study that shows that CITED2 may be inhibitory towards new cartilage and bone growth:

Identification of CITED2 as a negative regulator of fracture healing.

"The transcription regulator CITED2 (CBP/p300-Interacting-Transactivator-with-ED-rich-tail-2) is known to suppress genes mediating angiogenesis and extracellular matrix (ECM) remodeling. However, it is unclear whether CITED2 has a role in controlling skeletal repair or remodeling. We tested the hypothesis that CITED2 functions in bone fracture healing by suppressing the expression of genes critical to ECM remodeling, angiogenesis and osteogenesis, importantly the matrix metalloproteinases (MMPs). Three hours following mandibular osteotomy or sham surgery of adult rats, osteotomy fronts were harvested and the expression of CITED2 and genes associated with fracture healing was ascertained by quantitative PCR. In parallel, gain-of-function studies examined the effect of overexpressing CITED2 on the expression and activity of several MMPs. In the fractured mandible, CITED2 expression was inversely related to the expression of MMP-2, -3, -9, -13, VEGF, HIF-1alpha, M-CSF, RANK-L, and OPG. Consistent with this, the over-expression of CITED2 in osteoblasts inhibited the expression and activity of MMP-2, -3, -9, and -13. Taken together, the studies suggest that CITED2 is a critical upstream regulator of fracture healing. The suppression of CITED2 early after fracture may allow an optimal initiation of the healing response."

Since fracture healing often involves endochondral ossification, perhaps CITED2 inhibits new endochondral ossification from occurring. However, fracture healing usually requires catabolic activity to remodel bone. New height growth requires catabolic activity to make way for the new growth plates. Thus, LSJL likely requires high enough hydrostatic pressure to inhibit CITED2 activity.

"Early fracture healing is characterized by the initial formation of cartilage tissue in the callus, which is then resorbed by MMPs to allow for vascular invasion with the eventual replacement of cartilage with osseous tissue[so perhaps allowing for CITED2 may allow grow plates to stay active longer]. MMPs 9 and 13 are critical to normal skeletal development; most notably, MMP-9 deficiency delays fracture healing with poor cartilage resorption and impaired capillary and chondroclast invasion. MMP-2, in contrast, participates in cartilage degradation, while MMP-3 activates pro-MMP-9 during wound repair"

So inhibiting CITED2 may aid in the formation of new growth plates but CITED2 may help preserve existing growth plates.

Being Taller due to PEMF?

2011-12-15T12:58:00.000-08:00

Pulsed Electromagnetic Field Therapy is used to treat the healing of non-union fractures(fractures that don't heal). Now studying non-union fractures is very important to us height seekers as fractures heal as a result of endochondral ossification(stem cells, to chondrocytes, to hypertrophic chondrocytes, to dead chondrocytes, to being invaded by osteoblasts). The primary goal of Lateral Synovial Joint Loading, is to induce bone marrow stem cells to differentiate into chondrocytes from the epiphyseal bone marrow. Any technique with applications on non-union fractures(fractures where endochondral ossification does not occur) will have applications on Lateral Synovial Joint Loading.

Effects of Pulsed Electromagnetic Fields on Human Osteoblastlike Cells (MG-63): A Pilot Study.

"Although pulsed electromagnetic fields (PEMFs) are used to treat delayed unions and nonunions, their mechanisms of action are not completely clear. However, PEMFs are known to affect the expression of certain genes. QUESTIONS/PURPOSES: We asked (1) whether PEMFs affect gene expression in human osteoblastlike cells (MG63) in vitro, and (2) whether and to what extent stimulation by PEMFs induce cell proliferation and differentiation in MG-63 cultures. METHODS: We cultured two groups of MG63 cells. One group was treated with PEMFs for 18 hours whereas the second was maintained in the same culture condition without PEMFs (control). Gene expression was evaluated throughout cDNA microarray analysis containing 19,000 genes spanning a substantial fraction of the human genome. RESULTS: PEMFs induced the upregulation of important genes related to bone formation (HOXA10, AKT1), genes at the transductional level (CALM1, P2RX7), genes for cytoskeletal components (FN1, VCL), and collagenous (COL1A2)[COL1A2 is the gene coding fibrous cartilage which isn't as important for height growth as hyaline cartilage but could be useful nonetheless] and noncollagenous (SPARC) matrix components. However, PEMF induced downregulation of genes related to the degradation of extracellular matrix (MMP-11, DUSP4). CONCLUSIONS AND CLINICAL RELEVANCE: PEMFs appear to induce cell proliferation and differentiation. Furthermore, PEMFs promote extracellular matrix production and mineralization while decreasing matrix degradation and absorption. Our data suggest specific mechanisms of the observed clinical effect of PEMFs, and thus specific approaches for use in regenerative medicine."

PEMF's can induce cellular proliferation and differentiation. PEMF's alter genetic expression of genes related to height growth. Being taller thanks to PEMF is a definitive possibility. Even though this study showed the effects of PEMF on osteoblastic cells there may be spillover benefits on chondrogenic cells like the degradation of MMP-11. Also DUSP4 inhibits cellular proliferation so that again can have spillover chondrocyte and stem cell benefits.

"PEMFs determine signal transduction by means of intracellular release of Ca²⁺ leading to an increase in cytosolic Ca²⁺ and an increase in activated cytoskeletal calmodulin. PEMFs induce a dose-dependent increase in bone and cartilage differentiation, and upregulation of mRNA expression of extracellular matrix molecules, proteoglycan, and Type II collagen"<-dose dependent means that the stronger the stimulus, the more that the positive height increase benefits will be magnified.

Effect of pulsed electromagnetic field on the proliferation and differentiation potential of human bone marrow mesenchymal stem cells.

"Pulsed electromagnetic fields (PEMFs) have been used clinically to slow down osteoporosis and accelerate the healing of bone fractures for many years. The aim of this study is to investigate the effect of PEMFs on the proliferation and differentiation potential of human bone marrow mesenchymal stem cells (BMMSC). PEMF stimulus was administered to BMMSCs for 8 h per day during culture period. The PEMF applied consisted of 4.5 ms bursts repeating at 15 Hz, and each burst contained 20 pulses. Results showed that about 59% and 40% more viable BMMSC cells were obtained in the PEMF-exposed cultures at 24 h after plating for the seeding density of 1000 and 3000 cells/cm2, respectively. Although, based on the kinetic analysis, the growth rates of BMMSC during the exponential growth phase were not significantly affected, 20-60% higher cell densities were achieved during the exponentially expanding stage. Many newly divided cells appeared from 12 to 16 h after the PEMF treatment as revealed by the cell cycle analysis. These results suggest that PEMF exposure could enhance the BMMSC cell proliferation during the exponential phase and it possibly resulted from the shortening of the lag phase. In addition, according to the cytochemical and immunofluorescence analysis performed, the PEMF-exposed BMMSC showed multi-lineage differentiation potential similar to the control group."

PEMFs can affect stem cell proliferation and this study shows that it can directly affect bone marrow stem cell proliferation and differentiation. In this study, only osteogenic differentiation was analyzed but PEMF helping with chondrogenic differentiation is likely.

Cytokine release from osteoblasts in response to different intensities of pulsed electromagnetic field stimulation.

"We use an in-vitro osteoblast cell culture model to investigate the effects of low-frequency (7.5 Hz) pulsed electromagnetic field (PEMF) stimulation on osteoblast population, cytokines (prostaglandin E(2) (PGE(2))[PGE2 is bad for height growth], transforming growth factor beta1(TGFbeta1), and alkaline phosphatase (ALP) activity to find the optimal intensity of PEMF for osteoblast growth. The results demonstrate that PEMF can stimulate osteoblast growth, release of TGFbeta1, and, in addition, an increase of ALP activity. The synthesis and release of PGE(2) in the culture medium are reduced with increasing numbers of cells. Higher intensity does not necessarily mean increased osteoblast growth, and the most efficient intensity is about 2 mV/cm in this case. Although the lower intensities of the PEMF are yet to be determined, the results of this study can shed light on the mechanisms of PEMF stimulation on non union fracture therapy and osteoporosis prevention in the future."

So PEMF has effects on transforming growth factor beta too? This looks really promising for height growth. The effect on cytokines may be a necessary side effect. It's likely that for chondrogenic differentiation less than 2mV/cm will be used. PEMF is really understudied in terms of inducing chondrogenic differentiation. Right now, how much PEMF can help with growing taller is unknown. TGF-Beta1 is what is responsible for initial chondrogenic differentiation which is key for height growth.

PEMF is for sale but I couldn't find a lot of options. I know Amazon has a very strict refund policy that favors the consumer HealFast Therapy Equine PEMF Square Patch. I can't verify how good this product is. I also found Magnetic Therapy Set, Large. It's cheap and it has pieces that are ideal of putting on the epiphysis of the bones. You can see that they have one piece for the ankle and the other for the knee.

Here's a patent related to an electrical stimulation device. The diagrams do show growth plate stimulation but they don't show if the device can get around chondrocyte finite proliferative capacity. Two ways to get around it would be methods involving ECM secretion and inducing other stem cells not already a part of the growth plate to differentiate into chondrocytes.

The patent mentions periosteal irritation, medullary plugging[likely refers to the medulla of the bone marrow so a hydrostatic pressure method], creation of an arteriovenous fistula[likely a hydrostatic pressure method], sympathetic denervation[removal of part of the sympathetic nervous system], heat, and foreign objects inserted into the epiphysis(like epiphyseal distraction) as previous attempts to grow taller. We'll have to do independent investigations on these things later.

The study states that bimetallic strips were effective in inducing longitudinal growth[bimetallic strips convert temperature into mechanical stimuli]. A pulsed magnetic field of 1000mV was effective in inducing growth activity in a chick. An enhanced incorporation of 3H-thymidine is present in chondrocytes at 1166 V/cm^2 oscillating at 5 Hz.

The invention involves placing electrodes around the epiphysis and is AC[stands for alternating current which means that the current periodically changes direction likely to evade actin cytoskeleton adaptation] stimulation signal. Growth rate increase was demonstrated with the invention experimentation but it's uncertain whether that translates into higher adult height.

Here's a study about MSC's becoming proliferative due to electric currents and this may translate to it being easier to induce chondrogenic differentiation and therefore height growth:

Degenerate wave and capacitive coupling increase human MSC invasion and proliferation while reducing cytotoxicity in an in vitro wound healing model.

"Non-unions pose complications in fracture management that can be treated using electrical stimulation (ES). Bone marrow mesenchymal stem cells (BMMSCs) are essential in fracture healing; however, the effect of different clinical ES waveforms on BMMSCs cellular activities remains unknown[this is what we are looking for, what effect does electrical stimulation have on MSCs?]. We compared the effects of direct current (DC), capacitive coupling (CC), pulsed electromagnetic field (PEMF) and degenerate wave (DW) on cellular activities including cytotoxicity, proliferation, cell-kinetics and apoptosis by stimulating human-BMMSCs 3 hours a day, up to 5 days. In addition, migration and invasion were assessed using fluorescence microscopy and by quantifying gene and protein expression. We found that DW had the greatest proliferative and least apoptotic and cytotoxic effects compared to other waveforms. DC, DW and CC stimulations resulted in a higher number of cells in S phase and G(2)/M phase as shown by cell cycle analysis. CC and DW caused more cells to invade collagen and showed increased MMP-2 and MT1-MMP expression[remember MT1-MMP is responsible for the formation of new cartilage canals]. DC increased cellular migration in a scratch-wound assay and all ES waveforms enhanced expression of migratory genes with DC having the greatest effect. All ES treated cells showed similar progenitor potential as determined by MSC differentiation assay[so all forms of electrical stimulation increase the likelihood of chondrocyte differentiation which is good for a method like LSJL that wants to induce chondrocyte differentiation]. All above findings were shown to be statistically significant (p<0.05). We conclude that ES can influence BMMSCs activities, especially DW and CC, which show greater invasion and higher cell proliferation compared to other types of ES. Application of DW or CC to the fracture site may help in the recruitment of BMMSCs to the wound that may enhance rate of bone healing at the fracture site."

Degenerate wave mixing involves using all three electromagnetic waves. So we wouldn't just want PEMF but the other two forms of waves as well.

"Recent studies have illustrated the invasive capacity of human MSCs requiring MMP-2 and MT1-MMP"<-So in order to form new cartilage canals and growth plates you likely need MMP-2 and MT1-MMP.

"DC treated cells also significantly over expressed several migratory genes including SDF-1/CXCR4, PDGFB-R and TGF-β1-R, IGF-1 and IGF-1R (p<0.01)"<-So electrical stimulation increases the number of TGF-Beta and IGF-1 receptors meaning that the stem cells are more sensitive to anabolic height growth proteins like TGF-Beta and IGF-1.

Although the stem cells were obtained from patients undergoing hip replacement surgery so there is a chance that this electrical stimulation may not increase the proteins of healthy bones in the same way.

Grow Taller with Growth Hormone Binding Protein

2011-12-12T10:21:00.000-08:00

In our study of Growth Hormone, we found that(in mice at least) that Growth Hormone and IGF-1 increased levels of GHR and GHBP. And, that things that reduced height like dexamethasone lowered levels of GHBP and that things that increased height like thyroid hormone increased levels of GHBP.

Here's a study that shows which proteins active GHBP(and thus potentially which proteins we can manipulate):

The Extracellular Domain of the Growth Hormone Receptor Interacts with Coactivator Activator to Promote Cell Proliferation

"The presence of GH receptor (GHR) in the cell nucleus correlates with cell division[cell division is really good for growth plate chondrocytes and means more height growth], and targeting the GHR to the nucleus results in constitutive proliferation and transformation because of increased sensitivity to autocrine GH. Here we have sought additional mechanisms that might account for the enhanced proliferation seen with nuclear GHR, commencing with a yeast two-hybrid (Y2H) screen for interactors with the extracellular domain of the GHR [GH-binding protein (GHBP)]. We find that the GHBP is a transcriptional activator in yeast and mammalian cells, and this activity resides in the lower cytokine receptor module. Activity is dependent on S226, the conserved serine of the cytokine receptor consensus WSXWS box. By using parallel GHBP affinity columns and tandem mass spectrometry of tryptic digests of proteins bound to wild-type GHBP and S226A columns, we identified proteins that bind to the transcriptionally active GHBP. These include a nucleoporin and two transcriptional regulators, notably the coactivator activator (CoAA), which is also an RNA binding splicing protein. Binding of CoAA to the GHBP was confirmed by glutathione S-transferase pulldown and coimmunoprecipitation, and shown to be GH dependent in pro-B Ba/F3 cells. Importantly, stable expression of CoAA[the protein that binds to GHBP] in Ba/F3 cells resulted in an increased maximum proliferation in response to GH, but not IL-3[therefore if we stabilize the CoAA protein we will increase the cell response to GH]. Because CoAA overexpression has been identified in many cancers and its stable expression promotes cell proliferation and cell transformation in NIH-3T3 cells, we suggest CoAA contributes to the proliferative actions of nuclear GHR by the hormone-dependent recruitment of this powerful coactivator to the GHR."

So increase levels of CoAA to grow taller.

According to the study "The Coactivator activator CoAA regulates PEA3 group member transcriptional activity.": "Five CoAA interactors have been identified previously: the coactivator TRBP and the histone acetyltransferase CBP, the proto-oncogene coactivator SYT, the extracellular domain of the GH (growth hormone) receptor called GHBP (GH-binding protein), and the transcription factor RUNX2"<-So a likely way to manipulate CoAA is via RUNX2. Diosgenin increases RUNX2 levels. Parathyroid Hormone inhibits Zfp521 which allows for higher RUNX2.

Unfortunately it seems more likely that CoAA stimulates Runx2 than the other way around:

Co-activator activator (CoAA) prevents the transcriptional activity of Runt domain transcription factors.

"Runx proteins are essential for a number of developmental processes and are aberrantly expressed in many human cancers. Runx factors bind DNA and co-factors to activate or repress genes crucial for bone formation, hematopoiesis, and neuronal development. Co-activator activator (CoAA) is a nuclear protein that regulates gene expression, RNA splicing and is overexpressed in many human tumors. In this study, we identified CoAA as a Runx2 binding protein. CoAA repressed Runx factor-dependent activation of reporter genes in a histone deacetylase-independent manner. CoAA also blocked Runx2-mediated repression of the Axin2 promoter, a novel Runx target gene. The carboxy-terminus of CoAA is essential for binding the Runt domains of Runx1 and Runx2. In electophoretic mobility shift assays, CoAA inhibited Runx2 interactions with DNA. These data indicate that CoAA is an inhibitor of Runx factors and can negate Runx factor regulation of gene expression. CoAA is expressed at high levels in human fetal osteoblasts and osteosarcoma cell lines. Suppression of CoAA expression by RNA interference reduced osteosarcoma cell viability in vitro, suggesting that it contributes to the proliferation and/or survival of osteoblast lineage cells."

So CoAA stimulates osteoblasts as well as chondrocytes. Unfortunately, no way of increasing serum levels of the CoAA protein is known(which would stimulate GHBP).

Are bone marrow cells capable of chondrogenesis?

2011-12-08T10:58:00.000-08:00

The goal of LSJL is to induce chondrogenic differentiation of the mesenchymal stem cells in the epiphyseal bone marrow. The chondrogenic potential of bone marrow cells is therefore of interest to us. Are adult bone marrow cells capable of differentiating into chondrocytes?

Chondrogenesis of mesenchymal stem cells: role of tissue source and inducing factors.

"the cardio-vascular reparative effects attributed to MSCs appear to be mediated predominantly through the secretion of factors targeting cells at the site of repair"<-so what MSCs differentiate into depends on what factors are secreted at certain sites. So at the sites where we want to grow taller we want our cells to secrete pro-chondrogenic factors. LSJL encourages secretion of TGF-Beta which is pro-chondrogenic.

"MSC populations are heterogeneous cell populations whose composition depends on isolation methods and expansion conditions that differ largely among investigators. A recent publication on cloned populations of MSCs showed that nearly 50% of CFU-Fs from BM were tripotent MSCs while the remaining population of cells showed varied phenotypes"<-So the ability of MSCs to undergo chondrogenesis may vary by individual.

"A more extended analysis of clonal populations of SM MSCs distinguished two populations: 30% of cells were tripotent while the remainder displayed only osteo-chondral differentiation potential"<-so all synovial membrane cells have the ability to differentiate into chondrocytes. However, the synovial membrane lines the articular cartilage and does not necessarily have access to epiphyseal bone marrow.

"Large-scale analysis of DNA methylation in embryonic and adult stem cells has shown that embryonic stem (ES) cells can clearly be discriminated from MSCs by specific hypermethylation of numerous genes. In contrast, the comparison of AT and BM MSCs revealed few differences. A comparison of DNA methylation profiles in MSCs from AT, BM and muscle and in HSCs also revealed specific hypermethylation of numerous genes in HSCs while the methylation patterns of MSCs from different sources were very similar"<-So DNA Methylation affects the differences between embryonic and adult stem cells much more than methylation differences between individuals. We need to find out which specific methylations affect height.

" This suggests that promoter hypomethylation is not predictive for the differentiation potential of cells, while hyper-methylation sets restrictions that define frames for differentiation potentials"<-so we may want to un hyper-methylate some genes.

"two cytosines in the COL10A1(Collagen Type X or terminal differentiation) promoter were consistently hypomethylated in MSCs in comparison with articular chondrocytes, correlating to the inducibility of COL10A1 expression and hypertrophy during in vitro chondrogenesis of MSCs "->so the hypomethylation of the COL10A1 gene in articular chondrocytes could be why articular chondrocytes do not ossify and undergo terminal differentiation. For LSJL to work properly we need to ensure that the COL10A1 gene is hypomethylated.

"Histone modifications and histone-modifying molecules are regulated, while MSCs enter senescence in vitro and could be involved in the ensuing loss of differentiation potential. They are also actively involved in differentiation. Several studies have indicated that histone deacetylases, in particular HDAC4, may represent important regulators of chondrogenesis "<-So we need to worry about histones as well for inducing chondrogenesis.

"During embryogenesis the development of cartilage is initiated by a phase of condensation of mesenchymal precursor cells, and the cell-cell contact arising from condensation appears to be crucial for the onset of chondrogenesis. N-cadherin seems to be involved in cell-cell contact in pre-cartilage condensations, and functional N-cadherin was necessary for chondrogenesis of chick limb mesenchymal cells in vitro and in vivo. In human MSCs, N-cadherin is strongly up-regulated during the condensation phase during the first few days of chondrogenic induction in vitro . When MSCs are submitted to chondrogenic conditions in monolayer culture, they begin to condensate in response to the stimulus and form high-density three-dimensional cell aggregates . However, proper chondrogenic differentiation occurs also for MSCs embedded in gel-like biomaterials that keep cells apart from each other and thus limit direct cell-cell contact. This suggests that, although cell-cell contact facilitates chondrogenic induction of MSCs compared with monolayer culture, it does not represent an absolute requirement for in vitro chondrogenic differentiation of human MSCs in a three-dimensional structure."<-So cells may be able to differentiate into chondrocytes without contact with other MSCs. This is good for LSJL as we only have to encourage chondrogenesis(and subsequent endochondral ossification) of one cell rather than several.

So evidence supporting chondrogenesis in epiphyseal bone marrow. We need to learn more about methylation and histones that encourage height growth as we can manipulate those to grow taller.

Limb Lengthening Surgery

2011-11-15T15:12:00.000-08:00

Limb Lengthening Surgery is the premier method for gaining height. It is the only acknowledged method for height gain(even though physiologically other methods are possible). There are three steps involved in limb lengthening: fracturing the bone, lengthening the bone(distraction), and healing of the bone(osteogenesis). The bone is lengthening at a rate of 1mm a day(so about 25 days to grow taller by an inch). It may even be possible for spinal limb lengthening in the future. Scientists have also explored the possibility of facial limb lengthening.

Studying limb lengthening is important as it may have implications. The Periosteum is the primary source of stem cells for the endochondral ossification portion of distraction osteogenesis. Although there are other types of ossification involved in DO. DO(Distraction Osteogenesis) may provide us with insight into entirely new height increase methods like hypertrophy of osteoblast mitochondria.

Limb lengthening does not work by causing a macrofracture in the bone. So we can not apply distraction osteogenesis to microfracutres. Limb lengthening works by stretching the bony callus that is formed at the fractured ends of the bones. This stimulates bone growth. Microfractures may not necessarily form this bony callus.

Another interesting fact about limb lengthening is they don't stretch the fibula. Maybe they don't do that on purpose to discourage "excessive" height gain.

Here's a study that explains the mechanobiology of distraction osteogenesis. Let's look at how distraction osteogenesis causes height gain:

Mechanobiology of mandibular distraction osteogenesis: finite element analyses with a rat model.

"Three-dimensional finite element (FE) analyses were performed to characterize the local mechanical environment created within the tissue regenerate during mandibular distraction osteogenesis (DO) in a rat model. Finite element models were created from three-dimensional computed tomography image data of rat hemi-mandibles at four different time points during an optimal distraction osteogenesis protocol (i.e., most successful protocol for bone formation): end latency (post-operative day (POD) 5), distraction day 2 (POD 7), distraction day 5 (POD 10), and distraction day 8 (POD 13). A 0.25 mm distraction was simulated and the resulting hydrostatic stresses and maximum principal tensile strains were determined within the tissue regenerate[Limb Lengthening involves hydrostatic pressure and tensile strain which are two modalities we have been trying to use to induce height gain]. When compared to previous histological findings, finite element analyses showed that tensile strains up to 13% corresponded to regions of new bone formation and regions of periosteal hydrostatic pressure with magnitudes less than 17 kPa corresponded to locations of cartilage formation[So limb lengthening does involve cartilage formation rather than purely intramembranous ossification, 17 kPa is about 127 mmHg which isn't much at all]. Tensile strains within the center of the gap were much higher, leading us to conclude that tissue damage would occur there if the tissue was not compliant enough to withstand such high strains, and that this damage would trigger formation of new mesenchymal tissue. These data were consistent with histological evidence showing mesenchymal tissue present in the center of the gap throughout distraction. Finite element analyses performed at different time points during distraction were instrumental in determining the changes in hydrostatic stress and tensile strain fields throughout distraction, providing a mechanical environment rationale for the different levels of bone formation in end latency, and distraction day 2, 5, and 8 specimens."

A diagram in the study states that compression(like the lateral compression of LSJL) induces more cartilage formation whereas tensile strain induces more bone formation. The type of tension: hydrostatic pressure or tensile strain determined the type of bone formation. Chondrogenic differentiation from the periosteum with hydrostatic pressure and bone formation from the callus. So there are essentially two possibilities to make limb lengthening work: break the bone and then generate hydrostatic pressure in the bony callus or break the bone and then stretch the bony callus. Both seem to be effective in generating height growth. Generating a bony callus and then stretching it may be a method of height growth worth considering if we can do it without fracturing the bone of course.

Bone lengthening (distraction osteogenesis): a literature review.‏

"During a DO procedure, tissues are subjected to steady and constant tension and become metabolically activated. New bone formation occurs along the distraction stress line from both extremities of the distracted segment, on the cut ends of the two bony segments[so bone forms at the ends of the bones rather than within]. The proximal and distal parts of the osteotomized bone participate equally in bone regeneration. During this regeneration process, bone formation may show a rate of linear bone formation as high as 200-400 μm/day which is four to eight times faster than physiological physeal growth.

Distraction osteogenesis can be divided into three temporal phases: a latency period of 5 to 10 days, a distraction phase and a consolidation phase. The latency phase allows for the initial trauma response to take place. It starts immediately following the transverse osteotomy and extends until the beginning of distraction. Events taking place during this phase are basically the same as those in the early stages of fracture repair.

During the distraction phase, tensile forces are applied to the callus with a specific rate and rhythm by the distraction device[distraction osteogenesis applies a stretching force to the bony callus at the end of the bone].

As the primitive callus is stretched, a central fibrous zone called the fibrous interzone (FIZ) forms. It is rich in chondrocyte-like cells, fibroblasts and oval cells, which are morphologically intermediate between fibroblasts and chondrocytes[so distraction osteogenesis is not really like a growth plate]. The differentiating osteoblasts at the fibrous interzone deposit osteoid along collagen bundles. They subsequently undergo mineral crystallization parallel to the collagen bundles, forming a zone called the microcolumn formation zone (MCF). Microcolumns resemble stalagmite and stalagtites and have been identified as cones of 150-200 μm. Mineralization proceeds both longitudinally along collagen bundles, parallel to the distraction forces, and transversely as more collagen fibers incorporate[so the type II collagen fibers direct the mineralization]. In between the fibrous interzone and the microcolumn formation zone, a zone of highly proliferating cells, called the primary mineralization front (PMF), is observed.

Once the desired bone length is achieved, distraction ceases, marking the beginning of the consolidation phase, where bone and extensive amounts of osteoid undergo mineralization and remodeling.

Bone regeneration during distraction osteogenesis is believed to occur in response to the longitudinal mechanical strain applied to the callus during healing[distraction osteogenesis involves stretching the callus not the bone]. The exact mechanism by which strain stimulates bone formation remains unclear. It has been suggested that living tissues become metabolically activated by slow, steady traction, a phenomenon called "mechano-transduction", characterized by the stimulation of proliferative, secretory and biosynthetic cellular functions. The structural changes in the cells provide the basis for tissue regeneration under mechanical stress. Mitochondria in skeletal muscle hypertropy, showing evidence of increased volume with multiple cristae, and the functional activity of the nuclei was also increased during DO[skeletal muscle mitochondria hypertrophying causes increased volume perhaps an increase in the functional activity of the nuclei in the bone mitochondria can increase height]. Smooth muscle cells in the middle layer of the vessel walls were also activated, their nuclei were hypertrophied, and active euchromatin appeared in the nuclei.

Histological changes occurring in the regenerate under the tensile forces have been widely studied. Three different modes of ossification are identified and implicated in bone formation during DO.

Membranous ossification is the predominant mechanism of ossification during DO, particularly during late stages. Histological observations reveal that cells represent a continuum between fibroblasts, pre-osteoblasts and osteoblasts arranged longitudinally in order of differentiation. The different types of cells are seen along the bone trabeculae oriented along the tension vector within the MCF.

Endochondral ossification occurs during early stages of DO and is characterized by a cartilage tissue transition from fibrous tissue to bone. Ossification occurs through a cartilage intermediate. A hypertrophic cartilaginous callus is progressively invaded by capillaries and new bone will deposit on the surface of eroded cartilage. Enchondral ossification has been identified during distraction and consolidation phases. Enchondral ossification is usually seen at the junction of the FIZ and the newly mineralized membranous bone emanating from the cut ends. This mode of ossification which is characteristic of bone fracture repair has been identified in various experimental models of long bone DO (sheep, dogs, rabbits.) and in mandibular distraction. The ratio of membranous on enchondral ossifications in DO is close to 5/1.

A third mode of ossification called "transchondroid bone formation" has been described as part of DO histological events. During transchondroid ossification, chondroid bone is formed directly by chondrocyte-like cells, with a gradual transition from fibrous tissue to bone (chondroid bone)[so you can grow taller from fibrous tissue]. Transition from fibrous tissue to bone occurs gradually without capillary invasion. Chondrocyte-like cells undergo some kind of an osteogenic differentiation with type I and type II collagen fibres identified in hypertrophic chondrocytes and APL activity present in cartilage matrix in transitional region. Cartilage that forms during DO is usually observed at the level of the periosteum, but not between the cut ends of the cortices within the distraction gap[so stem cells from the periosteum are responsible for any endochondral ossification].

Tissue regeneration within the distraction gap is inevitably consecutive to changes in cellular morphology and function. There is hyperplasia of cell organelles including mitochondria, endoplasmic reticulum, Golgi complex in skeletal muscle, and blood vessels at the ultrastructural level, under mechanical tension. Proliferation of osteoblasts is increased by mechanical force. Immunohistochemical analysis showed that proliferating cell nuclear antigen was expressed during the initial period of distraction, indicating the active stimulation of cell proliferation by tension, which was coincident with the appearance of large numbers of fibroblasts in the distraction gap on histological examination . More recently, a very well-documented experimental study analysing the ultrastructural changes occurring within cells under tensile forces in a goat mandibular distraction model clearly showed morphological changes occurring within the cells during the distraction process. In the distraction gap, cells are seen longitudinally oriented along the distraction force 8 days after loading. A week later, at 16 days, cells in the distraction gap begin to differentiate into osteoblasts, showing changes in both protein synthesis and the energy-supplying system. At an ultrastructural level, these cells are hyperplastic in rough endoplasmic reticulum[so hyperplasia of osteoblastic cells can cause height growth?]. Active secretion of collagen fibers in the extracellular matrix is identified. Finally at 32 days, the main ultrastructural character was biosynthesis and secretion of the extracellular matrix. The cells showed numerous rough endoplasmic reticula and abundant mitochondria, smooth membrane vesicles and well-developed Golgi complexes indicating active synthetic and secretory capabilities. Cells were less likely to proliferate and osteoblasts on the surface of newly formed bone secreted collagenous fibres directly on to the matrix surface. In the 48 day group, the bony matrix was more mature and mineralised. Osteoblasts around the bony trabeculae secreted matrix on to the trabeculae, which may help new bone to be modelled.

Recent molecular investigations have also indicated that the molecular signaling cascade plays an important role in the relationship between induced strain and bone regeneration. The molecular signals that drive the regenerative process of DO are similar to those characterizing fracture repairs and include the pro-inflammatory cytokines, the transforming growth factor beta superfamily and angiogenic factors. Various studies have reported that among growth factors, bone morphogenetic proteins (BMPs) may play a central role in the molecular signaling cascade leading to bone renegeration and remodeling in a DO procedure. "

Although hyperplasia of muscle cells causes an increase in volume does it cause an increase in length? It would seem to be no but it's possible there is an increase in length but there's just no room for more muscle. Since bone is the limiting factor if hyperplasia of osteoblasts caused an increase in length this wouldn't be an issue. Analyzing hyperplasia of osteoblast mitochondria may be a method of height growth worth studying.

Molecular mechanisms controlling bone formation during fracture healing and distraction osteogenesis.

Stage of Fracture Repair	Biological Processes	Expression of Signaling Molecules and their Proposed Functions
Inflammation	Hematoma	IL-1, IL-6, and TNF-α play a role in initiating the repair cascade.
	Inflammation	TGF-β, PDGF, and BMP-2 expression increases to initiate callus formation.
	Recruitment of mesenchymal stem cells	GDF-8 is restricted to day 1, suggesting its role in controlling cellular proliferation.[GDF-8 is myostatin, so myostatin limits how much cellular proliferation you get during healing, so myostatin alters the effectiveness of limb lengthening and many other height growth mechanisms]

Cartilage Formation and Periosteal Response	Chondrogenesis and endochondral ossification begins	TGF-β2, -β3, and GDF-5 peak due to their involvement in chondrogenesis and endochondral bone formation.
	Cell proliferation in intramembranous ossification	BMP-5 and -6 rise.
	Vascular in-growth	Angiopoietins and VEGFs are induced to stimulate vascular in growth from vessels in the periosteum.
	Neo-angiogenesis

Cartilage Resorption and Primary Bone Formation	Phase of most active osteogenesis	TNF-α rises in association with mineralized cartilage resorption. This promotes the recruitment of mesenchymal stem cells and induces apoptosis of hypertrophic chondrocytes.[maybe TNF-alpha should not be inhibited. Inflammatory cytokines do cause DNA damage so there is likely a better way to induce recruitment of MSCS]
	Bone cell recruitment and woven bone formation	RANKL and MCSF rise in association with mineralized cartilage resorption.
	Chondrocyte apoptosis and matrix proteolysis
	Osteoclast recruitment and cartilage resorption	BMP-3, -4, -7, and -8 rise in association with the resorption of calcified cartilage. They promote recruitment of cells in the osteoblastic lineage.
	Neo-angiogenesis	BMP-5 and -6 remain high during this stage, suggesting a regulatory effect on both intramembranous and endochondral ossification.
		VEGFs are up-regulated to stimulate neo-angiogenesis.

Secondary Bone Formation and Remodeling	Bone remodeling coupled with osteoblast activity	IL-1 and IL-6 rise again in association with bone remodeling, whereas RANKL and MCSF display diminished levels.
Secondary Bone Formation and Remodeling	Establishment of marrow	Diminished expression of members of the TGF-β superfam

"Interleukins-1 and -6 (IL-1 and IL-6) and TNF-α have been shown to play a role in initiating the repair cascade. They induce a downstream response to injury by recruiting other inflammatory cells, enhancing extracellular matrix synthesis, and stimulating angiogenesis. They are secreted at the injury site by macrophages, inflammatory cells, and cells of mesenchymal origin."<-this could potentially make anti-oxidents bad by lowering the extracellular matrix synthesis. However, it may be possible to bypass inflammatory cytokines and go straight to BMP-2 and TGF-Beta1.

"In addition to stimulating osteoclast function, TNF-α promotes the recruitment of mesenchymal stem cells and induces apoptosis of hypertrophic chondrocytes during endochondral bone formation. Its absence delays the resorption of mineralized cartilage and, consequently, prevents the formation of new bone. In situations where TNF-α is over-expressed, such as diabetic healing, there is premature cartilage removal that is associated with deficient bone formation and healing"<-Other studies have shown that TNF-alpha inhibits chondrogenesis. It's possible you want only a minimal amount of TNF-alpha for maximal height growth. Just enough for new bone formation to occur.

"Bone regeneration during distraction osteogenesis is believed to occur in response to the longitudinal mechanical strain applied to the callus during healing"<-would the bone increase in length if there was no gap and you just stretched the cells of the callus.

To test if the fracture gap is needed for distraction osteogenesis height growth you would need to cause a fracture in a non-longitudinal direction. Then apply a tensile strain force to the callus. If the bone grows longitudinally then stretching the bony callus must provide a signal for the bone to increase in volume on a cellular signaling level(such as increasing osteoblast mitochondrial size).

This study describes the cells of the callus.

Bone remodeling during fracture repair: The cellular picture.

"Here's the inflammatory stage that proceeds callus formation:

The extravasation (bleeding) within the fracture site is contained by the surrounding tissue and develops into a hematoma. Degranulating platelets, macrophages, and other inflammatory cells (granulocytes, lymphocytes, and monocytes) infiltrate the hematoma between the fractured fragments and combat infection, secrete cytokines and growth factors, and advance clotting into a fibrinous thrombus . Over time, capillaries grow into the clot, which is reorganized into granulation tissue. Macrophages, giant cells and other phagocytic cells clear degenerated cells and other debris.

This cellular response is coordinated by and involves the secretion of a range of cytokines and growth factors including transforming growth factor-β (TGF-β), platelet-derived growth factor (PDGF), fibroblast growth factor-2 (FGF-2), vascular endothelial growth factor (VEGF), macrophage colony stimulating factor (M-CSF), interleukins-1 and -6 (IL-1 and -6), bone morphogenetic proteins (BMPs), and tumor necrosis factor-α (TNF-α). This factors facilitate the recruitment of additional inflammatory cells in a positive feedback loop, and also the migration and invasion of multipotent mesenchymal stem cells. Stem cells originating from the periosteum, bone marrow , circulation, and the surrounding soft tissues have been implicated in bone formation and repair."<-So a bony callus begins as a hematoma with inflammatory cytokines and traditional bone forming compounds(FGF-2, TGF-Beta, PDGF, BMPs, and VEGF).

For the soft callus:

"Chondrocytes derived from mesenchymal progenitors proliferate and synthesize cartilaginous matrix until all the fibrinous/granulation tissue is replaced by cartilage. Where cartilage production is deficient, fibroblasts replace the region with generalized fibrous tissue. Discrete cartilaginous regions progressively grow and merge to produce a central fibrocartilaginous plug between the fractured fragments that splints the fracture. In the final stages of soft callus production, the chondrocytes undergo hypertrophy and mineralize the cartilaginous matrix before undergoing apoptosis."<-We can get chondrocytes and fibrous tissue without a fracture based hematoma. Say within the bone marrow. If we proceed to stretch this region will it stimulate bone volume(and therefore bone height increase)?

" hard callus can form in the absence of a cartilaginous template in intramembranous bone formation (during conditions of high mechanical stability) or in appositional bone growth, where bone forms directly adjacent to an existing mineralized surface. However, in the majority of orthopaedic instances, some level of endochondral ossification is present."<-There has to be some limiting factor on appositional bone growth because why don't the tips of your fingers constantly grow(if they do then it is incredibly slow)? Stretching this hard callus must too stimulate height growth. It's unlikely for appositional bone growth to occur at a fracture gap as then it would be possible for the bones to become two separate bones.

"The initial woven bone matrix contains a combination of proteinaceous and mineralized extracellular matrix tissue. This is synthesized by mature osteoblasts, which differentiate from osteoprogenitors in the presence of osteogenic factors. Members of the BMP family are critical mediators of this process, and have been shown to be sufficient for de novo bone formation"<-If the hard callus is mostly type I collagen then we have tons of type I collagen in the bone to stretch. Either the loads people have been using to stretch bone have not been sufficient to stretch Type I collagen or stretching Type I Collagen alone does not cause height growth.

What part of stretching the callus makes you taller is unknown as stretching the components of the callus like cartilage or Type I collagen has not been enough to induce height growth(Now of course the stretching force may have never been enough). The hydrostatic pressure generated by a hematoma may definitely play a role in soft callus fracture healing but hydrostatic pressure is not needed for the hard callus. There would need to be a control study of stretching type I collagen by 1 mm a day with no fracture.

Here's a study that shows the formation of cartilage islands and bands during distraction osteogenesis. This is of relevance to LSJL, as cartilage islands and bands are likely to be what's formed when chondrogenic differentiation is achieved.

Bone lengthening osteogenesis, a combination of intramembranous and endochondral ossification: an experimental study in sheep.

"Endochondral ossification from the central fibrous tissue has been shown in the distraction gap in experimental models of distraction osteogenesis in rabbits. On the other hand, intramembranous ossification has been proposed to result when a low distraction rate under stable external fixation is applied"<-Maybe an initial fibrous tissue has to be formed within the bone marrow for chondrogenesis to proceed.

"At the proximal and distal ends of the fibrous tissue, chondrocytes became hypertrophic, and new bone trabeculae were formed through endochondral ossification. The cartilage tissue consisted of hypertrophic chondrocytes invaded by neovessels, and mesenchymal cells, abundant fibrous tissue and new bone gradually replaced the surface of the eroded cartilage . The fibrous tissue showed abundant vessels and mesenchymal cells in both forms of ossification"<-Everything else should be present in the bone marrow except for the abundant fibrous tissue.

Here's a diagram showing cartilage bands and islands within fibrous tissue:

"The cascade of endochondral bone development in association with the role of fibronectin has been described. During mesenchymal cell proliferation, fibronectin is present in a cottony array. During chondrogenesis, it is associated with the pericellular zone of chondrocytes. During chondrolysis, loss of proteoglycans unmasks the fibronectin in the hypertrophic cartilage matrix. This “exposed” fibronectin may then serve as nidus for osteoprogenitor cell attachment and differentiation into osteoblast. In the present study, we observed fibronectin in the cartilage tissue, the central region of the newly formed bone trabeculae, and some of the cells within the bone trabeculae."<-Fibronectin which is in fibrous tissue may be the key to grow taller.

Fibronectin may be the missing link to growing taller

2011-11-15T11:46:00.000-08:00

In the article on distraction osteogenesis, an observation was made that endochondral ossification occurred within the fibrous tissue. Fibronectin which is produced by fibrous tissue is key to endochondral ossification. Fibrous tissue is formed in the distraction gap in distraction osteogenesis. Does LSJL produce fibrous tissue? Is there anything we can do to encourage the production of fibrous tissue in the bone marrow?

Fibronectin- and collagen-mimetic ligands regulate bone marrow stromal cell chondrogenesis in three-dimensional hydrogels.

"While adhesion to RGD peptides has been shown to inhibit in vitro chondrogenesis, the effects of extracellular matrix (ECM)-mimetic ligands with complex secondary and tertiary structures are unknown. This study aimed to determine whether collagen- and fibronectin-mimetic ligands would retain biologic functionality in three-dimensional (3D) hydrogels, whether different ECM-mimetic ligands differentially influence in vitro chondrogenesis, and if effects of ligands on differentiation depend on soluble biochemical stimuli. A linear RGD peptide, a recombinant fibronectin fragment containing the seven to ten Type III repeats (FnIII7-10) and a triple helical, collagen mimetic peptide with the GFOGER motif were covalently coupled to agarose gels using the sulfo-SANPAH crosslinker, and bone marrow stromal cells (BMSCs) were cultured within the 3D hydrogels. The ligands retained biologic functionality within the agarose gels and promoted density-dependent BMSC spreading. Interactions with all adhesive ligands inhibited stimulation by chondrogenic factors of collagen Type II and aggrecan mRNA levels and deposition of sulfated glycosaminoglycans. In medium containing fetal bovine serum, interactions with the GFOGER peptide enhanced mRNA expression of the osteogenic gene osteocalcin whereas FnIII7-10 inhibited osteocalcin expression. In conclusion, modification of agarose hydrogels with ECM-mimetic ligands can influence the differentiation of BMSCs in a manner that depends strongly on the presence and nature of soluble biochemical stimuli."

"integrin-mediated adhesion to fibronectin is required for precartilage condensation of limb bud cells"<-Fibronectin may be critical for chondrogenesis. RGE was the most chondrogenic of all the ligands. RGE is the non-adhesive version of the RGD integrin.

"Our data provide additional support for the role of the cytoskeleton and suggest that cell shape, more
so than the specific integrin ligand, is a key regulator of chondrogenesis"<-this is good for LSJL as hydrostatic pressure may induce a more pro-chondrogenic cell shape.

It may be that Fibronectin is key but that it's key for it to be non-adhesive to allow for chondrogenesis. Perhaps, the adhesive form of Fibronectin is produced post cessation of endochondral ossification and that's what inhibits growth.

However, TGF-Beta1 may stimulate fibronectin and TGF-Beta1 is very pro-chondrogenic.

Fibronectin regulates proteoglycan production balance in transforming growth factor-β1-induced chondrogenesis.

"Transforming growth factor (TGF)-β and bone morphogenetic protein (BMP) induce a cartilage-specific extracellular matrix (ECM) gene, aggrecan, in a chondrogenic cell line, ATDC5. The results of our recent study show that TGF-β1, but not BMP-4, strongly induces an ECM gene, fibronectin, during chondrogenesis. However, the role of fibronectin in chondrogenesis is unclear. In the current study, our results showed that TGF-β1, but not BMP-4, led to versican-dominant proteoglycan production during chondrogenesis of ATDC5 cells. siRNA-mediated reduction of fibronectin and interference in the liaison between fibronectin and integrins by the Arg-Gly-Asp-Ser (RGDS) peptide increased aggrecan expression, and decreased versican expression by TGF-β1 stimulation[so reduction of fibronectin stimulated chondrogenesis, maybe fibronectin is a negative feedback mechanism by TGF-Beta1 and there's other height stimulating effects of TGF-Beta1 that are not fibronectin]. These data suggest that fibronectin is a critical mediator for TGF-β-specific production balance of 2 major proteoglycans, aggrecan and versican, during chondrogenesis."

"These data suggest that increased expression of versican in the cartilage correlates with fibrosis and decreases the water-holding capacity, and TGF-βs, which induce versican expression, may be key molecules that increase the degree of fibrosis during chondrogenesis."<-So Aggrecan is likely better than versican for height growth as more water equals a larger growth plate.

Fibroconectin signaling may be modulated by magnetic fields. PEMFs have been discussed before.

Mechanical integrin stress and magnetic forces induce biological responses in mesenchymal stem cells which depend on environmental factors.

"The control of mesenchymal stem cells (MSC) by physical cues is of great interest in regenerative medicine. Because integrin receptors function as mechanotransducers, we applied drag forces to β1 integrins on the apical surface of adherent human MSC. In addition to mechanical forces, the technique we used involved also the exposure of the cells to an inhomogeneous magnetic field. In order to assess the influence of the substrate on cell adhesion, cells were cultured on plain tissue culture polystyrene (TCP) or on coated well plates, which allowed only adhesion to embedded fibronectin or RGD peptides. We found that the expression of collagen I, which is involved in osteogenesis, and VEGF, a factor which stimulates angiogenesis, increased as a result of short-term mechanical integrin stress. Whereas, collagen I expression was stimulated by mechanical forces when the cells were cultured on fibronectin and RGD peptides but not on TCP, VEGF expression was enhanced by physical stimulation on TCP. The study further revealed that magnetic forces enhanced Sox 9 expression, a marker of chondrogenesis, and reduced the expression of ALP[magnetic forces help enhance Sox9 which helps you grow taller]. Concerning the intracellular mechanisms involved, we found that the expression of VEGF induced by physical forces depended on Akt activation. Together, the results implicate that biological functions of MSC can be stimulated by integrin-mediated mechanical forces and a magnetic field. However, the responses of cells depend strongly on the substrate to which they adhere and on the cross-talk between integrin-mediated signals and soluble factors."

"Mesenchymal stem cells are also able to sense the elasticity of a substrate, which determines the direction of differentiation or maintains their quiescence"<-We can manipulate this. Remember, bone becomes more elastic when exposed to acid for instance. How to do this safely is another question.

"The forces subjected to one bead were adjusted to 2 × 10⁻¹⁰ N. Because of the varying number and location of beads attached to one cell, differential strains across the cell can occur. A cyclic stress of 1 Hz (0.5 s on, 0.5 s off) was applied for 15 min."<-this seems pretty small so it's promising for potential application.

"On TCP, cells can adhere to a mix of extracellular matrix proteins both produced by the cells and contained in the culture medium" The increase in SOX9 was only observed on Tissue Cultured Polystyrene. So SOX9 was only induced when cells were free not to bind to Fibronectin and RGD peptides.

So Fibroconectin may be a key to growing taller but not by stimulating it. The breaking of the bone may loosen fibrous tissue and decrease the integrin binding to fibroconectin allowing for chondrogenesis. Acidifying the bone or applying magnetic fields may be possible ways to weaken integrin signaling to allow for chondrogenesis.

Does the structure of bone provide insight into growing taller?

2011-11-08T12:28:00.000-08:00

Stuart J Warden was a friend of CH Turner who developed part of LSJL. SJ Warden published a new paper and it provides some insight into bone and possibly insights into height growth.

Specialized Connective Tissue: Bone, the Structural Framework of the Upper Extremity

"Bone is a connective tissue containing cells, fibers, and ground substance. There are many functions in the body in which the bone participates, such as storing minerals[bone is not hydrophillic like cartilage, so if there are high quantities of water it makes sense for stem cells to differentiate into chondrocytes to store water, although water is not a mineral], providing internal support, protecting vital organs, enabling movement, and providing attachment sites for muscles and tendons. Bone is unique because its collagen framework absorbs energy, whereas the mineral encased within the matrix allows bone to resist deformation."<-In biology, structure correlates with function. Can there be any instances where a longer bone would improve bones function? Yes, if bone were longer it would serve better at enabling movement in some cases. But, playing basketball doesn't make people taller. Neither do bigger muscles or tendons. Only storing water requires the formation of cartilage which is what enables growing taller via endochondral ossification.

"Cortical bone thins toward the expanded ends (epiphyses) and interposed developing region (metaphysis) of long bones where it plays a lesser, yet clinically significant mechanical role"<-Maybe thicker cortical bone is a blocker of endochondral ossification?

"The periosteum covers external surfaces of most bones and is divided into two distinct layers—an outer fibrous and inner cellular layer. The cellular or “cambium” layer is positioned in direct contact with the bone surface and is of particular interest as it contains mesenchymal stem cells (MSCs), which have the potential to differentiate into osteoblasts and chondrocytes, and differentiated osteogenic progenitor cells. The localization of these cell types has made the cellular layer a target for drug therapies and cell harvesting for tissue engineering purposes."<-Since periosteal stem cells are right there next to the bone, they are a good target for chondrogenic differentiation. Unfortunately, they are oriented on a longitudinal axis rather than a horizontal(except for the flat bone of the skull).

"The endocortical surface of a bone faces the medullary canal and is lined by the endosteum, a single thin layer of bone lining cells (mature osteoblasts) and osteoblasts, which form a membrane over endocortical and trabecular bone surfaces to enclose the bone marrow. The endosteum contains osteoprogenitor cells, but does not appear to contain either MSCs or hematopoietic stem cells (HSCs). However, a portion of HSCs can be found next to the endosteum suggesting reciprocal communication between cells within the endosteum and multipotent HSCs. The close relationship between the cells forms a so-called stem cell niche, whereby the cells of the endosteum physically support and influence stem cell activity."<-There's no endosteum in the epiphysis of the bone. Maybe the functions of the endosteum can be mimicked in the epiphysis to help form new cartilagenous growth plates there.

"Lamellae in cortical bone form osteons or bone structural units, which consist of a central canal enveloped in concentric lamellae of bone tissue. Outer lamellae form first along the boundary of the osteon known as the cement line, with each successive lamella being laid concentrically inside the preceding one. In trabecular bone, lamellae are stacked into saucer-shaped bone packets that are separated by cement lines. The first lamellae are formed toward the center of the trabeculae with each successive lamella being stacked in parallel layers toward the bone surface . Uniformly spaced throughout lamellae are lenticular cavities called lacunae from which branching canaliculae radiate in all directions. The canaliculae penetrate the lamellae of the interstitial substance to anastomose with canaliculae of neighboring lacunae to form a continuous network of interconnecting cavities."<-Thus bone has the ability to communicate to other parts of the bone.

"Without the addition of mineral to collagen, bone tissue would have properties similar to a rubber band, whereas without collagen, bone is brittle such as chalk."<-If bone had properties similar to a rubber band it would be much easier to make longer. Demineralizing the bone is a potential way for height growth to occur.

"Osteoclasts are large, multinucleate cells that exclusively mediate the process of bone resorption. Osteoclastogenesis begins when a HSC is stimulated to generate mononuclear cells, which then become committed preosteoclasts and are introduced into the blood stream[so the bone has a way for stem cells to enter the blood steam we just have to make those cells chondrocytes instead of osteoclasts]. The circulating precursors exit the peripheral circulation at or near the site to be resorbed, and fuse with one another to form a multinucleated immature osteoclast. Mature osteoclasts establish a microenvironment between themselves and the underlying bone by peripherally attaching to the matrix using integrins. The attachment creates a compartment between the ruffled basal border of the osteoclast and the bone surface that is isolated from the general extracellular space. An electrogenic proton pump transports in H⁺ ions to acidify the compartment, which acts to mobilize the mineralized component of bone. This exposes the organic matrix, which is subsequently degraded using proteases. The end result is the removal of bone matrix and the development of characteristic shallow cavities known as Howship’s lacunae."<-So osteoclasts attach to bone matrix using integrins. Can we use similar integrins to encourage chondrocytes to attach to the site of a new growth plate? Osteoclasts acidifing the compartment is also useful in making the collagen more elastic. Elastic bone is much easier to stretch. However, once bone is degraded by protease there is none to stretch.

"Osteoblasts are bone-forming cells and develop locally after proliferation of MSCs residing in the bone marrow stroma and periosteum. Mature osteoblasts express the matrix proteins type I collagen and osteocalcin, and alkaline phosphatase—a key enzyme in the mineralization process. Rows of active osteoblasts secrete unmineralized matrix (osteoid) before becoming either bone lining cells or incorporated into the bone matrix. Cells that become incorporated into the matrix gradually develop long cytoplasmic processes to remain in communication with surrounding cells and are considered immature osteocytes. As the matrix matures and mineralizes, and the osteoid seam moves further away, the osteocyte becomes entombed in a bony matrix."<-If we could get osteoblasts to secrete matrix on the longitudinal ends of bones then we could become taller.

"Osteocytes are the most numerous bone cells and are dispersed throughout the matrix where they occupy lacunae[although LSJL is about chondrogenic differentiation of mesenchymal stem cells, osteocytes are likely a part of the process]. Lacunae are interconnected by an elaborate network of thin tunnels called canaliculi through which osteocytes pass cytoplasmic or dendritic processes[we can determine which cytoplasmic and dendritic processes help with height growth and mimic them to grow taller]. These processes connect individual osteocytes with neighboring cells via gap junctions to facilitate both the transport of nutrients for osteocyte viability and the conveying of intercellular messages[so osteocytes have the ability to communicate with other cells including possible communicating with mesenchymal stem cells to differentiate into chondrocytes]. Intercellular communication is also facilitated by the osteocytic release of signaling molecules into the extracellular fluid, which flows through the lacuna-canalicular system[LSJL increases this fluid flow thus possibly amplifying these signaling molecules]. Osteocyte function remains unclear; however, their principal role appears to be the sensing of mechanical stimuli. In addition, recent evidence has also found osteocytes have the capacity to regulate mineral metabolism and alter their surrounding matrix."

"Bones of the upper extremity predominantly develop by endochondral ossification wherein condensations of mesenchymal cells differentiate into chondrocytes to form a cartilaginous template (or “anlage”). Exceptions are parts of the clavicles and scapulae, which form via intramembranous ossification that does not involve a cartilaginous precursor. In the anlage, chondrocytes hypertrophy and an ossification center forms by neovascularization of the initially avascular cartilaginous template. Osteoblasts associated with the newly developed vasculature begin secretion and mineralization of a type-I collagen-containing extracellular matrix. As development continues, the ossification center propagates toward the epiphyseal growth plates."<-This mentions the ossification center propagating toward the epiphyseal growth plates this would of course mean that inhibiting estrogen and bone growth would help height growth by giving more time for chondrocytes to grow. However, this has been found not to be the case and that only very low or high levels of estrogen are detrimental to growth. If there is an ossification center, the rate of it's growth does not seem to affect height growth. In fact, it's likely a byproduct of normal endochondral ossification. Also, if trying to form a new growth plate with LSJL the region should already be vascularized which may prove problematic with forming a new ossification center.

"[Motor paralysis] interferes with skeletal growth and modeling leading to the development of bones with reduced length, mass, and size"<-So exercise definitely affects growth but there may be diminishing returns as exercise does not seem to positively correlate with height past a point.

This study finds a correlation though between muscle size and bone size(although a shared variable like serum levels of myostatin may be involved):

Mechanical loads and cortical bone geometry in healthy children and young adults

"Muscle torque was positively associated with tibia length and muscle CSA[cross sectional area], independent of age, sex, and race."<-Increasing tibial length increases muscle torque.

"muscle CSA was positively associated with endosteal circumference"<-If the endosteum does help with height growth then this could be a mechanism to how muscle CSA helps with height growth.

"the study had limited power to detect significant differences in muscle–bone relations in the mature vs. growing skeleton"<-the more mature bone is similar to growing bone the more likely that mature bone is able to grow longer.

"changes in estimated bone strength during growth are highly correlated with changes in lean mass and muscle cross-sectional area (CSA) but not fat mass. When the growing bone is not subject to mechanical loading, muscle size and function are reduced and bone lacks the shape necessary for its function. In a study of arm side-to-side differences in growing tennis players, greater muscle size induced by exercise was positively correlated with changes in bone mass, size, and strength. However, the greater muscle size only accounted for 12%–16% of the side-to-side variance in bone outcomes, suggesting that other mechanical factors may contribute to bone adaptation during growth."<-So muscle size may play a role in up to 12% of bone length increasing.

"Tibia length is highly associated with Zp and with muscle CSA."<-Tibial length is correlated to muscle CSA.

"muscle CSA was positively associated with Zp, explaining 85% of the variance in Zp. Conversely, tibia length alone was significantly associated with greater Zp, explaining 84% of the variance in Zp."<-The fact that percentage of variance explained in bone strength is so similar between muscle and bone means that Bone Length and Muscle CSA are likely connected.

Remember, that muscular activity lowers myostatin levels which could trickle into effecting the bone.

So, potential mechanisms to manipulate height growth are altering osteocyte communication(done by LSJL which increases fluid flow), making the bone more acidic so it's more elastic(but how to do this safely?), and integrin binding(would need to involve cellular engineering). Also, the endosteum is linked to muscle CSA and the endosteum is involved with bone marrow stem cell differentiation so maybe the endosteum is linked to myostatin which is linked to bone muscle and bone marrow stem cells.

Hydrostatic Pressure may not increase height growth for everyone

2011-11-03T12:17:00.000-07:00

Hydrostatic pressure is an important basis for the theory of LSJL. Could some individuals cells be responsive to hydrostatic pressure while others are not? Note in the following study 10 MPa is used which is far greater than what can be achieved by a clamp.

The effect of cyclic hydrostatic pressure on the functional development of cartilaginous tissues engineered using bone marrow derived mesenchymal stem cells.

"Mechanical signals can play a key role in regulating the chondrogenic differentiation of mesenchymal stem cells (MSCs)[We are trying to use the mechanical signal of LSJL to induce chondrogenic differentiation of mesenchymal stem cells in the epiphyseal bone marrow]. The objective of this study was to determine if the long-term application of cyclic hydrostatic pressure could be used to improve the functional properties of cartilaginous tissues engineered using bone marrow derived MSCs. MSCs were isolated from the femora of two porcine donors[so pig donors], expanded separately under identical conditions, and then suspended in cylindrical agarose hydrogels. Constructs from both donors were maintained in a chemically defined media supplemented with TGF-β3 for 42 days. TGF-β3 was removed from a subset of constructs from day 21 to 42. Loaded groups were subjected to 10 MPa of cyclic hydrostatic pressurisation at 1 Hz for one hour/day, five days/week. Loading consisted either of continuous hydrostatic pressure (CHP) initiated at day 0, or delayed hydrostatic pressure (DHP) initiated at day 21. Free swelling (FS) constructs were cultured in parallel as controls. Constructs were assessed at days 0, 21 and 42. MSCs isolated from both donors were morphologically similar, demonstrated comparable colony forming unit-fibroblast (CFU-F) numbers, and accumulated near identical levels of collagen and GAG following 42 days of free swelling culture[So the MSCs from different donors responded differently to other variables than hydrostatic pressure]. Somewhat unexpectedly the two donors displayed a differential response to hydrostatic pressure. For one donor the application of CHP resulted in increased collagen and GAG accumulation by day 42, resulting in an increased dynamic modulus compared to FS controls. In contrast, CHP had no effect on matrix accumulation for the other donor[So for one donor the hydrostatic pressure did not work at enhancing chondrogenic differentiation]. The application of DHP had no effect on either matrix accumulation or construct mechanical properties for both donors[So Hydrostatic Pressure works better at initiating chondrogenic differentiation which is what we're working towards with LSJL]. Variability in the response to hydrostatic pressure was also observed for three further donors. In conclusion, this study demonstrates that the application of long-term hydrostatic pressure can be used to improve the functional properties of cartilaginous tissues engineered using bone marrow derived MSCs by enhancing collagen and GAG accumulation. The response to such loading however is donor dependent, which has implications for the clinical utilisation of such a stimulus when engineering cartilaginous grafts using autologous MSCs."

Since the bone marrow was isolated from the bone itself, it cannot be bone properties that altered the resistance of stem cells to hydrostatic pressure. Some stem cells must be resistant to the stimulus of hydrostatic pressure to induce cartilage growth.

"Cyclic hydrostatic pressure has been shown to enhance chondrogenesis of MSC aggregates, as evidenced by increases in type II collagen and aggrecan mRNA expression and/or proteoglycan and collagen accumulation"<-MSC aggregates means before any signs of chondrogenesis is detected. Just if there are sufficient number of MSCs aggregated in a certain area. Hydrostatic Pressure has the ability to initiate chondrogenesis where there is none which is why it's so powerfully potential to induce height growth.

"It has also been demonstrated that the magnitude of hydrostatic pressure (0.1, 1 or 10 MPa) differentially regulates chondrogenesis of MSC aggregates, with greater type II collagen mRNA expression and collagen accumulation at higher pressures"<-Unfortunately, we are limited in how much hydrostatic pressure we can induce but we will still get some Type II collagen mRNA expression and collagen accumulation.

"In contrast, other studies report that hydrostatic pressure has little or no effect on chondrogenic gene expression or matrix accumulation in MSC aggregates, in either the presence or absence of TGF-β1 or BMP-2. Furthermore, hydrostatic pressure has been shown to have no effect on aggrecan and collagen II mRNA expression for MSCs embedded in agarose hydrogels"<-So hydrostatic pressure does not help induce height growth sometimes.

"The response of stem cells to mechanical signals [may be individual] dependent."<-Some stem cells may not send out chondrogenic signals to hydrostatic pressure which would mean that LSJL would not work for those people.

"Our hypothesis was initially motivated by our previous findings that other forms of mechanical stimulation, specifically dynamic compression, can inhibit chondrogenesis of MSCs if applied before chondrogenesis has occurred"<-Weight lifting is dynamic compression so weight lifting could inhibit chondrogenesis. This does not affect pubertal growth as the MSCs have already engaged in chondrogenesis although there may be some MSCs that have not yet.

" It may be that 3 weeks of delayed hydrostatic pressure was of insufficient duration, as previous studies have also suggested that multiple days of hydrostatic pressure may be required to enhance chondrogenesis of bone marrow derived MSCs"<-So it's possible that some people may need a longer duration of hydrostatic pressure to experience results.

" this result is to be expected given the well documented variability in the response of MSCs from different donors to cytokine induced chondrogenic differentiation, and the fact that animal model studies investigating mechanically induced chondrogenesis in vivo often report dramatic donor dependent response to loading"<-MSCs respond differently to various cytokines and other mechanical signals so responding differently to hydrostatic pressure is not out of the question.

So LSJL may not be effective for some individuals based on the responsiveness of their MSCs to hydrostatic pressure. Now this might not be solely genetic(which can be altered by the environment as well) but environment. Some MSCs could become conditioned to hydrostatic pressure or some MSCs might not be sufficiently primed for hydrostatic pressure by exposure to compounds like IGF-1 and Hyaluronic Acid.

We really have to know why some MSCs don't respond to hydrostatic pressure to know how to fix it.

Grow Taller with Seaweed and Beta-Glycerophosphate

2011-11-01T11:49:00.000-07:00

In our study of the periosteum, we learned that chondrocytes had difficulty fully ossifying into bone when they were not adjacent to the periosteum. Now there could be things present in the body that were not accounted for in the study but the lack of periosteum in the epiphysis means that endochondral ossification will be harder to occur there. Alginate is a part of seaweed and is very similar to Hyaluronic Acid. It is hydrophillic like Hyaluronic Acid. Alginate may be able to mimic the beneficial effects of hyaluronic acid but may be different enough to bypass the negative feedback mechanisms against hyaluronic acid. This supplement contains Alginic Acid and Sodium Alginate: Rx Vitamins - Acid Block formerly GES-5 - 60 Chewtabs.

There's always the issue though of whether Sodium Alginate can get to where it needs to go(the epiphysis of the bone). But Sodium Alginate is able to enhance quality chondrogenic differentiation(however the chondrocytes were still not ossifying as they were not adjacent to periosteum). Chondrocytes can still ossify of course when not adjacent to periosteum like in the case of osteoarthritis but that does not seem to make you taller.

Chondrogenesis of hMSC in affinity-bound TGF-beta scaffolds.

"Herein we describe a bio-inspired, affinity binding alginate-sulfate scaffold, designed for the presentation and sustained release of transforming growth factor beta 1 (TGF-β1), and examine its effects on the chondrogenesis of human mesenchymal stem cells (hMSCs). When attached to matrix via affinity interactions with alginate sulfate, TGF-β1 loading was significantly greater and its initial release from the scaffold was attenuated compared to its burst release (>90%) from scaffolds lacking alginate-sulfate[So some of the benefits could be due to prolonging the release of TGF-Beta1 and not alginate sulfate directly]. The sustained TGF-β1 release was further supported by the prolonged activation (14 d) of Smad-dependent (Smad2) and Smad-independent (ERK1/2) signaling pathways in the seeded hMSCs. Such presentation of TGF-β1 led to hMSC chondrogenic differentiation; differentiated chondrocytes with deposited collagen type II were seen within three weeks of in vitro hMSC seeding. By contrast, in scaffolds lacking alginate-sulfate, the effect of TGF-β1 was short-term and hMSCs could not reach a similar differentiation degree. When hMSC constructs were subcutaneously implanted in nude mice, chondrocytes with deposited type II collagen and aggrecan typical of the articular cartilage were found in the TGF-β1 affinity-bound constructs[so alginate sulfate helps us get to articular cartilage but not growth plate cartilage]. Our results highlight the fundamental importance of appropriate factor presentation to its biological activity, namely - inducing efficient stem cell differentiation."

"Alginate-sulfate, obtained by sulfation of the uronic acid on alginate, has been shown to bind heparin-binding proteins with equilibrium binding constants of the same order of magnitude as those of their binding to heparin"<-heparin is a GAG like chondroiton and Hyaluronic Acid so it operates similarly to those compounds.

The sustained release properties of alginate sulfate may be beneficial to LSJL as alginate sulfate may absorb the elevated TGF-Beta1 levels after a bout of LSJL and then sustain it over time though there is no guarantee that alginate sulfate will form a scaffold in the bone marrow.

"TGF-β1 is a well known heparin-binding protein and [has a] strong but reversible binding to alginate-sulfate"<-thus if you get alginate sulfate(or hyaluronic acid) into the bone marrow TGF-Beta1 will bind to it and will sustain it's release over time ensuring that chondrocytes don't de-differentiate.

This doesn't help with the periosteum problem but this study was able to induce endochondral ossification without periosteum. Glycerophoshate is also available in an acid relief supplement: Prelief Dietary Supplement Acid Relief Tablets - 60 ea

In-vivo generation of bone via endochondral ossification by in-vitro chondrogenic priming of adult human and rat mesenchymal stem cells.

"embryonic stem cells can form bone via the endochondral pathway, thereby turning in-vitro created cartilage into bone in-vivo. In this study we investigated the potential of human adult mesenchymal stem cells to form bone via the endochondral pathway.
MSCs were cultured for 28 days in chondrogenic, osteogenic or control medium prior to implantation. To further optimise this process we induced mineralisation in the chondrogenic constructs before implantation by changing to osteogenic medium during the last 7 days of culture.
After 8 weeks of subcutaneous implantation in mice, bone and bone marrow formation was observed in 8 of 9 constructs cultured in chondrogenic medium. No bone was observed in any samples cultured in osteogenic medium[note that the osteonic medium likely did not involve periosteum]. Switch to osteogenic medium for 7 days prevented formation of bone in-vivo[so if chondrocytes are exposed to periosteum loss bone before being implanted in bone with periosteum they may not form bone]. Addition of β-glycerophosphate to chondrogenic medium during the last 7 days in culture induced mineralisation of the matrix and still enabled formation of bone and marrow in both human and rat MSC cultures[So Beta-glycerophosphate may be the key to growing taller with periosteum]. To determine whether bone was formed by the host or by the implanted tissue we used an immunocompetent transgenic rat model. Thereby we found that osteoblasts in the bone were almost entirely of host origin but the osteocytes are of both host and donor origin.
The preliminary data presented in this manuscript demonstrates that chondrogenic priming of MSCs leads to bone formation in vivo using both human and rat cells. Furthermore, addition of β-glycerophosphate to the chondrogenic medium did not hamper this process[So B-glycerophosphate does not prevent stem cells to undergo a chondrogenic lineage]. Using transgenic animals we also demonstrated that both host and donor cells played a role in bone formation. In conclusion these data indicate that in-vitro chondrogenic differentiation of human MSCs could lead to an alternative and superior approach for bone tissue engineering."

So Beta-Glycerophosphate may be a very promising compound in enabling endochondral ossification to occur when not adjacent to the periosteum such as within epiphyseal bone marrow.

So Alginate Sulfate is a promising height increase supplement by potentially allowing you to double up on the beneficial effects of Hyaluronic Acid. However, Alginate Sulfate is available in foods, it's unknown whether oral Alginate Sulfate supplementation increases serum levels of Alginate Sulfate in the bone marrow(studies have shown that oral Hyaluronic Acid sulfate supplementation does increase serum levels though), and whether Alginate Sulfate and Hyaluronic Acid are affected by the same negative feedback mechanisms.

Beta-Glycerophosphate is also a promising as it may allow for endochondral ossification to occur without needing for the chondrocytes to be adjacent to periosteum. It is unclear whether oral supplementation increases serum levels of Beta-Glycerophoshate. The two supplements are designed for acid relief so they are designed to head into the digestive system. Maybe higher molecular weight doses of these two compounds are needed to avoid digestion.

Grow Taller by your periosteum?

2011-10-25T14:50:00.000-07:00

We already know it's possible to grow taller by increasing the periosteal width of your certain bones that have periosteum oriented in the longitudinal direction such as the flat bone of the skull . We also know that the periosteum is key in distraction osteogenesis surgery. Tissue damage is highly anabolic. How does muscular hypertrophy occur? By damage to the myosin-actin bridge. Does this damage just repair what was done before? No it increases the size of your muscle according to the cellular signals as regulated by myostatin(testesterone inhibits myostatin) plus other factors. Some of the cells that are released from damage to the muscle tissue go to repair but others go to build new muscle.

There are studies that show that bone can increase in size. Tissue damage is highly anabolic and bones can increase in size, the periosteum is a tissue and has been shown to be highly important in limb lengthening surgery, therefore the periosteum is likely to have anabolic effects on your bone. The periosteum contains progenitor cells which are like stem cells.

Even though the progenitor cells from the periosteum are not as potent as the stem cells from trabecular, it still has anabolic effects. The periosteum also contains fibroblasts which are anabolic for connective tissues and what can possibly account for the increase in periosteal width in runners.

One problem is the location of the most easily accessible periosteum(in the tibia) which damage to the tissues should only increase bone width unless the periosteal progenitor cells somehow differentiate into chondrocytes. Lateral Synovial Joint loading would definitely cause shear strain on the periosteum thus causing anabolic effects on the periosteum that way. However, it's unclear whether LSJL would cause hydrostatic pressure in the periosteum as the periosteum is far more malleable than the hard tissue of the bone surrounding the bone marrow.

Here's an article about the direct effect of the periosteum on growth plate development:

Tissue engineering models of human digits: effect of periosteum on growth plate cartilage development.

"Tissue-engineered middle phalanx constructs of human digits were investigated to determine whether periosteum wrapped partly about model midshafts mediated cartilage growth plate formation. Models were fabricated by suturing ends of polymer midshafts in a human middle phalanx shape with polymer sheets seeded with heterogeneous chondrocyte populations from bovine articular cartilage. Half of each midshaft length was wrapped with bovine periosteum[if periosteum was wrapped on the midshaft ends would the bone than grow longer?]. Constructs were cultured, implanted in nude mice for up to 20 weeks, harvested and treated histologically to assess morphology and cartilage proteoglycans. After 20 weeks of implantation, chondrocyte-seeded sheets adjacent to periosteum-wrapped midshaft halves established cartilage growth plates resembling normal tissue in vivo. Sheets adjacent to midshafts without periosteum had disorganized cells and no plate formation. Proteoglycans were present at both midshaft ends. Periosteum appears to guide chondrocytes toward growth plate cartilage organization and tissue engineering provides means for carefully examining construct development of this tissue."

So the periosteum is needed to from growth plates. Chondrocytes not near periosteum will not form growth plates and will not make you taller. Adults have periosteum so this is a good sign for the potential for adult growth plates. There is usually no periosteum surrounding the epiphysis of the bone which could make it difficult to form growth plates there as an adult. But there is periosteum at the end of the epiphysis when it becomes the diaphysis, so it may be close enough to direct the formation of new growth plates.

"After 20 weeks of implantation, engineered human middle phalanx models were found to have glistening, firm and well defined cartilage on both ends of their individual midshaft regions. The portion of midshaft covered with periosteum consisted of essentially clear tissue having a few red-colored areas over its surface indicative of vascular formation. The midshaft region left unwrapped was notably reddened and vascularized[so the periosteum does not seem to have an effect on growth plate vascularization]. X-ray radiography revealed marked mineral deposition within the midshafts of the models only where periosteum had been placed and sutured. No mineral formation was detectable in the cartilage regions at the ends of the models."

So it could be the periosteum that affects the distinction between articular and growth plate cartilage. The reason that articular cartilage usually does not ossify could be that it's too far away from periosteum.

"Over identical implantation times, chondrocyte-seeded PGA sheets adjacent to the half of the same model midshafts left uncovered by periosteum had disorganized cells and no growth plate formation or mineralization"<-So you need both chondrocytes and periosteum to grow taller. And the chondrocytes need to be pretty close to the periosteum as the chondrocytes adjacent to the periosteum did not form growth plates.

"These results support the possibility that periosteal tissue mediates growth plate cartilage formation, perhaps by synthesis and secretion of growth factors and other proteins that provide diffusion-limited regulation and control of neighboring cartilage."<-So we could mimic the benefits of periosteal tissue by increasing serum levels of growth factors and proteins. It would be hard to mimic the diffusion regulation and control of neighboring cartilage.

Shear Strain from lateral synovial joint loading may help spread periosteal growth factors to the epiphysis. Periosteum also has the ability to lengthen.

The fact that LSJL targets height growth by stimulating cell differentiation in the epiphysis into chondrocytes and that chondrocytes will not form growth plates unless adjacent to periosteum(and the epiphysis usually has no periosteum) means that it is likely that the periosteum has to be addressed to maximize gains from LSJL.

Here's the diagram of the study of the portions of the bone attached to periosteum:

Since the scientists attached the periosteum themselves it is likely that this does not completely represent a periosteal distribution pattern but you can see some periosteum at the very end of the epiphysis. Any stem cells that differentiate into chondrocytes at this end zone could have sufficient access to periosteum. Any other stem cells that differentiate in the remainder of the epiphysis will not form growth plates. Since only a small portion of the epiphysis is covered by periosteum, this makes only a small portion of stem cells successfully differentiated by LSJL increase height.

This could explain LSJL stagnation as well. In the beginning, individuals epiphysis may be well oriented to the periosteum making it easy for the chondrocytes to find surrounding periosteum. However, growing taller changes the epiphysis and periosteum thus perhaps making it harder for chondrocytes to be located next to periosteum.

Also, it was noted that people who performed LSJL got a larger epiphysis. It was then theorized that this could be due to chondrogenesis in the epiphysis. This is now not possible as growth plates cannot form unless adjacent to periosteum. The enlarged epiphysis is likely due to a direct increase in the width of individual osteons and direct bone deposition by osteoblasts.

There are two ingredients to growing taller: Stem Cells differentiating into chondrocytes and those chondrocytes being adjacent to periosteum. LSJL and some supplements that increase TGF-Beta1 and BMP-2 can help with the former, now we need to deal with the latter.

Growing Taller by Increasing the Periosteal Width

2011-10-24T16:04:00.000-07:00

We know that it's possible to increase the size of the periosteum. Flat bones are not completely covered by periosteum but if the flat bone is in the right location it is people to use an increase in the size of the periosteum to increase height. Some irregular bones and short bones are also partially covered by periosteum. For example, the tips of the fingers may have some periosteum allowing you to get some increase in wing span there.

To date most studies have shown increases in periosteal width to be almost insignificant as a result of exercises. And, periosteal width increases tend to need more stimulus to increase than trabecular or cortical bone size. Sprinting increases the size of the periosteum of the tibia by putting lots of shearing and compression forces on the bone(shearing forces in a way cause microfractures on the periosteum).

The most likely way to effectively increase periosteal width is by the usage of heavy weights(such as deadlifts+deadlift variations) which means that growing taller via periosteal width is not the right method if per say a girl wants to maintain her secondary sex characteristics(her femininity).

A workable method to increase torso length can likely be found in some sort of LIPUS method by increasing hydrostatic pressure in a method similar to LSJL. The vertebral bones do not have periosteum on the top and bottom.

Increases in periosteal width may play a role however by making you taller via the flat bone of the skull and the calcaneus. And, perhaps, if we optimize the exercises we perform we might be able to increase periosteal width by a lot more than 4%. Although, admittedly this is unlikely as sprinting is already incredibly effective at causing shearing forces on the periosteum of the tibia although the calcaneus may benefit from sprinting.

Growth Hormone, however, may be minorly effective on increasing body height via increasing periosteal width(on the flat bone of the skull) even if it cannot increase height on the long bones without a mutation.

This study shows that the periosteum can induce TGF-Beta which is a boon for chondrogenesis so the periosteum may have a secondary effect on height if not a primary one.

Coculture between periosteal explants and articular chondrocytes induces expression of TGF-beta1 and collagen I.

"Micromass pellets of human articular chondrocytes were cocultured for up to 28 days with human periosteal explants either with physical contact or separated by a membrane allowing paracrine interactions only. Quantitative reverse transcription (RT)-PCR, ELISA, immunohistochemistry and collagen isolation were used to analyse the expression and secretion of TGF-beta1, collagens I and II and chondrogenic differentiation markers such as MIA (CD-RAP) and aggrecan.
TGF-beta1 gene expression was induced significantly in paracrine cocultures in periosteum, whereas it was repressed in physical contact cocultures. However, a higher TGF-beta1 secretion rate was observed in physical contact cocultures compared with periosteal monocultures. The expression of COL2A1, melanoma inhibitory activity (cartilage-derived retinoic acid-sensitive protein) [MIA (CD-RAP)] and aggrecan was mainly unaffected by culture conditions, whereas COL1A1 gene expression was increased in periosteal paracrine cocultures. Collagen I staining was induced in micromass pellets from paracrine cocultures, whereas it was repressed in chondrocytes from physical contact cocultures.
We found evidence for a bidirectional regulating system with paracrine signalling pathways between periosteum and articular chondrocytes[extremely likely that this signaling pathway is shared by epiphyseal chondrocytes as well]. Stimulation of TGF-beta1 and COL1A1 gene expression in periosteal paracrine cocultures and the increased release of TGF-beta1 protein in physical contact conditions indicate an anabolic, and not merely chondrogenic micro-environment in this in vitro model for periosteal-based ACI."

"periosteum carries a thin proliferative cambium layer containing mesenchymal cells with chondrogenic and osteogenic potential which contribute to repair tissue formation"<-Periosteum is attached to the bone with sharpey's fibers too so it's possible that the periosteum contributes some mesenchymal cells for usage during LSJL and it's also possible that deformation of the periosteum during LSJL could lead to some of the height growth.

"While BMPs promote hypertrophy, signalling by TGF-βs favours a stable chondrogenic phenotype and inhibits or delays hypertrophy"<-TGF-Beta helps get stem cells to chondrocytes. Chondrocytes formed in bone make you taller.

Influence of cyclic bending loading on in vivo skeletal tissue regeneration from periosteal origin.

"Periosteum osteogenic and chondrogenic properties stimulate the proliferation then differentiation of mesenchymal precursor cells originating from its deeper layers and from neighboring host tissues[controlling the periosteum is key to controlling the chondrocyte differentiation that we're trying to induce with LSJL]. The local mechanical environment plays a role in regulating this differentiation of cells into lineages involved in the skeletal regeneration process[we can alter this local mechanical environment with stimuli like LSJL].
The aim of this experimental animal study is to explore the influence of cyclic high amplitude bending-loading on skeletal tissue regeneration[LSJL likely applies some degree of bending and bending may generate some hydrostatic pressure so it may have similar effects to LSJL]. The hypothesis is that this mechanical loading modality can orient the skeletogenesis process towards the development of anatomical and histological articular structures.
A vascularised periosteal flap was transferred in close proximity to each knee joint line in 17 rabbits[so they're moving the periosteum to a location not seen in normal development]. On one side, the tibiofemoral joint space was bridged and loading occurred when the animal bent its knee during spontaneous locomotion. On the other side, the flap was placed 12 mm distal to the joint line producing no loading during bending. Tissue regeneration was chronologically analyzed on histologic samples taken from the 4th day to the 6th month.
The structure and mechanical behavior of regenerating tissue evolved over time. As a result of the cyclic bending-loading regimen, cartilage tissue was maintained in specific areas of the regenerating tissue. When loading was discontinued, final osteogenic and fibrogenic differentiation occurred in the neoformed cartilage[the periosteum resulted in new cartilage formation and the cartilage underwent osteogenic differentiation]. Fissures developed in the cartilage aggregates resulting in pseudo-gaps suggesting similar processes to embryonic articular development. Ongoing mesenchymal stem cells stimulation was identified in the host tissues contiguous to the periosteal transfer[the periosteum stimulated MSC development as well]."

Since the periosteum is so important to chondrogenic differentiation and mesenchymal stem cell stimulation, it's likely that increasing periosteal width has benefits as well.

"mechanotransduction modulates the metabolism and synthesis of immature cells as well as their differentiation into different cell lineages."<-LSJL involves mechanotransduction

"High local strain directs precursor cell differentiation into fibrous tissue. On the other hand, mild stress directs precursor cell differentiation into osteochondrogenic cells with direct ossification associated with weak hydrostatic stresses while cartilage growth is favored by higher compressive stresses"<-With LSJL we are going for highly compressive stresses with the clamp. Direct osteochondrogenic growth likely results in no height growth as there is probably no chondrocyte hypertrophy or apoptosis which is likely what is responsible for the actual change in bone size.

"The mesenchymal precursor cells brought to the surgical bed by the periosteum and the host tissues proliferate before differentiating"<-the wider the periosteum likely the more mesenchymal precursor cells that are available.

"Significant proliferation of precursor cells constituting an undifferentiated blastema in the area of flap, and the first step in cell differentiation was found in both groups on the 4^th day. In the “control” group, the development of neotissue was observed along the medial gastrocnemius. In the “loaded” group, it developed on the medial side of the knee, and remained separate from the intact joint capsule"

"After the 4th day, chondrogenic differentiation of mesenchymal precursor cells, which is a key step in enchondral ossification, was similar in both experimental groups. In the “control” group, a process of ossification of the neotissue matrix gradually replaced all of the cartilage with bone. Between the 15th and 30th day, all the cartilage had disappeared and was replaced either with bone or fibrous tissue. After the 30th day, a segment of long bone, whose mean length was identical to that of the flap (27–32 mm), had formed in the posterior compartment of the muscle[a new segment of long bone formed identical to the length of the periosteum, stretch the periosteum to grow taller? Limb lengthening surgery does involve stretching the periosteum]. A medullary cavity had developed and usually included bone marrow. Osteoclasts were identified on the surfaces of newly formed bone. At 6 months, the regenerated tissue was composed of 90% bone and 10% fibrous tissue.
In the “loaded” group cartilage and fibrocartilage, differentiation continued until the 3rd month. The presence of cartilage was gradually limited to the ends and to the middle of the newly formed tissue[just like in endochondral ossification with the primary ossification center in the middle and the secondary ossification centers in the end at the epiphysis]. These areas extended to the initial junction with the support bone and to the tibiofemoral joint space, respectively. After the 3rd month, the newly formed skeletal tissue was detached from at least one of its points of attachment to the support bone. Knee bending no longer caused the regenerated tissue to bend. The cartilage had completely disappeared from the newly formed tissues. At 6 months, a bone segment with a medullary cavity had finally developed on the medial side of the knee. It barely interfered with articular range of motion because it was structurally separate from its initial support bone."<-so the formation of skeletal tissue adapts to movement which means that a method like LSJL which alters skeletal formation will not cause problems as the body adapts. There were no knee bending problems despite the formation of new bone.

" In the earliest stage (4^th day), lytic activity was observed in the tissues in contact with the transfer. This corresponded to necrosis of the superficial layers of muscle in immediate contact with the periosteum. This first stage was followed by a process of muscular regeneration which systematically resulted in complete repair without scar tissue in less than 14 days."<-muscle will adapt to the formation of new bone by remodeling.

"Variations in hydrostatic pressure influence the mechanisms that regulate the proliferation and differentiation of mesenchymal precursor cells. They stimulate proliferation in vitro, while in vivo, they redirect differentiation of precursors of bone tissue towards a cartilage phenotype[<-Why LSJL makes you taller].
Differentiation into chondrogenic cell lines is favored by a local mechanical environment associating high hydrostatic pressures and mild strains[In the LSJL rat study they used relatively low microstrain, maybe it's ideal for LSJL's effectiveness to minimize the microstrain while maximizing the hydrostatic pressure]. High amplitude strain inhibits angiogenesis thus influencing enchondral ossification"

"In our experimental protocol, loading of neotissue by cyclic bending generated a complex mechanical environment which could be described by numerous physical variables such as strain, variations in pressure or fluid as well as shear stress or movements at the cell/matrix and cell/cell interfaces"<-Cyclic bending generated changes in hydrostatic pressure just like LSJL.

"The structure and mechanical response of regenerating tissue evolves over time. As it matures, the regenerating tissue ossifies and mineralisation occurs so that it gradually becomes rigid. This process, which is incompatible with high amplitude knee movements, caused the regenerating tissue to break off from the anchor points of its support bone so that bending-loading no longer occurred[this shouldn't be a problem for us as we are striving for new cartilage formation in the epiphysis not the knee]. We then observed the disappearance of neocartilage, although it had been maintained until this event at the 3^rd month. Thus, the process of enchondral ossification was interrupted, and the cells did not finish their differentiation into cartilage. Nevertheless the deep layer of the periosteum contains cell precursors which are engaged in chondrogenic differentiation, and which form cartilage during monoclonal cell cultures"

"the segments of new cartilage sandwiched between two ossifying structures were not in a physiochemical environment that favored the stability of the cartilage phenotype. The molecular constituents of the extracellular matrix send signals of differentiation to its mesenchymal precursor cells. Thus, although the environment of the articular cavity and the new cartilaginous tissue are chondrogenic, contact with the extracellular bone matrix directs precursors towards osteogenic differentiation"<-This is a problem with LSJL as the stem cells are in an extracellular bone matrix.

" the maintenance of the cartilage phenotype became dependent upon continued cyclic mechanical loading"<-so the frequency of LSJL may have to increase to maintain cartilage phenotype.

Conclusion: The periosteum is a key source of mesenchymal precursor cells thus increasing periosteal width may help you indirectly grow taller. The stem cells being activated in LSJL are in an extracellular bone matrix thus it may be necessary to load more frequently to maintain a cartilage phenotype.

Here's an article about direct hypertrophy of periosteal cells which would likely increase both periosteal width and length:

Remodeling of Actin Cytoskeleton in Mouse Periosteal Cells under Mechanical Loading Induces Periosteal Cell Proliferation during Bone Formation.

"The adaptive nature of bone formation under mechanical loading is well known; however, the molecular and cellular mechanisms in vivo of mechanical loading in bone formation are not fully understood. To investigate both mechanisms at the early response against mechanotransduction in vivo, we employed a noninvasive 3-point bone bending method for mouse tibiae. It is important to investigate periosteal woven bone formation to elucidate the adaptive nature against mechanical stress. We hypothesize that cell morphological alteration at the early stage of mechanical loading is essential for bone formation in vivo.
We found the significant bone formation on the bone surface subjected to change of the stress toward compression by this method. The histological analysis revealed the proliferation of periosteal cells, and we successively observed the appearance of ALP-positive osteoblasts and increase of mature BMP-2[remember BMP-2 can help with chondrogenic differentiation as well], resulting in woven bone formation in the hypertrophic area. To investigate the mechanism underlying the response to mechanical loading at the molecular level, we established an in-situ immunofluorescence imaging method to visualize molecules in these periosteal cells, and with it examined their cytoskeletal actin and nuclei and the extracellular matrix proteins produced by them. The results demonstrated that the actin cytoskeleton of the periosteal cells was disorganized, and the shapes of their nuclei were drastically changed, under the mechanical loading. Moreover, the disorganized actin cytoskeleton was reorganized after release from the load. Further, inhibition of onset of the actin remodeling blocked the proliferation of the periosteal cells[so altering the actin cytoskeleton of the periosteum affects the hypertrophy of the periosteum].
These results suggest that the structural change in cell shape via disorganization and remodeling of the actin cytoskeleton played an important role in the mechanical loading-dependent proliferation of cells in the periosteum during bone formation."

"The periosteum is a membrane that lines the outer surface of all bones, except at the joints of long bones. This membrane, which consists of dense irregular connective tissue, is divided into an outer fibrous layer and an inner osteogenic layer. The fibrous layer contains fibroblasts, whereas the osteogenic layer contains the progenitor cells that develop into osteoblasts. In the observation of molecular and cellular phenomena acted by mechanical stress in vitro, the mechanical stress causes remodeling of cell-matrix adhesions, in which the cytoskeleton rapidly responds to external force by actin assembly"

" Upon detailed analysis, we observed that the mechanical loading rapidly decreased the quantity of stress fibers of the actin cytoskeleton and changed the nuclear shapes in the periosteal cells, and then disorganized actin cytoskeleton was remodeled in a time-dependent manner."<-Since mechanical loading decreases the number of stress fibers this could indicate a need for a deconditioning period to allow for new stress fibers to form.

"In addition, to identify the character of the hypertrophic periosteum, we performed anti-periostin antibody staining, since periostin is a typical marker of periosteum"

"At day 3, we found periostin to be expressed throughout the side opposite to the loading point in hypertrophic periosteum"<-So periosteum hypertrophies at the side opposite of the loading point. So if we want periosteum to increase in length we need to load the ends of the periosteum.

"and this signal had decreased in intensity at day 7 because of the reduced area of periosteum by woven bone formation"<-Periosteum reduces itself when it forms new woven bone, this could explain why adults stop growing taller. Periosteum reduces it's own area when it secretes new woven bone eventually periosteum is no longer next to the growth plate and growth plates need to be next to periosteum thus growth stops.

"The nucleus itself has been proposed to act a cellular mechanosensor, with alterations in nuclear shape causing conformational changes in chromatin structure and organization and directly affecting transcriptional regulation. By this machinery, extracellular forces can be transmitted across the cytoskeleton to the nucleus, resulting in intranuclear deformations; and the actin cytoskeleton is thought to provide protrusive and contractile forces and compressive bearing microtubules to from a polarized network allowing organelle and protein movement throughout the cell. In fact, compressive stress induces shape changes in chondrocyte nuclei; and collagen synthesis is strongly correlated with nuclear shape"<-So if we induce stress in a certain way we can change nuclei to be more chondrogenic. This is done by causing hydrostatic pressure like with LSJL.

Increase Bone Length by Stretching?

2011-10-20T09:03:00.000-07:00

I'm not talking about ordinary stretching as in the Grow Taller 4 Idiot's Program, Yoga, or Pilates. I'm talking more about Sky from Easy Height's new limb center. Before my research stumbled upon the lateral synovial joint loading system, I believed that the best way to gain height was by stretching the bones. You see bone is elastic, by the very definition of microstrain theory the bone is constantly changing in length. If the bone did not change in length then over time bone density would decrease. One thousand units of microstrain is equivalent to 0.1% change in length. If our bones did not lengthen or compress by 0.1% every day they would fall into disuse according to mechanostat theory.

The problem with stretches like the medieval rack or sitting/sleeping with ankle weights is that they stretch more the cartilage and ligaments than the bones themselves. Sky of easy height was working on a program that stretched the bone. Unfortunately, Sky disappeared before sharing his data.

If your tie a rope around the top of your ankle and then load the bottom of the rope with iron plates, your leg is being stretched down and out(being lengthened). If you then tie a rope around the bottom of the ankle and then tie the middle of the rope around a bar; and then load that rope with iron plates your leg is being stretched upwards and out. If you do both of these at the same time with equal amounts of force then the up and down forces cancel out and your bone is only being lengthened outwards.

You could also alternate between pulling your ankle up and down which would proceed to lengthen your bone in a zig-zag motion. We know that bone is elastic and that bone has the ability to microfracture. If you stretch your bone and microfractures occur in a stretched state then the bone should maintain some of that elasticity. Pull a pencil apart, when you do microscopic damage occurs in the length of the pencil. The pencil is now longer than before. Unlike a pencil however, bone has the ability to heal those microfractures. So you distract the bone cause microfractures, the microfractures heal, and then you distract the bone again. Gradually, becoming taller and taller over time.

One element of Sky's multitude of experiments that he has kept in his new Shinbone Version 2011 is cycling with ankle weights(with a raised saddle). The hypothesis of why cycling with ankle weights would work is that you are stretching your leg bone forcing your leg bone to reach lower and lower. Now if Sky does have a method of performing cycling to properly put a stretching force on the leg bone then it could work.

There are a couple of problems involved with the heavy iron plates method however. You are putting load on your tendons and ligaments. You have to find some way to nullify the tendons and ligaments. If you perform this method say around the ankle then you are probably only going to stretch the tibia and not the fibula. The iron plates method would probably best be used in parts of the limb where there is only one bone such as the femur and humerus.

Essentially, a bone stretching method could work. In contrast to LSJL, you would want the load slightly below the growth plate(you only want to stretch your cortical bone in contrast to LSJL where you are trying to deliver red bone marrow stem cells into your growth plate). You would then have to find a way to perform it without putting all the strain on your tendons/ligaments. You would then have to find a way to equally push the bone down from the top and the bottom. You would have to make sure that you were causing microfractures during the process. If you read the last study, you can see why it may be worth it to perform bone stretching while doing LSJL.

Osteodistraction of the maxilla in transverse deficiency in adults: Analysis of the literature and clinical case.

"Osteogenic distraction is a bone regeneration and reconstruction technique. [Osteogenic distraction is] "the process of creating new bone by stretching"[bone stretching can best be analyzed by tensile strain, however this seems to be a different form of stretching]. Disjunction entails separating two anatomical structures at their junction system and, therefore, at a suture[so they're not stretching the bone they're separating the junctions between the bone]. Usually, it involves separating two semi-maxillae in the transverse dimension by means of an osteotomy[osteotomy refers to bone cutting so they are cutting the bone but now it seems like they are separating two separate bones and not cutting the bones itself]. Transverse maxillary distraction appears to offer an alternative of choice to orthognathic surgery alone, which is frequently prone to relapse. The greatest benefit of osteogenic distraction lies in its greater potential for expansion and concurrent growth of the soft tissues. Among other things, this technique increases arch length, thus precluding tooth extractions in cases of maxillary crowding, and appears to provide more stable results than conventional surgical intermaxillary disjunction."

If osteodistraction does work by stretching between the bones then perhaps something that stretches the cartilagenous area of the knee could help you to grow taller.

Effects of osteoinduction on bone regeneration in distraction: results of a pilot study.

"Rate and frequency of distraction as well as stimulatory effects transmitted by growth factors and local gene therapy have a decisive influence on bone regeneration. In a pilot study we tested the effect of four different morphogenetic and mitotic proteins and a genetically transferred vector system on bone healing in continuous osteodistraction in a large animal experiment on 24 Goettingen mini-pigs. For this purpose bone morphogenetic protein (BMP-2), BMP-7, TGF-beta, IGF-1 and a liposome vector were instilled into the distraction gap. The animals were killed after 1-4 weeks of consolidation. Histological and radiological evaluations showed maximum bone formation after the application of BMP-2/7, whereas the application of TGF-beta, IGF-1 and the liposomal vector had only a limited effect on bone regeneration. The quantitative analysis demonstrated an average amount of bone in the distraction gap of 50% and 61% after instillation of BMP-2 and 7, respectively. The BMP-2 expression, however, was maximal after induction with the non-viral vector. Only after BMP-2/7 application could physical, radiographic and histological evidence of bone union be detected. In bone distraction with a short observation period the application of morphogenetic proteins seems to enhance bone regeneration significantly. Before application in humans further studies are necessary to measure the dose-effect relationship, the mode of application and the efficacy of different inductive proteins. The combination of osteodistraction with osteoinduction, however, could shorten treatment times dramatically."

If you look in this picture you can see that the bone grew back without any compounds like BMP-2/7 or TGF-Beta1

A1 is one week after distraction A2 is two weeks. So the bone does grow without chemicals and it also looks like there was no periosteum involved in contrast to distraction osteogenesis. However the periosteal progenitor cells are capable of migration.

"Within the context of ossification, cellular elements with increased BMP-2 expression were found both in the distraction zone, and in the consolidated osseous area close to the osteotomy region. A reduced BMP-2 expression was found in the central distraction zones of those animals, where induction did not stimulate bone regeneration in the distraction region"<-so bone does not seem to form without BMP-2 in the distraction region in this study

"Labelled bone marrow stem cells are systematically mobilized and attracted to fracture sites from remote cell depots"<-Stem cells are still involved. Also remember that a callus might generate hydrostatic pressure.

In the article on limb lengthening surgery, we learned that stretching Type I Collagen might cause a mechanotransduction based signal that results in the increase of the size of the micronuclei of bone cells thus causing bone hypertrophy. This would mean that there would be no need for the fracture and that all you'd need is for tensile strain on Type I Collagen.

Limb bud mesenchyme cultured under tensile strain remodel collagen type I tubes to produce fibrillar collagen type II.

"In this work, we studied the effects of tensile strain on limb bud mesenchymal cells (MSC) cultured on a collagen type I tubular scaffold. A novel bioreactor was designed to culture the cells while subjecting the tubular scaffold to tensile stress and strain. Control samples included unseeded and MSC-seeded tubes cultured for 2 weeks under unloaded, no-strain conditions, and unseeded tubes subjected to prolonged tensile stress and strain. Mechanical properties of tube specimens were measured under oscillatory compressive stress. Following mechanical testing, scaffolds were fixed for immunohistochemistry or frozen for mRNA extraction. The storage modulii of both seeded/unstrained and seeded/strained tubes were significantly less than that of unseeded tubes, suggesting that MSC disrupted the structure and elasticity of the tubes' collagen type I. At a frequency of 1.0 Hz, the loss tangent of seeded/strained tubes was more than 2.5 times greater than that of seeded/unstrained tubes, and almost 6 times greater than that of unseeded tubes. Confocal microscopy and qRT-PCR results demonstrated that collagen type II and aggrecan expression was upregulated in the seeded/strained tubes. Culture under tensile strain induces MSC to remodel the collagen type I tube with collagen type II and aggrecan expression into fibrils dispersed throughout the matrix[so basically tensile strain on the mesenchyme encourages removal of bone and the creation of cartilagenous, possibly growth plate like structures. Note there is mesenchymal tissue in the bone marrow]. The seeded/unstrained tubes manifested less collagen type II with a more random expression pattern. Compared to seeded/unstrained tubes, qRT-PCR for collagen type II in the seeded/strained tubes showed a 4-fold increase in the message for collagen type II and a 13-fold increase in the message for aggrecan. These results demonstrate that MSC cultured for at least some period under tensile strain are able to remodel collagen type I scaffolds to produce a more viscous construct having many of the mechanical and biological features of engineered cartilage."

"In the knee, static compression of the joint creates hydrostatic stress, and movement of the joint creates shear stresses. However, because the knee is a non-conformal surface, the cartilage will also experience a directional tensile stress and strain during use"<-We use static compression in LSJL to create hydrostatic pressure on the bone marrow of the epiphysis.

So tensile strain involved in limb lengthening surgery may stimulate cartilage formation by tensile strain on the mesenchymal tissue itself. Here's the effects of tensile strain directly on the Type I Collagen.

Deformation-dependent enzyme mechanokinetic cleavage of type I collagen.

"Collagen is a key structural protein in the extracellular matrix of many tissues. It provides biological tissues with tensile mechanical strength and is enzymatically cleaved by a class of matrix metalloproteinases known as collagenases. Collagen enzymatic kinetics has been well characterized in solubilized, gel, and reconstituted forms. However, limited information exists on enzyme degradation of structurally intact collagen fibers and, more importantly, on the effect of mechanical deformation on collagen cleavage. We studied the degradation of native rat tail tendon fibers by collagenase after the fibers were mechanically elongated to strains of epsilon=1-10%. After the fibers were elongated and the stress was allowed to relax, the fiber was immersed in Clostridium histolyticum collagenase and the decrease in stress (sigma) was monitored as a means of calculating the rate of enzyme cleavage of the fiber. An enzyme mechanokinetic (EMK) relaxation function T(E)(epsilon) in s(-1) was calculated from the linear stress-time response during fiber cleavage, where T(E)(epsilon) corresponds to the zero order Michaelis-Menten enzyme-substrate kinetic response. The EMK relaxation function T(E)(epsilon) was found to decrease with applied strain at a rate of approximately 9% per percent strain, with complete inhibition of collagen cleavage predicted to occur at a strain of approximately 11%[but is inhibition of collagen cleavage good or bad for height growth?]. However, comparison of the EMK response (T(E) versus epsilon) to collagen's stress-strain response (sigma versus epsilon) suggested the possibility of three different EMK responses: (1) constant T(E)(epsilon) within the toe region (epsilon<3%), (2) a rapid decrease ( approximately 50%) in the transition of the toe-to-heel region (epsilon congruent with3%) followed by (3) a constant value throughout the heel (epsilon=3-5%) and linear (epsilon=5-10%) regions. This observation suggests that the mechanism for the strain-dependent inhibition of enzyme cleavage of the collagen triple helix may be by a conformational change in the triple helix since the decrease in T(E)(epsilon) appeared concomitant with stretching of the collagen molecule."

"Collagen degradation is a mechanism for extracellular matrix (ECM) remodeling and maintenance[the possibility for remodeling Type I Collagen into Type II Collagen which is the cartilage of the growth plate], and in response to trauma, disease and inflammation. Collagenases-1, 2 and 3 are the primary enzymes that act to degrade interstitial collagens (types I, II and III) in humans and animals. These collagenases are part of a larger family of enzymes (matrix metalloproteinases or MMPs) characterized by a zinc dependency for catalytic activity. MMPs are secreted by the cell as inert zymogens in response to the cell being activated by inflammatory cytokines, such as growth factors (interleukin-1) and mechanical loads. In order for collagen cleavage to occur, the collagenase (MMPs-1, 8 and 13, respectively) gains access to the collagen triple helix by binding to the enzyme’s attachment domain along the α-chains, followed by separation (unwinding) of the α-chains to expose the cleavage site, and then cleavage of the α-chain by the enzyme’s catalytic domain[tensile strain of 11% or greater(which means that the bone is stretched to 11% of it's original length) results in the in ability for collagen cleavage]. Collagenases contain two protein domains joined by a linker (hinge), a hemopexin C domain to which the collagen molecule attaches, and a catalytic domain responsible for the α-chain cleavage. MMP-1, 8 and 13 will cleave all three α-chains of interstitial collagens by a single scission at a specific site, located 3/4 from the N terminal and 1/4 from the C terminal, which is characterized by a Gly⁷⁷⁵-Ile⁷⁷⁶ or Gly⁷⁷⁵-Leu⁷⁷⁶ peptide bond, resulting in two fragments of the collagen molecule. Following this initial cleavage other MMPs (mainly gelatinases and stromelysins) can collectively further degrade the collagen fragments. However, the mechanism of the initial cleavage of the collagen molecule must originate with collagenase binding, triple helix unfolding and ¾-¼ scissoring."

"increasing tensile strain up to 4% (grip-to-grip) resulted in a decrease in the rate of enzymatic degradation, while strains above this (to 7%) caused an increase in the rate"<-Thus perhaps why limb lengthening surgery only stretches by 1mm a day which is well below 4%.

"mechanical deformation of type I collagen fibers caused by an axial strain (elongation) applied to the fiber will result in a significant decrease in the rate of collagen degradation by bacterial collagenase"<-This could cause height growth, if production of Type I Collagen outweighs degradation of Type I Collagen then bone could become longer.

It's possible that hydrostatic pressure is involved and that decreased cleavage results in increased hydrostatic pressure.

"Due to the self assembly nature of collagen, there appears to be a long range attraction which prevents molecules from coming too far apart and which induces the self assembly where hydrogen water bridges surrounding the molecule act as specific recognition sites for attracting other collagen molecules[So collagen attracts water to form water bridges]. More important, however, is that an exponential increase in the interaction energy (forces between triple helices) occurs as the α-chain separation distance decreases[as the Type I collagen fibers get farther apart the interaction cost increases]. Heightened hydration interaction forces are observed nearing the last 10-20 angstroms of α-chain separation (osmotic stress as high as 1000 MPa have been measured)[Increased hydration interaction forces due to increased distrace results in increased hydrostatic pressure]. This increased interaction force is [possibly] due to the energy required to rearrange the hydrogen bonding network near the molecular surfaces of macromolecules, such as might occur as the collagen molecule’s diameter is reduced as the molecule is stretched in response to an axial tensile load[tensile strain lowers the collagen molecule's diameter thus increasing the energy for hydrogen bond interaction]. Alterations in the ionic strength will also effect electrostatic interactions (<1 MPa at 15-60 Å). When the collagen molecules come into close proximity the van der Waals forces (<1 MPa at 10-25Å separation) result in an attraction or repulsion dynamic. Thus, a high concentration of repulsion ionic character will position the molecules further away from neighboring molecules creating a greater separation distance and ultimately a larger diameter. Conversely, attractive ionic character would link the molecules with a greater affinity, resulting in smaller diameters."

Stretching Type I Collagen lowers the diameter resulting in an increased energy required to rearrange the hydrogen bonding network. This increases hydrostatic pressure. So again it all leads to hydrostatic pressure.

So bone stretching may help during LSJL by lowering Type I Collagen fibril diameter thereby increasing hydrostatic pressure.

Gain in Height by decreasing bone marrow fat content

2011-10-18T15:52:00.000-07:00

It was once speculated that bone marrow turned to fat post epiphyseal fusion. Later it was learned that that is based on nutrition and that if the bone marrow turns to fat then bone marrow can be restored with proper nutrition again. Stem cell count decreases with age. Since our goal with Lateral Synovial Joint Loading is to get stem cells to differentiate into chondrocytes(via hydrostatic pressure, which we induce by compression by a table clamp or dumbell) it behooves us to try to increase our stem cell count as much as possible. How do we increase our stem cell count so we have more stem cells available for differentiation? We already know that mechanical stimulation increases mesenchymal stem cell count. One way is to discourage stem cells from differentiating into fat leaving more to differentiate into chondrocytes and osteoblasts.

Human blood and marrow side population stem cell and Stro-1 positive bone marrow stromal cell numbers decline with age, with an increase in quality of surviving stem cells: Correlation with cytokines.

"Hematological deficiencies increase with aging leading to anemias, reduced hematopoietic stress responses and myelodysplasias. This study tested the hypothesis that side population hematopoietic stem cells (SP-HSC) would decrease with aging, correlating with IGF-1 and IL-6 levels and increases in bone marrow fat. Marrow was obtained from the femoral head and trochanteric region of the femur at surgery for total hip replacement (N=100). Whole trabecular marrow samples were ground in a sterile mortar and pestle and cellularity and fat content determined. Marrow and blood mononuclear cells were stained with Hoechst dye and the SP-HSC profiles acquired. Marrow stromal cells (MSC) were enumerated flow cytometrically employing the Stro-1 antibody, and clonally in the colony forming unit fibroblast (CFU-F) assay. Plasma levels of IGF-1 (ng/ml) and IL-6 (pg/ml) were measured by ELISA. SP-HSC in blood and bone marrow decreased with age but the quality of the surviving stem cells increased. MSC decreased non-significantly. IGF-1 levels (mean=30.7, SEM=2) decreased and IL-6 levels (mean=4.4, SEM=1) increased with age as did marrow fat (mean=1.2mmfat/g, SEM=0.04). There were no significant correlations between cytokine levels or fat and SP-HSC numbers. Stem cells appear to be progressively lost with aging and only the highest quality stem cells survive."

So increasing IGF-1 levels and decreasing IL-6 levels may increase stem cell count. IL-6 is an inflammatory cytokine so it likely "kills" stem cells. However, the last sentence of the study indicates that there is no correlation between IL-6 and IGF-1 and stem cell count and rather that it's some other process related to aging. This could be something like Methylation Status or Telomere length[although the effectiveness of both is in question]. Supplementing with B-6, B-12, or Folic Acid will work for the former unless for some reason you are non responsive and then it would be best to take S-Adenosyl Methionine. Telomere length involves supplements like Astragalus and you may increase telomere length with weight lifting also. None of those will work unless your body is deficient as you can't make your body methylate cells it doesn't want to by supplementing with SAM-e[and SAM-e is an expensive experiment].

It's very likely that increased IL-6 and decreased IGF-1 levels are just the by-product of some pathway that happens to decrease MSC number rather than increased IL-6 reducing MSCs.

The pathophysiology of the aging skeleton.

"In recent decades the population of both elderly men and women has grown substantially worldwide. Aging is associated with a number of pathologies involving various organs including the skeleton. Age-related bone loss and resultant osteoporosis put the elderly population at an increased risk for fractures and morbidity. Fortunately, in parallel our understanding of this malady has also grown substantially in recent years. A number of clinical as well as translational studies have been pivotal in providing us with an understanding of the pathophysiology of this condition. This article discusses the current concepts of age-related modulation of the skeleton involving intrinsic factors such as genetics, hormonal changes, levels of oxidative stress[IL-6, TNF-alpha], and changes in telomere length, as well as extrinsic factors such as nutritional and lifestyle choices. It also briefly outlines recent studies on the relationship between bone and fat in the marrow as well as the periphery."

"There has been much recent interest in the relationship between fat and the aging skeleton. This is very important because both bone-forming osteoblastic cells and fat-forming adipocytic cells arise from a common progenitor in the marrow. Another exciting and recent area of investigation is the central regulation of bone mass and how this might be affected by age. Finally, a number of recent studies point to telomere shortening and its association with age-related bone loss."<-So osteoblastic cells compete with adipocytic cells for progenitors more than chondrogenic cells although adipocytic cells may still leave a smaller pool of progenitors for potential chondrocytes.

"With aging, hematopoietic tissue is replaced by fatty bone marrow, with consequent reduction in osteoblast number and function. Further, there appears to be a predominant differentiation of MSCs into adipocytes at the expense of osteoblasts"<-Again this likely applies to chondrocytes as well but the effect is not being reported.

"More recent studies found that a higher percentage of body fat was associated with a higher risk for osteoporosis, osteopenia, and non-spine fractures, as well as an inverse relationship between fat mass and bone mass after adjusting for the mechanical loading effects of body weight has been reported"<-so overall fat mass does reduce the available pool of bone marrow progenitors for chondrogenic and osteoblastic activities. This can be reversed by mechanical loading somewhat but it's better to maximize mechanical loading and ensure that the amount of fat is not so great as to strikingly inhibit osteoblastic and chondrogenic stem cell differentiation.

PPARγ: a circadian transcription factor in adipogenesis and osteogenesis.

"Peroxisome proliferator-activated receptor γ (PPARγ) is a critical factor for adipogenesis and glucose metabolism, but accumulating evidence demonstrates the involvement of PPARγ in skeletal metabolism as well. PPARγ agonists[agonist means that it activates], the thiazolidinediones, have been widely used for the treatment of type 2 diabetes mellitus owing to their effectiveness in lowering blood glucose levels. However, the use of thiazolidinediones has been associated with bone loss and fractures. Thiazolidinedione-induced alterations in the bone marrow milieu-that is, increased bone marrow adiposity with suppression of osteogenesis-could partially explain the pathogenesis of drug-induced bone loss. Furthermore, several lines of evidence place PPARγ at the center of a regulatory loop between circadian networks and metabolic output. PPARγ exhibits a circadian expression pattern that is magnified by consumption of a high-fat diet. One gene with circadian regulation in peripheral tissues, nocturnin, has been shown to enhance PPARγ activity. Importantly, mice deficient in nocturnin are protected from diet-induced obesity, exhibit impaired circadian expression of PPARγ and have increased bone mass. This Review focuses on new findings regarding the role of PPARγ in adipose tissue and skeletal metabolism and summarizes the emerging role of PPARγ as an integral part of a complex circadian regulatory system that modulates food storage, energy consumption and skeletal metabolism."

PPAR-lambda may be what's involved in "turning" bone marrow into fat. PPAR-lambda's effects are augmented by a high-fat diet and a gene called nocturnin. So, you can inhibit PPAR-lambda by not eating a high fat diet. Also, ways of decreasing the expression of nocturnin will decrease the number of MSC's turning into fat.

The Effects of Native and Synthetic Estrogenic Compounds as well as Vitamin D Less-Calcemic Analogs on Adipocytes Content in Rat Bone Marrow.

"We demonstrated previously that phytoestrogens and vitamin D analogs like estradiol-17betaf0 (E2) modulate bone morphology in rat female model. Aim: We now analyze the effects of phytoestrogens, E2, SERMs and the lesscalcemic analogs of vitamin D: JKF1624F2-2 (JKF) or QW1624F2-2 (QW) on fat content in bone marrow (BM) from long bones in ovariectomized female rats (OVX). Materials and Methods: OVX rats were injected with treatments known to affect bone formation, 5 days per week for 2.5 month for analysis of fat content in BM. Results: In OVX young adults there is a decreased bone formation and a 10 folds increase in fat cells content in BM. Treatment with E2, raloxifene (Ral) or Femarelle (DT56a) resulted in almost completely abolishment of fat cells content. Daidzein (D) decreased fat cells content by 80%, genistein (G) or biochainin A (BA) did not change fat cells content and carboxy BA (cBA) had a small but significant effect. JKF or QW did not affect fat cells content, whereas combined treatment of JKF or QW with E2 resulted in complete abolishment of fat cells content. These changes in fat cells content are inversely correlated with changes in bone formation. Conclusions: Our results demonstrate that adipogenesis induced by OVX is a reversible process corrected by hormonal treatments. The awareness of a relationship between fat and bone at the marrow level might provide a better understanding of the pathophysiology of bone-loss as well as a novel approach to diagnosis and treatment of post-menopausal osteoporosis."

So, Vitamin D will help prevent bone marrow from turning into fat.

One way to increase stem cell count is to reduce bone marrow fat content. To do that you can lower the amount of fat in your diet and make sure you have enough Vitamin D. You can also increase stem cell count by mechanical stimulation and various supplements.

Bone marrow fat content may not be the only thing that changes but also the sub-populations of the bone marrow itself.

The composition of the mesenchymal stromal cell compartment in human bone marrow changes during development and aging.

"Life-long hematopoiesis depends on the support by mesenchymal stromal cells within the bone marrow. Therefore, changes in the hematopoietic compartment that occur during development and aging probably correlate with variation in the composition of the stromal cell microenvironment[if we can alter the composition of the stromal cell microenvironment than we can reverse some of the changes that occur during development and aging]. Mesenchymal stromal cells are a heterogeneous cell population and various subtypes may have different functions. In accordance with others, we show that CD271 and CD146 define distinct colony-forming-unit-fibroblast containing mesenchymal stromal cell subpopulations. In addition, analysis of 86 bone marrow samples revealed that the distribution of CD271brightCD146- and CD271brightCD146+ subsets correlates with donor age. The main subset in adults was CD271brightCD146-, whereas the CD271brightCD146+ population was dominant in pediatric and fetal bone marrow[increasing the amount of CD271brightCD146+ population may help increase the differentiative potential of stem cells into chondrocytes]. A third subpopulation of CD271-CD146+ cells contained colony-forming-unit-fibroblasts in fetal samples only[Thus fetal marrow has even better differentiation potential with CFUFs in the subpopulation of CD271-CD146+ cells]. These changes in composition of the mesenchymal stromal cell compartment during development and aging suggest a dynamic system, in which the subpopulations may have different functions."

"Mesenchymal stromal cells (MSC) cultured from adult and fetal tissues constitute a heterogeneous cell population. Although a panel of markers, including CD105 (Endoglin) and CD90 (Thy-1), was introduced to define cultured MSC, the cells initiating the culture remain unidentified. Recently, the low-affinity nerve growth factor receptor CD271 and melanoma cell adhesion molecule CD146 were described for prospective isolation of MSC with colony forming unit-fibroblast capacity."<-The presence of CD271 and CD146 means that the MSC population has good differentiation capabilities.

"These subsets had a similar capacity to differentiate and to support hematopoiesis, but their localization in human BM was different. CD271+/CD146-lo cells were bone-lining, while CD271+CD146+ had a perivascular localization."<-perivascular means surrounding a blood vessel. Adults have more of the bone-lining stem cells which would explain why there's more apopsitional growth. Whereas youthful individuals have perivascular stem cells which will enable more interstitial(height gaining) growth.

"CD271+CD146-/lo and CD271+CD146+ respectively localize to endosteal or perivascular niches in vivo"<-Thus even if there's growth stimulation for a perivascular region a bone lining cell will still localize to a bone-lining(appositional thereby non-height gaining region). Thus, perivascular localizating bone marrow may be essential for height growth.

Alternatively, the number of subpopulations may be a function of need. There is far perivascular growth without growth plates thus it makes sense that there are less mesenchymal stem cells localized to perform perivascular growth.

Can Forskolin help with increasing height?

2011-10-06T11:48:00.000-07:00

Over at the Hakker forum, I saw that he was recommending Forskolin as a possible way of increasing height. Forskolin is available for sale: Life Extension Forskolin 10mg Capsules, 60-Count. Can Forskolin help increase your height? He lists the beneficial effects as boosting cAMP, GH, and lowering SOCS. SOCS increases FGFR3 levels which have been implicated in dwarfism. FGFR3 inhibits chondrocyte differentiation which means that high SOCS and/or FGFR3 levels may reduce LSJL effectiveness by making it harder for stem cells to differentiate into chondrocytes. That means that Forskolin can increase the effectiveness of LSJL by decreasing the minimum hydrostatic pressure required to induce chondrogenic differentiation.

Previously, we also learned that cAMP and in turn Forskolin helped regulate osteoblast adhesion to cell surfaces. Hyaluronic Acid at a high molecular weight inhibits cAMP activity. If cAMP does help with height increase then if you are taking Hyaluronic Acid you may wish to take Forskolin as well. However, Hyaluronic Acid is usually not available at the 300 molecular weight that they found inhibits cAMP activity.

cAMP may be important to height growth by another mechanism than osteoblast adhesion and may enhance production of pro-chondrogenic molecules like aggrecan.

Forskolin Stimulates Aggrecan Gene Expression in Cultured Bovine Chondrocytes.

"Prostaglandins are autacoids that elevate intracellular 3prime prime or minute:5prime prime or minute-cyclic adenosine monophosphate (cAMP) levels in chondrocytes and other cells in culture. To facilitate intracellular cAMP accumulation, bovine chondrocytes were incubated with forskolin alone or forskolin and isobutylmethylxanthine. Both significantly increased proteoglycan synthesis, which was inhibited by the cAMP-dependent protein kinase inhibitor H89[meaning that the Forskolin enhancement of proteoglycan's is mediated by CAMP]. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS/PAGE) on 3--16% gels revealed the presence of two large proteoglycan core proteins which migrated more slowly than the 200-kDa marker protein and two small proteoglycan core proteins which migrated slightly slower than the 46-kDa marker. Northern blot hybridization, employing (32)P-labeled cDNA probes, showed that aggrecan steady-state mRNA levels were increased by forskolin and isobutylmethylxanthine after 1 h and 5 h incubation. Decorin and type II collagen mRNA levels were not altered under these conditions. Link protein mRNA levels were slightly elevated, but only at the 5-h time point. These results indicated that stimulation of intracellular cAMP accumulation by forskolin or forskolin and isobutylmethylxanthine resulted in augmented proteoglycan synthesis via increased steady-state aggrecan mRNA levels. Suppression of proteoglycan synthesis by the cAMP-dependent protein kinase inhibitor H89 suggested that cAMP-dependent protein kinase may also play a role in regulating the synthesis and completion of newly synthesized proteoglycans."

Forskolin increases Aggrecan mRNA synthesis and this synthesis dependent on molecules related to cAMP. Aggrecan in addition to Type II Collagen and Sox9 is a marker of chondrogenesis.

This study found that high doses of Forskolin inhibits proteoglycan synthesis:

Metabolic effects of forskolin in chick chondrocytes.

"The effects of forskolin on parameters of energy metabolism and proteoglycan synthesis have been investigated in chick embryo sternal chondrocyte cultures. After 8 h exposure to 100 microM forskolin, ATP levels and oxygen consumption were unaltered. Protein synthesis was unaffected up to 50 microM forskolin and protein degradation was unaffected by forskolin up to 100 microM. In contrast, incorporation of the proteoglycan precursors, 35SO4 and [3H]glucosamine, was more sensitive to forskolin. Inhibition was linear with dose between 10 and 100 microM, reaching 70% at 100 microM. Incorporation of 35SO4 into glycosaminoglycan chains initiated on an artificial beta-xyloside acceptor was inhibited in the same manner. cAMP accumulation was maximal at 10 microM forskolin, a concentration which did not alter proteoglycan synthesis. We conclude that a major, acute effect of forskolin in these short-term experiments is inhibition of proteoglycan synthesis in a cAMP-independent manner."

So for chicks at least you don't want more than 10 microM of forskolin. Forskolin also slows down maturation of chondrocytes so it may help you grow taller for longer.

Extracellular calcium and parathyroid hormone-related peptide signaling modulate the pace of growth plate chondrocyte differentiation.

"An adequate supply of Ca2+ is critical for normal growth plate development. Previous studies suggest that changes in extracellular [Ca2+] ([Ca2+]e) modulate the function of chondrocytes with high [Ca2+]e promoting cell differentiation[calcium encourages chondrocytes to further hypertrophy until apoptosis]. In contrast, signal transduction by the PTH/PTHrP type I receptor (PTH1R) slows down chondrocyte differentiation[Teriparatide has been shown to enhance chondrogenesis]. This study addressed whether changes in [Ca2+]e modulate the differentiation of mouse growth plate chondrocytes by interacting with PTHrP/PTH1Rs. Raising [Ca2+]e from 0.5-3.0 mM dose-dependently promoted the development of mouse growth plate chondrocytes as indicated by decreases in proteoglycan accumulation and in the expression of early differentiation marker genes and by increases in mineral deposition and in the expression of markers of terminal differentiation[Forskolin by slowing the development of growth plate chondrocytes may enable for more division before terminal differentiation]. The effects of high [Ca2+]e on gene expression and matrix synthesis were blunted by incubating cells with PTHrP and vice versa. High [Ca2+]e also suppressed the expression of PTH1Rs. Chronic stimulation of PTHrP/PTH1R signaling by adenoviral expression of constitutively active human PTH1Rs (223hPTH1Rs) reduced the effects of high [Ca2+]e on proteoglycan synthesis and gene expression. Similar results were seen when we treated cells with forskolin[Forskolin reduces the effects of high calcium on inhibiting proteoglycan synthesis] or 8-bromo-cAMP. Taken together, these data support the idea that the pace of chondrocyte differentiation depends on a balance of interactions between PTHrP/PTH1R and extracellular Ca2+ signaling and that high [Ca2+]e promote cell differentiation potentially by reducing the availability of PTH1Rs and the level of cAMP-dependent signal transduction."

"Maturing and upper hypertrophic chondrocytes express the type X collagen [α₁(X)] and alkaline phosphatase (ALP) as well as the PTH1R[So PTH1R would seem to counteract the effects of Type X collagen and alkaline phosphatase. Terminally differentiated chondrocytes in the lower hypertrophic zone (or mineralization zone) demonstrate high levels of the osteopontin (OP), osteonectin (ON), and osteocalcin (OC) expression and deposit Ca²⁺- and phosphate-containing mineral (i.e. hydroxyapatite) into the matrix"

"PTH (1–34), induced apoptosis in a dose-dependent manner. This observation supports our finding in mGPCs overexpressing hPTH1Rs that demonstrated increased apoptosis. We, however, did not observe apoptotic responses in uninfected cultures continuously treated with PTHrP with concentrations as high as 10⁻⁷ m. We think this is due to the ability of ligand to desensitize endogenous PTH1Rs"<-So too much Parathyroid Hormone may be bad by inducing too much apoptosis. Continuous treatment of Teriparatide may not induce apoptosis but continuous treatment also means that the body has adapted to Teriparatide and no longer responding to Teriparatide.

Thus, Forskolin can help increase height during development by stimulating the cAMP pathway(which slows down terminal differentiation) and by inhibiting SOCS which stimulates FGFR3 which has been implicated in dwarfism.

Forskolin can help increase height with LSJL by encouraging stem cells to differentiate into chondrocytes.

Why don't people with Osteoarhritis grow taller?

2011-10-04T12:15:00.000-07:00

Osteoarthritis involves endochondral ossification. If endochondral ossification results in increased height then why doesn't osteoarthritis increase height? A main difference is the environment, the growth plates are inside a long bone whereas the endochondral ossification in osteoarthritis occurs in the synovial joint. Height growth is not a symptom of osteoarthritis, however joint deformation is which indicates growth. A likely possibility is that an eventual outcome of osteoarthritis is that the ends of the bones bump into each other. The contact between bones could prevent further growth. Another possibility for the bone to bone contact is gravity pushing the bones together with a lack of cartilage to cushion the bones from happening.

It's possible that height growth is an unreported symptom of osteoarthritis. But, if osteoarthritis could cause height growth then it could lead to a liability issue if doctors failed to list it as a symptom. So why doesn't osteoarthritis make you taller?

Normal cartilage is a lot like endochondral ossification anyways with four zones including: superficial, medial, deep, and calcified cartilage. People have often stated that calcified cartilage is not bone. The cartilage in the synovial joint may undergo all stages of endochondral ossification except for the ossification phase. Since this stage only involves calcified cartilage in the synovial joint rather than ossification like in the growth plate you do not grow taller. This may mean that osteoblasts are of key importance to height growth as other parts of the phases of endochondral ossification like ECM degradation and chondrocyte apoptosis still likely occur in articular artilage.

Chondrocyte hypertrophy is a part of osteoarthritis. Chondrocytes do not hypertrophy when collagen is prevented from degradation. The degradation of collagen changes the osmotic gradient and results in chondrocyte apoptosis.

TGF-Beta1 is important for cartilage homeostasis and deregulation of it's signaling is part of osteoarthritis. Chondrocyte cultures under TGF-Beta1 will usually maintain a chondrogenic phenotype whereas chondrocyte cultures exposed to BMP-2 will undergo endochondral ossification. Endoglin enhances the Smad 1/5 phosphorylation which promote mineralization whereas they inhibit Smad 2 phosphorylation which encourages more Type II Collagen production. Endoglin has been associated with osteoarthritis. Therefore, it is not likely a lack of involvement of chemical signaling that prevents chondrocytes from fully undergoing an osteogenic phenotype. Remember that bone begins as a completely cartilagenous template and yet manages to become bone. Therefore, even though chondrocytes in articular cartilage are not surrounded by bone like in the growth plate they should still be able to become bone.

The interplay of MMP-9, osteoclasts, and VEGF are essential for endochondral ossification. In Osteoarthritis, cartilage degrades usually by way of MMP-13 rather than osteoclasts. This distinction could be a key reason why osteoarthritis doesn't increase height.

Development of articular cartilage and the metaphyseal growth plate: the localization of TRAP cells, VEGF, and endostatin.

"During long bone development the original cartilaginous model in mammals is replaced by bone, but at the long bone endings the avascular articular cartilage remains. Before the articular cartilage attains structural maturity it undergoes reorganization, and molecules such as vascular endothelial growth factor (VEGF) and endostatin could be involved in this process. VEGF attracts blood vessels, whereas endostatin blocks their formation. The present study therefore focused on the spatio-temporal localization of these two molecules during the development of the articular cartilage. Furthermore, we investigated the distribution of the chondro/osteoclasts at the chondro-osseous junction of the articular cartilage with the subchondral bone. Mice served as our animal model, and we examined several postnatal stages of the femur starting with week (W) 4. Our results indicated that during the formation of the articular cartilage, VEGF and endostatin had an overlapping localization. The former molecule was, however, down-regulated, whereas the latter was uniformly intensely localized until W12[So VEGF is downregulated in articular cartilage making it less vascular]. At the chondro-osseous junction, the number of tartrate-resistant acid phosphatase (TRAP)-positive chondro/osteoclasts declined with increasing age[TRAP is expressed by chondroclasts and osteoclasts but decreased with age. Meaning that lower osteoclast levels could be a reason why you don't grow taller with osteoarthritis]. Until W3 the articular cartilage was not well organized but at W8 it appeared structurally mature. At that time only a few TRAP cells were present, indicative of a low resorptive activity at the chondro-osseous junction. Subsequently, we examined the metaphyseal growth plate that is closed when skeletal maturity is attained. Within its hypertrophic zone, localization of endostatin and VEGF was observed until W6 and W8, respectively. At the chondro-osseous junction of the growth plate, chondro/osteoclasts remained numerous until W12 to allow for its complete resorption[growth plates had many osteoclasts]. According to former findings, VEGF is critical for a normal skeleton development, whereas endostatin has almost no effect on this process. In conclusion, our findings suggest that both VEGF and endostatin play a role in the structural reorganization of the articular cartilage and endostatin may be involved in the maintenance of its avascularity. In the growth plate, however, endostatin does not appear to counteract VEGF, allowing vascular invasion of hypertrophic cartilage and bone growth."

Osteoclasts are important for height growth and an osteoclast inhibitor Alendronate has been shown to reduce height growth.

"Angiogenesis, the formation of new blood vessels from preexisting capillaries, is triggered by the vascular endothelial growth factor (VEGF), and with the vessels, bone-forming cells and the chondro- as well as osteoclasts are recruited. In doing so, the cartilage matrix is remodelled, and newly formed bone leads to the establishment of the diaphyseal primary ossification centre (POC) followed by the epiphyseal secondary ossification centre (SOC)"<-Due to an insufficient supply of osteoclasts, the cartilage matrix is not remodeled in such a way as to increase height.

" Principally, the adult articular cartilage shows no evidence of hypertrophy. It is a stable avascular tissue, and VEGF is down-regulated during its development[lack of VEGF could be another reason for lack of complete endochondral ossification in cartilage] and nearly undetectable under normal conditions. Only in patients suffering from osteoarthritis is the angiogenic factor up-regulated in the joint system[however VEGF becomes present during osteoarthritis so VEGF could not be involved in the lack of osteoarthritic height growth], and during the progression of this degenerative disease, vessels from the subchondral bone occasionally grow into the calcified zone of the articular cartilage"

"Articular cartilage is not well structured [during early development], and numerous blood vessels as well as chondro/osteoclasts invade the mineralized hypertrophic layers indicative of an intense tissue resorption and epiphyseal bone formation. The articular cartilage represents a surface growth plate with a high level of activity at this developmental time."<-articular cartilage starts out like a growth plate. Decline of VEGF and osteoclast activity being a key reason for lack of growth plate function.

So, the reason that people with osteoarthritis don't grow taller may be due to a lack of osteoclast activity. Though VEGF helps recruit osteoclasts, there are not enough TRAP-positive osteoclasts to recruit. Remodeling by osteoclasts may play a key role in height growth. Osteoclasts may physically alter the growth plate in some way as to make you taller. If degradation of type II collagen was the important part of height growth so as to alter the osmotic gradient then MMP-13 would be enough to increase height and MMP-13 is present in osteoarthritis.

However, there does seem to be an upregulation of osteoclasts in normal arthritis.

Osteoclastogenesis and arthritis.

"There is emerging interest for osteoclasts as key players in the erosive and inflammatory events leading to joint destruction in chronic arthritis. In fact, chronic inflammatory joint diseases such as psoriatic arthritis and rheumatoid arthritis are often characterized by destruction of juxta-articular bone and erosions due to the elevated activity of osteoclasts, which are involved in bone resorption. The main step in inflammatory bone erosion is an imbalance between bone resorption and bone formation: osteoclast formation is enhanced by proinflammatory cytokines such as TNF-α, IL-1β, and IL-17 and is not balanced by increased activity of bone-forming osteoblasts. T-cells, stromal cells, and synoviocytes enhance osteoclast formation via expression of RANKL and, under pathologic conditions, of proinflammatory cytokines. In rheumatoid arthritis, accumulation of osteoclasts in synovial tissues and their activation associated with osteoclastogenic cytokines and chemokines at cartilage erosion sites suggest that they could be usefully selected as therapeutic target. In particular, in consideration of the primary role of RANKL and TNF-α in osteoclastogenesis, the control of the production of RANKL and the inhibition of TNF-α represent important strategies for reducing bone damage in this disease."

This is regular arthritis however, maybe in the presence of VEGF(osteoarthritis) and Osteoclasts(regular arthritis), you would grow taller.

" A physical contact of precursor cells with osteoblasts or other specific mesenchymal cells, such as stromal or synovial cells, is essential for osteoclastogenesis"<-Synovial joint cells have contact with synovial mesenchymal cells so osteoclastogenesis must be possible in the synovial joint.

This study shows though that height growth did not occur even with both VEGF and osteoclasts present:

Subchondral bone loss following orthodontically induced cartilage degradation in the mandibular condyles of rats.

"Osteoarthritis (OA) is a degenerative joint disease generally characterized by progressive cartilage degradation and subchondral bone changes. Subchondral bone changes have been proposed to initiate or accompany with cartilage degradation in OA. The purpose of this study was to characterize cartilage damage, subchondral bone remodeling, and the possible mechanism involved in these morphological changes in our reported rat model with OA-like lesions in the mandibular condyle. In experimental groups, the dental occlusion was orthodontically disturbed. By histological analysis, transmission electron microscopy (TEM), micro-CT scanning and serum tests, changes in condylar cartilage and subchondral bone were analyzed at 8 and 12 weeks after treatment. The mRNA and protein levels of bone pro-resorptive and pro-formative factors by chondrocytes were investigated. Increased degraded cartilage areas and obvious cartilage calcification were observed in 8- and 12-week treated (EXP) groups compared to the age-matched controls. Subchondral bone loss, characterized as decreased bone mineral density (BMD), bone volume fraction (BV/TV) and trabecular thickness (Tb.Th), but increased trabecular separation (Tb.Sp), was observed in the 12-week but not the 8-week EXP group, respectively, versus their age-matched controls. The subchondral bone loss in the 12-week EXP group was accompanied with decreased new bone formation rate, but increased serum carboxy terminal telopeptides (CTXs), and increased osteoclast numbers and proportion of surface area in the subchondral bone regions. Increased mRNA and protein levels of M-CSF, VEGF, RUNX and RANKL/OPG ratio[So VEGF increased as well], but decreased OPG, were found in condylar cartilage in the 12-week EXP group versus its age-matched controls, and those of RANKL/OPG ratios were significantly higher in the 12-week EXP group than the 8-week EXP. In addition, increased mRNA levels of VEGF, RUNX and RANKL/OPG ratio, but decreased OPG, were also found in condylar cartilage in the 8-week EXP group versus its age-matched controls (All P<0.05). This study demonstrated that obvious subchondral bone loss followed cartilage degradation in the mandibular condyles in the present rat models and suggested that the imbalance of chondrocyte-secreted regulatory factors within the degraded cartilage may play a role in the osteoclastogenesis, and thus leading to the subchondral bone loss in OA."

Now it could be that the osteoclasts were active at the wrong area. The osteoclasts were reabsorbing at the subchondral plate rather than in the hypertrophic zone of the articular cartilage.

"articular cartilage is non-vascularized and does not contain mononuclear phagocytes, apoptotic products would not be cleared out effectively, leading to the excessive inorganic pyrophosphate and calcium precipitate within the cartilage"<-This could be the key to way you don't grow taller in osteoarthritis. Chondrocytes undergo apoptosis but their products are not cleared out effectively leading to calcium precipitate(which causes calcified cartilage) but not growing taller as maybe the apoptotic byproducts inhibit bone formation. However, could the presence of VEGF in this study enable for the cleaning of these apoptotic products. In the study referenced, by this statement, normal articular cartilage was used.

In this study, the subchondral bone loss study, the bones do look bigger and taller. However, no official measurements were made. An increase in bone mineral density for example could be caused by longitudinal bone growth as the bone is now spread over a larger area. An increase in trabecular spacing supports this theory as there is now a larger bone for the same number of trabeculae.

The reason that people with osteoarthritis don't grow taller involves VEGF and osteoclasts. Lack of osteoclasts(but having VEGF present) seems to cause osteoarthritis whereas lack of VEGF(with osteoclasts present) causes degenerative arthritis. But the presence of both VEGF and osteoclasts did not clearly lead to bone growth in the one study that had both factors(although bone growth is still possible but not measured). The presence of cartilage calcification in the subchondral bone study indicates that the apoptotic byproducts were not disposed of efficiently despite the presence of VEGF. Maybe the increase of VEGF was not sufficient and higher VEGF levels would result in an increase in height or something else is needed to get rid of those byproducts that may inhibit the final stages of endochondral ossification and height growth.

This study states that osteocytes may be a key in the final stage of endochondral ossification and thereby why osteoarthritis doesn't make you taller:

The turnover of mineralized growth plate cartilage into bone may be regulated by osteocytes.

"During endochondral ossification, growth plate cartilage is replaced with bone. Mineralized cartilage matrix is resorbed by osteoclasts, and new bone tissue is formed by osteoblasts. As mineralized cartilage does not contain any cells, it is unclear how this process is regulated. We hypothesize that, in analogy with bone remodeling, osteoclast and osteoblast activity are regulated by osteocytes[articular cartilage does not have nearby osteocytes to regulate this activity], in response to mechanical loading. Since the cartilage does not contain osteocytes, this means that cartilage turnover during endochondral ossification would be regulated by the adjacent bone tissue. We investigated this hypothesis with an established computational bone adaptation model. In this model, osteocytes stimulate osteoblastic bone formation in response to the mechanical bone tissue loading. Osteoclasts resorb bone near randomly occurring microcracks that are assumed to block osteocyte signals. We used finite element modeling to evaluate our hypothesis in a 2D-domain representing part of the growth plate and adjacent bone. Cartilage was added at a constant physiological rate to simulate growth. Simulations showed that osteocyte signals from neighboring bone were sufficient for successful cartilage turnover, since equilibrium between cartilage remodeling and growth was obtained. Furthermore, there was good agreement between simulated bone structures and rat tibia histology, and the development of the trabecular architecture resembled that of infant long bones. Additionally, prohibiting osteoclast invasion resulted in thickened mineralized cartilage, similar to observations in a knock-out mouse model. We therefore conclude that it is well possible that osteocytes regulate the turnover of mineralized growth plate cartilage."

So osteocytes may be needed for height growth as well as VEGF and osteoclasts.

" It is known that in certain situations, osteoblasts seem capable of forming bone in the absence of osteocyte signals, for example when the first osteoid is synthesized in the anlage. Such independent osteoblast activity could be involved during endochondral ossification close to the epiphyseal plate as well. However, in the presence of adjacent osteocytes, osteoblast activity seems to become regulated by osteocyte signals."<-Since bone begins as a cartilagenous template there are no osteocytes. However, endochondral ossification seems to occur in that stage. However, at that stage maybe purely remodeling occurs and not actual height growth until osteocytes are in place to regulate it.

" the simulated development of the trabecular architecture with increasing distance from the growth plate resembled that of infant bones"<-So, the articular cartilage may be too far away to receive signals from the osteocytes thus limiting it's growth potential.

Note that the factors inhibiting height growth in osteoarthritis(an other -arthritis) do not affect LSJL like osteocytes, VEGF, and osteoclasts.

Why don't osteoblasts make us taller?

2011-09-27T12:47:00.000-07:00

In our discussion on water and height, we went over the differences between chondrocytes and osteoblasts and tried to determine why chondrocytes can cause interstitial growth whereas osteoblasts cannot. Both chondrocytes and osteoblasts secret ECM(osteoblasts secret Type I collagen) but cartilage is far more hydrophillic(water loving). Since cartilage is more hydrophillic it is more prone to hypertrophy thus that could be a key to why chondrocytes are a key to growing taller. Do osteoblasts undergo hypertrophy and if they do then why don't they make you taller like chondrocytes? Osteoblasts also undergo apoptosis but maybe water release from apoptotic chondrocytes can help you grow taller.

Chronic hyperglycemia modulates osteoblast gene expression through osmotic and non-osmotic pathways.

"Insulin dependent diabetes mellitus (IDDM; type I) is a chronic disease stemming from little or no insulin production and elevated blood glucose levels. IDDM is associated with osteoporosis and increased fracture rates. The mechanisms underlying IDDM associated bone loss are not known. Previously we demonstrated that osteoblasts exhibit a response to acute (1 and 24 h) hyperglycemia and hyperosmolality[so there is a high number of solute in the body so osteoblasts release water and shrink]. Here we examined the influence of chronic hyperglycemia (30 mM) and its associated hyperosmolality on osteoblast phenotype. Our findings demonstrate that osteoblasts respond to chronic hyperglycemia through modulated gene expression. Specifically, chronic hyperglycemia increases alkaline phosphatase activity and expression and decreases osteocalcin, MMP-13, VEGF and GAPDH expression. Of these genes, only MMP-13 mRNA levels exhibit a similar suppression in response to hyperosmotic conditions[MMP-13 degrades extracellular matrix, so hyperosmotic conditions suppress degradation of the extracellular matrix] (mannitol treatment). Acute hyperglycemia for a 48-h period was also capable of inducing alkaline phosphatase and suppressing osteocalcin, MMP-13, VEGF, and GAPDH expression in differentiated osteoblasts. This suggests that acute responses in differentiated cells are maintained chronically. In addition, hyperglycemic and hyperosmotic conditions increased PPARgamma2 expression[PPARgamma is usually associated with increased adipocyte differentiation and reduced osteoblast differentiation], although this increase reached significance only in 21 days chronic glucose treated cultures. Given that osteocalcin is suppressed and PPARgamma2 expression is increased in type I diabetic mouse model bones, these findings suggest that diabetes-associated hyperglycemia may modulate osteoblast gene expression, function and bone formation and thereby contribute to type I diabetic bone loss."

So, hyperosmotic conditions are catabolic to bone cells. So osteoblasts do respond to water much like chondrocytes.

"increased expression of PPARγ2, aP2 and resistin in streptozotocin-induced diabetic mice corresponded with increased adipocyte maturation and suggested the possibility that IDDM may also affect lineage selection of mesenchymal stem cells, leading to adipocyte rather than osteoblast maturation."

"cells can also respond to hyperglycemia through an osmotic response. Because osteoblasts express glucose transporters, GLUT-1 and -3, with low K_m (1–2 mM and <1 mM, respectively), glucose transport is maximal at euglycemic[normal blood glucose levels] state (glucose concentration of 3–5.5 mM) so an increase in extracellular glucose could be an osmotic stress." Since cells are transporting less glucose there is more glucose outside a cell therefore water leaves the cell to restore concentration to normal.

"During osmoadaptation to extracellular hyperosmotic conditions, virtually all cells undergo a volume change and shrink"<-therefore osteoblasts should be capable of undergoing hypertrophy as well.

"Osteoblast morphology and number does not change under chronic hyperglycemia"<-remember hyperglycemia causes hyperosmolarity as well. So hyperosmolarity does not cause osteoblast hypertrophy(which would be a part of morphology). So even though water is leaving the cell, osteoblasts do not stay shrunken in size.

"During this period osteoblasts undergo immediate volumetric changes (cell shrinking) induced by hyperglycemia-associated hyperosmolality"<-Hypertonic means water leaving the cell so it makes sense for cells to shrink

So osteoblasts are osmotically sensitive like chondrocytes. Do osteoblasts swell(hypertrophy) in response to hyposmotic(water enters the cell) solutions?

Regulation of cell volume and intracellular pH in hyposmotically swollen rat osteosarcoma cells.

"The maintenance of cell volume involves transduction of a volume-sensing signal into effectors of volume-regulatory transporters. After exposure to anisotonic conditions, cells undergo compensatory volume changes that are mediated by active transport and passive movement of ions and solutes. Intracellular pH (pHi) homeostasis may be compromised during these processes. We have studied pHi and some of the signal transduction mechanisms involved in the regulatory volume decrease (RVD) that occurs after exposure to hypoosmolar conditions in rat osteosarcoma cells, ROS 17/2.8. Cells were loaded with BCECF; pHi and cell volume were estimated by dual excitation ratio fluorimetry. Swelling of cells in 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffered hypotonic medium induced a rapid cell swelling followed by an incomplete RVD of approximately 30% in suspended (i.e., round) cells and approximately 60% in attached (i.e., spread) cells that was independent of subpassage number[so osteoblast cells did cell and the swelling did not return to normal after time as shown by the incomplete regulatory volume decrease]. RVD was inhibited by ouabain, valinomycin, and high external [K+], all of which should reduce the cell membrane electrochemical gradient for K+. Inhibition of RVD was induced also by decreasing intracellular [Ca2+] with BAPTA-AM and by depletion of Cl-, indicating the role of calcium-regulated K+ and Cl- efflux during RVD. Depolymerization of actin filaments by cytochalasin D prolonged the RVD three-fold and nonspecific activation of GTP-binding proteins up-regulated RVD. In attached cells the hypoosmolar-induced swelling caused a large reduction in pHi (approximately 0.7 units), which was sustained as long as cells were in hypoosmotic medium. The reduction of pHi induced by cell swelling was inhibited by Na(+)-free extracellular medium, ouabain, the tyrosine kinase inhibitor genistein, and to a lesser extent by Cl(-)-free medium. However, amiloride failed to inhibit the hypoosmolar-induced reduction of pHi. Collectively these data indicate that RVD of ROS 17/2.8 cells in HEPES-buffered medium is dependent on conductive efflux of K+ and Cl- that is regulated by cell shape, actin, and GTP-binding proteins. The sustained inhibition of pHi homeostasis induced by cell swelling may reflect the existence of cell volume sensing mechanisms that operate through tyrosine kinases to regulate pHi."

It could be a location issue that chondrocytes are in a better position to increase height than osteoblasts. However, there are osteoblasts at the surface of the bone with potential for hypertrophy and the ability to secrete extracellular matrix. However, key components to chondrocyte hypertrophy may not be due to osmotic swelling and may be due to other factors. It could be these other forms of hypertrophy that are responsible for height growth.

Since cartilage is hydrophillic, chondrocytes are a lot better at manipulating water levels since it can store water in it's ECM. Osteoblasts do not have water stored in it's ECM. Osmotic lysis is more likely to occur in osteoblasts than chondrocytes as chondrocytes have the water storage ability of the cartilage. Since chondrocytes do have water stored in the ECM, they can better orchestrate the osmotic lysis of the cells thus resulting in an explosion pushing the bone apart.

Hyperosmotic conditions were found to result in more chondrocyte apoptosis. Water leaving the cell resulted in apoptosis rather than water flooding into the chondrocyte. During terminal differentiation, cartilage is absorbed leading to less water outside the cell therefore chondrocytes release water to result in osmotic balance this results in chondrocyte apoptosis. This response did not seem to occur in osteoblast cells which only exprienced decreased expression of MMP-13 and increased levels of PPARgamma2 in response to a hyperosmotic environment like those experienced by chondrocytes who just had their ECM degraded.

There is evidence that there is an active role of chondrocyte apoptosis in endochondral ossification and that active role may play a role in how physically growth plates make you taller. Other cells do have the ability to influence body shape for example you could have swollen skin. Osteoblasts have the ability to influence shape too but only by secreting new matrix beneath the periosteum. This however does not involve hypertrophy or apoptosis but is only the result of matrix secretion.

Therefore, matrix secretion may only be able to cause apopsitional growth but hypertrophy and apoptosis may be needed for interstitial growth.

Growth plate chondrocytes have to do something else besides proliferate, divide and secrete ECM to make us taller. Lots of cells perform those functions and don't make us taller. There needs to be a force generated pushing the bone apart from within to make room for new bone. Choreographed chondrocyte apoptosis has the ability to do that like a string of dynamite. Osteoclasts can degrade ECM all at once like triggers resulting in chondrocytes going off at once resulting in a big "explosive" force.

If the force isn't big enough like isolated osteoblast apoptosis you won't grow taller.