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

Flurbiprofen(Grow Taller by manipulating Lymphocyte count)

2013-04-29T14:28:00.001-07:00

This is a compliment to the analysis by Natural Height Growth on Flurbiprofen.

What's interesting about Flurbiprofen is that usually things that increase growth plate height as a result of inhibiting cartilage or bone degraders usually decrease growth such as MMP13 inhibitors.

My hypothesis is that alters bone growth by inhibiting lymphocyte activity. There needs to be an equilibrium amount of lymphocyte activity as confirmed by the highest dose of Flurbiprofen decreasing longitudinal bone growth. Based on whether the amount of lymphocytes you have is above or below equilibrium taking Flurbiprofen will either increase or decrease your height.

Lymphocytes can be measured on a blood sample. Unfortunately, we don't know the optimal lymphocyte count for maximizing height growth so we don't know what the target goal would be with Flurbiprofen.

Flurbiprofen-induced stimulation of periosteal bone formation and inhibition of bone resorption in older rats.

"The skeletal effects of flurbiprofen (Fb), a nonsteroidal anti-inflammatory drug, was studied by histomorphometry in 9-month-old retired female breeder, Sprague-Dawley rats. Flurbiprofen was given subcutaneously at 0, 0.2, 0.1, 0.5, 2.5, or 5 mg/kg/d for 21 days. Flurbiprofen had no effect on longitudinal growth, but stimulated radial growth (+200%){maybe this could give a little skull and calcaneus height?} over controls. In the tibial shaft, Fb stimulated the mineral apposition rate (+25%), mineral bone formation rate (+100%), and periosteal labeling length (+64%) at the 2.5 and 5.0 mg Fb/kg dose levels, and had no effect on marrow cavity size compared to controls. However, these changes were insufficient to increase cortical bone mass. In the proximal tibial metaphysis, Fb suppressed osteoclasts/mm2 of metaphyseal tissue (-47%), osteoclasts/mm of bone surface (-46%), and the osteoclast/osteoblast ratio (-50%), increased the calcified cartilage core population (+100%), and had no effect on osteoblast numbers at all dose levels{osteoclasts may be good for height during development and may allow for the formation of cartilage canals to form new growth plates}. There was an insignificant increase in metaphyseal cancellous bone mass. Flurbiprofen-stimulated periosteal bone growth was due to direct stimulation of osteoblast recruitment and activity independent of longitudinal bone growth."

"retired female breeder, Sprague-Dawley rat, with a 5 mcm/day longitudinal growth rate of the proximal tibia and a 3.6 mcm/day periosteal bone apposition rate of the tibia) shaft ."<-I'm not sure what mcm is relative to units of measurement but 9 month old rats are still growing.

"increased longitudinal bone growth and growth plate thickness in the weanling rat [who took flurbiprofen], while the size of the hypertrophic cells and the cartilage cell production rate did not differ from the controls ."

In older rats, there was an increase in the size of the calcified cartilage core.

Flurbiprofen enhances growth and cancellous and cortical bone accumulation in rapidly growing long bones.

"The effects of flurbiprofen, a non-steroidal anti-inflammatory drug, on bone growth was studied by static and dynamic histomorphometry in immature (28 days old) male Sprague-Dawley rats. Flurbiprofen at 0, 0.02, 0.1, 0.5 or 2.5 mg/kg/d doses was given subcutaneously daily for 21 days. The 0.1 and 0.5 mg/kg/d doses were most effective in stimulating longitudinal and radial bone growth and enhancing the accumulation of cancellous and cortical bone{so it seems there is an equilibrium quantity}. Proximal tibial longitudinal bone growth rate, growth plate thickness, and periosteal bone formation rate were increased 30-40%, while cortical bone (tibial shaft) and cancellous bone (proximal tibial metaphysis) accumulated 12% and 90% more bone than controls, respectively. Enhanced accumulation of cortical bone was attributed to stimulated periosteal bone formation without accompanying marrow cavity enlargement. Enhanced accumulation of cancellous hard tissue was postulated to be due to reduced trabecular bone resorption and no effect on bone formation. The cell counts support these conclusions. There was a decrease in osteoclast numbers (-62 to -70%), an insignificant decrease in osteoblast numbers (-5 to -30%) per mm of bone surface and a decrease in osteoclast to osteoblast ratio (-35 to -56%). The findings presented are compatible with the conclusion that flurbiprofen, induced changes in rapidly growing long bones by reducing osteoclast activity and recruitment, stimulating longitudinal and radial growth, increasing the cortical bone mass by stimulated periosteal bone growth and depressed endosteal resorption, and increasing cancellous bone mass by depressed trabecular bone resorption without affecting bone formation."

The increase in longitudinal growth was fairly significant from about 165 mcm/day to 204 mcm/day for 0.5mg/d group. The longitudinal growth was virtually the same from 0.1mg/d to 0.5mg/d. The percentage of increase of growth plate height was approximately the same as the increase in longitudinal growth about 25%. The trends of maximal chondrocyte hypertrophy size and rate of chondrocyte proliferation were less consistent and dramatic. Longitudinal growth was less at 2.5mg per day than control 159 mcm/day to 165 mcm/day.

Here's a drug that's sort of the inverse to Flubiprofen:

Effects of phytohemagglutinin-P (PHA-P) on bone of the growing rat.

"The effects of phytohemagglutinin-P, (PHA-P), a mitogen known to selectively stimulate cells of hematogenous or lymphoid monocytic origin, 25 and 50 mg/kg/day administered for 15 days on proximal tibiae of growing male Sprague-Dawley rats, were studied. The general effect of PHA-P was to decrease the amount of cartilage, hard tissue, and longitudinal growth in the proximal tibial metaphysis. A decrease in longitudinal bone growth, in the number of chondrocytes, in the thickness of cartilage plate, in the metaphyseal mass of hard tissue, in the percentage of calcified cartilage core, and in the number of osteoblasts per mm of bone surface was observed. Additionally, PHA-P increased the number of osteoclasts, the number of labeled osteoclastic nuclei, and the average number of nuclei per osteoclast. There was a significant decrease in the time to the first appearance of labeled osteoclastic nuclei as the dose of PHA-P increased. Thus, PHA-P treatment leads to the dominance of osteoclastic over chondroblastic and osteoblastic activity and results in a hard tissue deficit in a growing skeleton. The data indicate that PHA-P administration selectively increases osteoclast numbers by elevating osteoclastic progenitor cell proliferation and enhancing their fusion and differentiation to osteoclasts."

So PHA-P decreases osteoblasts and increases the number of osteoclasts and decreases longitudinal bone growth. PHA-P is part of the red kidney bean. PHA-P stimulates proliferation of lymphocytes.

According to Flurbiprofen and immunosuppression of Trypanosoma brucei infection in the goat., Flurbiprofen inhibits T-lymphocytes.

This study provides a possibly model for how lymphocytes may affect longitudinal bone growth:

Growth plate compressions and altered hematopoiesis in collagen X null mice.

"A variable skeleto-hematopoietic phenotype was observed in collagen X null mice which mirrored the defects in transgenic (Tg) mice with dominant interference collagen X mutations. Specifically, perinatal lethality was seen in approximately 10.8% of null mutants at week three after birth, and in another subset by 12 wk. In perinatal lethal mutants, growth plates were compressed, trabecular bone reduced, and hematopoietic aplasia and erythrocyte-filled vascular sinusoids were apparent in marrows. Lymphatic organs, reduced to approximately 80% that of controls, displayed altered architecture and lymphocyte content. In thymuses, a paucity of cortical CD3(+)/CD4(+)/CD8(+) lymphocytes was consistent with the marrow's inability to replenish maturing T cells. In spleens, an unaltered T cell distribution was coupled with diffuse staining for IgD(+)/B220(+) B cells, whose reduction was prominent in poorly organized lymphatic nodules. Disorderly arrays of splenic macrophages surrounding periarteriolar lymphatic sheaths and a red pulp depletion further complemented the Tg perinatal lethal phenotype. Moreover, subtle growth plate compressions and hematopoietic changes were seen in all null mice{this is inconsistent with the larger growth plates seen in flurbiprofen treated mice}."

So either lymphocytes are not the cause of flurbiprofen elevated height growth or there is an equilibrium quantity of lymphocytes for optimal height growth(supported by there being an optimal dosage of flurbiprofen).

Inflammatory cytokines may have effects like lymphocytes.

Interleukin-6 modulates trabecular and endochondral bone turnover in the nude mouse by stimulating osteoclast differentiation.

"We have chosen the immunologically compromised athymic mouse, which demonstrate sclerotic features in its trabecular bone, as the animal model for assessment of possible modulation effects of interleukin-1alpha (IL-1alpha) and interleukin-6 (IL-6) on bone and cartilage metabolism. The cytokines were applied by daily subcutaneous injections for 3 consecutive days. Histomorphometry, measuring epiphyseal trabecular bone volume (ETBV), metaphyseal trabecular bone volume (MTBV), and the width of the growth plate, and tartrate-resistant acid phosphatase (TRAP) histochemistry were used to assess parameters of bone turnover in the proximal tibia. IL-6, but not IL-1alpha, reduced ETBV and MTBV. Both IL-6 and IL-1alpha reduced the width of the growth plate. IL-6, but not IL-1alpha, increased the number of chondroclasts and osteoclasts in the primary spongiosa of the proximal tibia, as well as the number of nuclei. The resultant bone resembled that of the wild-type mouse. The results point to IL-6 as a possible regulator of bone turnover in vivo. It is suggested that the athymic mouse has a deficiency somewhere in the cascade of events leading to the production of IL-6 or, alternatively, that IL-6 replaces other factors that are supplied by T lymphocytes directly or indirectly. As T lymphocytes interact with B lymphocytes it is suggested that the athymic mouse might be appropriate for studying the in vivo effects of the immune system on normal bone metabolism."

"hematopoietic cells and lymphocytes residing in the bone marrow may have effects on bone, especially in the pathological state."

On lymphocytes affecting height:

Growth patterns in pubertal HIV-infected adolescents and their correlation with cytokines, IGF-1, IGFBP-1, and IGFBP-3.

"This study aims to describe the final adult height (FAH) and pubertal growth patterns in human immunodeficiency virus (HIV)-infected adolescents and to compare these to an age-matched population of seroreverting HIV-exposed, uninfected (HEU) adolescents. It further aims to evaluate the interplay of proinflammatory cytokines with insulin-like growth factor 1 (IGF-1), insulin-like growth factor binding protein 3 (IGFBP-3), and IGFBP-1 during the pubertal growth spurt. Methods: HIV-infected and HEU adolescents who had achieved FAH were evaluated. Auxologic data, viral load, CD4+ T-lymphocyte (CD4) count, and the use of highly active antiretroviral therapy were obtained via a retrospective chart review. Serum interleukin (IL)-1α, IL-6, tumor necrosis factor (TNF)-α, IGFBP-1, IGFBP-3, and IGF-1 were assessed. The mean FAH standard deviation score for the HIV-infected group was -0.78 (±1.1) compared to 0.05 (±0.78) for the HEU{so being exposed to HIV but not being infected slightly increased height}. There was a positive correlation between CD4 count and FAH{But this correlation occur indefinately}. The mean age and magnitude of peak growth velocity (GV) was within normal limits. IL-1α, IL-6, TNF-α, IGFBP-3, and IGF-1 were not significantly correlated with HIV RNA or height. IGFBP-1 was detectable in 100% of poorly controlled HIV-infected patients and 25% of the HEU cohort. The FAH of HIV-infected patients was significantly shorter than that of HEU patients, and it positively correlated with CD4 count. Our cohort demonstrated normal timing and magnitude of peak GV during puberty."

Adult Height Achieved was linearly correlated to CD4+ lymphocyte count to 2000.

Bone lengthening in response to stress?

2013-04-22T19:42:00.000-07:00

Ctrl-F (*NEW*). Presented are the findings that tennis players do have longer stroke arms than the contralateral arm. This does not seem to be due to a selection bias as the mean contralateral arm of the tennis player is the same as the control arm for non-players. Thus there does not seem to be a selection bias for arm length as there does for say basketball and height.

The changes in the tennis player seem to be throughout the entire bone rather than just the ends of the bones. If the changes were due to the growth plate you'd expect the changes to be constrained to near to the ends of the bones but since the changes are throughout the entire bone it's more consistent with plastic deformation.

However, the changes in bone length are small and it's hard to create a serving/throwing motion with your legs or spine. And the studies are not perfect for our purposes as we'd want to look at more longitudinal studies and people without present growth plates.

This still provides evidence that very high forces could generate active tensile induced longitudinal growth in bone. However, the difficult in reproducing the throwing/serving motion in other bones and the relatively minor amount of growth in general means that such a method is not a practical method of gaining height.

Basepall pitcher's pitching arm tends to be longer than their non-pitching arms. Many have speculated that this may be due to the stress that pitching arm undergoes. However, many have retorted that people with one arm longer than the other may just be better pitchers. That problem does not exist with instrument players. Longer fingers do not make people better guitar or violin players.

I found this as a science fair project for the California state science fair. Looks like students are the only ones willing to do height increase research thanks to the fact that grow taller is a dirty phrase.

The Fingers of Isaac Stern: Will Constant Stress Affect the Development of Phalanges?

Here's a page that considers a similar project.

Objectives/Goals
The objective is to determine if violinists have longer phalanges in their left hand than their right hand compared to non-violinist. I believe violinists have longer left hand fingers due to the stress on the bones.
Methods/Materials
Methods: 6 steps: 1)Design a questionnaire 2)Define samples. 3)Select two groups: violinists and control group,each with twenty four people,divide evenly into four sub-groups: male, female, adult and young adult.(12 & up) 4)Define uncontrolled variables. 5)Conduct a personal interview and measure the index, middle, ring finger & pinky. 6)Analyze data.
Materials: A specially made ruler is used. It has a moveable piece of cardboard on the ruler for easy reading and maximum accuracy.
Results
The violinist group has much longer phalanges in their left hand by as much as 0.6 cm. The non-violinists left hand four fingers are significantly shorter than the right hands' by as much as 0.9 cm. The data show no significant difference between both adults and young adults, male and female group.
Conclusions/Discussion
Conclusions: My hypothesis is correct. The violinists' left hand fingers are longer than their right hand. This might be due to the stress they put on their bones during years of practice.
Next question: I would like to know if my research would help any medical study. Especially for the handicapped with two legs of different length.

Now, unlike the baseball pitcher, longer fingers do not make for better violin players. The non-violinists had longer fingers in their right hand and since most people are right handed, using a hard more would indicate finger length. There is still the possibility that left handed people may be better violin players and that left handed people have longer left hands that right hands.

What is interesting is that playing the violin there are no microfractures and there are no epiphyseal distraction forces. The phalanges are long bones in the finger. The mechanism as to how playing the violin would result in long bone lengthening is unknown. One would wonder what the effect of bone lengthening would be in regards to typing which puts the same kind of stresses on finger bones, doesn't select versus right or left hand(okay qwerty is more left handed and dvorak is more right handed), and also doesn't cause microfractures or major distraction forces.

Unfortunately this one kids research paper is the only piece of information I could find on the subject. Hopefully this kid will became an important scientist and help us find way to grow taller naturally.

Mechanical stresses and endochondral ossification in the chondroepiphysis.

"The ossific nucleus appears in an area of high shear (deviatoric) stresses; The edge of the advancing ossification front (zone of Ranvier or ossification grove) also experiences high shear stresses; and the joint surface, where articular cartilage forms, is exposed to high-magnitude hydrostatic compression. Intermittently applied shear stresses (or strain energy) promote endochondral ossification and that intermittently applied hydrostatic compression inhibits or prevents cartilage degeneration and ossification."

LSJL likely applies both types of stresses which is why it is both pro-chondrogenic and pro-endochondral ossification.

"pressure caused cartilage formation in the perichondrium and periosteum as did tensile stresses acting at right angles to the perichondrium fiber direction."

"Pressure and tensile stresses imposed on cartilage caused the “disintegration of the hyaline substance and its replacement by a fibrillar system”"

"adventitious cartilage arises in response to intermittent pressure and tension accompanied by movement. Immobilization caused the transformation of this cartilage into “bonelike” tissue."

"mechanical pressure and avascularity have similar effects in that both conditions favor differentiation

of cartilage rather than bone from precursor cells."

"(a) deviatoric (distortional or shear) stress and (b) dilatational (or hydrostatic) stress. Deviatoric stresses cause material distortions with no change in volume. Dilatational stresses are pure hydrostatic (compression or tension) stresses that do not distort but will cause volume changes if the material is compressible. The stored strain energy is the sum of the deviatoric and dilatational energy. Materials like cartilage, which are nearly incompressible, will store negligible dilatational energy since negligible volume change occurs, regardless of the magnitude of the dilatational stress. In such materials, therefore, the shear stress distributions will reflect the distribution of strain energy density."

"deviatoric stresses (which are accompanied by elongation or tensile strains in some direction) [are] a specific stimulus for the development of collagenous fibrils and hydrostatic pressure [is] responsible for chondrogenesis."

"The vascular supply pattern to the femoral head was found to correlate with regions that were not exposed to high magnitudes of intermittent hydrostatic compression."

Hydrostatic stress places stress more directly into the epiphysis of the bone than other forms of stress.

Short-term and long-term site-specific effects of tennis playing on trabecular and cortical bone at the distal radius

"Epiphyseal bone enduring longitudinal growth showed a great capacity to respond to mechanical loading in children"

"In children, no significant difference was observed between the dominant and nondominant forearm lengths (21.6 cm on both sides). In adults, the respective values were 25.3 ± 1.6 cm and 25.0 ± 1.6 cm, with a significant side-to-side difference"

(*NEW*)

Stimulation of Bone Growth Through Sports: A Radiologic Investigation of the Upper Extremities in Professional Tennis Players

"Does unilateral sports-specific strain affect the skeletal system of the athlete? Specifically, can any differences be found in longitudinal growth of the bones of the forearm and hand in professional tennis players between the stroke arm and the contralateral arm? An investigation
was conducted involving 20 high-ranking professional tennis players (12 male and eight female players) between 13 and 26 years of age as well as 12 controls of the same age range. The radiologic examinations of the bones of the forearm and hand yielded an increase in density of bone substance and bone diameter as well as length in the stroke arm as compared with the contralateral arm. This change in bone structure and size can be attributed to two factors: mechanical stimulation and hyperemia{increase in blood flow} of the constantly strained extremity."

"we observed a highly significant difference in ulnar length between the two arms in the tennis players. Differences ranged from 0.2 to 1.3 cm"

The mean difference in ulna length in control group was 0.17mm but this could be a correlational rather than causal relationship. People with a dominant longer arm may select tennis as a sport.

The mean length for the contralateral arm was 270mm which was the same as the control group but the mean length for the stoke arm was 278mm.

They also found an increase in the length of the second metacarpal of the playing arm of the hand of 4.1mm. Average lengthening of the second metacarpal was 2.7mm.

In all likelihood, I think it's more likelihood that the tennis caused the overgrowth rather than being a correlational effect.

The phenomenon of twisted growth: humeral torsion in dominant arms of high performance tennis players

There's a lot of stuff in this article about the physics of the forces generated during a serve.

"twisted humeral geometry at different stages of development could be attributed to muscle forces inducing a torsional load."

"characteristic twisted bone growth profile was found in tennis players, baseball pitchers and handball players."

"Predominant axial loading by deltoid induces humeral hypertrophy with pronounced bone growth along the longitudinal axis."

"During ball impact, muscle forces are aligned with the longitudinal axis of the humerus."

"During the serve, the entire upper limb is subject to tremendous loads"

Right arm versus left arms of professional tennis player. You can kind of see a twisted nature of the bone and the twist seems to go throughout the whole bone and not just the growth plate region.

According to Humeral torsion and passive shoulder range in elite volleyball players, "the dominant arm [is] on average 9.6° more retroverted than the non-dominant arm" for volleyball players.

Increase your Stature with Plastic Deformation

2013-04-16T13:17:00.000-07:00

Ctrl-F (*NEW*) for the related content. Recently, someone asked if it was possible to grow taller while running with ankle weights and natural height growth recently posted about tensile stress for height growth. The goal for tensile stress for height growth is to induce enough stress in the bone to induce plastic deformation in the bone to make it longer. The point at which this occurs is the yield stress. Cortical bone is the limiting factor for this to occur. Ultimate stress is the point where the bone breaks. According to this engineering page, the ultimate stress for bone is 133MPa whereas the yield stress is 115MPa. It would be hard to generate this kind of stress in the bone but it's possible that there may be some residual strain that occurs at the nanometer level. Can we generate enough residual strain via tensile forces on the bone to eventually reach the plastic deformation range? According to LOAD TRANSFER ACROSS THE PELVIC BONE DURING NORMAL WALKING, 20-67MPa stresses were generated in the hip bone during walking. So it's conceivable that a tensile force of 115MPa could be generated by a physiological loading regime. I couldn't find how much tensile stress is generated by running.

Plastic deformation refers to residual strain whereas elastic strain refers to strain that has no residual strain. So a potential treatment would involve using say a tensile strain mechanism to stretch the bone to such a degree that it retains some of the stretch after the load is removed.

Plastic deformation seems to take a high level of strain to occur.

Orientation dependence of progressive post-yield behavior of human cortical bone in compression.

"[We] determine the effect of loading direction on the evolution of post-yield behavior of bone using a progressive loading protocol. To do so, cylindrical compressive bone samples were prepared each in the longitudinal, circumferential and radial directions, from the mid-shaft of cadaveric femurs procured from eight middle-aged male donors (51.5 ± 3.3 years old). These specimens were tested in compression in a progressive loading scheme. The elastic modulus, yield stress, and energy dissipation were significantly greater in the longitudinal direction than in the transverse (circumferential and radial) directions. However, no significant differences were observed in the yield strain as well as in the successive plastic strain with respect to the increasing applied strain among the three orientations. The initiation and progression of plastic strain are independent of loading orientations, thus implying that the underlying mechanism of plastic behavior of bone in compression is similar in all the orientations."

Therefore we can see what loads are required in other orientations like compressive loading and use those some loads to induce tensile strain to such a degree as to induce plastic strain.

"the post-yield behavior of bone is associated with an exponential decay of elastic modulus (microdamage accumulation), linear plastic deformation, and an acute saturation of viscous behavior of the tissue"

Cooperation of length scales and orientations in the deformation of bovine bone.

"Combined wide angle X-ray diffraction and small angle X-ray scattering were used together with in situ tensile testing to investigate the deformation and failure mechanisms of bovine cortical bone at three material levels: (1) the atomic level, corresponding to the mineral crystal phase; (2) the nano level, corresponding to the collagen fibrils; (3) the macroscopic level. Deformation was linear at all three levels up to a strain of 0.2% in the longitudinal tensile direction. At this critical strain a sudden 50% decrease in the fibrillar and mineral strains was observed. The presence of partial local damage leads to inhomogeneous strain distributions within the probed gauge volume. This gives rise to diffraction peak broadening in the mineral phase, as well as strain relaxation at the nanoscale. Above the critical strain the longitudinally oriented strains below the nanoscale remained constant at a reduced level until failure. The lateral orientation of the nanostructures toughens the bone, while a higher material level dominated the subsequent deformation process, either by sliding between the lamellar layers or by the growth of microcracks. The bone has compressive residual stress in the crystal phase."

"At low stresses the bone behaves linear elastically with stiffness primarily coming from the mineral phase. Physiological loading generally falls in this elastic region. The mineral phase that provides rigidity is proposed to carry the load, while the soft matrix transfers the load to neighboring mineral crystals by shear. Yielding is known to be the start of damage, when strain reaches a critical level and starts initiating crack formation. In the post-yield region cortical bone experiences large plastic deformation while absorbing large amounts of energy prior to fracture"

"The post-yield deformation involves a combination of slippage at cement lines, which reduces strain energy and slows down crack propagation by deflecting the crack path, and discontinuity of microcracks, that greatly reduces the stress intensity at the crack tip. At the nanoscopic level, breaking of sacrificial bonds at the fibrillar level dissipates energy, while long mineral platelets delocalize the crack-tip deformation"<-maybe inducing other forms of slippage of cement lines will also permanently make the bone grow longer without the high levels of tensile strain required for plastic strain.

" Upon tensile loading the gap zones between the fibrils were stretched and the change in the dimensions of the gap zones is a measure of fibrillar strain."

"Fracture surface of the bone captured by an optical camera during tensile testing."<-So the stretching did induce microfractures in such a way as to kind of lengthen the bone.

How tough is bone? Application of elastic-plastic fracture mechanics to bone.

"bone contains a high volumetric percentage of organics and water that makes it behave nonlinearly before fracture. We applied elastic-plastic fracture mechanics to study bone's fracture toughness. The J integral, a parameter that estimates both the energies consumed in the elastic and plastic deformations, was used to quantify the total energy spent before bone fracture. Twenty cortical bone specimens were cut from the mid-diaphysis of bovine femurs. Ten of them were prepared to undergo transverse fracture and the other 10 were prepared to undergo longitudinal fracture. The specimens were prepared following the apparatus suggested in ASTM E1820 and tested in distilled water at 37 degrees C. The average J integral of the transverse-fractured specimens was found to be 6.6 kPa m, which is 187% greater than that of longitudinal-fractured specimens (2.3 kPa m). The energy spent in the plastic deformation of the longitudinal-fractured and transverse-fractured bovine specimens was found to be 3.6-4.1 times the energy spent in the elastic deformation. The toughness of bone estimated using the J integral is much greater than the toughness measured using the critical stress intensity factor."

"bone contains water that can affect the properties of collagen"

"60% of water was bonded to collagen [in dog bones]"

Mechanisms of short crack growth at constant stress in bone.

"Slow, stable crack growth occurred at a rate and angle which were dependent on the orientation of the sample: tests were conducted with the loading axis both parallel and perpendicular to the longitudinal axis of the bone. All cracks showed intermittent growth in which periods of relatively rapid propagation alternated with periods of temporary crack arrest or relatively slow growth. In some cases crack arrest could be clearly linked to microstructural features such as osteons or Volkmann's canals, which acted as barriers to crack growth. Crack-opening displacement increased over time during the arrest periods. The growth of small cracks in bone at constant stress, [involves] microstructural barriers, time-dependent deformation of material near the crack tip and strain-controlled propagation."

Progressive post-yield behavior of human cortical bone in shear.

" the shear modulus of bone decreased with respect to the applied strain, but the rate of degradation was about 50% less than those previously observed in compression and tension tests. In addition, a quasi-linear relationship between the plastic and applied strains was observed in shear mode, which is similar to those previously reported in tension and compression tests. However, the viscous responses of bone (i.e. relaxation time constants and stress magnitude) demonstrated slight differences in shear compared with those observed in tension and compression tests."

"After a preload of 10 N in compression, each specimen was loaded using the cyclic loading protocol. In each cycle, the specimens were loaded under the displacement control with a rate of 0.005 mm/s, held at the displacement level for 120 s, unloaded to 25 N, and held at the 25 N for 120 s. The dwelling time (120 s) was determined through pilot studies to ensure that the specimens reach to a quasi-equilibrium condition"

"The shear yield strain and yield stress of the bone specimens were 0.88 ± 0.18% and 35.7 ± 9.88 MPa, respectively"<-These were bones from 80 year olds though.

" the yield strain in shear observed in this study was about 0.88 ± 0.18% (N = 6), which is higher than those in compression (0.71 ± 0.07%, N = 8) and in tension (0.39 ± 0.03%, N = 8) "

Traumatic plastic deformation of the tibia: case report and literature review.

" a 10-year-old girl who, after falling down a slope, came to a sudden stop when her right foot hit a rock. This resulted in a fracture of the fibula and bowing of the tibia."

"Plastic deformation refers to the deformation of a bone, without fracture of its cortices, that persists once the deforming force has been removed. It has been reported most commonly in the forearm, with 58 of a review of 74 cases involving the forearm."

Mechanical and morphological aspects of experimental overload and fatigue in bone

"long bone fatigue is produced in 30 pairs of dog ulnas by applying opposing forces at both extremities thereby causing a strain. On a force-deformation curve the force axis indicates a zone of load, an intermediate zone of fatigue and a zone of overload; the deformation axis shows an elastic zone and a plastic zone"

"the plastic phase is short for dense cortical bone"

Guided growth: recent advances in a deep-rooted concept.

"Guiding growth by harnessing the ability of growing bone to undergo plastic deformation is one of the oldest orthopaedic principles."

"Bracing for adolescent idiopathic scoliosis does not influence vertebral development."

(*NEW*)

Damage accumulation in vertebral trabecular bone depends on loading mode and direction.

"251 cylindrical samples (8×18-25mm) were obtained from 50 male and 54 female fresh frozen human vertebrae (T1-L3) of 65 (21-94) years. Vertebrae were randomly assigned to three groups cranial-caudal, anterior-posterior and latero-lateral. Specimens were mechanically loaded in compression, tension or torsion in five load steps at a strain rate of 0.2%/s. Three conditioning cycles were driven per load step. Stress-strain curves were reconstructed from the force-displacement or from the moment-twist angle curves. Damage accumulated from 0 to 86% in compression, from 0 to 76% in tension and from 0 to 86% in torsion through the five load steps. Residual strains accumulated from 0 to -0.008mm/mm in compression, 0 to 0.006mm/mm in tension and 0 to 0.026rad/rad in torsion. Significantly less damage but not residual strains accumulated in transverse directions."

Lots of alterations were made to the bone so that may have had effects on physiological loading properties.

"Cortical bone shows qualitatively similar damaging behaviour as trabecular bone"

"substantial damage occurs at the nanometer level "

"Cracks and diffuse damage that accumulate within trabeculae cause reductions in apparent modulus prior to failure of whole trabeculae"<-see above where the scientists stated that cortical bone has similar damaging properites as trabecular bone.

Comparison of the elastic and yield properties of human femoral trabecular and cortical bone tissue

"Effective tissue modulus and yield strains were calibrated for cadaveric human femoral neck specimens taken from 11 donors, using a combination of apparent-level mechanical testing and specimen-specific, high-resolution, nonlinear finite element modeling. The trabecular tissue properties were then compared to measured elastic modulus and tensile yield strain of human femoral diaphyseal cortical bone specimens obtained from a similar cohort of 34 donors. Cortical tissue properties were obtained by statistically eliminating the effects of vascular porosity. Results indicated that mean elastic modulus was 10% lower (p<0.05) for the trabecular tissue (18.0±2.8 GPa) than for the cortical tissue (19.9±1.8 GPa){so it actually doesn't take that much more stress to induce plastic deformation in the cortical bone than in the trabecular bone(cortical bone is the limiting factor for lengthening)}, and the 0.2% offset tensile yield strain was 15% lower for the trabecular tissue (0.62±0.04% vs. 0.73±0.05%, p<0.001). The tensile–compressive yield strength asymmetry for the trabecular tissue, 0.62 on average, was similar to values reported in the literature for cortical bone. We conclude that while the elastic modulus and yield strains for trabecular tissue are just slightly lower than those of cortical tissue, because of the cumulative effect of these differences, tissue strength is about 25% greater for cortical bone."

The yield modulus is the point where looking for as that is when the bone deforms plastically.

0.2% offset yield stress (MPa) for cortical bone 107.9±12.3

Offset yield means that the extract stress needed is inexact.

107.9 MPa is equal to 107.9N/millimeter^2. So the larger area over which the force is applied, the less force overall is generated.

Do fats and sugars affect your height gain?

2013-04-11T17:50:00.000-07:00

Considering that there are several Vitamin D related genes that influence height but there are some instances where Vitamin D intake does not affect adult height. Although, the levels of glucose consumed versus starch and fructose affect the pathways related to Vitamin D rather than just Vitamin D levels. Therefore, it is likely that glucose versus starch and fructose consumption may affect adult height rather than just a temporary decrease in growth rate.

Eating high glucose foods versus high fructose and to a much less extent high starch foods will only affect people with open growth plates but could result in a little bit more adult height.

Ctrl-F (*NEW*) for the new content.

People are always looking for a quick fix. Rather than hard, strenuous exercise to increase height people want to take some height increase pill. In this blog entry, I'm going to look at if dietary factors can affect human height.

Now, I'm considering "normal" foods. Anything that you put in your body could be considered part of your body. The definition of diet as per this article is any chemical that could be found regularly in food(so no supplements, although they do have chondroitin and glucosamine in liquid form now but it still isn't what I would consider mainstream food).

High-fat, sucrose diet impairs geometrical and mechanical properties of cortical bone in mice.

"Exposure to diets high in fat and sucrose can induce hyperinsulinaemia, affect Ca and Mg metabolism, and alter bone mineralisation and mechanical properties."

One possible explanation for how diets high in fat and sucrose alter bone mechanical properties is that unesterified long-chain saturated fatty acids have a melting point above body temperature and, with sufficient calcium in the intestinal lumen, form insoluble calcium soaps.

So, sugar and fat competes with bone for calcium absorption. So, a very high diet with high fat and glucose levels could impair height gain. Remember, that reduced sensitivity to insulin has been associated with possible height gain.

"The present study assessed morphological and mechanical changes in a murine model exposed to a high-fat/sucrose (HFS) diet, as well as corresponding molecular and endocrine markers of bone turnover. "

Bone turnover however doesn't necessarily have an affect on human height. The old confusion between bone modeling and remodeling(neither of which can increase height) is an example of how things can be misconstrued. Bone turnover can affect the rate at which microfractures heal however and microfractures in the cortical bone can be possibly used to help you grow taller.

"Both body mass and percentage body fat were greater in mice fed HFS diet. After adjusting for body mass, tibial structural and morphological properties were adversely affected in the HFS cohort. Cortical thickness, cross-sectional area, and load at maximum were all significantly lower in mice fed HFS diet. Receptor activator of nuclear factor kappabeta ligand (RANKL) mRNA was significantly upregulated in HFS mice, but osteoprotegerin/RANKL mRNA ratio remained unchanged between cohorts[So OPG increased to compensate for the increase in RANKL leading to a change in bone turnover] . Moreover, cyclo-oxygenase-2[also known as COX2] mRNA tended to be increased in HFS. Thus, ingestion of an HFS diet had a significant adverse effect on mouse bone morphology and mechanics, and these effects were likely due to elevated osteoclast activity associated with the inflammatory state of obesity, and not necessarily osteoclast recruitment/proliferation."

The investigators in this study theorized that obesity caused the change in cortical thickness not the high fat/fructose diet. Any diet with a caloric surplus could have potentially caused the same effect. A high caloric diet may be beneficial but a high fat diet may cause additional inflammatory factors that can be bad for height growth.

Study on the effect of T-2 toxin combined with low nutrition diet on rat epiphyseal plate growth and development.

"The purpose of this study was to observe early lesions of rat epiphyseal plates and metaphysis caused by T-2 toxin and T-2 toxin combined with a low nutrition diet to determine possible pathogenic factors of Kashin-Beck disease (KBD). Ninety Wistar rats were divided into three groups. Group A was fed with a normal diet as control; group B was fed with a normal diet and T-2 toxin; and group C was fed with a low nutrition diet and T-2 toxin."

T-2 toxin is a mold byproduct of a fungus.

"After two weeks, the epiphyseal plate showed necrosis of chondrocytes in groups B and C. After four weeks, more obvious chondrocyte necrosis appeared. The positive rate of Lamellar necrosis in group C was significantly higher than that in groups B and A (P < 0.01). Metaphyseal trabecular bone showed sparse disorder and disruption in group C. T-2 toxin combined with a low nutrition diet could lead to more serious chondrocyte necrosis in the epiphyseal plate and disturb metaphyseal trabecular bone formation."

So, the T-2 toxin has the potential to decrease height by destroying chondrocytes. Chondrocytes are the basis for height growth in the growth plates. This shows you how detrimental toxins can be in terms of growing taller.

Dietary patterns in Canadian men and women ages 25 and older: relationship to demographics, body mass index, and bone mineral density.

"The objective of the study was to determine whether dietary patterns in men (ages 25-49, 50+) and women (pre-menopause, post-menopause) are related to femoral neck bone mineral density (BMD) independently of other lifestyle variables, and whether this relationship is mediated by body mass index."

The BMI is a perfect example of how people cling to something because it's the popular thing rather than because it's the correct thing. The BMI is only useful for populations as the deviations average out. Their is too much internal differences in bone size within individuals for a tool like the BMI to be useful. Further, the BMI doesn't account for things like on average people's wingspans being larger than their height.

"We identified two underlying dietary patterns using factor analysis and then derived factor scores. The first factor (nutrient dense) was most strongly associated with intake of fruits, vegetables, and whole grains. The second factor (energy dense) was most strongly associated with intake of soft drinks, potato chips and French fries, certain meats (hamburger, hot dog, lunch meat, bacon, and sausage), and certain desserts (doughnuts, chocolate, ice cream). The energy dense factor was associated with higher body mass index independent of other demographic and lifestyle factors, and body mass index was a strong independent predictor of BMD. Surprisingly, we did not find a similar positive association between diet and BMD. In fact, when adjusted for body mass index, each standard deviation increase in the energy dense score was associated with a BMD decrease of 0.009 (95% CI: 0.002, 0.016) g/cm2 for men 50+ years old and 0.004 (95% CI: 0.000, 0.008) g/cm2 for postmenopausal women. In contrast, for men 25-49 years old, each standard deviation increase in the nutrient dense score, adjusted for body mass index, was associated with a BMD increase of 0.012 (95% CI: 0.002, 0.022) g/cm2."

BMD density was measured by dual x-ray absorptiometry so bone size could have been increased by increased diet. Eating more was associated with increased BMD. Now BMD may not be a causal way to increase height but it is a good measure of anabolism in the bone. The reason that age had the affect of lowering BMD instead of racing BMD with energy dense score could possibly be that men over 50 had lower activity levels.

Regulation of Mesenchymal Stem Cell Chondrogenesis by Glucose through Protein Kinase C/Transforming Growth Factor Signaling.

"The extent of chondrogenesis of hMSCs previously cultured with different concentrations of glucose was evaluated. Transforming growth factor-beta (TGF-β) signaling molecules and protein kinase C (PKC) were analyzed to identify the role of these molecules in the regulation of glucose on chondrogenesis. In addition, hMSCs in high-glucose expansion culture were treated with the PKC inhibitor to modulate the activity of PKC and TGF-β signaling molecules.
High-glucose maintained hMSCs were less chondrogenic than low-glucose maintained cells upon receiving differentiation signals. High-glucose culture increased the phosphorylation of PKC and expression of type II TGF-β receptor (TGFβRII) in pre-differentiation hMSCs. However, low-glucose maintained hMSCs became more responsive to chondrogenic induction with increased PKC activation and TGFβRII expression than high-glucose maintained hMSCs during differentiation. Inhibiting the PKC activity of high-glucose maintained hMSCs during expansion culture upregulated the TGFβRII expression of chondrogenic cell pellets, and enhanced chondrogenesis."

"During chondrogenic induction, high-glucose medium enhances chondrogenesis of chick mesenchymal cells, in comparison with low-glucose medium"

"high-glucose expansion culture reduces the proliferation of hMSCs"

"TGF-β ligand binds to type II TGF-β receptor (TGFβRII) to form a heterodimeric complex with type I TGF-β receptor (TGFβRI), which phosphorylates downstream signaling molecule Smad2/3. Phosphorylated Smad2/3 forms a heteromeric complex with Smad4, acting as a transcriptional activator to regulate the activity of TGF-β-responsive genes, including Sox9 for chondrogenesis"

"Human MSCs transfected with the TGF-β1 or TGF-β2 gene have been shown to induce chondrogenesis with the production of cartilage-related collagen type II."

"Human bone marrow-derived MSCs were isolated from femoral heads of 3 patients between 25 to 50 years of age who underwent total hip arthroplasty"

"[The human MSCs] expressed CD73, CD90, and CD105, but not CD34 and CD45"

"HGMCs grew slower than LGMCs"

"at day 9, the levels of mRNA expression of cartilage-related markers Sox9 and aggrecan of HGMC pellets were significantly downregulated, and at day 22, the expression levels of aggrecan and collagen type II of HGMC pellets were also significantly decreased, compared to those of LGMC pellets."

During differentiation TGFBRI expression was barely detectable in either mesenchymal group and TGFBRII was downregulated in the High-Glucose group versus the Low-Glucose group. Smad3-p and PKC-p were lower in HGMC pellets than LGMC.

Pre-differentiation PKC-p was actually higher in HGMC than LGMC. Inhibition of PKC during pre-differentiation culture can increase PKC and TGFBRII levels during chondrogenesis. At 14 days of chondrogenesis, pre-differentiation chondrocytes treated with PKC inhibitor had higher levels of Acan, Col2, and Col9.

"high-glucose chondrogenic culture is essential for maintaining matrix structural integrity"

Glucose: an energy currency and structural precursor in articular cartilage and bone with emerging roles as an extracellular signaling molecule and metabolic regulator.

"Glucose is vital for osteogenesis and chondrogenesis, and is used as a precursor for the synthesis of glycosaminoglycans, glycoproteins, and glycolipids. Glucose sensors are present in tissues and organs that carry out bulk glucose fluxes (i.e., intestine, kidney, and liver). The beta cells of the pancreatic islets of Langerhans respond to changes in blood glucose concentration by varying the rate of insulin synthesis and secretion. Neuronal cells in the hypothalamus are also capable of sensing extracellular glucose. Glucosensing neurons use glucose as a signaling molecule to alter their action potential frequency in response to variations in ambient glucose levels. Bone cells can influence (and be influenced by) systemic glucose metabolism. Cartilage and bone cells are sensitive to extracellular glucose and adjust their gene expression and metabolism in response to varying extracellular glucose concentrations."

"The transport of sugar across the plasma membrane of mammalian cells is mediated by members of the GLUT/SLC2A family of facilitative sugar transporters and the SGLT/SLC5A family of Na+-dependent sugar transporters"

"GLUT1, GLUT3, and GLUT4 are high-affinity transporters whereas GLUT2 is a low-affinity transporter; GLUT5 is primarily a fructose carrier " GLUT1 is expressed in articular cartilage and IVD cells.

"IVD is anatomically and functionally very similar to cartilage although in contrast to cartilage it develops from notocordal cells rather than mesenchymal cells"

"chondrocytes express multiple isoforms of the GLUT/SLC2A family"

"Chondrocytes are capable of adjusting to high and low glucose concentrations by changing the protein levels of GLUT1"

"OA chondrocytes exposed to high glucose were unable to down-regulate GLUT1. OA-derived chondrocytes accumulated more glucose and produced more ROS."

GLUT1 and GLUT4 are expressed in murine endochondral bone formation.

"high d(+)glucose may alter RANKL-induced osteoclast formation by inhibiting redox-sensitive NF-kappaB activity through an anti-oxidative mechanism."

"Mature osteoclasts rely on the citric acid cycle and mitochondrial respiration to generate high levels of ATP production for acid secretion and bone resorption."

" glucose metabolism is increased during osteoclast differentiation resulting in a metabolic shift toward accelerated glucose metabolism at an early stage of RANKL-stimulated osteoclast differentiation. Increased mitochondrial oxidative phosphorylation will then result in elevated ATP production and enhanced osteoclast differentiation."

"osteocalcin{up in LSJL} [is a] regulator of pancreatic insulin production and glucose metabolism"

"osteocalcin deficiency in knockout mice leads to decreased insulin and adiponectin secretion, insulin resistance, higher serum glucose levels, and increased adiposity"

Perinatal maternal dietary supplementation of ω3-fatty acids transiently affects bone marrow microenvironment, osteoblast and osteoclast formation, and bone mass in male offspring.

[Omega3-fatty acids]

"[Does] maternal supplementation with ω3-polyunsaturated fatty acids (n3FA) [improve] offspring bone growth and adult bone mas?. Female rats were fed a diet containing 0.1% (control, n = 10) or 1% (n3FA, n = 11) docosahexanoic acid (DHA) during pregnancy and lactation. Offspring were weaned onto a control rat chow diet. Tibial growth plate and metaphysis structure, osteoblast/osteoclast density and differentiation, and gene expression were assessed in offspring at 3 wk (weaning), 6 wk (adolescent), and 3 months (adult). Maternal n3FA supplementation elevated offspring plasma n3FA levels at 3 and 6 wk. Although total growth plate heights were unaffected at any age, the resting zone thickness was increased in both male and female offspring at 3 wk. In n3FA males, but not females, bone trabecular number and thickness were increased at 3 wk but not other ages. The wk 3 n3FA males also exhibited an increased bone volume, an increased osteoblast but decreased osteoclast density, and lower expression of osteoclastogenic cytokines receptor activator of nuclear factor-κB ligand, TNF-α, and IL-6. No effects were seen at 6 wk or 3 months in either sex. Thus, perinatal n3FA supplementation is associated with increased bone formation, decreased resorption, and a higher bone mass in males, but not in females, at weaning; these effects do not persist into adolescence and adulthood and are unlikely to produce lasting improvements in bone health."

"Despite n3FA supplementation being ceased at weaning, increased DHA and total n3FA levels persisted until 6 wk of age but had returned to control levels by 3 months of age."

"maternal n3FA supplementation did not alter body length or body weight of the offspring at the ends of critical growth periods (3 wk, 6 wk, and 3 months of age)."

"eicosapentaenoic acid (EPA) and DHA are never completely absent from breast milk, and the level is largely determined by the mother's diet"

"feeding postweaning male Fisher rats DHA substantially increased bone marrow cell number"<-more bone marrow cells means more possibilities for mesenchymal condensation and more ability to induce chondrogenesis.

Glucose reduction prevents replicative senescence and increases mitochondrial respiration in human mesenchymal stem cells.

"During in vitro expansion of MSCs, replicative senescence may occur and will compromise the quality of the expanded cells. Because calorie restriction has been shown to effectively extend the life span of various organisms, the purpose of this study is to investigate the effect of glucose reduction on MSCs and the coordinated changes in energy utilization. It was found that the frequency of cycling cells was significantly increased, while senescence markers such as β-galactosidase activities and p16(INK4a) expression level were markedly reduced in MSCs under low-glucose culture condition. MSCs [maintained chondrogenic differentiation potential] after low-glucose treatment. Interestingly, the ability of osteogenic lineage commitment was improved, while the ability of adipogenic lineage commitment was delayed in MSCs after glucose reduction. We observed decreased lactate production, increased electron transport chain complexes expression, and increased oxygen consumption in MSCs after glucose reduction treatment. Increased antioxidant defensive responses were evidenced by increased antioxidant enzymes expression and decreased superoxide production after glucose reduction. MSCs utilize energy more efficiently under restricted glucose treatment and exhibit greater self-renewal and antisenescence abilities, while their differentiation potentials remain unaffected."

"CR induces SIR2 family gene expression to regulate the downstream stress resistance reaction and to slow the aging processes"

"during cell proliferation an increase in lactate production will occur when there is excessive amount of glucose"

E and F are chondroinduction metrics to progressively higher concentrations of glucose(left to right).

(*NEW*)

Excessive fructose intake causes 1,25-(OH)2D3-dependent inhibition of intestinal and renal calcium transport in growing rats.

"chronic high fructose intakes by lactating rats prevented adaptive increases in rates of active intestinal Ca2+ transport and in levels of 1,25-(OH)2D3, the active form of vitamin D. Since sufficient Ca2+ absorption is essential for skeletal growth, our discovery may explain findings that excessive consumption of sweeteners compromises bone integrity in children. We tested the hypothesis that 1,25-(OH)2D3 mediates the inhibitory effect of excessive fructose intake on active Ca2+ transport. First, compared with those fed glucose or starch, growing rats fed fructose for 4 wk had a marked reduction in intestinal Ca2+ transport rate as well as in expression of intestinal and renal Ca2+ transporters that was tightly associated with decreases in circulating levels of 1,25-(OH)2D3, bone length, and total bone ash weight but not with serum PTH. Dietary fructose increased the expression of 24-hydroxylase (CYP24A1) and decreased that of 1α-hydroxylase (CYP27B1), suggesting that fructose might enhance the renal catabolism and impair the synthesis, respectively, of 1,25-(OH)2D3. Serum FGF23, which is secreted by osteocytes and inhibits CYP27B1 expression, was upregulated, suggesting a potential role of bone in mediating the fructose effects on 1,25-(OH)2D3 synthesis. Second, 1,25-(OH)2D3 treatment rescued the fructose effect and normalized intestinal and renal Ca2+ transporter expression. The mechanism underlying the deleterious effect of excessive fructose intake on intestinal and renal Ca2+ transporters is a reduction in serum levels of 1,25-(OH)2D3."

"1,25-(OH)2D3 is one of the key hormones controlling intestinal active Ca2+ transport, mainly by regulating TRPV6 and CaBP9k expression"

"Expression levels of TRPV5 and CaBP28k decreased in the fructose-fed compared to the glucose- and starch-fed rats"

The glucose fed group had the highest Vitamin D and PTH levels.

Glucose had the most bone length. 34.4mm for glucose versus 32.4mm for fructose. Although we can't be sure if this decrease in growth rate translates into decreased adult height.

Glucose diet was slightly superior than starch diet as well.

Foods high in Glucose:
Vegetables, Fruits, Breads, Grains, Dairy, Meats

Foods high in fructose:
Mostly processed foods

Foods high in starch:
Potatoes, bread, rice, cereal,

Micro-Growth Plates by LSJL

2013-04-05T09:52:00.000-07:00

It's unlikely that LSJL can re-establish a whole entire new growth plate. It's far more probable that LSJL can produce smaller growth plates that can each contribute a little to longitudinal bone growth. Here I present a study that provides evidence of the microgrowth plate theory.

The Interplay between Chondrocyte Redifferentiation Pellet Size and Oxygen Concentration.

"Chondrocytes dedifferentiate{in endochondral ossification chondrocytes die out rather than transdifferentiate into bone type cells so it's unlikely that there's any remnants of dedifferentiated or transdifferentiated chondrocytes that retain some chondrocyte epigenetic material, for LSJL we have to rely on regular mesenchymal stem cells} during ex vivo expansion on 2-dimensional surfaces. Aggregation of the expanded cells into 3-dimensional pellets, in the presence of induction factors, facilitates their redifferentiation and restoration of the chondrogenic phenotype. Typically 1×10(5)-5×10(5) chondrocytes are aggregated, resulting in "macro" pellets having diameters ranging from 1-2 mm. These macropellets are commonly used to study redifferentiation, and recently macropellets of autologous chondrocytes have been implanted directly into articular cartilage defects to facilitate their repair. However, diffusion of metabolites over the 1-2 mm pellet length-scales is inefficient, resulting in radial tissue heterogeneity. Herein we demonstrate that the aggregation of 2×10(5) human chondrocytes into micropellets of 166 cells each{it's probable to be able to induce an aggregate of 166 cells with LSJL stimuli, likely more cells are needed as we are dealing with MSCs rather than dedifferentiated chondrocytes}, rather than into larger single macropellets, enhances chondrogenic redifferentiation. In this study, we describe the development of a cost effective fabrication strategy to manufacture a microwell surface for the large-scale production of micropellets. The thousands of micropellets were manufactured using the microwell platform, which is an array of 360×360 µm microwells cast into polydimethylsiloxane (PDMS), that has been surface modified with an electrostatic multilayer of hyaluronic acid and chitosan to enhance micropellet formation{Both chitosan and hyaluronic acid are supplements, whether they can help create some kind of basement membrane or scaffold for growth is unclear}. Such surface modification was essential to prevent chondrocyte spreading on the PDMS. Sulfated glycosaminoglycan (sGAG) production and collagen II gene expression in chondrocyte micropellets increased significantly relative to macropellet controls, and redifferentiation was enhanced in both macro and micropellets with the provision of a hypoxic atmosphere (2% O2). Once micropellet formation had been optimized, we demonstrated that micropellets could be assembled into larger cartilage tissues{so micro-growth plates can combine to become larger growth plates}."

"The reduced diameter of the micropellets[100 micrometers each] mitigated diffusion gradients, enhanced MSC chondrogenic differentiation and generated a more uniform cell product"

"Hypoxic micropellets assembled into macrotissues. Alcian blue staining for hypoxic micropellets assembled at different time points (indicated days). The total culture duration was 21 days. Scale bars: 100 µm."

Key Differences between this study and LSJL:
*Articular Cartilage was used and not Growth Plate cartilage
*Dedifferentiated Chondrocytes were used and not mesenchymal stem cells.
*The micro-cartilage was uniform here. In LSJL, the microgrowth plates would not be uniform.

Regardless it still provides evidence that MSCs could potentially form microgrowth plates and those growth plates could combine to form larger growth plates. It seems that the higher oxygen concentration was of a more endochondral rather than chondral environment which is good considering that adult bone is vascularized.

How they treat short stature in medicine

2013-04-04T11:48:00.000-07:00

Clinical practice. Short stature in childhood--challenges and choices.

"Treatment with recombinant human growth hormone can increase the adult height of children with idiopathic short stature by 1.2 to 2.8 in. (3.0 to 7.1 cm), with wide variation in the incremental gain."<-10,000 to $60,000 per patient per year

"human growth hormone therapy in children with idiopathic short stature increases the growth rate and mean adult height by 1.2 to 2.8 in., or approximately 0.4 in. (1.0 cm) per year of human growth hormone treatment."

"Human growth hormone is administered subcutaneously at a dose of 0.2 to 0.375 mg per kilogram of body weight per week. Daily administration of human growth hormone is superior to less frequent administration. Dose modulation may influence the effect; doses at the higher end of this range and adjustment of the dose to achieve high-normal IGF-I levels lead to faster growth and perhaps to taller adult height"

"For short peripubertal[early stages of puberty boys, growth-promoting alternatives to human growth hormone are low-dose androgen therapy with injectable testosterone and low-dose androgen therapy with oral oxandrolone (e.g., 1.25 to 2.5 mg per day). Both regimens are relatively low in cost, and though they are not FDA-approved for growth acceleration, they increased the growth rate by 1.2 to 2.0 in. (3.0 to 5.1 cm) per year for 1 to 3 years in controlled trials."

"To avoid accelerated estrogen-mediated epiphyseal maturation, oxandrolone (not aromatized to estrogen) is theoretically preferred over testosterone when the bone age is less than 11 years. Oxandrolone is usually discontinued after a documented increase in endogenous testosterone; long-term follow-up studies indicate that treatment is followed by normal pubertal growth and eventual attainment of an adult height equal to or slightly greater than the predicted height before treatment "

"Aromatase inhibitors (which reduce estrogen production and delay skeletal maturation) have been used experimentally in boys to prolong pubertal growth and increase height, but they are more expensive and have less of a growth-accelerating effect than androgens, and actual adult height gains have fallen short of prior predictions of 1.6 to 2.4 in. (4.1 to 6.1 cm)."

Few Notes:

* Pediatric Treatment is extremely narrow: Only HGH and Testosterone. This is likely because they want the most expensive option and don't want treatments that could additionally help by say 0.1cm.
* Oxandrolone is the best testesterone supplement as it does not convert to estrogen
* These studies look at a wholistic view i.e. how much does this supplement increase the final height rather than looking at what happens directly at the cellular level.

CTGF(CCN2), height increase target gene

2013-04-02T11:00:00.000-07:00

CCN2 could increase adult height by accelerating the rate of endochondral ossification but keeping the epiphyseal growth plate the same size resulting in more growth. CCN2 is also associated with the height gene IGF2.

CTGF meets the criteria for a height growth target where overexpression increases height and underexpression decreases height. However this was only for cartilage specific expression of CTGF. Overexpression of CTGF has been found to have catabolic effects on muscle.

Glucosamine and CLA are potential ways to increase CTGF levels however can a global increase of CTGF increase height like a cartilage specific one? Does anyone know any other supplements that can increase CTGF? Prefereably only in the cartilage.

It should be noted that CTGF is only a promising target for people with open growth plates.

Cartilage–Specific Over-Expression of CCN Family Member 2/Connective Tissue Growth Factor (CCN2/CTGF) Stimulates Insulin-Like Growth Factor Expression and Bone Growth

"CCN family member 2/connective tissue growth factor (CCN2) promotes the proliferation, differentiation, and maturation of growth cartilage cells in vitro. We generated transgenic mice overexpressing CCN2 and analyzed them with respect to cartilage and bone development. Transgenic mice were generated expressing a ccn2/lacZ fusion gene in cartilage under the control of the 6 kb-Col2a1-enhancer/promoter. Changes in cartilage and bone development were analyzed. Primary chondrocytes as well as limb bud mesenchymal cells were cultured and analyzed for changes in expression of cartilage–related genes, and non-transgenic chondrocytes were treated in culture with recombinant CCN2. Newborn transgenic mice showed extended length of their long bones, increased content of proteoglycans and collagen II accumulation. Transgenic bones indicated increases in bone thickness and mineral density. Chondrocyte proliferation was enhanced in the transgenic cartilage. In in vitro short-term cultures of transgenic chondrocytes, the expression of col2a1, aggrecan and ccn2 genes was substantially enhanced; and in long-term cultures the expression levels of these genes were further enhanced. Also, in vitro chondrogenesis was strongly enhanced. IGF-I and IGF-II mRNA levels were elevated in transgenic chondrocytes, and treatment of non-transgenic chondrocytes with recombinant CCN2 stimulated the expression of these mRNA. The addition of CCN2 to non-transgenic chondrocytes induced the phosphorylation of IGFR, and ccn2-overexpressing chondrocytes showed enhanced phosphorylation of IGFR. The observed effects of CCN2 may be mediated in part by CCN2-induced overexpression of IGF-I and IGF-II. CCN2-overexpression in transgenic mice accelerated the endochondral ossification processes, resulting in increased length of their long bones."

" At 8 weeks, the majority of the transgenic mice were about 12% larger than their wild-type littermates"

"Safranin-O staining indicated consistently an enhanced density of proteoglycans in the transgenic cartilage in comparison with cartilage of wt littermates"

"type II collagen [had] an enhanced reaction in resting chondrocytes and in the growth plate [of the transgenic mice]" CCN2 enhanced MMP9, Col10a1, VEGF, aggrecan, and Col2a1 expression.

" the enhanced matrix deposition did not result in an increase in the size of the cartilaginous epiphysis; rather, the extended bone length was the result of an elongated bony shaft of the diaphysis."

"Staining of the skeleton of transgenic embryos with type X collagen antibodies indicated that the hypertrophic zone was shorter in the transgenic embryos than in their wt littermates"<-So does CTGF Col2a1 specific overexpression increase adult height or just accelerate growth rate?

"Chondrogenic differentiation of limb-bud mesenchymal cells from CCN2 transgenic animals was greatly enhanced as compared with that of their wild-type counterparts"<-this could result in increased adult height.

Loss of function of CCN2 resulted in impaired endochondral ossification.
According to Magnesium supplementation prevents angiotensin II-induced myocardial damage and CTGF overexpression., in one instance Magnesium inhibited CTGF.

According to Paracrine role for TGF-β-induced CTGF and VEGF in mesangial matrix expansion in progressive glomerular disease., TGF-B induced upregulation of CTGF in one instance.

According to Performance of repetitive tasks induces decreased grip strength and increased fibrogenic proteins in skeletal muscle: role of force and inflammation., exercise can increase CTGF levels. CTGF seemed to reach maximal levels of 6 weeks of the exercise in the exercise group of a high repetition, minimal force task(target reach rate of 4 reaches/min and <5% maximum pulling force). The other group with high repetition and high force(target rate of 4 reaches/min and 60% maximum pulling force. ) had higher increases in CTGF and continued even into the 9th week. The increase in CTGF seemed to be regulated by TNFa and TGFb. The increase in CTGF was inhibited by anti-inflammatory drugs.

According to Oral glucosamine increases expression of transforming growth factor β1 (TGFβ1) and connective tissue growth factor (CTGF) mRNA in rat cartilage and kidney: implications for human efficacy and toxicity., glucosamine increases CTGF in cartilage. The increase in CTGF was in articular but not growth cartilage. It wasn't huge but it was a significant increase 2.3-fold.

In some cases, mechanical load can decrease CTGF by decreasing MMP3. Cyclic tension increased CTGF expression.

Creating a pro-chondrogenic microenvironment

2013-03-31T10:39:00.000-07:00

Ctrl-F for (*NEW*) for the new information. Several times cellulose is used in biomaterial scaffolds to induce chondrogenesis. Cellulose is also known as fiber and is not digested. The question is can we ingest enough of it to build up enough fiber in the bone marrow to build a pro-chondrogenic microenvironment? Since fiber is something that is studied in the mainstream and not just in the bubble of height increase I open the subject to all of you.

How much fiber can build up in the bone marrow?

How do we create a pro-chondrogenic microenvironment within the epiphyseal bone marrow to allow for the formation of new growth plates? The goal of LSJL is to induce that environment.

Creation of an in vitro microenvironment to enhance human fetal synovium-derived stem cell chondrogenesis.

"[We] assess the feasibility of the sequential application of extracellular matrix (ECM) and low oxygen to enhance chondrogenesis in human fetal synovium-derived stem cells (hfSDSCs). Human fetal synovial fibroblasts (hfSFs) were characterized and found to include hfSDSCs, as evidenced by their multi-differentiation capacity and the surface phenotype markers typical of mesenchymal stem cells. Passage-7 hfSFs were plated on either conventional plastic flasks (P) or ECM deposited by hfSFs (E) for one passage. Passage-8 hfSFs were then reseeded for an additional passage on either P or E. The pellets from expanded hfSFs were incubated in a serum-free chondrogenic medium supplemented with 10 ng/ml transforming growth factor-β3 under either normoxia (21% O(2); 21) or hypoxia (5% O(2); 5) for 14 days. Pellets were collected for evaluation of the treatments (EE21, EE5, EP21, EP5, PE21, PE5, PP21, and PP5) on expanded hfSF chondrogenesis. Compared with seeding on conventional plastic flasks, hfSFs expanded on ECM exhibit a lower expression of senescence-associated β-galactosidase and an enhanced level of stage-specific embryonic antigen-4. ECM-expanded hfSFs show increased cell numbers and an enhanced chondrogenic potential. Low oxygen (5% O(2)) during pellet culture enhances hfSF chondrogenesis. The presence of stem cells in hfSFs, and that modulation of the in vitro microenvironment can enhance hfSDSC chondrogenesis."

The two elements of the microenvironment identified as being pro-chondrogenic are hypoxia and ECM. LSJL heavily upregulates ECM molecules.

"Adult MSCs cultured in vitro lack telomerase activity"<-in contrast to fetal MSCs which have higher telomerase activity and longer telomeres.

Group PE5 had the most positive chondrogenic markers. Which would be groups first plated on conventional plastic flasks than transferred to ECM by synovial fibroblasts in an hypoxic environment. EE5 which is ECM for the whole time also had positive measures on all chondrogenic factors.

The reason that Plastic followed by ECM was better than pure ECM could be due to a catch-up growth phenomenon so ECM may be still better at all stages for chondrogenesis overall.

Cellular Response to Hypoxia("Any process that results in a change in state or activity of a cell (in terms of movement, secretion, enzyme production, gene expression, etc.) as a result of a stimulus indicating lowered oxygen tension. Hypoxia, defined as a decline in O2 levels below normoxic levels of 20.8 - 20.95%, results in metabolic adaptation at both the cellular and organismal level. ") genes for mus musculus also altered in LSJL:
Adam8{down}
Gnb1{down}

ECM stiffness primes the TGFβ pathway to promote chondrocyte differentiation.

"We investigate mechanisms by which chondrocytes generate an integrated response to ECM stiffness and transforming growth factor β (TGFβ), a potent agonist of chondrocyte differentiation. Primary murine chondrocytes and ATDC5 cells{ATDC5 cells are progenitor cells to chondrocytes} grown on 0.5-MPa substrates deposit more proteoglycan and express more Sox9, Col2α1, and aggrecan mRNA relative to cells exposed to substrates of any other stiffness. The chondroinductive effect of this discrete stiffness, which falls within the range reported for articular cartilage, requires the stiffness-sensitive induction of TGFβ1. Smad3 phosphorylation, nuclear localization, and transcriptional activity are specifically increased in cells grown on 0.5-MPa substrates. ECM stiffness also primes cells for a synergistic response, such that the combination of ECM stiffness and exogenous TGFβ induces chondrocyte gene expression more robustly than either cue alone through a p38 mitogen-activated protein kinase-dependent mechanism."

"Upon integrin binding to ECM ligands and the generation of internal cell tension, cells develop focal adhesions, a highly ordered array of proteins including focal adhesion kinase (FAK), talin, vinculin, and α-actinin. These proteins interact with small GTPases (i.e., Rho, Rac) and other signaling pathways, facilitating changes in cytoskeletal organization, actinomyosin contractility, and cell shape with even small changes in matrix compliance"

"The activated TβRI phosphorylates Smad2 and Smad3 on the C-terminal domain, causing heteromerization with Smad4 and preferential retention in the nucleus, where Smads act as transcription factors. In chondrocytes, phosphorylated Smad3 recruits CBP to activate Sox9-mediated transcription of Col2α1"

"Cells respond to substrate stiffness by increasing internal cellular tension through stress fiber formation and cell spreading"

"Rho and ROCK participate in stiffness sensing in part through stress fiber formation"

"TGFβ rapidly activates the p38 pathway through the MAPK kinase kinase TAK1 activation of MKK3/6. Phospho-p38 was increased on 0.5-MPa substrates relative to plastic"

"there is an optimal level of ROCK activity on 0.5-MPa substrates that activates chondroinduction, in part, through the induction of TGFβ1 expression on compliant substrates."

"BMP-inducible nuclear translocation of Smad1/5/8 requires sufficient ROCK-dependent cytoskeletal tension. ROCK can also enhance the activity of both Smad3 and Sox9 by phosphorylation of the Smad3 linker region or Sox9 on serine 181"

Mesenchymal stem cells and their microenvironment.

"MSCs are stromal-like cells that are characterized by a CD105+ /CD73+ /CD90+ /CD45- /CD34- /CD11b- /CD19- /HLA-DR- cell surface signature"

"MSCs [may] exist in a perivascular[near or around blood vessels] location and share a number of cell surface markers with pericytes"

"MSC show a greater propensity to differentiate to cartilage and bone, if they are bone marrow derived and to differentiate to fat, if isolated from adipose tissue"

"n order to self renew, stem cells need to be protected from differentiation signals and from apoptosis, and the niche provides the adhesion molecules, soluble factors and conditions that allow this in a concerted fashion. These soluble factors and ECM components activate various kinase cascades including the ERK1/2 MAPK and the PI-3K pathways"

"EGF, which stimulates the EGFR1 on MSCs, strongly activates the MAP kinases ERK1/2 and Jnk1, the Stat3 and PKC pathways and weakly stimulates the PI-3K pathway, and high concentrations of EGF induce osteogenic differentiation in MSCs "

"high concentrations of covalently tethered EGF, which restrict signaling to the cell surface, result in increased osteogenic differentiation of MSCs, while low concentrations of soluble EGF, which induce receptor internalization, are anti-osteogenic"

"concomitant PI-3K stimulation prevents ERK1/2-dependent osteogenic differentiation."<-LSJL likely stimulates both PI3K and ERK1/2 which may be why it is more pro-chondrogenic than osteogenic.

"MSCs can produce VEGF, bFGF{up}, PDGF, angiopoietin, CXCL8/IL-8 and other angiogenic factors"

"[MSCs] express Oct4, Nanog, Sox2, SSEA3, SSEA4, Rex1, c-myc, nucleostemin, Nodal, Sca1{only expressed by mice and not humans}, Snail2"

"when chondrogenesis was assessed in cross-linked methacrylated hyaluronic acid hydrogels, the macromer density influenced MSC chondrogenesis: high density macromers resulted in increased chondrogenesis, but of inferior quality than seen with lower density gels"

"early MSC progenitors, defined by their smaller size and expression of podocalyxin-like protein (PODXL), selectively express alpha4 and alpha6 integrins, which are lost during culture. Freshly isolated MSC do not express the vitronectin receptor alphav beta5, but express low levels of the fibronectin receptor alpha5beta1 and the collagen receptors alpha1beta1, alpha2beta1 and alpha3beta1. Upon culture alphavbeta5 is up-regulated. Furthermore, increased expression of alphavbeta5, and of alpha5beta1 is observed during chondrogenic differentiation"

"MSCs do not seem to circulate in the vasculature under physiological conditions, it seems likely that they are released into the vasculature, when there is increased demand for these cells during any kind of tissue injury "

"But although MSC accumulate in damaged tissue to some degree, recruitment of circulating MSC is very inefficient"

"MSCs seem to be resistant in general to certain apoptotic pathways perhaps due to very low expression of caspase 8 and caspase 9 "

"all MSC express cell surface receptors for C3a and C5a"

"genes expressed in MSCs specifically involved in the HSC niche including galectin-1, fibronectin-1, osteopontin, CXCL12, thrombospondin-1 and -2, TGF-beta 2, Angiopoietin-1, ILGFBP-4, FGF-7, SFRP-1 and -2, VCAM-1, and BMPR type 1a"

Redox regulation of stem/progenitor cells and bone marrow niche.

"Bone marrow (BM)-derived stem and progenitor cell functions including self-renewal, differentiation, survival, migration, proliferation, and mobilization are regulated by unique cell-intrinsic and -extrinsic signals provided by their microenvironment, also termed the "niche." Reactive oxygen species (ROS), especially hydrogen peroxide (H(2)O(2)), play important roles in regulating stem and progenitor cell functions in various physiologic and pathologic responses. The low level of H(2)O(2) in quiescent hematopoietic stem cells (HSCs) contributes to maintaining their "stemness," whereas a higher level of H(2)O(2) within HSCs or their niche promotes differentiation, proliferation, migration, and survival of HSCs or stem/progenitor cells. Major sources of ROS are NADPH oxidase and mitochondria. In response to ischemic injury, ROS derived from NADPH oxidase are increased in the BM microenvironment{mechanical loading stimulates ROS release from mitochondria creating a more hypoxic microenvironment}, which is required for hypoxia and hypoxia-inducible factor-1α expression and expansion throughout the BM. This, in turn, promotes progenitor cell expansion and mobilization from BM, leading to reparative neovascularization and tissue repair. In pathophysiological states such as aging, atherosclerosis, heart failure, hypertension, and diabetes, excess amounts of ROS create an inflammatory and oxidative microenvironment, which induces cell damage and apoptosis of stem and progenitor cells."

"ROS such as O2•− and H2O2 are generated from a number of sources including mitochondria, NADPH oxidases (NOXs), xanthine oxidase, cytochrome P450, and nitric oxide synthase (through its uncoupling). Because O2•− is produced from oxygen, the oxygen concentration or hypoxic condition has a significant impact on the total amount of ROS. The O2•− reacts with nitric oxide (NO) to generate peroxynitrite (OONO−), thereby inhibiting endothelial function, whereas it can be quickly converted to H2O2 by superoxide dismutases (SODs) such as MnSOD (SOD2) or Cu/ZnSOD (SOD1) or extracellular SOD (SOD3). H2O2 is catalyzed by catalase, glutathione peroxidases (GPx′s), and the thioredoxin–peroxiredoxin system to nonreactive water"

"growth factor signaling is mediated through H2O2 production."

"Different from phagocytic NADPH oxidase that is normally quiescent but generates a large burst of O2•− (the “oxidative burst”) upon activation, the NOXs constitutively produce low levels of O2•− or H2O2 intracellularly in the basal state and are further stimulated acutely by various agonists and growth factors. NOXs are now recognized to have specific subcellular localizations, which is required for localized H2O2 production and activation of specific redox signaling pathways to mediate various functions" NOXs are located in MSCs.

"Once O2•− is generated, it is immediately converted into H2O2 by MnSOD or Cu/ZnSOD"

"To avoid the potential damaging effects of H2O2, mitochondria express other antioxidant enzymes such as peroxiredoxin (Prx) 3 and Prx5 and glutathione peroxidase."

"hypoxic conditions increase mitochondrial ROS production, which stabilizes HIF-1α protein expression. "

"optimal levels of ROS are required for normal responses, whereas excess or insufficient levels of ROS are associated with cellular dysfunction and reduced growth factor signaling, respectively"<-So we want to maximize ROS signaling before cellular dysfunction.

"PTEN is a negative regulator of the PI3K–Akt pathway and contains catalytic cysteine residues that are highly susceptible to oxidation by H2O2. Therefore, PTEN inhibition stabilizes the active phosphorylated form of Akt. "

(*NEW*)
Directing chondrogenesis of stem cells with specific blends of cellulose and silk.

"We systematically prepared cellulose, blends with silk at different compositions using an environmentally benign processing method based on ionic liquids as a common solvent. We tested the effect of blend compositions on the physical properties of the materials as well as on their ability to support mesenchymal stem cell (MSC) growth and chondrogenic differentiation. The stiffness and tensile strength of cellulose was significantly reduced by blending with silk. The characterised materials were tested using MSCs derived from four different patients. Growing MSCs on a specific blend combination of cellulose and silk in a 75:25 ratio significantly upregulated the chondrogenic marker genes SOX9, aggrecan and type II collagen in the absence of specific growth factors{so the stiffness of this environment is likely optimal for chondrogenesis}. This chondrogenic effect was not found with neat cellulose or cellulose/silk 50:50 blend composition. No adipogenic or osteogenic differentiation is detected on the blends suggesting that cellulose/silk 75:25 blend induces specific stem cell differentiation into the chondrogenic lineage without addition of the soluble growth factor TGF-β."

"Cellulose is a linear homopolymer of glucose." Cellulose is also called fiber. It can't be digested so if you can get it to your bone marrow it may help create a pro-chondrogenic microenvironment.

"Cellulose, which comprises three hydroxyl groups per repeating unit, is theoretically a good choice as an initiator of chondrogenesis"

"silane-treated glass surfaces functionalized with carboxyl (–COOH) and hydroxyl (–OH) groups initiated chondrogenic marker mRNA expression in MSCs in the absence of chondrogenic growth factors, whereas amine (–NH2) functional groups encouraged osteogenic differentiation of stem cells in the absence of osteogenic supplements"

Compounds with Hydroxyl groups:
Alcohol
Sugars

Compounds with Carboxyl groups:
Sugars
Vinegar
Goat fat
Breast milk
Coconut/Palm/Peanut oil
Nutmeg

Amino acids contain the amine functional group.

The study Galactooligosaccharides improve mineral absorption and bone properties in growing rats through gut fermentation. found that "Galactooligosaccharides (GOS), prebiotic nondigestible oligosaccharides derived from lactose" had no effect on femur length.

According to Dietary cellulose, zinc and copper: effects on tissue levels of trace minerals in the rat., Fiber only increased tibia dry weight with a diet deficient in zinc and copper. Dry weight should but not necessarily correlate with bone length. The mice were below 9 weeks old so they were growing. The study was done in 1979 so there's no hope to contact for length data.

Lengthening your Body with a Hyperbaric Chamber?

2013-03-28T15:26:00.000-07:00

Request for help: Does anyone know of any athletes who regularly use hyperbaric chambers so we can study changes in their bone phenotype?

The hyperbaric chamber makes you breathe solely oxygen and increases the atmospheric pressure surrounding you. Now cartilage is an avascular tissue but can hyperbaric chamber treatment increase chondroinduction or increase the growth from growth plates?

Many athletes use hyperbaric chambers so if hyperbaric chambers could chondroinduce on their own there would be some sort of anecdotal evidence.

Hyperbaric chambers may be an interesting possible way to enhance growth but are too cost prohibitive.

The effect of hyperbaric oxygen and air on cartilage tissue engineering.

"Under hyperbaric oxygen and air stimulation, the cell number of chondrocytes in cartilage matrix was not significantly increased, but the glycosaminoglycans syntheses markedly increased compared to the control group. The chondrogenic-specific gene expression of SOX9, aggrecan, and COL2A1 were compared respectively. Within the limitation of this study, it was concluded that 2.5 atmosphere absolute oxygen and air may provide a stress environment to help cartilage tissue engineering development."

"In approximately 2.5 ATA[absolute atmosphere] HBO–treated group, the SOX9 and aggrecan expressed significantly at days 9 and 12, but there was no increase with the type I collagen–related gene COL1A2. Alternately, in 2.5 ATA hyperbaric air–treated group, the SOX9 increased with the time and type II collagen–related gene COL2A1 showed significant over expression at days 6, 9, and 15, with no manifest increase with the COL1A2 "

Since hyperbaric chambers go to 2-3 times normal pressure maybe this would would be sufficient.

"Fifteen hASCs contained biocomposites and were equally separated into 3 groups and subsequently treated with 1 ATA air (as control), 2.5 ATA HBO, and 2.5 ATA hyperbaric air operations which were performed in a hyperbaric chamber (MEDITT, Republic of China). The duration of treatment was 1 hour a day for 5 days, and samples were observed at days 6, 9, 12, and 15, respectively, after induction."

Equine peripheral blood-derived mesenchymal stem cells: isolation, identification, trilineage differentiation and effect of hyperbaric oxygen treatment. states that hyperbaric oxygen increased the concentration of mesenchymal stem cells. Mesenchymal condensation is an important part of new growth plate formation. Increased MSC concentration would facilitate mesenchymal condensation.

Osteodistraction of a previously irradiated mandible with or without adjunctive hyperbaric oxygenation: an experimental study in rabbits. states that mandibular osteodistraction resulted in cartilagenous tissue in all the experimented groups. Only irradiation increased the size of chondroid islands and not hyperbaric oxygen.

According to Hyperbaric oxygen-stimulated proliferation and growth of osteoblasts may be mediated through the FGF-2/MEK/ERK 1/2/NF-κB and PKC/JNK pathways., stimulates the growth and proliferation of osteoblasts but can that apply to chondrocytes or stem cells?

Age-dependent response of murine female bone marrow cells to hyperbaric oxygen.

"We treated 2- and 18-month old C57BL/6 female mice by HBO[Hyperbaric Oxygen]. Hematopoietic stem cells and progenitors, enumerated as colony-forming units in culture, were doubled only in peripheral leukocytes and BM cells of young mice receiving HBO. In old mice colony-forming unit fibroblast numbers, a measure of mesenchymal stromal cells (MSCs) from BM, were high but unaffected by HBO. To further explore this finding, in BM-MSCs we quantified the transcripts of adipocyte early-differentiation genes peroxisome proliferator-activated receptor-γ, CCAAT/enhancer binding protein-β and fatty-acid binding protein 4; these transcripts were not affected by age or HBO. However, osteoblast gene transcripts runt-related transcription factor 2, osterix (OSX) and alkaline phosphatase (AP) were twofold to 20-fold more abundant in MSCs from old control mice relative to those of young control mice. HBO affected expression of osteoblast markers only in old MSCs (OSX gene expression was reduced by twofold and AP expression was increased threefold)."

Unfortunately no chondrogenic markers were measured. The study did say the HBO increased the mobilization of MSCs.

Grow Taller by your periosteum?

2013-03-25T14:50:00.000-07:00

In the *NEW*(ctrl-f for *NEW*) section of this article I provide articles supporting Natural Height Growth's theory that periosteal stripping could help increase height. When cartilage first condenses in the embryo there is no periosteum therefore the periosteum could serve as a mechanism to inhibit growth related to maturity. Can rapid repetive loading(like via a chisel and hammer method) be akin to periosteal stripping? One of the papers mentioned in the aforementioned page is on adult rats and seems to possibly have longitudinal bone growth.

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.

Here's a study that shows how the periosteum can cause bone regeneration:

A novel osteogenesis technique: The expansible guided bone regeneration

"Guided bone regeneration is a unique osteogenesis technique that requires a barrier membrane under periosteum to create space for bone regeneration[if we extend the periosteum over the longitudinal ends of the bones and create a barrier membrane then we can grow taller, also we can grow taller when periosteum is already at the longitudinal location of the bone such as the flat bone of the skull]. However, creating sizeable spaces is clinically not commonly feasible. A titanium plate and a thin silicone membrane were surgically layered on each calvaria of eight rabbits. Then, the periphery of the silicone membrane was fixed by a plastic ring to the underlying bone using titanium micro screws. After 1 week, a 5-mm-length titanium screw was used to elevate the titanium plate, which in turn elevated the silicone membrane together with overlying soft tissue in a rate of 1 mm/day for 5 days to create a secluded space. Animals were killed at 2 months (n = 4, group 1) and 4 months (n = 4, group 2) after the elevation. Histological and microradiographical analyses demonstrated creation of an amount of de novo bone formation (68.2 ± 22 mm3 in group 1 and 70.3 ± 14 mm3 in group 2) in the sizeable created spaces (207.1 ± 31 mm3 in group 1 and 202 ± 21 mm3 in group 2) without exposure of the device. This novel osteogenesis technique, “expansible guided bone regeneration,” created a substantial in vivo incubator without applying growth factors or osteoprogenitor cells. Creating a growing space over the secluded surface allowed the development of normal biological healing process occurring on the bone surface into a regenerative process, generating bone outside the genetically determined skeletal bone[so what we can do is create a growing space between the articular cartilage and the subchondral ends of bone]. This technique is a new tissue engineering approach stimulating endogenous tissue repair without applying cells or factors exogenously."

"large volumes of bone can be produced in a predictive manner without exogenously applying the three key players, if the space is provided by injecting biocompatible gel under periosteum."<-space plus periosteum equals bone growth. The problem is there's no periosteum at the longitudinal ends of bones.

"More recently, we and others have been reported that gradual periosteum elevation creating a space over bone surface results in new bone formation in this space"<-How do we elevate and stretch the periosteum?

"the invasion of the created space with highly competitive nonosteogenic soft tissue and poor quality of the newly formed bone are the main drawbacks of this technique"

In the study they use an elevation screw to lift the periosteum. Maybe we can mimic this with mechanical stimuli somehow.

"Upon activation of bone surface, biological healing process taking place on the activated surface is kept confined to the surface during the first week. After that the elevation plate is set to move upward, the membrane is gradually elevated and the space attains its maximum size in 5 days."

Now we we need find mechanical stimuli that can stretch periosteum over the longitudinal ends of bones and that can elevate the periosteum.

The nature and role of periosteum in bone and cartilage regeneration.

"[Can] periosteum from different bone sources in a donor [result] in the same formation of bone and cartilage? In this case, periosteum obtained from the cranium and mandible (examples of tissue supporting intramembranous ossification) and the radius and ilium (examples of tissues supporting endochondral ossification) of individual calves was used to produce tissue-engineered constructs that were implanted in nude mice and then retrieved after 10 and 20 weeks. Specimens were compared in terms of their osteogenic and chondrogenic potential by radiography, histology, and gene expression levels. By 10 weeks of implantation and more so by 20 weeks, constructs with cranial periosteum had developed to the greatest extent, followed in order by ilium, radius, and mandible periosteum. All constructs, particularly with cranial tissue although minimally with mandibular periosteum, had mineralized by 10 weeks on radiography and stained for proteoglycans with safranin-O red (cranial tissue most intensely and mandibular tissue least intensely). Gene expression of type I collagen, type II collagen, runx2, and bone sialoprotein (BSP) was detectable on QRT-PCR for all specimens at 10 and 20 weeks. By 20 weeks, the relative gene levels were: type I collagen, ilium >> radial ≥ cranial ≥ mandibular; type II collagen, radial > ilium > cranial ≥ mandibular; runx2, cranial >>> radial > mandibular ≥ ilium; and BSP, ilium ≥ radial > cranial > mandibular. The osteogenic and chondrogenic capacity of the various constructs is not identical and depends on the periosteal source regardless of intramembranous or endochondral ossification. Cranial and mandibular periosteal tissues appear to enhance bone formation most and least prominently, respectively."

Only the madible had no signs of cartilage proteoglycans.

"These results indicate that osteoblasts and chondrocytes derived from sutured periosteum remain viable during implantation and migrate into the constructs. The cells proliferate and secrete matrix that leads to new bone and mineral formation (osteoblasts) and new cartilage (chondrocytes) in interior spaces of the scaffolds as well as in the tissue over the scaffolds"<-With LSJL we have no scaffold. We're trying to use endogenous tissues as a scaffold.

Multiple exostosis: a short study of abnormalities near the growth plate.

"The pathogenesis of multiple exostosis has been controversial with many theories put forward including the structural/mechanical theory, which emphasizes that the osteochondroma arises in the displaced growth plate cartilage penetrating a defective periosteum. Recently, molecular genetics has offered the neoplastic model with tumor suppressor genes implicated in the development and pathogenesis of exostosis. In this study, we demonstrated the spectrum of histological abnormalities in the developing exostosis present on the surface of the bone at the physis. Seven skeletally immature patients with multiple exostoses were used in this study. The patients' families were advised of and consented to the proposed study. Coincident with removal of symptomatic exostoses that was adjacent to the physis, a thin strip of bone with overlying periosteum was removed to include the edge of the physis. This was followed by formalin fixation and routine paraffin embedding. We demonstrated the earliest lesion as a microchondroma within the periosteum adjacent to the normal physis (also called the 'groove of Ranvier'). More mature progressively larger lesions showing enchondral ossification were seen distally. The periosteum and the perichondrium were intact with normal physis. Our observations give support to the fact that precursor cells in the periosteum adjacent to the physis (also called the 'groove of Ranvier') gives rise to the chondrocytes that clonally expands and develops into exostosis."

"the cause of exostoses was a ‘fault of the epiphyseal plate, nests of cartilage being misplaced’. He indicated that ‘fragments of cartilage around the epiphyseal line become isolated on the surface of the metaphysis, proliferate, and form exostosis’. ‘The periosteum, which is incomplete at the sites of these cartilaginous nests, fails to model the metaphysis in a normal manner’."

"Multiple noncontiguous clusters of cartilage cells of increasing size were found on the surface of the bone. The chondromas increased in size as the distance from the physis increases."

In vivo generation of cartilage from periosteum.

"Damaging the periosteum may be a way to generate ectopic cartilage or bone, which may be useful for the repair of articular cartilage and bone defects. Periosteum was bilaterally dissected from the proximal medial tibia of New Zealand White rabbits. Reactive periosteal tissue was harvested 10, 20, and 40 days postsurgery and analyzed for expression of collagen types I, II, and X, aggrecan, osteopontin, and osteonectin and collagen types I and II. Reactive tissue was present in 93% of cases. Histologically, this tissue consisted of hyaline cartilage at follow-up days 10 and 20. Expression of collagen type II and aggrecan was present at 10 and 20 days postsurgery. Highest expression was at 10 days. Expression of collagen type X increased up to 20 days. No significant changes in the mRNA expression of osteopontin or osteonectin were observed. Cartilage [was present], which was positive for collagen types I and II at 10 days and only for collagen type II at 20 days. At 20 days postsurgery the onset of bone formation was also observed. At 40 days postsurgery, the reactive tissue had almost completely turned into bone."

"cells in the cambial layer of the periosteum have chondrogenic potential in vitro and in vivo"

The ectopic cartilage is at the longitudinal ends of the bones so maybe it can increase height.

*NEW*

Regulation of endochondral cartilage growth in the developing avian limb: cooperative involvement of perichondrium and periosteum.

"To determine if the perichondrium and periosteum regulate growth through the production of diffusible factors, we have tested various conditioned media from these tissues for the ability to modify cartilage growth in tibiotarsal organ cultures from which these tissues have been removed. Both negative and positive regulatory activities were detected. Negative regulation was observed with conditioned medium from (1) cell cultures of the region bordering both the perichondrium and the periosteum, (2) co-cultures of perichondrial and periosteal cells, and (3) a mixture of conditioned media from perichondrial cell cultures and periosteal cell cultures. Positive regulation was observed with conditioned media from several cell types, with the most potent activity being from articular perichondrial cells and hypertrophic chondrocytes."

"At the point where the boney shaft borders the cartilage, the perichondrium (PC) differentiates into the periosteum (PO), whose cells have osteoblastic potential"

"PC/PO-free long bones [had an] increase in overall length of the cartilage [resulting from] increases in the sizes of both the proliferative and hypertrophic zones"

Multiple mechanisms of perichondrial regulation of cartilage growth.

"he perichondrium (PC) and the periosteum (PO) negatively regulate endochondral cartilage growth through secreted factors. Conditioned medium from cultures of PC and PO cells when mixed (PC/PO-conditioned medium) and tested on organ cultures of embryonic chicken tibiotarsi from which the PC and PO have been removed (PC/PO-free cultures) effect negative regulation of growth. Of potential importance, this regulation compensates precisely for removal of the PC and PO, thus mimicking the regulation effected by these tissues in vivo. We have now examined whether two known negative regulators of cartilage growth (retinoic acid [RA] and transforming growth factor-beta1 [TGF-beta1]) act in a manner consistent with this PC/PO-mediated regulation. The results suggest that RA and TGF-beta1, per se, are not the regulators in the PC/PO-conditioned medium. Instead, they show that these two factors each act in regulating cartilage growth through an additional, previously undescribed, negative regulatory mechanism(s) involving the perichondrium. When cultures of perichondrial cells (but not periosteal cells) are treated with either agent, they secrete secondary regulatory factors into their conditioned medium, the action of which is to effect precise negative regulation of cartilage growth when tested on the PC/PO-free organ cultures. This negative regulation through the perichondrium is the only activity detected with TGF-beta1. Whereas, RA shows additional regulation on the cartilage itself. However, this regulation by RA is not "precise" in that it produces abnormally shortened cartilages. Overall, the precise regulation of cartilage growth effected by the action of the perichondrial-derived factor(s) elicited from the perichondrial cells by treatment with either RA or TGF-beta1, when combined with our previous results showing similar--yet clearly different--"precise" regulation by the PC/PO-conditioned medium suggests the existence of multiple mechanisms involving the perichondrium, possibly interrelated or redundant, to ensure the proper growth of endochondral skeletal elements."

"RA has been reported to be both an inhibitor and promoter of cartilage development. In developing embryos of various species, both hypervitaminosis A and hypovitaminosis A greatly disturb the organization of the growth plate. In cell cultures, low doses (50 nM) of RA promote cartilage differentiation. However, in organ cultures, the addition of RA produces the opposite effect: a dose-dependent inhibition of longitudinal bone growth. This finding is due to decreases in both chondrocyte proliferation and hypertrophy"<-one difference between an organ culture and cell culture is the presence of the periosteum.

"RA treatment of the intact cultures produced a reduction in cartilage length from 3.77 mm for the controls to 2.96 mm for the RA-treated. This reduction of 0.81 mm is an even greater overcompensation than for the PC/PO-free cultures, suggesting that RA must have another mechanism of action in addition to that which acts directly on cartilage"

"TGF-β1 showed negative regulation only with the intact organ cultures—not with the PC/PO-free ones. When 10 ng/ml TGF-β1 was added to the intact cultures, the lengths of the cartilage was reduced to 3.48 mm for the treated vs. 3.81 mm for the controls. However, the PC/PO-free organ cultures showed no response to TGF-β1 treatment, with both treated and untreated cultures growing to 4.0 mm"

"FGF-2 acts solely on the cartilage, resulting in identical cartilage lengths between intact and PC/PO-free cultures when treated with FGF-2."

"one of the three nuclear RARs (RARβ) has been shown to be expressed at high levels in the perichondrium, as is RA itself; the remaining two RARs (RARα and RARγ) are expressed in cartilage."

Intracellular tension in periosteum/perichondrium cells regulates long bone growth.

"erichondrium/periosteum cells were cultured on substrates with different stiffness. The medium produced by these cultures was added to embryonic chick tibiotarsi from which perichondrium/periosteum was either stripped or left intact. After 3 culture days, long bone growth was proportionally related to the stiffness of the substrate on which perichondrium/periosteum cells were grown while they produced conditioned medium. A second set of experiments demonstrated that the effect occurred through expression of a growth-inhibiting factor, rather than through the reduction of a stimulatory factor. Finally, evidence for the importance of intracellular tension was obtained by showing that the inhibitory effect was abolished when perichondrium/periosteum cells were treated with cytochalasin D, which disrupts the actin microfilaments. Modulation of long bone growth occurs through release of soluble inhibitors by perichondrium/periosteum cells, and that the ability of cells to develop intracellular tension through their actin microfilaments is at the base of this mechano-regulated control pathway."

"periosteum [may regulate] growth via a direct mechanical feedback mechanism where pressure in growing cartilage, balanced by tension in the periosteum, [modulating] growth processes of chondrocytes. "

" after 3 days of culture, distal cartilage length was significantly longer in stripped versus intact tibiotarsi in non-conditioned medium and in conditioned medium obtained from periosteum/perichondrium cell cultures on 3, 14, 21, and 48 kPa stiff substrates. The difference in distal length between stripped and intact tibiotarsus decreased with increasing stiffness and was no longer significant on 80 kPa gels and on glass"<-Thus the stiffness of the periosteum may affect the height reduction.

"Both the variations in substrate stiffness and applying cytochalasin D in culture modulated the ability of periosteum cells to actively develop intracellular tension via their actin microfilament network."

Stripped periosteum cartilage was about 33% higher than intact periosteum.

You can see here that axial loading increases periosteal thickness(From Cortical and trabecular bone adaptation to incremental load magnitudes using the mouse tibial axial compression loading model). This serves to contrast LSJL images here where there is less visible periosteal thickness in the without drilling mice. One reason for this difference could be that LSJL involves less force as in the axial loading study periosteal thickness increased with increasing force. So less increase of periosteal thickness could be one possibility in why LSJL can increase height but why axial loading does not. Which leads to the question of whether axial loading can increase height with periosteal stripping.

The Resting Zone of the Growth Plate

2013-03-07T15:19:00.000-08:00

Within the resting zone of the growth plate are stem-like cells which means they are like stem cells but only have limited proliferative capacity. If we can characterize these resting zone cells we can know more about how to initiate the first stage of the growth plate.

Identification of target genes for wild type and truncated HMGA2 in mesenchymal stem-like cells.

"The HMGA2{up in LSJL} gene [codes] for an architectural transcription factor involved in mesenchymal embryogenesis.
We have over-expressed wild type and truncated HMGA2 protein in an immortalized mesenchymal stem-like cell (MSC) line, and investigated the localisation of these proteins and their effects on differentiation and gene expression patterns.
Over-expression of both transgenes blocked adipogenic differentiation of these cells, and microarray analysis revealed clear changes in gene expression patterns, more pronounced for the truncated protein. Most of the genes that showed altered expression in the HMGA2-overexpressing cells fell into the group of NF-kappaB-target genes, suggesting a central role for HMGA2 in this pathway. Of particular interest was the pronounced up-regulation of SSX1, already implicated in mesenchymal oncogenesis and stem cell functions, only in cells expressing the truncated protein. Furthermore, over-expression of both HMGA2 forms was associated with a strong repression of the epithelial marker CD24, consistent with the reported low level of CD24 in cancer stem cells.:
We conclude that the c-terminal part of HMGA2 has important functions at least in mesenchymal cells, and the changes in gene expression resulting from overexpressing a protein lacking this domain may add to the malignant potential of sarcomas."

"There were several genes up-regulated by HMGA2WT and down- regulated in cells expressing the truncated form, such as FGF13, EHF, HCLS1, MEST, G0S2 and PTPRN2."<-Since the truncated form of HMGA2 can increase height these genes may be important.

Genes downregulated in HMGA2WT-transgenic also downregulated in LSJL:
IL6{up}
Ces1
Thbs2{up}
S100a4{up}
JunB{up}
Has1{up}
Ptgs2{up}
Kynu{up}
Oasl

Upregulated:

Genes downregulated in HMGA2Ttruncated also downregulated in LSJL:
LAMA4{up}
Thbs2{up}
S100a4{up}
JunB{up}
Has1{up}
Ptgs2{up}
Kynu{up}
Oasl

Upregulated:
MMP3
Edn1
Hapln1

"over-expression of truncated HMGA2 induces a more mesenchymal (stem-like) phenotype, characterized by resistance toward differentiation, over-expression of SSX1, lost expression of certain epithelial markers and strengthened expression of mesenchymal markers."

Differential expression of phenotype by resting zone and growth region costochondral chondrocytes in vitro.

"Chondrocytes derived from the resting cell zone and adjacent growth zone of rat costochondral cartilage were compared for retention of phenotype in culture. At third passage confluence, two cell populations differ morphologically and biochemically. Resting zone cells are fibroblast-like, with smooth cell membranes and little rough endoplasmic reticulum. Growth zone cells are more polygonal, smaller in diameter, with numerous cytoplasmic extensions of the plasma membranes and abundant rough endoplasmic reticulum. Both cell populations produce matrix vesicles that are comparable morphologically to matrix vesicles isolated enzymatically from epiphyseal cartilage. While membrane vesicles are released into the media by cells derived from the resting zone as well as from the growth cartilage, alkaline phosphatase activity is enriched in media vesicles produced by growth cartilage cells. Alkaline phosphatase enriched vesicles appear to be preferentially incorporated into the extracellular matrix. Both the plasma membrane marker enzyme activity and the membrane phospholipid composition are differentially expressed in matrix vesicles and plasma membranes and are cell specific. Matrix vesicles produced by resting zone cells are enriched in alkaline phosphatase, 5'-nucleotidase, ouabain sensitive Na+/K+ ATPase and cardiolipin when compared to the cell membrane. In addition, the plasma membranes of these cells contain more phosphatidylcholine plus sphingomyelin than do growth cartilage plasma membranes. Resting zone cell matrix vesicles have less phosphatidylethanolamine than do vesicles from growth cartilage cultures. Matrix vesicles produced by growth cartilage cells contain one proteolipid at 43,000 Mr which comigrates with plasma membrane proteolipid and an additional proteolipid at approximately 3,000 Mr. These data indicate that both cells retain differential expression of phenotype in culture and that one expression of this phenotype is production of specific extracellular matrix vesicles."

"Growth cartilage chondrocyte plasma membranes exhibit higher 5'-nucleotidase activity than do resting cell membranes"

"The resting zone cells membranes contain more phosphatidylcholinc plus sphingomyelin than do the growth zone chondrocyte membranes"

Transforming growth factor-beta1 regulation of resting zone chondrocytes is mediated by two separate but interacting pathways.

" transforming growth factor-beta1 (TGF-beta1) stimulates protein kinase C (PKC) via a mechanism that is independent of phospholipase C or tyrosine kinase, but involves a pertussis toxin-sensitive G-protein. Maximal activation occurs at 12 h and requires new gene expression. To understand the signaling pathways involved, resting zone chondrocytes were incubated with TGF-beta1 and PKC activity was inhibited with chelerythrine, staurosporine or H-7. [(35)S]Sulfate incorporation was inhibited, indicating that PKC mediates the effects of TGF-beta1 on matrix production. However, there was little, if any, effect on TGF-beta1-dependent increases in [(3)H]thymidine incorporation, and TGF-beta1-stimulated alkaline phosphatase was unaffected, indicating that these responses to the growth factor are not regulated via PKC. TGF-beta1 caused a dose-dependent increase in prostaglandin E(2) (PGE(2)) production which was further increased by PKC inhibition. The increase was regulated by TGF-beta1-dependent effects on phospholipase A(2) (PLA(2)). Activation of PLA(2) inhibited TGF-beta1 effects on PKC, and inhibition of PLA(2) activated TGF-beta1-dependent PKC. Exogenous arachidonic acid also inhibited TGF-beta1-dependent increases in PKC. The effects of TGF-beta1 on PKC involve genomic mechanisms, but not regulation of existing membrane-associated enzyme, since no direct effect of the growth factor on plasma membrane or matrix vesicle PKC was observed. TGF-beta1 modulates its effects on matrix production through PKC, but its effects on alkaline phosphatase are mediated by production of PGE(2) and protein kinase A (PKA). Inhibition of PKA also decreases TGF-beta1-dependent proliferation. We have previously shown that PGE(2) stimulates alkaline phosphatase through its EP2 receptor, whereas EP1 signaling causes a decrease in PKC. Thus, there is cross-talk between the two pathways."

"Resting zone chondrocytes synthesize TGF-β1 in latent form and store it in their extracellular matrix as a 290 kDa complex consisting of latent TGF-β1, latent TGF-β1 binding protein-1 and the latency-associated peptide. Extracellular matrix vesicles produced by these cells can activate latent TGF-β1 when they are exposed to 1,25-(OH)2D3. The interrelationship of TGF-β1 action and vitamin D metabolites is also demonstrated by the fact that TGF-β1 causes resting zone cells to produce increased 1,25-(OH)2D3 within 1 h and increased 24,25-(OH)2D3 at 24 h, which is correlated with TGF-β1-dependent downregulation of the 1α-hydroxylase and upregulation of the 24-hydroxylase in these cells"

"PGE2 has multiple effects on the chondrocytes, promoting differentiation and anabolic responses via cAMP production and PKC activity"

" Resting zone cells have both EP1 and EP2 receptors, as well as an EP1 variant, EP1v. The increase in cAMP leads to increased PKA activity. The importance of this pathway in the response to TGF-β1 is evident in the decrease in proliferation following treatment of the cells with TGF-β1 and the PKA inhibitor, H-8."

Direct effects of 1,25-dihydroxyvitamin D3 and 24,25-dihydroxyvitamin D3 on growth zone and resting zone chondrocyte membrane alkaline phosphatase and phospholipase-A2 specific activities.

"1,25-Dihydroxyvitamin D3 [1,25-(OH)2D3] and 24,25-(OH)2D3 differentially affect the specific activity of alkaline phosphatase (ALPase) and phospholipase-A2 (PLA2) of plasma membranes and extracellular matrix vesicles produced by costochondral reserve zone and growth zone cartilage chondrocytes in culture. In the present study, growth zone and cartilage and reserve zone matrix vesicles and plasma membranes were isolated from confluent chondrocyte cultures and incubated with hormone for 3 and 24 h in vitro. Addition of 1,25-(OH)2D3 to GC matrix vesicles and plasma membranes resulted in dose-dependent increases in ALPase and PLA2 specific activities in both membrane fractions. Addition of 24,25-(OH)2D3 to RC membrane fractions stimulated matrix vesicle ALPase at 10(-7) and 10(-8) M and plasma membrane ALPase at 10(-8) M only. However, 24,25-(OH)2D3 inhibited matrix vesicle and plasma membrane PLA2 activity. The effects of the vitamin D metabolites were noticed after both 3 and 24 h. Neither hormone metabolite had any effect on these enzymes in membrane fractions from cultures of neonatal rat muscle mesenchymal cells, which do not calcify their matrix in vivo. 1,25-(OH)2D3 and 24,25-(OH)2D3 can directly affect chondrocyte membrane enzymes without genomic influence or protein synthesis and that membrane response depends on the stage of chondrocyte differentiation. Changes in PLA2 activity may change membrane fluidity and may be a mechanism by which the hormones affect cell membranes."

"Enzymes present in membranes isolated from the less differentiated mesenchymal cells do not respond to either vitamin D3 metabolite tested, although both metabolites stimulate ALPase gene expression in cultures of these cells"

Treatment of resting zone chondrocytes with bone morphogenetic protein-2 induces maturation into a phenotype characteristic of growth zone chondrocytes by downregulating responsiveness to 24,25(OH)2D3 and upregulating responsiveness to 1,25-(OH)2D3.

"To determine if bone morphogenetic protein-2 (BMP-2) can induce the endochondral maturation of resting zone (RC) chondrocytes, confluent fourth-passage cultures of these cells were pretreated for 24, 36, 48, 72, or 120 h with recombinant human BMP-2. At the end of pretreatment, the media were replaced with new media containing 10(-10)-10(-8) M 1,25-(OH)2D3 or 10(-9)-10(-7) M 24,25-(OH2)D3 and the cells incubated for an additional 24 h. This second treatment was chosen, because prior studies had shown that the more mature growth zone (GC) chondrocytes and RC cells respond to 1,25-(OH)2D3 and 24,25-(OH)2D3 in distinctly different ways with respect to the parameters examined. The effect of BMP-2 pretreatment on cell maturation was assessed by measuring alkaline phosphatase specific activity (ALPase). In addition, changes in matrix protein production were assessed by measuring collagen synthesis, as well as [35S]-sulfate incorporation into proteoglycans. When RC cells were pretreated for 72 or 120 h with BMP-2, treatment with 1,25-(OH)2D3 caused a dose-dependent increase in ALPase specific activity and collagen synthesis, with no effect on proteoglycan sulfation. RC cells pretreated with 1,25-(OH)2D3 responded like RC cells that had not received any pretreatment. RC cells normally respond to 24,25-(OH)2D3; however, RC cultures pretreated for 72 or 120 h with BMP-2 lost their responsiveness to 24,25-(OH)2D3. These results indicate that BMP-2 directly regulates the differentiation and maturation of RC chondrocytes into GC chondrocytes. These observations support the hypothesis that BMP-2 plays a significant role in regulating chondrocyte maturation during endochondral ossification."

"Resting zone cells exhibit greater sensitivity to BMP-2 than do cells derived from the prehypertrophic and upper hypertrophic zones"

Treatment of resting zone chondrocytes with 24,25-dihydroxyvitamin D3 [24,25-(OH)2D3] induces differentiation into a 1,25-(OH)2D3-responsive phenotype characteristic of growth zone chondrocytes.

"rat costochondral cartilage chondrocytes isolated from the growth zone (GC) respond to 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3], whereas those from the resting zone (RC) respond to 24,25-(OH)2D3[the inactive form of Vitamin D]. The aim of the present study was to determine whether 24,25-(OH)2D3 induces differentiation of RC cells into a 1,25-(OH)2D3-responsive GC phenotype. To do this, confluent, fourth passage RC chondrocytes were pretreated for 24, 36, 48, 72, and 120 h with 10(-7) M 24,25-(OH)2D3. The medium was then replaced with new medium containing 10(-10) to 10(-8) M 1,25-(OH)2D3, and the cells were incubated for an additional 24 h. At harvest, DNA synthesis was measured as a function of [3H]thymidine incorporation; cell maturation was assessed by measuring alkaline phosphatase (ALPase) specific activity. Incorporation of [3H]uridine was used as a general indicator of RNA synthesis. Matrix protein synthesis was assessed by measuring incorporation of [3H]proline into collagenase-digestible protein (CDP) and collagenase-nondigestible protein (NCP) as well as 35SO4 incorporation into proteoglycans. When RC cells were pretreated for 24 h with 24,25-(OH)2D3, they responded like RC cells that had received no pretreatment; further treatment of these cells with 1,25-(OH)2D3 had no effect on ALPase, proteoglycan, or NCP production, but CDP production was inhibited. However, when RC cells were pretreated for 36-120 h with 24,25-(OH)2D3, treatment with 1,25-(OH)2D3 caused a dose-dependent increase in ALPase, CDP, and proteoglycan synthesis, with no effect on NCP production. RC cells pretreated with 1,25-(OH)2D3 responded like RC cells that had not received any pretreatment. To determine whether these responses were specific to chondrocytes in the endochondral pathway, cells were isolated from the xiphoid process, a hyaline cartilage. In these cells, 1,25-(OH)2D3 inhibited ALPase, whereas 36 h of pretreatment with 24,25-(OH)2D3 caused these cells to lose their response to 1,25-(OH)2D3. 24,25-(OH)2D3 can directly regulate the differentiation and maturation of RC chondrocytes into GC chondrocytes, as evidenced by increased responsiveness to 1,25-(OH)2D3. 24,25-(OH)2D3 also promotes differentiation of cells derived from xiphoid cartilage, resulting in the loss of 1,25-(OH)2D3 responsiveness."

The 24,25-form tends to correlate with chondrogenesis whereas the 1,25 form tends to correlate with osteogenesis. The 24,25-form downregulates it's own production in resting zone chondrocytes but upregulates the active form by growth chondrocytes.

Monocarboxylate transporter 10 functions as a thyroid hormone transporter in chondrocytes.

"untreated congenital hypothyroidism is marked by severe short stature. The monocarboxylate transporter 8 (MCT8) is a highly specific transporter for thyroid hormone. The hallmarks of Allan-Herndon-Dudley syndrome, caused by MCT8 mutations, are severe psychomotor retardation and elevated T(3) levels. However, growth is mostly normal. We therefore hypothesized that growth plate chondrocytes use transporters other than MCT8 for thyroid hormone uptake. Extensive analysis of thyroid hormone transporter mRNA expression in mouse chondrogenic ATDC5 cells revealed that monocarboxylate transporter 10 (Mct10) was most abundantly expressed among the transporters known to be highly specific for thyroid hormone, namely Mct8, Mct10, and organic anion transporter 1c1. Expression levels of Mct10 mRNA diminished with chondrocyte differentiation in these cells. Accordingly, Mct10 mRNA was expressed most abundantly in the growth plate resting zone chondrocytes in vivo. Small interfering RNA-mediated knockdown of Mct10 mRNA in ATDC5 cells decreased [(125)I]T(3) uptake up to 44% compared with negative control. Moreover, silencing Mct10 mRNA expression abolished the known effects of T(3), i.e. suppression of proliferation and enhancement of differentiation, in ATDC5 cells. Mct10 functions as a thyroid hormone transporter in chondrocytes and can explain at least in part why Allan-Herndon-Dudley syndrome patients do not exhibit significant growth impairment."

"TH inhibits proliferation and promotes differentiation of chondrocytes and is indispensable for normal growth"

"The SLC16A10 gene, which encodes MCT10, localizes to 6q21-q22 [and is associated with height growth]"

"in RZ chondrocytes, TH exerts its actions via TRα1."

[Histology and proliferative capability of thoracic vertebral body growth plates of rats at different ages].

"The thoracic VBGPs obtained from rats aged 1 day and 1, 4, 8, 16 and 28 weeks were identified using safranin O-fast green staining, and the height of the hypertrophic zone, proliferative zone, and resting zone were measured. The chondrocytes were isolated from these VBGPs with a modified trypsin-collagenase type II digestion method for primary culture in vitro. The expressions of proliferating cell nuclear antigen (PCNA) mRNA and protein was detected by real time-PCR and Western blotting, respectively.

The 1-day- and 1-week-old rats showed significantly greater hypertrophic zone and proliferative zone in the VBGPs than older rats; the proliferative zone was significantly greater in rats aged 4 weeks than in those aged 28 weeks. The resting zone was obviously greater in rats aged 1 day and 1 week than in older rats, and also greater in rats aged 4 weeks than in those aged 16 and 28 weeks. Obvious ossification in the resting zone occurred at 16 weeks, and most of the resting zone became ossified at 28 weeks. The expression of PCNA decreased at both the mRNA and protein levels as the rats grew.

The 3 zones of VBGPs are greater in rats aged 1 day and 1 week than in older ones. Ossification in the resting zone begins at 16 weeks, and till 28 weeks, most of the resting zone is ossified. The proliferation ability of VBGP chondrocytes decreases with the increase of age of the rats."

Study is in a foreign language unfortunately.

Distribution of type I and type II collagen gene expression during the development of human long bones.

"The temporal and spatial gene expression of collagen type I and type II during the development of the human long bones was studied by the technique of in situ hybridization covering the period from the cartilagenous bone anlage to the formation of a regular growth plate in the newborn. Analysis of the early stages around the seventh week of gestation revealed for type II collagen a strong hybridization signal limited to the chondrogenic tissue. The surrounding connective tissue and the perichondrium showed weak type I collagen expression, while the zones of desmal ossification like the clavicle gave a strong signal. Beginning with the eighth week of gestation, type I collagen mRNA was detectable in newly formed osteoblasts at the diaphysis and appeared along with the formation bone marrow, in the areas of enchondral ossification. Parallel to the development of the different zones of cartilage differentiation, a specific pattern of type II expression could be observed: type II was mainly found in the chondrocytes of the hypertrophic zone and to a lesser degree in the zone of proliferation, while the resting zone and the zone of provisional calcification showed little activity. This segregation of type II expression was most pronounced in the early stages of cartilage calcification and in the growth plate of the newborn."

"As prechondrogenic mesenchyme cells develop to chondrocytes, a dramatic increase in the cytoplasmatic volume, the rough endoplasmatic reticulum and the Golgi apparatus takes place. This is

paralleled by the switch from collagen type I, the predominant collagen of fibroblasts, to collagen type II, the major collagen found in cartilage"

"Limbs of human fetuses between the 7th and 15th menstrual weeks" Mature chondrocytes never displayed Type I Collagen activity. Type II collagen negative cells occur at the osteochondral junction.

Mesenchymal chondroprogenitor cell origin and therapeutic potential.

"In embryonic limb development, FGF-4 stimulates Sonic hedgehog (Shh) expression in a positive feedback loop that coordinates proximal-distal and anterior-posterior patterning of the cartilaginous anlagen"

According to the paper 20-50% of cells in the bone marrow have the ability to differentiate into chondrocytes.

"progenitor cells with chondrogenic capacity have been isolated from the superficial zone of articular cartilage"

"chondroprogenitors have been identified in arthritic cartilage after their migration from the bone marrow through breaks in the tidemark and into the diseased cartilage"<-meaning chondroprogenitors exist in the bone marrow.

Morphogenetic and regulatory mechanisms during developmental chondrogenesis: new paradigms for cartilage tissue engineering.

Some great diagrams in this paper.

"When the cells aggregate, MCs[mesenchymal cells] begin to produce collagen I, fibronectin, and proteoglycans. The result of the strong interactions that cells establish with their environment is the formation of a dense mass of MCs that immediately begins to differentiate into chondroblasts. Condensed MCs start expressing mainly the transcription factor Sox9 that controls downstream genes involved in chondrogenesis, promoting these progenitor cells to secrete cartilage-specific ECM molecules"

"MMP1 and MMP2 have the capacity to degrade cartilage matrix, and they are characterized as the MMPs that are involved in earlier chondrogenesis. Specifically, blockage of MMP2 function supports precartilage condensation and chondrogenesis, and MMP1 knockout mice show decreased chondrocyte proliferation in the proliferative zone of the growth plates of long bones." MMP2 is increased in LSJL so perhaps we should find a way to decrease it's expression.

"overexpression of human Sox9 in murine ESCs (mESCs) leads to upregulated expression of the cartilage markers collagen IIA, aggrecan, and pax1 even in undifferentiated ESCs"

"fibroblasts can undergo spontaneous chondrogenesis in simple three-dimensional culture conditions"

LSJL progress update 2-23-13

2013-03-07T11:16:00.000-08:00

Unfortunately on one has been able to explain how I can retrieve x-rays from Sharp-Reese Steeley Online and the internet has been no luck either. The threshold required for growth to show up via x-ray is pretty high. No micro-growth plates are going to show up. So until someone can explain how I can access my x-ray records via the Sharp Reese Steeley medical system there can be no X-rays for now. I've been using an allen wrench and a hammer to test out Natural Height Grow's Pick Axe method in addition to LSJL: Bondhus 12116 1/2-Inch Long Hex L-Wrench. Since Michael is now performing the LSJL method maybe that is the solution to proving the LSJL method. My tests so far show that the allen wrench + hammer has been effective in creating sensations in the bone. In fact, I was worried that doing this may actually fracture the bone but I think it would take tons of hard taps to create enough residual strain to fracture the bone. And it may be necessary to fracture the bone as there is a different microenvironment involved in a microfracture versus a macrofracture. A microenvironment that is more chondrogenic. It's unclear whether this method has synergy with LSJL as drilling the bone in LSJL has been shown to reduce LSJL effectiveness. However this method may be a way to induce (-micro)fractures without penetrating the skin. There might be a region between micro- and macro-fracture that can generate a pro-chondrogenic microenvironment without the disability caused by a full blown macro-fracture.

The current progress pic is above.
Here's the last set of pictures.

I know it's grainy but if you look at these pics from 2010:

So here's me trying to match that measurement today at 13 1/4 inches:

The ruler was too far to the side so I tried to move it in:

You can see that 13 1/4 no longer quite covers end of the ankle to where the calf stops sloping in and where the next muscle slopes out. And I think this is true even if you account for the account that the tibia is rotated slightly outward in the present pics.

You can also see an increase in ankle width even if the foot in the before pic is rotated slightly inward. So I definitely gained some height with LSJL.

How much does frequency matter for LSJL?

2013-03-06T13:44:00.000-08:00

In the LSJL lengthening studies a frequency of 5 Hz is used. Frequency is the hardest thing to mimic in our home made clamping method. Even though this is an axial loading study it is still by the LSJL scientist Hiroki Yokota and it'll provide insights on how important frequency is for LSJL results.

Resonance in the mouse tibia as a predictor of frequencies and locations of loading-induced bone formation

"we conducted axial tibia loading using low, medium, or high frequency to the mouse tibia. The experimental data demonstrated dependence of the maximum bone formation on location and frequency of loading{But does frequency influence chondrogenic differentiation?}. Samples loaded with the low-frequency waveform exhibited peak enhancement of bone formation in the proximal tibia, while the high-frequency waveform offered the greatest enhancement in the midshaft and distal sections. Furthermore, the observed dependence on loading frequencies was correlated to the principal strains in the first five resonance modes at 8.0–42.9 Hz. Collectively, the results suggest that resonance is a contributor to the frequencies and locations of maximum bone formation."

"When loading is applied to such a material at or near its resonant frequencies, additional energy is absorbed and the material tends to vibrate at greater amplitude than when loading is applied at other frequencies. These vibrations propagate through the material in specific ways, or modes, based on the geometry and characteristics of the material."

"he tibia is composed of a shell of dense, stiff cortical bone that is thinnest on the outside of each epiphysis and thickest throughout the diaphysis. Inside the epiphysis a matrix of less dense, weaker trabecular bone is present. An epiphyseal plate is found at the border between the each epiphysis and diaphysis, which consists of hyaline cartilage. Each type of tissue likely contributes to the frequency response of the tibia."<-So when the epiphyseal plate is absent that affects the optimal frequency for chondroinduction.

"(C57BL/6 male, ∼13 weeks old) were used in this study."<-So these mice were definitely growing.

7N were used versus 0.5N in LSJL.

Low Frequency: 1-17Hz<-So LSJL is in this range
Medium Frequency: 18-34Hz
High Frequency: 35-51Hz

The animals experienced all 200 repetitions at each of the frequencies within the range.

So 8% region would be the region closest to the growth plate range and that was the range the responded most to low frequency.

It's hard to tell how chondrogenesis was affected in the 8% region.

"When a periodic load is applied at or near one of an object’s resonant frequencies, it tends to absorb more energy and oscillate at greater amplitudes than at other loading frequencies. In the case of the tibia, loading at frequencies near the resonant frequencies of the bone may be causing more energy to be dissipated and larger displacements in certain areas of the bone than loading at other frequencies with equal amounts of force. This may lead to increased strain rates, amplified intramedullary fluid flow, increased fluid shear stresses on bone cells, and enhanced cellular response in areas that absorb the most energy"<-So the correct frequency is a bonus but is not required.

LSJL Prototype Device designed by Yokota/Zhang

2013-03-04T12:39:00.000-08:00

This study was actually published in 2005 but it's in an obscure space journal so I didn't find it until now.

Development of a Knee-Loading Joint Supporter for Potential Use in Preventing Bone Loss during Spaceflight/Aging

14 week old female C57/BL6 mice were used. Knee loading was applied for 3 minutes for 3 consecutive days. Peak force of 0.5N was used. Groups were 5, 10, 15Hz. 5Hz was the one used to generate the most bone formation which doesn't mean it will translate into the most length but 5 Hz was used in the lengthening study.

Fig1 provides a diagram with a loaded mouse.

In Figure 3 they give the knee prototype. In this study they blocked copy and pasting so you'll have to read the full study.

In the device there is a pad to avoid a local stress concentration on the knee. Perhaps for LSJL foam could be placed in between the clamp and the knee. Although the study above did not study any lengthening effects of LSJL.

Yokota/Zhang mention 10N being what is required for humans but again this was before they noted any LSJL lengthening effects.

Usage mentioned being approx. 30 min daily...per knee. In this study the the outer part of the knee was the one loaded. Here's how a gear/cam mechanism works:

This ratcheting motion is very similar to a clamp.

Grow taller by eating deer antlers?

2013-02-21T11:49:00.000-08:00

Deer antler extract is available for sale: 3 Pack Deer Antler Velvet 2 oz. Liquid Extract.

Deer antler has been reported to have anecdotal uses on dogs. I'm bumping this study due to this topic being a possible field of interest for an upcoming Natural Height Growth Podcast. Deer Antler has had positive effects on chondrogenesis but only in vitro(in cell cultures). So it's unknown whether digestion ruins the pro-chondrogenic stimulatory effects. Only few in vivo(in live animals) studies have been done on bones and deer antler but found that bone and deer antler did have a stimulatory effect on bone formation. So deer antler can affect the bone but it's still unclear whether the chondrogenic effects can remain unmolested.

So Deer Antler could possibly play a role in inducing ectopic chondrogenesis or enhancing the growth plate in the developing. But it may not do anything at all but it shouldn't hurt or inhibit any pro-chondrogenic actiions.

Pilose antler polypeptides promote chondrocyte proliferation via the tyrosine kinase signaling pathway.

"Pilose antler polypeptides (PAP) have been reported to promote chondrocyte proliferation. [We] investigate the effects of PAP on the proliferation of chondrocytes.
Chondrocytes isolated from the knee of Zealand white rabbits were cultured. The second generation chondrocytes were collected. The chondrocytes were divided into the following 4 groups including serum-free, PAP, genistein (an inhibitor of tyrosine kinases), and PAP plus genistein group. Cell viability was analyzed. The cell cycle distribution of the chondrocytes was analyzed. The expression levels of cyclin A was detected.
No significant difference was observed between serum-free and genistein group. Treatment of the cultures with PAP produced a significant dose-dependent increase in cell viability, the percentage proportion of chondrocytes in the S phase and Cyclin A expression as well. However, the promoting effect of PAP on chondrocyte proliferation were dose-dependently inhibited by genistein, whereas genistein alone had no effect on proliferation of isolated chondrocytes.
PAP promotes chondrocyte proliferation with the increased cell number, percentage proportion of chondrocytes in S phase and expression of protein cyclin A via the TK signaling pathway."

"Genistein (4,7,4'-trihydroxyisoflavone), a major isoflavone from soybean, has been proven as a specific inhibitor of TK. Genistein, which block kinase ATP-binding sites, specifically inhibit phosphorylation of tyrosine residues, thereby inhibiting cells growth"

"The higher concentration of PAP added the [higher the] number of cultured chondrocytes". Genistein inhibited this increase in proliferation.

"Compared to control group, the mean proportion of cells in S phase increased sharply from 6.4% to 35.2% after adding PAP."<-The S phase is the DNA replication phase of the cell cycle.

"cyclin A expression increased to 50% after adding PAP"

"level of cyclin A correlates directly with the proliferative state of cells"

It should be noted that this may only increase growth rate in growth plates and may not increase adult height. It should also be noted that the PAP was added directly to the cartilage so it doesn't show whether or not deer antler supplemenation can bypass digestion.

[Role of pilose antler polypeptides on replicative senescence of rat chondrocyte].

"3rd generation chondrocytes were divided into blank group, and PAP groups with three different concentration of PAP which were passaged to the 4th generation. Meanwhile, the 2nd generation of chontrocytes were used as control group. The chondrocytes in different groups were detected with the method of histochemistry for S-A-beta-gal, flow cytometry for cell life cycle and proliferation index, alcian blue test for the content and structure of GAG of ECM, and RT-PCR for type II collagen and Aggrecan. Then PAP's function was observed regarding the appearance and functional status in the process of chondrocyte's senescence.
PAP significantly inhibited chondrocyte's express of S-A-beta-gal, promoted chondrocyte's proliferation, reduced cell content on G1 phase, enhanced the content of GAG, type II collagen and Aggrecan of ECM."

So deer antlers are pro chondrogenic but the study was not done on live rats so no idea whether it gets past digestion. This was a chinese study so couldn't get access to the full version.

Effect of Pilose Antler Polypeptides on Cataplasia and Senescence of Rat Chondrocyte in Vitro

" 1. The rat articularchondrocytes were isolated with the method of enzyme digestion.The 3rdpassage chondrocytes were divided into blank group, different concentration PAP groups,different concentration glucosaminsalfate groups and were sequently passaged to 4thgeneration. The 2nd passage chondrocytes was contrasted as young cells group. Thechondrocytes of different groups were detected with the methods of histochemistry forS-A-β-gal, and with alcian blue test for the content and constructure of GAG of ECM,immuocytochemistry for typeⅡcollagen and PCNA, MTT assay for proliferation, RT-PCRfor typeⅡcollagen and Aggrecan, flow cytometry for cell life cycle and proliferationindex,by which to observe PAP’s function regarding to the appearance and functional status inthe process of chondrocyte’s cataplasia and senescence. 4. The successive tert-generation (2ndpassage, 3rd passage, 4th passage) chondrocytes and the 4th passage cells intervented by PAPwere studied for senenscence mechanism. In this course, immuocytochemistry was applied for p16, pRb, E2F, CyclinD, CDK4 and TRAP-ELISA was applied for telomerase activation toobserve targets’ changing regarding to cataplasia and senescence. The function of PAP wasdetected too. Results: 1. The method of enzyme digestion is practicable for harvesting considerable and better activity cells, which were identified that had good phenotype anddifferentiation. 2. From 4th passage, the chondrocytes emerging some cataplasia-senescence changes such as the expression of S-A-β-gal raising to large extent, cell life cycle being detented on G1 phase, dedifferentiation and so on. 3. PAP has better anti-senenscence function than GS on several respect such as inhibiting express of S-A-β-gal, promoting chontrocyte proliferating, reducing cell content on G1 phase, promoting cell energy metabolism, makingcell growth active, enhancing chondrocyte differentiating and so on. 4. In the course ofchondrocyte’s cataplasia and senescence, factors controlling cell life cycle and cell growth changes as follow: p16↑—pRb↑—E2F↓—CyclinD↑—CDK4↓—telemorase"

Effect of pilose antler polypeptides on chondrogenic phenotype differentiation of bone marrow-derived mesenchymal stem cells in vitro

"third passage BMSCs from [6-month old] rabbits were randomly divided into control group cultured in ordinary medium, induced group cultured in defined medium, and PAP group cultured in defined medium containing 10 mg/L PAP. An equal volume of articular chondrocytes were selected from rabbits as articular cartilage group. The cellular morphological and functional characteristics were observed after 1, 2, 3 weeks in centrifuge tubes by histological, biochemical and reverse transcription-polymerase chain reaction (RT-PCR) technique. Cell masses in the control group gradually crumbled after 2 weeks, and hematoxylin-eosin staining could not be done. Cell masses in the induced and PAP groups were semitransparent, but slightly contracted. A part of these cells were round or oval with a high density distribution at the surface. The content of GAG and mRNA expression of typeⅡ collagen in the induced and PAP groups were increased with culture time, and higher than those in the control group at different time points. The content of GAG and mRNA expression of type Ⅱ collagen in the PAP group were higher than those in the induced group, but lower than those in the articular cartilage group. BMSCs can differentiate into chondrogenic phenotype in the defined medium, and PAP can significantly enhance chondrogenic phenotype differentiation of BMSCs."

TGFB1 was present in the serum.

[The initial mechanism's investigation of pilose antler polypeptides resisting replicative senescence of rat chondrocyte].

"The successive tert-generation (2nd passage, 3rd passage, 4th passage) chondrocytes and the 4th passage cells intervented by PAP were studied for senenscence mechanism. In this course, immunocytochemistry was applied for pl6, pRb, E2F, CyclinD, CDK4 and TRAP-ELISA (telomerase repeat amplification protocol assay-enzyme linked immunosorbent assay) was applied for telomerase activation to observe targets' changing regarding to senescence and the function of PAP.
Along with cell's replicative senescence, pl6, pRb and Cyclin D express significantly rised, while E2F, CDK4 and telomerase express significantly lowerd. Meanwhile, in PAP interfered group compared with which in 4th passage group, pl6, pRb and Cyclin D express significantly lowered, while E2F, CDK4 and telomerase express significantly increased.
PAP postpones chondrocyte senenscence{thus it could possibly keep growth plates open longer}."

[Comparison of protein composition and activities of pilose antler processed by different methods].

"To elucidate the influence of processing conditions on pilose antlerś therapic effects, the protein composition and activities were compared on three kinds of pilose antler processed by lyophilization, freezing and traditional short-time heating, respectively. The concentration of the water soluble protein in freeze-dried pilose antler was 126.54 mg/g (Folin-Phenol assay), which was 13.1 times higher than that of heating processed antler. These proteins distributed widely in SDS-PAGE electrophoresis and the protein band between 50.0 kDa approximately 60.0 kDa achieved the highest concentration. The water extract of freeze-dried antler promoted the proliferation and IGF-I secretion of rat osteogenic-like cell UMR-106 by 245.25% ( MTT assay) and 66.36 ng/ml, which was respectively 2.2 times and 1.2 times of those of heating processed antler. The same candidate inhibited the growth of human hepatic carcinoma cell BEL-7402 by the highest rate of 47.64% , which was 1.4 times of heating processed antler. The activities of frozen fresh pilose antler were lower than those of its freeze-dried counterpart, but were much higher than those of heating processed antler. The results indicated that lyophilization help to remain the activity of pilose antlerś proteins as much as possible and improve its efficacy."

This study could be informative but can't get access.

[Effect of pilose antler polypeptides on the apoptosis of rabbit marrow mesenchymal stem cells differentiated into chondrogenic phenotype in vitro].

"The MSCs were separated from the nucleated cells fraction of autologous bone marrow by density gradient centrifuge and cultured in vitro. The MSCs were induced into chondrogenic phenotype by transforming growth factor beta1 (TGF-beta1) and basic fibroblast growth factor (bFGF). According to different medias, the MSCs were randomly divided into four groups: group A as black control group, group B (100 ng IL-1beta), group C (10 microg/ml PAP + 100 ng IL-1beta) and group D (100 ng/ml TGF-beta1 + 100 ng IL-1beta). The samples were harvested at 24, 48 and 72 hours.
The intranuclear chromatin agglutinated into lump and located under nuclear membranes which changed into irregular shape at 24 hours. The intranuclear chromatin agglutinated intensified at 48 hours. Then the nuclear fragments agglutinated into apoptosis corpuscles at 72 hours in group B. The structure change of cells in groups C and D was later than that in group B, and the number of cells changed shape was fewer than that in group B. The structure change of cells in group A was not significant. The apoptosis rate of cells, the mRNA expression of Caspase-3 and the enzymatic activity of Caspase-3 gradually increased in group B, and there were significant differences compared with groups A, C and D.
Caspase-3 is involved in apoptosis of the MSCs differentiated into chondrogenic phenotype cultured in vitro. PAP could prevent from or reverse apoptosis of these MSCs by decreasing the expression of Caspase-3 and inhibiting the activity of Caspase-3."

Adult stem cells and mammalian epimorphic regeneration-insights from studying annual renewal of deer antlers.

"[Some] animals have the ability to reprogram phenotypically committed cells at the amputation plane toward an embryonic-like cell phenotype (dedifferentiation). Deer antlers are the only mammalian appendages capable of full renewal. Following casting of old hard antlers, new antlers regenerate from permanent bony protuberances, known as pedicles. Antler renewal is markedly different from that of amphibian limb regeneration (dedifferentiation-based), being a stem cell-based epimorphic process. Antler stem cells reside in the pedicle periosteum. We envisage that epimorphic regeneration of mammalian appendages, other than antler, could be made possible by recreating comparable milieu to that which supports the elaboration of that structure from the pedicle "

Deer antler regeneration: a stem cell-based epimorphic process.

"Antler regeneration takes place in yearly cycles from its pedicle, a permanent protuberance on the frontal bone. Both growing antlers and pedicles consist of internal (cartilage and bone) and external components (skin, blood vessels, and nerves). The regeneration of both internal and external components relies on the presence of pedicle periosteum (PP){the properties of the pedicle periosteum can provide insight into how to grow taller}. PP cells express key embryonic stem cell markers (Oct4, Nanog, and SOX2) and are multipotent, so are termed antler stem cells. Now it is clear that proliferation and differentiation of PP cells directly forms internal antler components. The full regenerative ability of external antler tissue components is achieved through PP-derived chemical induction and PP-derived mechanical stimulation: the former triggers the regeneration of these external components, whereas the latter drives their rapid elongation."

"Antlers, despite being called head pieces, do not regenerate directly from the head of a deer but instead from the permanent cranial bony outgrowths, known as pedicles. Deer are not born with pedicles; instead, these start to develop from frontal crests (behind and above the eye sockets) when deer approach puberty"

" Initially, the developing pedicles are covered by typical scalp skin. When they have grown to their species-specific height (around 5–6 cm in red deer), first antlers begin to generate spontaneously from the apices of these pedicles. This development can be seen externally by a change in the appearance of the skin from the typical scalp skin to a velvet-like soft pelage, called velvet skin or velvet. When the rutting season approaches, the antlers become fully calcified and the blood supply is occluded, which causes the demise of the velvet skin. The dead velvet is subsequently shed to expose the bare bone of the hard antlers. These are cast in the following spring, and regeneration of the second set antlers from their living pedicle stumps is immediately initiated. From then on, annual renewal of subsequent antlers enters a well-defined cycle: casting of previous hard antler and regeneration of a new soft antler in spring, with rapid antler growth (up to 2 cm/day) and maturation in summer, then full antler calcification and velvet shedding in autumn, followed by the bare bony antler phase in winter"

" Immediately after a hard antler falls off, the rim of pedicle skin surrounding the distal end of a pedicle stump encroaches upon the bone margin, the space that was formerly occupied by the periphery of the antler base. This rim of skin is shiny, only sparsely populated by hair, and it possesses the peculiar features of velvet skin, specifically a thicker epidermis and the de novo formation of hair follicles, which distinguishes it from the more proximal pedicle skin, typical of the scalp skin. Within days of hard antler casting, wound healing nears completion by centripetal growth of velvet skin over the cast plane of a pedicle. At the same time, the distal part of pedicle periosteum (PP) becomes thickened through the active division of cells resident within it. Toward the late wound healing stage, two crescent-shaped growth centers are formed directly from the thickening distal PP, one of which is located anteriorly and the other posteriorly. Each center is made up of cartilaginous clusters that are capped by a layer of hyperplastic PP/perichondrium. Further augmentation of each growth center pushes up the anterior and posterior portions of the pedicle stump and leaves the central region behind. These posterior and anterior growth centers are the centers for the formation of the antler “main beam” and “first tine”"

"both PP and antlerogenic periosteum, from which PP is derived, express the stem cell marker CD9 antigen [and have elevated telomerase and nucleostemin activity]"

"stretching the skin stimulates epidermal proliferation only sufficiently to relieve tension. "<-Maybe such a concept would work for the one if osteoblast proliferation was stimulated to relieve stretching tension in the bone.

"direct proliferation and differentiation of the PP cells cause the internal tissue components (cartilage and bone) of a regenerating antler to form, whereas close association with PP or PP-derived tissue is the prerequisite for regeneration of external pedicle components (including skin, blood vessels, and nerves) to take place."

Deer antler supplements could certainly be a source of embryonic-like stem cells.

Histological studies of bone formation during pedicle restoration and early antler regeneration in roe deer and fallow deer

"Initially, [during antler regeneration], bone formation occurs by intramembranous ossification, but early during the regeneration process cartilage is formed at the tips of the cranial appendages, and is subsequently replaced by bone in a process of endochodral ossification"

"the periosteum serves as a cell source for the bone-forming tissue covering the exposed pedicle bone."

"The early onset of chondrogenesis in the regeneration process is regarded as an adaptation to the necessity of producing a huge volume of bone within a short period. This parallels the situation in other cases of chondrogenesis in membrane bones."

"antler casting can be postponed by administration of testosterone or estradiol to deer carrying hard antlers"

"administration of compounds that inhibit LH and thus testosterone release (medroxyprogesterone acetate), or interfere with both the release of testosterone and its action at the receptor level (cyproterone acetate) [can cause premature antler casting]"

"Early regenerating antler of a roe buck. a: Middle portion of regenerated antler. The cartilage (asterisks) is lined by a perichondrium consisting of an inner cellular (C) and an outer fibrous layer (F). D, dermis; E, epidermis; H, hair follicle; S, sebaceous gland; arrows, vascular spaces. Specimen R4: Heidenhain's azan (×26, bar = 500 μm). b: Vertical columns of cartilage (C) separated by highly vascularized mesenchymal tissue. Arrowheads, vascular spaces. Specimen R4: Heidenhain's azan (×80, bar = 100 μm). c: Zone of cartilage resorption and replacement by bone. Asterisk, cartilage; arrows, newly formed bone; arrowheads, multinucleated chondro-/osteoclasts; I, intertrabecular tissue. Specimen R4: Heidenhain's azan (×160, bar = 50 μm)."

"Cells from the cellular condensations in the fibrous mesenchymal tissue of the fallow deer pedicle distal to the newly formed osseous trabeculae. The cells contain numerous mitochondria (M) and a prominent rough endoplasmic reticulum (ER). N, nucleus; asterisks, bundles of collagen fibers in the extracellular matrix cut at different angles (×4,800, bar = 2 μm)."

"Advanced stage of pedicle restoration in a fallow buck. a: Overview of regenerated cranial appendage. Newly formed slender osseous trabeculae (T) distal to the pedicle stump (asterisk). The zone of the slender trabeculae is capped by mesenchymal tissue (M). D, dermis; E, epidermis. The rectangles indicate the regions shown in b, c, and e. Specimen F: Heidenhain's azan (×6.4, bar = 1.25 mm). b: Higher magnification of the transition zone between the pedicle stump (asterisk) and the newly formed osseous trabeculae (T). The mesenchymal tissue (M) overlying the trabecular bone is seen in the upper left corner of the figure. Specimen F, Heidenhain's azan (×13.2, bar = 600 μm). c: Newly formed, mostly vertically oriented osseous trabeculae (T) lined by osteoblasts (arrow). Note the presence of numerous capillaries (asterisk) in the intertrabecular mesenchymal tissue. Specimen F: Heidenhain's azan (×53, bar = 150 μm). d: Higher magnification of a newly formed osseous trabecula with some recently incorporated cells (osteocytes, black arrow). White arrows, osteoblasts; asterisk, mineralized bone matrix. Specimen F: semithin section, toluidine blue-borax (×211, bar = 38 μm). e: Cellular condensations and accompanying reticular bundles of collagen fibers (arrow) in the richly vascularized mesenchymal tissue overlying the zone of the newly formed osseous trabeculae. Asterisk, vascular space. Specimen F: Heidenhain's azan (×53, bar = 150 μm)."

Evidence that the canonical Wnt signalling pathway regulates deer antler regeneration

"Immunocytochemistry was used to map the distribution of the activated form of β-catenin (aβCAT). A low level of aβCAT staining was detected in chondrocytes and in osteoblasts at sites of endochondral bone formation{Thus perhaps inhibiting Beta-Catenin activation can be a possible way to form ectopic growth plates]. However, aβCAT was localised in cellular periosteum and in osteoblasts in intramembranous bone, where it co-localised with osteocalcin. The most intense aβCAT staining was in dividing undifferentiated cells in the mesenchymal growth zone. Antler progenitor cells (APCs) were cultured from this region and when the canonical Wnt pathway was inhibited at the level of Lef/TCF by epigallocatechin gallate (EGCG), the cell number decreased. TUNEL staining revealed that this was as a result of increased apoptosis. Activation of the pathway by lithium chloride (LiCl) had no effect on cell number but inhibited alkaline phosphate activity (ALP), a marker of APC differentiation, whereas EGCG increased ALP activity."

"targeted deletion of β-catenin in head and limb mesenchyme prevents the trans-differentiation of osteoblasts into chondrocytes"

Localization and characterization of STRO-1 cells in the deer pedicle and regenerating antler.

"Cells positive for the mesenchymal stem cell marker STRO-1 [were present] in the chondrogenic growth zone and the perivascular tissue of the cartilaginous zone in primary and regenerating antlers as well as in the pedicle of fallow deer (Dama dama). In addition, cells positive for the stem cell/progenitor cell markers STRO-1, CD133 and CD271 (LNGFR) were isolated from the growth zones of regenerating fallow deer antlers as well as the pedicle periosteum and cultivated for extended periods of time. STRO-1(+) cells isolated from the different locations are able to differentiate in vitro along the osteogenic and adipogenic lineages."

"the growing tip of the deer antler contains proliferating perivascular cells and possible angioblastic precursors"

"In contrast to long bones, adipogenesis does not occur in regenerating antlers."

Exploring the mechanisms regulating regeneration of deer antlers.

" Molecules that we have identified as having potentially important local roles in antlers include parathyroid hormone-related peptide and retinoic acid (RA). Both are present in the blastema and in the rapidly growing antler where they regulate the differentiation of chondrocytes, osteoblasts and osteoclasts in vitro. Blockade of RA signalling can alter cellular differentiation in the blastema in vivo. The trigger that regulates the expression of these local signals is likely to be changing levels of sex steroids because the process of antler regeneration is linked to the reproductive cycle. The natural assumption has been that the most important hormone is testosterone, however, at a cellular level oestrogen may be a more significant regulator. Exogenous oestrogen acts as a 'brake', inhibiting the proliferation of progenitor cells in the antler tip while stimulating their differentiation, thus inhibiting continued growth. Deciphering the mechanism(s) by which sex steroids regulate cell-cycle progression and cellular differentiation in antlers may help to address why regeneration is limited in other mammalian tissues."

"The first set of antlers are shed in the spring when testosterone levels fall, a process known as ‘casting’. A blastema then forms on the exposed surface of the pedicle bone and from this the first set of ‘mature’ branched antlers regenerate"

"RXRB expression increases as cells differentiate from chondroprogenitors into mature chondrocytes."

"significant amounts of vitamin A (retinol) are present in antler tissues at all stages of differentiation."

"IGF-I and IGF-II receptors [are present] in the antler tip"

"[regenerating antlers express] Wnt 3a and the active form of Beta-catenin, leptin, the beta isoform of the leptin receptor, Msx-1 and Msx-2, FGF-4, cbfa1 and osterix"

Lentiviral-mediated RNAi knockdown of Cbfa1 gene inhibits endochondral ossification of antler stem cells in micromass culture.

"we silenced expression of Cbfa1 [in antler cartilage] , a key factor regulating endochondral ossification, using RNAi, and showed that expression of the downstream genes type I collagen and osteocalcin were suppressed which, in turn, inhibited endochondral ossification process taking place in the antler stem cell-formed nodules."

Neither chondrogenesis or osteogenesis occured in the Runx2(Cbfa1) knock down culture.

Gene expression dynamics in deer antler: mesenchymal differentiation toward chondrogenesis.

"To identify novel genes involved either in early events of mesenchymal cell specialization or in robust bone development, we have introduced a 3 K heterologous microarray set-up (deer cDNA versus mouse template). Fifteen genes were differentially expressed; genes for housekeeping, regulatory functions (components of different signaling pathways, including FGF, TGFbeta, Wnt), and genes encoding members of the Polycomb group were represented. Expression dynamics for genes are visualized by an expression logo. The expression profile of the gene C21orf70 of unknown function is described along with the effects when over-expressed; furthermore the nuclear localization of the cognate protein is shown. In this report, we demonstrate the particular advantage of the velvet antler model in bone research for: (1) identification of mesenchymal and precartilaginous genes and (2) targeting genes upregulated in robust cartilage development."

Skeletal development genes upregulated in gene antlers:
Sprouty 1 homolog
Gas2
Bmpr2
Glypican 3

Detailed comparison to LSJL genes to be done.

"unlike in the growth plate cartilage, the antler cartilage is densely vascularized. Expression of p311 may keep under control the proliferation of the myofibroblast-like cells surrounding the blood vessels "

Identification of differentially expressed genes in the developing antler of red deer Cervus elaphus.

"we have identified the expression patterns for 36 genes that were characteristic or dominant in the consecutive cell differentiation zones (mesenchyme, precartilage, cartilage) of the tip section of the developing velvet antler of red deer Cervus elaphus. Two major functional groups of these genes clearly outlined: six genes linked to high metabolic demand and other five to tumor biology. Our study demonstrates the advantages of the antler as a source of mesenchymal markers, for distinguishing precartilage and cartilage by different gene expression patterns and for identifying genes involved in the robust bone development, a striking feature of the growing antler. Putative roles for "antler" genes that encode alpha-tropomyosine (tpm1), transgelin (tagln), annexin 2 (anxa2), phosphatidylethanolamine-binding protein (pebp) and apolipoprotein D (apoD) in intense but still controlled tissue proliferation are discussed."

"In the antler mesenchyme, α-tropomyosin (tpm1) and less profoundly, transgelin, (tagln) slow down the vigorous cell proliferation and make conditions favorable toward differentiation. Downstream of this pathway, annexin 2 (anxa2) acts by channeling the cells toward chondrogenesis."

Comparison to LSJL genes to be done.

Deer antler base as a traditional Chinese medicine: A review of its traditional uses, chemistry and pharmacology.

"Both in vitro and in vivo pharmacological studies have demonstrated that deer antler base possess immunomodulatory, anti-cancer, anti-fatigue, anti-osteoporosis, anti-inflammatory, analgesic, anti-bacterial, anti-viral, anti-stress, anti-oxidant, hypoglycemic, hematopoietic modulatory activities and the therapeutic effect on mammary hyperplasia."

"Based on animal studies and clinical trials, deer antler base causes no severe side effects."

The Table summary of chemical constituients reveals no unusual compositions but those would not reveal things like the embryonic-like stem cells. And there may be some unique proteins.

"Deer antler base collagens (800 mg/kg/day, i.g., for 90 days)" had a stimulatory effect on bone formation parameters on rats in vivo(Therapeutic effects of collagen of antler base on osteoporosis in ovariectomized rats).

Effects of Velvet Antler with Blood on Bone in Ovariectomized Rats

" In traditional Chinese medicine (TCM), both velvet antlers (VA) and VA blood can tonify qi, essence, and marrow, nourish the blood, and invigorate bones and tendons. In TCM, the combination of VA and VA blood is believed to have superior pharmacological effects. The effectiveness of the combination therapy of VA middle sections (VAMs) and VA blood (VAM-B) was first examined in promoting proliferation of mouse osteoblastic cells (MC3T3-E1). The anti-osteoporotic activity of VAM-B (ratio of VAM:VA blood = 1:0.2) was evaluated with ovariectomized (OVX) rats at a dose of 0.2 g/kg. In VAM-B-treated OVX rats, the body weight decreased 10.7%, and the strength of vertebrae and the femur respectively increased 18.1% and 15.4%, compared to the control. VAM-B treatment also recovered the estrogen-related loss of the right tibial trabecular bone microarchitecture. Alkaline phosphatase (ALP) significantly decreased, but estradiol did not significantly change in serum of VAM-B-treated OVX rats."

"[In another study], long-term antler administration (13 months) moderated decreased plasma phosphorus and calcitonin levels and femoral bone density and calcium content, and increased plasma parathyroid hormone (PTH) and alkaline phosphates (ALP) activity levels associated with an ovariectomy (OVX) in 2 month-old senescence-accelerated mouse (prone-8, SAMP8)"

"VAM-B contained testosterone, estradiol, and IGF-1. The top three amino acids in VAM-B were in the order glutamic acid, glycine, and aspartic acid."

Growing Taller with Swimming?

2013-02-06T15:58:00.000-08:00

Got a full study ctrl-F (*NEW*)

Swimming to increase height has been a theory that has been bounced around for a while now within the grow taller community because of this guy:

Much has been made of Michael Phelps' long torso and has led many to investigate the possibility that swimming may increase height in the torso. And, it is true that since the bones of the spine are irregular an increase in periosteal width of these bones would increase overall body height. Swimming does put shearing forces on the spinal bones especially the freestyle stroke. It is possible therefore that swimming did increase Michael Phelps' spinal height. But, one thing that contraindicates that is that Michael Phelps has a long wingspan. Now there are some short and irregular bones in the hands that contribute to wingspan but Michael Phelps' hand size could not possibly account for his large wingspan(6'7").

Michael Phelps has large feet too(size 14) and the feet are made largely of irregular and short bones(the long bones of the feet do not account for a large amount of shoe size).

It entirely makes sense for certain individuals to have long torsos, large hands, and large feet but short arms and legs. The bones that make up the former are largely short and irregular bones and the bones of the latter are long bones. It does not make sense to have long arms and short legs. The explanation for this paradox could be key for height seekers. Their could be something unique in the growth plate histology of people with long arms and short legs that is essential to extending the growth phase.

Dear Michael Phelps,

Stop swimming and donate your body to science.

These next two studies were based on citations from the study "Endochondral bone growth, bone calcium accretion, and bone mineral density: how are they related?" which flat out stated that swimming and other non-impact exercises increase body height. I have also emailed that author for clarification and got this response "Thank you for your e-mail. Our opinion regarding the effect of swimming on bone length (mentioned in the perspective section of our review) is based on the previous study in young rats by Swissa-Sivan et al. (Bone Miner. 1989;7(2):91-105). Therein, rats were randomly divided into swimming (experimental) and sedentary (control) groups, and bone length was found to increase by approx. 3% after 20-week swimming. In our opinion, swimming did cause bone elongation (i.e., not correlational)."

Differential effects of swimming versus weight-bearing activity on bone mineral status of eumenorrheic athletes.

"To examine the role of skeletal loading patterns on bone mineral density (BMD), we compared eumenorrheic athletes who chronically trained by opposite forms of skeletal loading, intensive weight-bearing activity (gymnastics), and nonweightbearing activity (swimming) and 19 nonathletic controls. BMD (g/cm2) of the lumbar spine, femoral neck, trochanter, and whole body was assessed by dual energy X-ray absorptiometry (DXA). Subregion analysis of the whole body scan permitted BMD evaluation of diverse regions. Swimmers were taller, heavier, and had a greater bone-free lean mass than gymnasts and nonathletic controls. When adjusted for body surface area, there was no difference in lean mass between swimmers and gymnasts, and both were higher than controls. Gymnasts had a lower fat mass than swimmers and controls. There were no group differences for spine or whole body BMD, but gymnasts had higher spine BMD corrected for body mass than either swimmers or controls. Gymnasts had higher femoral neck BMD than controls, who were higher than swimmers (0.875 +/- 0.105). This result still applied when BMD was normalized for body weight and bone size. Trochanter BMD of gymnasts (0.898 +/- 0.130) was also higher than controls (0.784 +/- 0.097) and swimmers (0.748 +/- 0.085), and remained higher when corrected for body mass."

Unfortunately, I couldn't get the full study. So we can't analyze the parameters to see how much was the swimming causal for height growth versus correlational.

Effect of swimming on bone growth and development in young rats.

"The effect of chronic swimming on bone modelling was studied. Forty female Sabra rats (5 weeks old) were randomly assigned to the following experimental groups: 30 rats were trained to swim (water bath 35 +/- 1 degree C, one h daily, five times a week) for 20 weeks--20 of them loaded with lead weights (1% body weight) while the rest (10 animals) swam load free. Ten sedentary rats matched for age and weight served as controls. At the end of the twenty-week swimming period, all rats were sacrificed, both humeri bones were dissected and prepared for the following examinations: morphometric, bone density (BD), bone mineral content (BMC), compression tests and cross-sectional geometrical parameters, histomorphometry and biochemical analysis of minerals (Ca, Pi, Mg, Zn). All measured parameters were found to be significantly higher (P less than 0.05) in the swimming rats irrespective of load, as compared with the controls. Bone weight was higher by 19%, bone volume by 11%, bone length by 2.8%, cortical area by 16%, BD by 7% and BMC by 15%. The compression breaking force at the distal shaft of the humerus was higher by 24% in the trained group, while the ultimate compressive stress was not significantly different. Maximal and minimal moment of inertia at the distal diaphysis were 33.4 and 40% higher, respectively, for the swimming groups than the controls. Ca, Pi, Mg and Zn levels per total humeral bone were significantly higher in the exercising rats. The histomorphometry and cross-sectional data emphasize longitudinal and transversal growth. These data indicate that swimming exercise exerts a positive effect on bone growth and development in young rats."

(*NEW*)
"The forces applied on the limbs during any moment of running can reach values of 6-12 times body weight"

"During swimming no limb-ground pounding is exerted and body weight-bearing action is reduced signiticantly. The gravitational forces acting on the body are counteracted by buoyancy. Thus. the changes that might occur in the long bones could be attributed solely to the stresses applied by muscle contractions during intensive treading water action of the animal, trying to stay afloat."

Humeral Length: "30.34(Control) 30.852(Groups that swam unloaded) 31.20(groups loaded with 1% bodyweight)" So the groups with the extra load grew longer arms.

"The longitudinal growth of the humerus of trained animals was faster compared to controls. Bone length increased by 1.7 and 2.8% for [unloaded swimmers] and [loaded swimmer] groups, respectively, compared to control groups."

Calcium, Phosphate, Magnesium, and Zinc content was higher with increasing load to control group per bone volume and total bone but not per bone dry weight.

Exercising group has the larger growth plate. Note that this exercising rat growth plate is different from the LSJL growth plate. But note that the control growth plates are similar in both groups. So LSJL increases bone length in a different manner from swimming. The main difference being that LSJL growth plate is less straight and that the area surrounding the growth plate is more porous with LSJL.

Here's a study that uses similar variables as the above study:

Swimming Training Increases the Post-Yield Energy of Bone in Young Male Rats

"Male Wistar rats (7 week-old) were assigned to one baseline control group, one control group and two swimming training groups, which were trained with 2(LOW) and 4(HIGH)% body-weight mass added, respectively."

"Femoral length, mm
31.5 ± 0.1(BCON)

39.4 ± 0.2(CON)

37.6 ± 0.4(LOW)

38.4 ± 0.3(HIGH)"

"Femoral length (mm)[when adjusting for bodyweight]

38.2 ± 0.3(CON)

38.1 ± 0.2(LOW)

38.7 ± 0.2(HIGH)"

In this study, versus the other study there was a baseline control group in this group whereas in the other group the control group was rats matched for age and weight.

Statement by Narattaphol Charoenphandhu "I agree that these two papers used different method for choosing baseline control. In addition, the duration of swimming protocol in Swissa-Sivan et al. (20 weeks) was much longer than that in Huang et al. (8 weeks)." The duration was longer in the study that found growth.

I couldn't get the study The Sabra rat: definition of a laboratory animal. This study would help us learn when female Sabra rats tend to stop growing. The rats would be 25 weeks old at the end of the experiment. Which is about 6 months which is at the point where mouse rats reach growth plate senesence and stop growing which leads credence to the possibility that swimming increased rats adult bone length and didn't just increase growth rate.

Here's another study that while not cited by the author who claims that swimming can make you taller may provide insight:

Histomorphometry of long bone growth plate in swimming rats.

" We performed a histomorphometric study on the effect of swimming on the growth plate and subepiphyseal area of young adult rats. The experiments were carried out on 28 12-week-old albino Sabra rats. One group of 14 rats was trained to swim 1 hour/day, 5 days a week, for 12 weeks. Another group of 14 rats served as controls. The proximal femur and humerus of each animal were examined histomorphometrically. There was an increase in the subepiphyseal cancellous bone trabecullae of the femur. In the growth plate there was an increase in the number of column cells and proliferative cells. These changes were more pronounced in the femur than the humerus. Swimming induces an increase in subepiphyseal cancellous bone in young adult rats by enhancing growth plate activity."

Note in this study swimming did not increase the proximal length of the rat femur. In fact the rat femur was slightly shorter in length than the control group.

Effect of a five-week swimming program on rat bone: a histomorphometric study.

"To specify the exercise-induced changes on different skeletal sites, the effect of a 5-week endurance swim training was studied in rats. Eighteen Lyon strain (Sprague-Dawley) 5-week old female rats were divided into nine sedentary and nine swimming rats. Each swim training session was increased by 15 minutes from 2-6 hours per day. A histomorphometric study was performed at the primary and secondary spongiosa of the distal femur and at the secondary spongiosa of lumbar and thoracic vertebral bodies. After training, bone loss was observed in the secondary spongiosa of lumbar vertebral bodies (24.7%) and in the primary spongiosa of distal femur (15.2%). A tendency to bone loss was also detected in the secondary spongiosa of distal femur (10.8%), whereas no change was detected in thoracic vertebral bodies. In secondary spongiosa, bone loss was accompanied with a thinning of trabeculae. Total eroded surfaces and osteoid surfaces were significantly decreased in the three studied skeletal sites, suggesting a decreased bone turnover. The decreased thickness of osteoid seams in both lumbar vertebrae and distal femur could mean that the osteoblastic activity has also been altered at the cell level, leading to thinning of trabeculae. Five-week swim training with such duration and intensity of exercise appears unable to increase bone volume in rats and, therefore, causes adverse effects. The three studied bones seemed to adapt differently to experimental conditions. The lack of ground reaction forces induced by water immersion might have contributed to the observed bone loss. "Normal" gravity would be an important cofactor in the osteogenic effects of exercise."

The effects of exercise mode, swimming vs. running, upon bone growth in the rapidly growing female rat.

"The purpose of this study was to compare the effects of two programs of endurance training, of equal duration and intensity, on bone development in female rats. Thirty-eight female Wistar rats were randomly assigned to one of three groups: run-trained (RUN), swim-trained (SWIM) or control (CON). The RUN group ran at a speed of 27 m/min up an 8 degrees incline. Swim trained animals swam with 2% of body weight attached to their tails. Training sessions were 2 h/day, 5 days/week and were conducted over a 10-week period. Hindlimb and forelimb muscles were removed upon sacrifice and analyzed for citrate synthase (CS) activity, liver (LG) and muscle (MG) glycogen. The parametrial fat pads were removed, digested with collagenase, and 2-deoxy-D-[3H]glucose uptake measured in isolated cells. Bone weight, length, diameter, ponderal index and bone mineral content (BMC) were measured in the femur and humerus of each animal. The LG, MG, fat cell volume, glucose uptake of the adipocyte and adrenal weight data indicate that the training response was identical. The CS activity of the muscles indicated that mechanical and recruitment patterns of the upper and lower body differ and could be responsible for bone development patterns found in this study. Exercise had a minimal effect on bone growth in the run-trained animals but did stimulate development in the swim-trained animals. The humerus of the SWIM was significantly (P < 0.05) heavier, wider and had a greater BMC when compared with those of the RUN and CON rats. The results of this study indicate that the muscular forces applied by the swim training protocol produced greater bone adaptations than the forces applied by a running protocol of equal duration and intensity."

Both running and swimming increased the glycogen levels of muscles and increased glucose uptake by adipocytes. Running increased citrate synthesis activity more than swimming which is stilll more than control. Swimming caused a minor, statistically insignificant increase in femur and humerus length. Running was statistically insignificantly shorter in femur and humerus length than control.

The age of the rats wasn't specified.

Physical activity and bone mass: exercises in futility?

"In Frost's CGFR[chondral/growth force response curve], increasing compression to the right of the curve increases growth to a peak, after which loads begin to decrease growth (with large enough loads being sufficient to arrest it)."<-although LSJL has been proven effective on mice at 4 months old which are close to the four month senescence point but this theory could explain swimming induced longitudinal growth.

"When growing mice from 14 days of age up to 22 weeks were run at a speed of 18 m/rain for 80 rain each day for 12 weeks, larger and heavier femora were found. But when the duration was increased to 21 weeks, or for a duration of 120 min/day, femora were shorter and lighter than those in the control group."

High-impact exercise strengthens bone in osteopenic ovariectomized rats with the same outcome as Sham rats

"Forty-two 9-mo-old female rats were either sham-operated (Sham) or ovariectomized (OVX). Three months after surgery, the rats were divided into the following groups: Sham sedentary, Sham exercised, OVX sedentary, and OVX exercised. Rats in the exercise groups jumped 10 times/day, 5 days/wk, for 8 wk, with a jumping height of 40 cm. Less than 1 min was required for the jump training. "

"jump exercise generates high-impact loading before the rats leave the ground."

"OVX and training did not affect tibial length."

Physical Exercise Improves Properties of Bone and Its Collagen Network in Growing and Maturing Mice

" male C57BL/6J mice"

Running mice had access to running wheels in cages. The length of the femur began to decrease in the running group between 4-6 months of age. Bone length was the same for control and running groups between 1-4 months of age.

EFFECTS OF RUN TRAINING ON BONE DEVELOPMENT AND BONE MINERALIZATION IN GROWING MICE

"male Swiss Albino mice (4 weeks old)"

"The mice within their cage in the running training group were placed on the flat bed treadmill, the speed of which was constant at 15 m/min, on a plain floor. An electric grid at the rear of the belt was used to create a stimulus for the mice to run. All mice ran 5 days/week for 12 weeks. Each running training period lasted 30 minutes."

"The lengths of the femur and the tibia were significantly greater for mice in the running training group" The exercise group increased 50% more in femur length than the sedentary group.

Reverse Ossification

2013-01-28T11:01:00.000-08:00

This study seems like a game changer but unfortunately it's in persian:

Growth Plate Reappearance after Closure in Ankle Radiography for Trauma

"Bone growth plates or physis are present at the end of long bones and are responsible for longitudinal growth. These plates consist of 4 layers and are lucent in radiography as a line perpendicular to the longitudinal axis, Because of cartilage layer x-ray absorption is less than calcified bone. Gradually increases with age and bone maturity these line will be narrower and as longitudinal bone growth stops, the line disappears. This phenomenon occurs at different ages in different bones of the skeleton but with complete maturity at the age of 19, all growth plates are closed and sclerosed. Re-appearing after closing is uncommon. We introduce two young patients in this study due to trauma have been treated for an ankle cast and the growth plates of tibia and fibula in their control X-ray was re-appeared. Subchondrel Bone Resorbtion is a known phenomenon that will occur after 6 to 8 weeks immobility in any bone. The lucent line caused by imbalance in osteoblast and osteoclast activity and bone absorption. Re-appearing of growth plates can be caused by reversed ossification and bone absorption."

The cited studies may lead to clues on reversed ossification but I could not get access to them:

Effects of tiludronate on bone loss in paraplegic patients

The other links I could find don't seem to have a direct relation to reverse ossification just bone resorption.

Please help out if you know Persian or are an expert at translation documents(Google Translate doesn't like multiple columns).

Some selected translations(manually copying and pasting):

Figure 1: The graph occurs when the ankle and a half before casting

Figure 2: X-ray of the ankle occurred and half occurred after casting

Figure 3: X-ray event occurring half calf 78 days after casting

Figure 6: X-ray of the ankle occurred and half occurred after treatment

Figure 7: The graph and profile ankle occurred 45 days after treatment

Figure 4: X-ray of the ankle occurred before casting

Figure 5: X-ray of the ankle profile

Some notes in my feeble attempts to translate:

"The resurgence of growth plate can be absorbed by tissue"

"Treatment, the growth plates of the tibia and fibula appeared again."

"Patients can reverse the growth plate of the bone at the growth plate cartilage of the past"

LSJL Case Study: Felipe

2013-01-22T10:41:00.002-08:00

At the point of this x-ray Felipe has been performing LSJL for three months with no reported height gain but has reported an increase in knee thickness and width.

4 months after the x-ray's still no increase in height. He had been doing MENS routine for 2 years. MENS is just a fancy name for taking melatonin and high dose niacin.. It is not really a height increase routine.

He reports loading for 3 minutes for very high pressure. Of note that at this point he was loading at the bumpy part of the bone rather than at the synovial joint intersection.

Here's what open tibial growth plates look like. A growth plate shows up as a gap in the bone in the xray.

Here's what a normal tibial x-ray looks like.

That's what an enchondroma looks like.

That's what a chondrosarcoma looks like.

What's interesting to note is that while a growth plate just presents itself on a gap in the x-ray. An enchondroma(ectopic endochondral ossification) and a chondrosarcoma(ectopic cartilage formation) are both visible. However, both sarcomas consist of both cartilage/bone cells and tumor cells so maybe it's the tumor cells that are visible and not the cartilage cells.

Comparing the normal x-ray to felipe's x-ray:

Felipe's x-ray is much whiter on the perimeter of the bone than the normal x-ray(which is more gray) indicating enhanced bone density. This is despite the fact that the normal x-ray individual has a much more muscular calf than Felipe.

Felipe's x-ray is hollower in the middle of the bone. Note that in the LSJL study involving drilling, LSJL caused degradation in the middle of the bone.

The normal x-ray no longer has a visible epiphyseal line(scar) whereas Felipe does so the normal person is likely older than Felipe.

After the 3 months of these x-ray's Felipe loaded his knees for four minutes(high intensity) and loaded his ankles for two minutes(also high intensity).

Unfortunately these were the best x-ray's of the normal tibia I could find so if you could provide links to other x-rays that would be helpful. For example, this person may be fatter than Felipe which may present itself as adipose tissue in the bone marrow which would also explain why Felipe's x-ray is clearer. X-ray's of athlete tibia's would be helpful.

Since growth plate's present as a gap and it's likely that the visible tissue present in other forms of ectopic cartilage formation are other types of tumor cells. The hollower state of Felipe's bone indicates that there is room for growth plate formation. You can see clear through the fibula to the tibia in contrast to the normal xray. It could be due to x-ray quality but the quality of the muscle and fat seems to be the same on both x-rays.

Also, in Felipe's bone you can see streaks within the bone which may be signs that LSJL induced shear strain and fluid flow.

Even with shifting the load from the bone to more the synovial joints Felipe still didn't gain (visible) height and he reported increasing the load from three to four minutes.

He also stated that he did not use a deconditioning period.

So hypothesis from this case study:
Three minutes(high intensity) of LSJL is enough to degrade bone making room for growth plates.
It is not enough to complete formation of new growth plates.
Thus 5 minutes may be the minimum if not longer. Human bones are much longer than rat bones so it may take more time for fluid to travel within the bone and build up hydrostatic pressure to induce chondrogenesis.

egr1

2013-01-18T13:35:00.000-08:00

Egr1 is upregulated 4.973 fold by LSJL. Under normal axial loading on 20 week old mice, egr1 was downregulated which could mean that egr1 is a key reason as to why LSJL can increase chondrogenesis but axial loading cannot. Chondrocytic cells under hydrostatic pressure had egr1 upregulated. Egr1 was also upregulated by dynamic chondrocyte compression. LIPUS can also upregulate EGR1. Removing the sciatic nerve increased EGR1 levels. One study found that Egr1 was upregulated in older rat growth plates. Increases in HMGA2(which is linked to height and upregulated by LSJL) levels also increased EGR1 levels. Surprisingly Egr1 was downregulated in mesodermal progenitor cell differentiation to chondrocytes. Arachidonic acid can increase Egr1 levels.

The Immediate Early Gene Product EGR1 and Polycomb Group Proteins Interact in Epigenetic Programming during Chondrogenesis.

"Initiation of and progression through chondrogenesis is driven by changes in the cellular microenvironment. At the onset of chondrogenesis, resting mesenchymal stem cells are mobilized in vivo and a complex, step-wise chondrogenic differentiation program is initiated {our goal with LSJL is to induce chondrogenic differentiation of resting mesenchymal stem cells}. Differentiation requires coordinated transcriptomic reprogramming and increased progenitor proliferation; both processes require chromatin remodeling. The nature of early molecular responses that relay differentiation signals to chromatin is poorly understood. Immediate early genes are rapidly and transiently induced in response to differentiation stimuli in vitro {So does EGR1 cause the differentiation or is EGR1 the byproduct?}. Functional ablation of the immediate early factor EGR1 severely deregulates expression of key chondrogenic control genes at the onset of differentiation. In addition, differentiating cells accumulate DNA damage, activate a DNA damage response and undergo a cell cycle arrest and prevent differentiation associated hyper-proliferation. Failed differentiation in the absence of EGR1 affects global acetylation and terminates in overall histone hypermethylation. Polycomb associated histone H3 lysine27 trimethylation (H3K27me3) blocks chromatin access of EGR1. In addition, EGR1 ablation results in abnormal Ezh2 and Bmi1 expression. Consistent with this functional interaction, we identify a number of co-regulated targets genes in a chondrogenic gene network. EGR1 [is involved] in early chondrogenic epigenetic programming to accommodate early gene-environment interactions in chondrogenesis."

ATDC5 cells were used which are technically chondrocyte progenitor cells like those in the growth plate resting zone and not true mesenchymal stem cells.

"Progression through chondrogenesis is in part driven by interaction with a constantly changing microenvironment, which is defined by soluble growth and differentiation factors, hormones, oxygen tension, cell-cell and cell-ECM contacts"

"abnormal skeletogenesis [occurs] in PRC1 LOF[Loss of Function} mice"

"Egr1 mRNA induction in chondrogenesis is transient and precedes transcriptional upregulation of Sox9"

"Sox6 and Agc1-loci do not represent early EGR1-targets for transcriptional activation in chondrogenesis."<-Although aggrecan was upregulated by LSJL.

"Consistent with reduced chondrogenic capacity; shEgr1{silenced-Egr1} significantly reduced Col2a1 expression at the mRNA and protein levels"

"chondrogenic markers Sox9, Agc1, Col2A1 and Col10A1 showed a delayed (5–10 days) differentiation response [after Egr1 is silenced]" All of these genes were up in LSJL.

"Enchondral ossification pathway analysis for predicted EGR1 targets (green) and published PRC1 targets (blue)."

That Runx2 is an EGR1 target could explain how Egr1 could upregulate osteoblast differentiation as well.

There is evidence though that loss Egr1 can be compensated for.

"H3K27me3-decorated chromatin prevents EGR1 from accessing promoters."<-thus an H3K27me3 inhibitor may be a well to allow EGR1 to access chondrogenic promoters again.

Sox9 was not upregulated by Egr1 in H3K27me3 cells.

"EZH2 levels decrease in the context of replicative senescence"

"shEgr1 cultures do not reach super-confluence and do not form chondrogenic nodules"

EGFR ligands drive multipotential stromal cells to produce multiple growth factors and cytokines via early growth response-1. establishes a connection between MSCs and Egr1 but not a relationship between the two and chondrogenic differentiation.

Early growth response genes signaling supports strong paracrine capability of mesenchymal stem cells.

"EGF [facilitates] in vitro expansion of MSCs without altering multipotency. The molecular machinery underlying MSCs' strong paracrine capability lies downstream of EGFR signaling, and we focus on transcription factors EGR1 and EGR2. EGR1 regulates angiogenic and fibrogenic factor production in MSCs{fibrogenic factors are involved in chondrogenesis}, and an EGFR-EGR1-EGFR ligands autocrine loop is one of the underlying mechanisms supporting their strong paracrine machinery through EGR1. EGR2{also up in LSJL} appears to regulate the expression of immunomodulatory molecules."

Egr1 is expressed 3 fold higher in human MSCs than human fibroblasts.

So Egr1 likely plays a role in LSJL but unfortunately this was only established in chondrocyte progenitor cells and not MSCs directly.

Amomum Villosum, can it help you grow upwards?

2013-01-18T11:54:00.001-08:00

Amomum villosum is available for sale: Sha Ren(concentrated Extract Powder)(amomum Villosum Fruit)(amomum Fruit / Grains of Paradise) Mayway-5330c. A bunch of chinese studies where I can't get the full studies. It may seem like I'm constantly doing products. I actually do a lot of things but I only put things on the main page that people can take action on.

Amomum villosum induces longitudinal bone growth in adolescent female rats.

"Adolescent female Sprague-Dawley rats were divided into 3 groups and treated for 4 days: control (distilled water, p.o.), recombinant human growth hormone (rhGH; 100 microg/kg, s.c.), and A. villosum (500 mg/kg, p.o.) groups. On day 3, tetracycline (20 g/kg, i.p.) was injected for growth plate identification. On days 2, and 4, 5-bromo-2'-deoxyuridine (BrdU) (50 mg/kg, i.p.) was injected to label proliferating cells. On day 5, tibias were dissected.
The rate of bone growth in the A. villosum and rhGH groups increased to (410 +/- 44) and (389 +/- 46) microm/day, respectively, as compared with the control (330.7 +/- 34.7) microm/day. The thickness of the growth plates also increased to (591 +/- 37) and (598 +/- 32) microm, respectively, as compared with the control (524 +/- 89) microm. The number of BrdU-positive cells in the chondrocytes of the A. villosum and rhGH groups was also significantly higher (126 +/- 24) and (143 +/- 18) cells/mm2, respectively) than in the control (109 +/- 25) mm2. Insulin-like growth factor-1 and bone morphogenetic protein-2 in the A. villosum and rhGH groups were highly expressed in the growth plate as compared with the control samples, indicating increased bone formation."

Effects of Amomum villosum on longitudinal bone growth in adolescent rats

"The fruit of Amomum villosum has been used for an improvement of gastrointestinal motility in traditional Korean medicine. In Dongeuibogam, the traditional book of Korean medicine, there was mentioned that an herbal mixture containing A. villosum used as medicine for malnutrition associated with growth retardation. This study was aimed to investigate the effect of A. villosum on longitudinal bone growth in adolescent rats. A. villosum was extracted with water for 3h at 100°C in a reflux apparatus. The A. villosum treated group (500mg/kg) and the control group (vehicle) were administered orally twice daily for 4 days. On day 3, tetracycline (20mg/kg) was injected intraperitoneally to form a fluorescent band on the growth plates. On days 2-4, bromodeoxyuridine (BrdU) (50mg/kg) was injected intraperitoneally for labeling proliferating cells. A. villosum caused a significant acceleration of longitudinal bone growth, compared to control group. BrdU-positive cells were increased in the chondrocytes of the A. villosum group. The growth plate width was significantly increased, compared to control group. BMP-2 and IGF-1 were highly expressed in the hypertrophic and proliferative zone, respectively. A. villosum [may] increase longitudinal bone growth by stimulation of the chondrocyte hypertrophy and chondrogenesis, through regulation of IGF-1 and BMP signaling in the growth plate."

[Study on phenolic constituents of Amomum villosum].

"Five compounds were isolated from Amomum villosum. They were identified as: 3-ethoxy-hydroxy benzoic acid (1), vanillic acid-1-beta-D-glucopyranosyl ester (2), isorhamnetin-3-beta-D-glucoside (3), flavanocoumarin (4), isoflavanocoumarin (5)." Benzoic Acid is in Curculigo Orchiodes and Benzoic Acid may be a BMP-2 stimulator. Glucosides are derived from glucose.

According to Flavanocoumarins from Guazuma ulmifolia bark and evaluation of their affinity for STAT1., flavanocoumarin can inhibit Stat1. STAT1 is a catabolic compound that may inhibit height.

isoflavanocoumarin and the glucopyranosyl ester are uncharacterized.

Amomum Villosum may increase height via Benzoic acid upregulation of BMP-2 or flavanocoumarin inhibition of STAT1. Or, there may be some novel function of the other three compounds. Also, since we can't access the full study we can't know if the increase in growth rate is due to the nutritional value of Amomum Villosum since calories do increase growth rate.

Ectopic endochondral ossification genes versus LSJL

2013-01-15T13:16:00.000-08:00

A gene expression profile for endochondral bone formation: oligonucleotide microarrays establish novel connections between known genes and BMP-2-induced bone formation in mouse quadriceps.

"This study evaluated several aspects of the osteogenic effect of hBMP-2 protein injected into quadriceps of female C57B1/6J SCID mice. Mice were euthanized 1, 2, 3, 4, 7, and 14 days postinjection and muscles were collected for several methods of analysis. Hematoxylin and eosin-stained sections of muscles injected with formulation buffer showed no evidence of osteogenesis. In contrast, sections of muscles injected with hBMP-2 showed evidence of endochondral bone formation that progressed to mineralized bone by day 14. In addition, radiographs of mice injected with hBMP-2 showed that much of the quadriceps muscle had undergone mineralization by day 14. Labeled mRNA solutions were prepared and hybridized to oligonucleotide arrays designed to monitor approximately 1300 murine, full-length genes. Changes in gene expression associated with hBMP-2 were determined from time-matched comparisons between buffer and hBMP-2 samples. A gene expression profile was created for 215 genes that showed greater than 4-fold changes at one or more of the indicated time points. One hundred twenty-two of these genes have previously been associated with bone or cartilage metabolism and showed significant increases in expression, e.g., aggrecan (Agc1){up}, runt related transcription factor 2 (Runx2), bone Gla protein 1 (Bglap1), and procollagens type II (Col2a1){up} and X (Col10a1){up}. In addition, there were 93 genes that have not been explicitly associated with bone or cartilage metabolism. Two of these genes, cytokine receptor-like factor-1 (Crlf1){up} and matrix metalloproteinase 23 (Mmp23), showed peak changes in gene expression of 15- and 40-fold on days 4 and 7, respectively. In situ hybridizations of muscle sections showed that Mmp23 and Crlf1 mRNAs were expressed in chondrocytes and osteoblasts, suggesting a role for both proteins in some aspect of cartilage or bone formation"

"BMP-2-induced changes in the expression of gene"

Genes upregulated on Day2-3 versus LSJL:
Cdh11{down}
Cyr61
Bgn
Col5a2
Col3a1
Col12a1
Col6a1(no change on day 1) {up}
Lox
Ptgs2
Timp1
Serpina3n
MMP14
Serpine1
Csf2ra{down}
Junb
Scx
Ccl7
Sod3
Ccnb1{down}
Ccnb2{down}
Crlf1
Ccr1
Vav1{down}
Myog{down}

No Change Day1-3 with some transient upregulation(noted if occurred) versus LSJL:
Sdc2 {down}
Vcam1 (transient on day 2) {down}
Col6a2 {up and down}
Lum {up}
Bsp {up}
Col2a1 (transient decrease on day 3) {down}
Wisp2 {up}
Agc1 {up}
Cd9 {down}
Ptpra {down}
Anxa8 {up}
Socs3 (transient increase on day 2) {up}
Ptpn12 {down}

Generally Downregulated:
MMP2{up}
Col10a1 {up}
Col9a1 {up}
Col15a1 {up}
Eln {up}
Col9a3 {up}

"Ectopic cartilage induced: A histological time course of the effects of a single intramuscular injection of buffer or hBMP-2 protein into quadriceps muscles of C57B1/6J SCID females. A, C, E, G, and I correspond to muscles removed after 1, 3, 4, 7, and 14 days, respectively, following an injection of formulation buffer. B, D, F, H, and J correspond to muscles removed after 1, 3, 4, 7, and 14 days, respectively, following an injection of 50 μg of hBMP-2 protein. Muscles were fixed and stained with H&E as described under Methods. Abbreviations: M, normal skeletal muscle fiber; I, inflammatory cells; DM, degenerated skeletal muscle fiber; F, fibroplasia; Cb, chondroblast; C, chondrocyte; Ob, osteoblast; Oc, osteoclast; B, bone; BM, bone marrow. The bar in each panel = 0.1 mm."

"The expression profile for cytokine receptor-like factor 1 (Crlf1) is qualitatively similar to that of Cyr61 and peaks on day 4"<-Crlf1 and Cyr61 may be key components of the chondroinduction by LSJL.

This study has a lot of useful information which will neccessitate returning to it in the future.

AKG for height augmentation?

2013-01-14T10:11:00.002-08:00

According to Wikipedia AKG contains 2-oxyglutaric acid: Olympian Labs - A-Akg, 90 g powder. 2-oxyglutaric acid is a synonym for α-ketoglutarate.

The effect of dietary administration of 2-oxoglutaric acid on the cartilage and bone of growing rats.

"2-Oxoglutaric acid (2-Ox), a precursor to hydroxyproline, [proline, glutamate, arginine and asparagine], exerts protective effects on bone development during different stages of organism development. [What's] the influence of dietary 2-Ox supplementation on the growth plate, articular cartilage and bone of growing rats? A total of twelve male Sprague-Dawley rats were used in the study. Half of the rats received 2-oxoglutarate at a dose of 0·75 g/kg body weight per d in their drinking-water. Rats receiving 2-Ox had an increased body mass and absolute liver weight. Femoral length and bone mineral density, overall thickness of growth plate and the thickness of femoral articular cartilage{so 2-oxoglutaric acid could also cause a miniscule increase in height in adults} were also increased. Dietary supplementation with 2-Ox to growing rats exerts its effects mainly on cartilage tissue, having only a slight influence on bone."

"2-Ox, together with Fe2+, have been proposed to be active participants in the conversion of proline to hydroxyproline, the main amino acid of bone collagen. Moreover, 2-Ox acts as a cofactor for Fe2+ absorption from the intestine"<-Iron decreases FGF23, which affects height.

" resting and calcification zones of the femoral growth plate and calcification zone of the tibial growth plate were significantly thicker in the [supplement] group"

A stands for supplement group, C stands for control. Above is femoral growth plate.

Above is tibial growth plate.

Above is the increase in articular cartilage thickness and the difference is pretty big(28%). Unfortunately, articular cartilage thickness only makes a small difference in height. Perhaps if it affects the intervertebral discs as well.

"2-Ox is a N scavenger and a source and precursor of glutamine, synthesised in human skeletal muscles, which improves and stimulates protein synthesis and inhibits protein degradation in skeletal muscles"<-Thus another connection between muscle and height growth.

" The question that arises is whether 2-Ox is the cause of the cartilage thickening, in particular the articular cartilage, or whether the bone loading (by a heavier body mass) is the key factor stimulating better nutrient utilisation facilitated by 2-Ox abundance?"<-So the heavier bone mass is a confounding variable. Would anything that makes heavier bones stimulate GP's and AC's in the same way? It should be noted that LSJL makes bones heavier too but other effects unrelated to bone weight have been established due to LSJL such as fluid flow and an increase in pressure within the bone.

"Within the femur articular cartilage, the most loaded point of the knee joint, thickening was the predominant reaction to additional 2-Ox."

"Unlike articular cartilage, growth plate cartilage is a tissue which is sensitive to load and overload and may slow down the growth, which is mostly related to the hypertrophy zone of this particular tissue. And yet we observed an increase in growth plate thickness, in the present study, in the rats treated with 2-Ox. "

The question is also would byproducts of 2-Ox like arginine have similar effects and thus there would be no need for both. Since AKG is so promising in bodybuilding perhaps there are answers.

But so far, based on the results in this study it seems possible that AKG supplementation may increase height by miniscule amounts via articular cartilage thickness it may also help people grow taller via their growth plates.

Gene Expression Patterns of Bone under normal mechanical load versus LSJL

2013-01-10T12:19:00.000-08:00

Each day I go through studies to put into old posts and most of them are not that helpful but I found one that's full of one and insight's into the legendary microfracture theory sprouted by Sky and others. Search for (*NEW*) to find it. The study has a lot of great diagrams and the full study is available. It's a rich informative study so it would be beneficial for other people to look into it. Ultimately, you can see that the load required to induce a microfracture may be too much and if kicking with ankle weights induced the microfractures such as in this study you would notice them due to the massive appositional bone formation(thus if you did grow taller with such a method you'd have lots of bumps on your bones--chicks dig scars but do they dig microfractures?). However, it may be possible for tapping with a sufficient load enough times may be enough to induce such a fracture. I have no idea what that load is though.

CH Turner was one of the leading researchers behind Lateral Synovial Joint Loading before he passed away and he was the researcher who was most directly interested in bone(Hiroki Yokota for example was more interested in mechanotransduction).

CH Turner's studies are still coming out. Here's one about the gene expression of bone on mechanical loading. It will be interested to compare that to gene expression under LSJL.<-Read that study, it's important!

Gene expression patterns in bone following mechanical loading.

"The primary goal of this study was to determine the time sequence for gene expression in a bone subjected to mechanical loading during key periods of the bone-formation process, including expression of matrix-related genes, the appearance of active osteoblasts, and bone desensitization. A standard model for bone loading was employed in which the right forelimb was loaded axially for 3 minutes per day{so axial loading rather than our lateral loading, note that the epiphysis still gets loading under axial loading as there's no way to avoid it}, whereas the left forearm served as a nonloaded contralateral control. We evaluated loading-induced gene expression over a time course of 4 hours to 32 days after the first loading session. Six distinct time-dependent patterns of gene expression were identified over the time course and were categorized into three primary clusters: genes upregulated early in the time course, genes upregulated during matrix formation, and genes downregulated during matrix formation. Genes then were grouped based on function and/or signaling pathways. Many gene groups known to be important in loading-induced bone formation were identified within the clusters, including AP-1[The C-Fos and c-Jun complex]-related genes in the early-response cluster, matrix-related genes in the upregulated gene clusters, and Wnt/β-catenin signaling pathway inhibitors in the downregulated gene clusters. Chemokine-related genes, were upregulated early but downregulated later in the time course; solute carrier genes{these help with chondrocyte hypertrophy}, were both upregulated and downregulated; and muscle-related genes, were primarily downregulated{This is interesting, as many anabolic pathways are shared between muscle and bone; this gives weight to the theory that muscle and bone compete for resources}."

The Peak Load used was 13N. "Compressive load was applied as an oscillating Haversine waveform for 360 cycles at a frequency of 2 Hz" 24 hours between loading. Genes upregulated in supplementary material were taken from all time points from 4 hours to 32 days. Mice were 20 weeks old.

"Bone responds in an anabolic manner to physiologic dynamic loading. For example, the midshaft humerus in the throwing arm of baseball pitchers and catchers showed enhanced bone mass, structure, estimated strength, and resistance to torsion compared with the nonthrowing control arm.{bone length was measured but the comparative length was not presented, something to ask Stuart J. Warden} In contrast, bone mineral density (BMD) in astronauts decreased 1.0% and 1.5% in the spine and hip, respectively, per month of spaceflight{This is not that much considering astronauts are reported to gain 3 inches in space, this shows you the potential of your intervertebral discs in terms of height gain}"

"Mechanical loading uses pathways currently being investigated for new drug development, such as low density lipoprotein receptor–related protein 5 (LRP5) and sclerostin"<-supplements to look into

"New osteoblasts appear on the bone surface 24 to 48 hours after initiating mechanical loading, and bone formation is observed within 96 hours of loading. Bone formation increases between 5 and 12 days after starting loading, but after 6 weeks of loading, bone formation returns to baseline levels."

This is for osteoblasts not chondrocytes which is what we are mainly looking for with LSJL but it may take 12 days after starting LSJL to notice new osteoblast bone deposition(never mind that LSJL requires a chondrocyte phase beforehand). A month is not enough time to measure results.

The shaft of the bone was used so stem cell genes should be detected. So if any mesenchymal chondrogenesis occurred it should show up.

Looking at what was upregulated in axial Loading and Lateral Loading share many of the same pathways like TGF-Beta and WNT/B-Catenin in addition to many ECM related proteins.

Some genes involved in LSJL that were not involved in axial loading hyaluronan synthase(involved in hyaluronic acid). No MMP3 in axial loading in contrast to lateral loading(MMP-3 is stimulatory to chondrocytes).

And of course no induction of chondrogenic differentiation of stem cells in axial loading(that's more due to hydrostatic pressure though than genes). Though it should be represented by genes which I think it is given the upregulating of ECM genes. Chondrogenic differentiation produces ECM but ECM doesn't always indicate chondrogenic differentiation.

BMP-2 and TGF-Beta were produced by axial loading. Both of which can induce chondrogenic differentiation. Axial Loading + LIPUS may be enough to gain height.

Interleukin 1 receptor-like 1 was expressed by both axial and LSJL. Stat3 was expressed which induces Lin28B expression. It also downregulated FGF23 which may be involved in growth plate reactivation.

Upregulated Genes of Note(see supplementary material):
Acan(upregulated in LSJL)
ADAMTS1(up)
Adh7(up)
Angptl2(up)
Apcs(down)
Apln(up)
Arg1(up)
Bambi
Bgn(up)
BMP2(up)
c1qtnf5(up)
c3ar1(up)
Capn6(up)
Car8(up)
ccl2(up)
ccl7(up)
cd14(up)
cd276(up)
CCND1
Cdh15(down)
Cgref1(up)
Chgb(down)
Cit(down)
Cntn1(up)
Fn1
Follistatin
Cntn1(up in LSJL)
Col2A1 alpha1(up in LSJL)
Col3A1 alpha1(up in LSJL)
Col16A1(up)
crabp2(up)
creb3l1(up)
cspg4(up)
cthrc1(up)
cxcl1(up)
dbx1(down)
dlg4(up)
dnm1(up)
ENPP3(down)
fkbp10(up)
Gfpt2(up)
ggcx(up)
glrb(up)
GPR180(down in LSJL)
grin2d(up)
Hapln1(up in LSJL)
Hdlbp(down)
Hif1alpha
Hnf4a(down in LSJL)
HTRA1(up)
Id2(down in LSJL)
il1rl1(up)
IRS-1(down in LSJL)
Junb(also upregulated in LSJL)
Kcnn2(up)
Lepre1(up)
Leptin
lmna(up)
lox(up)
Lrat(up)
Mall(up)
Metrnl(down)
MMP2(up in LSJL)
MMP9
MMP14(up in LSJL)
NDRG4(up)
Neurod2(down)
Ninj1(down)
Nkx2.5
Nos3
Nr4a2(up)
Pacsin1(down)
Pcdhb2(up)
pcsk6(up)
pdgfc(up)
pdpn(up)
prrx1(up)
prss35(up)
PTHR1
PTGS2(up in LSJL)
PTN(up in LSJL)
RPL36al(down)
S100A4(up)
Scn1a(up)
Sct(down)
Serpina3n(up)
Serpine1(up)
Sept5(down)
Slc1a4(up)
Slc6a2(up)
Slc6a15(up)
Slco2a1(up)
Smad9(up)
Smpd3(down)
SOCS2(Anti-height gene)
SOCS3(upregulated in LSJL)
Sp7
Stat3
Syndecan 4(also upregulated in LSJL)
TGFbp1
TGFbp3
tnfrsf12a(up)
TIMP1(up in LSJL)
Vcan(up in LSJL)
VDR
Zfp36(upregulated in LSJL)

Axial loading upregulated a few chondrogenic genes like Acan and COL2A1 but nowhere near the amount of Collagens upregulated by LSJL which also upregulated Col9. Also key, is that Sox9 is not upregulated in axial loading whereas it is in LSJL.

Downregulated genes of note:
Acacb(down)
Acsl6(down)
Anxa3(down in LSJL)
Arl6ip1(down)
Asb2(down)
Asph(up)
BMPR1B(up in LSJL)
Btla(up)
ccnb1(down in LSJL)
ccr1(up)
dpp4(down)
Egr1(up in LSJL)
Fgr(down)
Fnbp1(down)
GADD45A
Galc(down)
Gas6
Ghitm(down)
GHR
IGFBP6(up in LSJL)
Kynu(up)
Leptin Receptor
Mkrn1(down)
Mrps18b(up)
Myl1(up)
Nexn(down)
Ntn1(up)
Pcsk1(up)
Pdlim3(up)
Pkia(up)
Plag1
Ppp1r3c(up)
Prkaa2(down)
Prkg2(up in LSJL)
Pygl(down)
Rsad2(down as Pcaf)
Sdpr(down)
Sla(down)
Slc16a1(down)
Slc25a30(down)
Sost
Srpkg3(down)
TGFBR3
Tnnt3(down)
Trim55(down)
Tsc22d3(down)
Ucp2(down)
Vav1(down)
Vcam1(down in LSJL)

The differential expression of Egr and BMPR1B between Axial Loading and Lateral Loading could be key to LSJL's ability to induce chondrogenesis.

The gene expression data for LSJL was taken 1 hour after the last loading and in this study genes were taken 4 hours after the first loading. So we can compare these early response genes to see how they compare to LSJL.

Upregulated:
Fosl1
Junb(up in LSJL)
Anxa2
S100A4(up)
S100A10
CCBP2
CCL2(up)
CCL7(up)
CXCL1(up)
CXCL13
IL1RL1(up)
IL1RL2
Osm
Osmr
Socs3(up)
Stat3
Tnfrsf12a(up)
Adamts1(up)
ECM1
Serpina3n(up)
Serpine1(up)
Tfpi2
CCND2
Clic1
Gpr1
KCNE4
Lep
Syndecan4 (up)

Regulatory mechanisms in bone following mechanical loading.

"The right forelimb [of rodents] was loaded axially for three minutes per day, while the left forearm served as a non-loaded, contralateral control. Animals were subjected to loading sessions every day, with 24 hours between sessions. Ulnas were sampled at 11 time points, from 4 hours to 32 days after beginning loading."

Mice were 20 weeks old.

"The peak load achieved during loading was 13 N"

Stat5B was upregulated 4 hours following loading. Stat5b is downregulated in LSJL

The expression of COL1 differs greatly with LSJL. At 14 days there was almost no COL1 expression whereas at 14 days there was still extremely high COL1 levels with Axial Loading.

"The CREB-related transcription factors are important for bone formation, specifically ATF4, which is required for collagen synthesis by mature osteoblasts. The CREB motif was predicted to be positive at 2d, 4d, 6d, and 8d. The transcription factors that bind to the CREB motif include cAMP responsive element binding protein 1 (CREB1), cAMP responsive element modulator (CREM), activating transcription factor 1 (ATF1), ATF2, ATF3, ATF4, and ATF7. The CREB motif was present in the promoter of an important matrix gene, fibronectin 1 (Fn1), and in genes that promote collagen construction and cross-linking, including Lox, prolyl 4- hydroxylase beta polypeptide (P4hb), and procollagen C-endopeptidase enhancer (Pcolce)"

"at 32d, the system was less responsive to loading and had shifted from bone forming to baseline bone maintenance"<-maybe every 32 days take a break from LSJL?

Collagen 1 alpha 1 did not begin to rise until two days after loading. The LSJL study took gene expression at 49 days after first loading. In the axial loading Col1A was upregulated 3-fold seven days after loading whereas with LSJL it was upregulated only 2 fold.

Alternative Splicing in Bone Following Mechanical Loading

Alternative splicing means that gene expression was altered in mRNA. The whole bone was ground.

"Compressive load was applied as an oscillating Haversine waveform for 360 cycles at a frequency of 2 Hz using a Bose ElectroForce 3200 Series electromechanical actuator"<-Peak Load was 13N. Axial loading was used.

"Rats were subjected to loading sessions every day, with 24 hours between sessions." Rats were 20 weeks old. Gene expression data was taken up to 4 hours to 32 days.

The greatest alteration of gene expression occurred at 16 days or about 2 weeks. Maybe this is when conditioning effect starts to inhibit gene expression?

According to this Study Sox9 mRNA was not altered at any time point. Col2a1and Acan mRNA were altered. Tgfbeta1 and Tgfbeta2 expression was altered. The key stature genes HMGA2 and Lin28b were altered in LSJL but not here.

Altered genes of note:

Akt1

Akt2

BMPr1a

BMPr1b{up in LSJL}

BMP2{up in LSJL}

BMP4

CNP

CREB3l1

Esrra

Esrrb

Esr2{up in LSJL}

FGF2{up in LSJL}
FGF4

FGF21

FGFR1{up}

FGFR3

GH1{down in LSJL}

GHR

GHRHR

GPC3

HMGA1

ID2{down}

ID4

IGF1

IGF1R

IGF2R

IGF2bp1

NPR1

NPR2

NPR3

PLAG1

PRKG2{up}
RARA

Runx3

Shh

SHOX2

Smad1{down}

Smad2

Smad3

Smad4

Smad5

Smad7(inhibits BMP signaling, Smad6 which inhibits TGF-Beta signaling is not altered)

Smad9

Sox10

Sox11

Syn3

Twist1

Wnt4

Wnt5a

Here's the Partek GSEA Analysis to compare to LSJL for chondrogenic related genes, Bold means the p-value < 0.05, no fold changes were given and no fold cutoff is used:

Chondroblast Differentiation(6.77) 100%:
RARA
FGF4
FGF2
Cyr61

Chondrocyte Differentiation(5.16) 45%:
Col2a1
Creb3l2
MAPK14
Col11a2
TGFB1
Mef2d
FGFR1
OSR1
FGF9

Cartilage Condensation(8.32) 58.32%:
THRA
COL2A1
Tgfb2
Uncx
Ctgf
Bmpr1b
Acan

Chondrocyte Development(1.80) 33.33%<-this could be a key between LSJL and axial loading which has an enrichment score of 3.80.

Cartilage Development(12.70) 44.2%

Cartilge Development Involved in Endochondral Bone Morphogenesis(2.10) 42.8%

Growth Plate Cartilage Development(0.27) 14.29%

Endochondral Ossification(1.66) 29.41%

Systemic effects of ulna loading in male rats during functional adaptation.

"The aim of this study was to determine the effects of loading of a single bone on adaptation of other appendicular long bones and whether these responses were neuronally regulated. Young male Sprague-Dawley rats were used. The right ulna was loaded to induce a modeling response. In other rats, a second regimen was used to induce bone fatigue with a mixed modeling/remodeling response; a proportion of rats from each group received brachial plexus anesthesia to induce temporary neuronal blocking during bone loading. Sham groups were included. Left and right long bones (ulna, humerus, tibia, and femur) from each rat were examined histologically 10 days after loading. In fatigue- and sham-loaded animals, blood plasma concentrations of TNF-α, RANKL, OPG, and TRAP5b were determined. Loading the right ulna induced an increase in bone formation in distant long bones that were not loaded and that this effect was neuronally regulated{LSJL increased length in bones not loaded}. Distant effects were most evident in the rats that received loading without bone fatigue. In the fatigue-loaded animals, neuronal blocking induced a significant decrease in plasma TRAP5b at 10 days. Histologically, bone resorption was increased in both loaded and contralateral ulnas in fatigue-loaded rats and was not significantly blocked by brachial plexus anesthesia. In young, growing male rats we conclude that ulna loading induced increased bone formation in multiple bones. "

"The periosteum is the skeletal tissue with the greatest density of sensory nerve fibers, which are arranged in a dense netlike meshwork that is optimized for detection of mechanical distortion. Nerve branches or single neurons enter the bone cortex, often in association with the microvasculature, and connect individual bone cells to the central nervous system via unmyelinated sensory neurons."

"In the load and block + load groups, loading was performed for 1500 cycles at 4 Hz, with an initial peak strain of −3,750 µɛ (−18 N entered into materials testing machine, −16.8 N applied to ulna). In the fatigue and block + fatigue groups, cyclic loading was performed at 4 Hz. Loading was initiated at −16 N, and the load applied to the ulna was increased incrementally until fatigue was initiated. Loading then was terminated when 40% loss of stiffness was attained."

"TRAP5b is expressed on both immature and mature osteoclasts; plasma TRAP5b concentrations are proportional to osteoclast number."

(*NEW*)

Healing of non-displaced fractures produced by fatigue loading of the mouse ulna.

"Using adult (5 month) C57Bl/6 mice, we first determined that cyclic compression of the forelimb under load-control leads to increasing applied displacement and, eventually, complete fracture. We then subjected the right forelimbs of 80 mice to cyclic loading (2 Hz; peak force approximately 4N) and limited the displacement increase to 0.75 mm (60% of the average displacement increase at complete fracture). This fatigue protocol created a partial, non-displaced fracture through the medial cortex near the ulnar mid-shaft, and reduced ulnar strength and stiffness by >50%. Within 1 day, there was significant upregulation of genes related to hypoxia (Hif1a) and osteogenesis (Bmp2, Bsp) in loaded ulnae compared to non-loaded, contralateral controls. The gene expression response peaked in magnitude near day 7 (e.g., Osx upregulated 8-fold), and included upregulation of FGF-family genes (e.g., Fgfr3 up 6-fold). Histologically, a localized periosteal response was seen at the site of the fracture; by day 7 there was abundant periosteal woven bone surrounding a region of cartilage. From days 7 to 14, the woven bone became denser but did not increase in area. By day 14, the woven-bone response resulted in complete recovery of ulnar strength and stiffness, restoring mechanical properties to normal levels. In the future, the fatigue loading approach can be used create non-displaced bone fractures in transgenic and knockout mice to study the mechanisms by which the skeleton rapidly repairs damage."

Monotonic loading: "Both forelimbs of five mice were loaded by a displacement ramp (0.5 mm/sec) to complete, displaced fracture in order to determine monotonic mechanical properties. Mice were euthanized immediately after loading. Ultimate force (mean ± SD) was 4.32 ± 0.21 N, and stiffness was 3.98 ± 0.26 N/mm."

Fatigue loading: "Both forelimbs of 14 mice were cyclically loaded at peak compressive forces (F) ranging from 2.1 to 3.5 N (50 to 80% of average ultimate force) until complete fracture. "

Force loading, to partial non-displaced fracture: "Right forelimbs of 80 mice were cyclically loaded at peak forces ranging from 3.75 to 4.10 N (70–75% of ultimate force) while displacement was monitored. Loading was terminated when peak displacement increased by 0.75 mm relative to the peak displacement at cycle 10."

The key here is to see if any chondrogenic genes were upregulated in partial, non -displaced fractures(so like a microcrack). Col2a1 was highly upregulated at day 7. BMP2[1.1-1.9 fold] and FGF2[1.1-1.8 fold] were moderately upregulated. FGF2 was more highly upregulated in LSJL than here whereas BMP2 was more highly upregulated here than LSJL. Hif1a the chondrogenically related transcription factor was more significantly upregulated peaking at 3.0 at day 3 and increasing before and decreasing after.

It should be noted that LSJL gene expression was done by microarray whereas this study was done with RT-PCR with the exception of BMP2 which was also done by PCR.

Here's what a bone microfracture-microcrack looks like:

M stands for marrow. CB stands for cortical bone. WB stands for woven bone.

"Longitudinal sections of fatigue-loaded ulnae (H&E) show that the fracture occurred as a non-displaced, oblique crack through the medial cortex (arrows). On day 1 after loading, a clot is seen on both ends of the crack. On day 3, the periosteum is expanded and filled with cellular, fibrovascular tissue; nascent woven bone is seen sub-periosteally. On days 7 and 11 there is abundant woven bone on the medial periosteum. In approximately one-half of specimens the callus contained no cartilage (not shown), but in the others there was cartilage (*) in the center of the woven bone."

"Cyclic loading of the rat forelimb (~18 N peak force) to 85% of fracture displacement resulted in a non-displaced fracture localized to the medial cortex of the ulna and an associated loss of ulnar strength and stiffness of 55 and 80%, respectively. Because the fracture in the rat ulna was partial and non-displaced, and because the repair process involved negligible cartilage formation, we referred to this as a “stress fracture”, consistent with descriptions by others"<-now we've considered that that 0.5N in LSJL is equivalent to 100N on a 200lbs human which is already a challenge. Imagine the challenge of generating 18N peak force. It's possible that less force may be needed if the force is applied cyclically over a long period of time as long as that force causes residual damage in the bone.

"Cartilage was often observed at 7 and 11 day timepoints and appeared only on the medial surface, corresponding to the periosteal fracture location. By comparison, in studies of complete fracture in mice cartilage is seen on both sides of the bone as well as between the fractured ends"

The fracture occurred on the medial side and although the majority of the activity is on the medial side there is some enhanced activity on the lateral side giving weight to the possibility of gradually lengthening the bone through microfracture(as the unfractured side does seem to adapt). Although the force required to induce a sufficient microfracture may be too large to be induced under normal physiological circumstances(and you would notice a bone adaptation as large as that depicted). That does not preclude the possibility that rapid loading that is large enough to induce residual damage to bone is enough to induce such a microfracture as well. Something like tapping.

Tibial loading increases osteogenic gene expression and cortical bone volume in mature and middle-aged mice.

"We examined this question in female BALB/c mice of different ages, ranging from young to middle-aged (2, 4, 7, 12 months). We first assessed markers of bone turnover in control (non-loaded) mice. Serum osteocalcin and CTX declined significantly from 2 to 4 months. There were similar age-related declines in tibial mRNA expression of osteoblast- and osteoclast-related genes, most notably in late osteoblast/matrix genes. For example, Col1a1 expression declined 90% from 2 to 7 months. We then assessed tibial responses to mechanical loading using age-specific forces to produce similar peak strains (-1300 µε endocortical; -2350 µε periosteal). Axial tibial compression was applied to the right leg for 60 cycles/day on alternate days for 1 or 6 weeks. qPCR after 1 week revealed no effect of loading in young (2-month) mice, but significant increases in osteoblast/matrix genes in older mice. For example, in 12-month old mice Col1a1 was increased 6-fold in loaded tibias vs. controls. In vivo microCT after 6 weeks revealed that loaded tibias in each age group had greater cortical bone volume (BV) than contralateral control tibias, due to relative periosteal expansion. The loading-induced increase in cortical BV was greatest in 4-month old mice (+13%). Non-loaded female BALB/c mice exhibit an age-related decline in measures related to bone formation. Yet when subjected to tibial compression, mice from 2-12 months have an increase in cortical bone volume. Older mice respond with an upregulation of osteoblast/matrix genes, which increase to levels comparable to young mice."

Unfortunately, no chondrogenic genes were studied.

Global gene expression analysis in the bones reveals involvement of several novel genes and pathways in mediating an anabolic response of mechanical loading in mice

"We applied mechanical loads[4-point bending] to the right tibias of the B6 mice at 9 N, 2 Hz for 36 cycles per day, with the left tibias used as unloaded controls"

"4 days of loading"

"Twenty-four hours after last stimulation"<-whereas LSJL was 1 hour after last stimulation.

"Ten-week-old C57BL/6J female mice"

Complete list of supplementary gene comparison to LSJL to be done. Gene comparison of just genes on main paper and spot comparisons done below.
Genes upregulated in bone to four point bending also upregulated by LSJL:
Ptn
Ogn{down}
Itm2a
Lepre1
Col6a3
Col14a1
Col18a1
Matn2
Lox
Gas1
Timp1
Acta2
Ppfibp1{down}
Fer1l3
Spon2
Wnt2
Lmna
Sgk
Odz3
Anxa8
Chl1
Adamts4
Tcf12{down}
Col4a2
Junb
Tnc{down}
Bgn
Egfr
MMP2
BSP

Downregulated:
Mkrn1

Grow Taller with Folinic Acid

2013-01-07T09:21:00.000-08:00

For the first time, there is evidence that Folinic Acid treatment may increase length in developing individuals see (*NEW*). Considering that Folinic Acid should not be harmful to length development, if an individual has active growth plates it's definitely a supplement one should add to their regime.

Hypermethylation of the growth plate chondrocytes may be a way to induce supranatural height growth. DNA Methyltransferase stopping the addition of methyl groups to stem cell chondrocytes in the resting zone may be an indication to cease height growth. High levels of HGH may also cause hypermethylation explaining gigantism(but why Gigantism is not inducible in all with HGH is unclear). Folinic Acid is available as a supplement:Folinic Acid 800 mcg.

Are there any other ways of inducing DNA Hypermethylation(Bare in mind that DNA Hypermethylation increases the spread of cancer as DNA Methylation is one of the negative feedback mechanisms on cell growth)? And since hypermethylation may not be possible to induce in growth plates general methods of increasing DNA synthesis like Folinic Acid may have similar effects.

Damaging effects of chronic low-dose methotrexate usage on primary bone formation in young rats and potential protective effects of folinic acid supplementary treatment.

"Methotrexate (MTX) is a most commonly used anti-metabolite in cancer treatment and as an anti-rheumatic drug. MTX chemotherapy at a high dose is known to cause bone growth defects in growing bones. We examined effects on bone growth of long-term MTX chemotherapy at a low dose in young rats, and potential protective effects of supplementary treatment with antidote folinic acid (given ip at 1 mg/kg 6 h after MTX). After two cycles of 5 once-daily MTX injections (at 0.75 mg/kg, 5 days on/9 days off/5 days on), MTX at this dose caused significant reduction in heights of growth plate and primary spongiosa bone on day 22 compared to controls. In contrast, a similar dosing regimen but at a lower dose (0.4 mg/kg) caused only slight or no reduction in heights of both regions. However, after the induction phase at this 0.4 mg/kg dosing, continued use of MTX at a low dose (once weekly at 0.2 mg/kg) caused a reduction in primary spongiosa height and bone volume on weeks 9 and 14, which was associated with an increased osteoclast formation and their bone surface density as well as a decreased osteoblast bone surface density in the primary spongiosa. Folinic acid supplementation was shown able to prevent the MTX effects in the primary spongiosa. Acute use of MTX can damage growth plate and primary bone at a high dose, but not at a low dose. However, long-term use of MTX at a low dose can reduce primary bone formation probably due to decreased osteoblastic function but increased osteoclastic formation and function, and supplementary treatment with folinic acid may be potentially useful in protecting bone growth during long-term low-dose MTX chemotherapy."

Folinic Acid(leucovorin) prevents damage to the bone marrow. It may help by reducing the number of micronuclei.

"Low-dose MTX is able to inhibit other folate-dependent enzymes such as thymidylate synthase, which further blocks the de novo purine synthesis by directly inhibiting the activity of 5-aminooimidazole-4-carboxamide ribonucleotide transformylase, causing an increase in both intracellular and extracellular adenosine (an potent anti-inflammatory mediator), and an increase in cAMP"

No data was taken on Folinic Acid treatment alone.

A comparison of vitamin A and leucovorin for the prevention of methotrexate-induced micronuclei production in rat bone marrow.

"Methotrexate [is] a folate antagonist. In rats, methotrexate is known to induce micronuclei formation, leading to genetic damage, while vitamin A is known to protect against such methotrexate-induced genetic damage. Leucovorin (folinic acid) is generally administered with methotrexate to decrease methotrexate-induced toxicity.
We aimed to determine whether vitamin A and leucovorin differed in their capacity to prevent formation of methotrexate-induced micronuclei in rat bone marrow erythrocytes. The present study also aimed to evaluate the effect of combined treatment with vitamin A and leucovorin on the formation of methotrexate-induced micronuclei.
Male and female Wistar rats were injected with 20 mg/kg methotrexate (single i.p. dose). The control group received an equal volume of distilled water. The third and fourth groups of rats received vitamin A (5000 IU daily dose for 4 successive days) and leucovorin (0.5 mg/kg i.p. dose for 4 successive days), respectively. The fifth and sixth groups of rats received a combination of vitamin A and a single dose of methotrexate and a combination of leucovorin and methotrexate, respectively. The last group of rats received a combination of leucovorin, vitamin A and single dose of methotrexate. Samples were collected at 24 hours after the last dose of the treatment into 5% bovine albumin. Smears were obtained and stained with May-Grunwald and Giemsa. One thousand polychromatic erythrocytes were counted per animal for the presence of micronuclei and the percentage of polychromatic erythrocyte was determined.
Comparison of methotrexate-treated rats with the control group showed a significant increase in the percentage of cells with micronuclei and a significant decrease polychromatic erythrocyte percentage. Combined methotrexate and vitamin A therapy and combined methotrexate and leucovorin therapy led to significant decreases in the micronuclei percentage and an increase in polychromatic erythrocyte percentage when compared to rats treated with methotrexate alone. Leucovorin was found to be more effective than vitamin A against the formation of methotrexate-induced micronuclei."

"rats treated with a combination of methotrexate and vitamin A had a significantly reduced frequency of micronuclei formation when compared to methotrexate (20mg/kg)-treated rats"

Leucovorin helps to prevent genetic damage. Does it do it in cases not caused by Methotrexate?

Inhibitory effect of folinic acid on radiation-induced micronuclei and chromosomal aberrations in V79 cells.

"Folinic acid (FA), clinically called leucovorin, has been widely used as a nutrient supplement in dietary intake and is capable of inhibiting cytotoxicity and chromosomal damage induced by chemicals. However, data on its antigenotoxic effect on radiation-induced chromosomal damage are limited. The present study was, therefore, performed to investigate the effect of FA on radiation-induced (X-rays and UV radiation) micronuclei (MN) and structural chromosomal aberrations (SCA) concurrently in V79 Chinese hamster lung cells. Exponentially growing cells were exposed to five doses of X-rays (1-12 Gy) and UV radiation (50-800 microJ x 10(2)/cm2) and post-treated with 5 or 50 micrograms FA/ml of culture medium for 16 h. The slides were analyzed for the presence of MN and SCA using standard procedures. X-ray treatment alone produced dose-related cytotoxicity. X-rays produced a clear dose-related clastogenicity as measured by percent of micronucleated binucleated cells (MNBN) (5-79%) and percent of aberrant cells (11-92%). FA at 5 micrograms/ml slightly decreased X-ray induced chromosomal damage in both assays; however, the inhibition was significant (12-46% of MNBN, 14-48% in aberrant cells) only when X-ray-treated cultures were post-treated with 50 micrograms FA/ml. Post-treatment of FA had no effect on X-ray induced cytotoxicity as measured by NDI and MI. A similar a dose-related increase in % MNBN (0.5-10.3%) and percent aberrant cells (6-35%) was produced by UV radiation treatment alone. There were significant percentages of MNBN and aberrant cell inhibitions at both 5 and 50 micrograms/ml in both assays. As in the case of X-ray-treated cells, there was a clear dose-related cytotoxicity in UV-treated cells alone. No reduction in NDI or MI was found when UV-exposed cells were post-treated with 5 or 50 micrograms of FA. FA [decreases] radiation-induced chromosomal damage."

"X-rays produce DNA double-strand breaks (DSBS), DNA single-strand breaks(SSBs), base damages and DNA-protein cross-links and that DSBs are the main lesions responsible for chromosome aberrations"

Folinic Acid protects against many sources of DNA damage(and DNA damage could include damage to DNA Methyltransferase explaining it's growth stunting properties).

Low expression of gamma-glutamyl hydrolase mRNA in primary colorectal cancer with the CpG island methylator phenotype.

"The CpG island methylator phenotype (CIMP+) in colorectal cancer (CRC) is defined as concomitant and frequent hypermethylation of CpG islands within gene promoter regions. We previously demonstrated that CIMP+ was associated with elevated concentrations of folate intermediates in tumour tissues[elevated folate levels may cause hypermethylation]. In the present study, we investigated whether CIMP+ was associated with a specific mRNA expression pattern for folate- and nucleotide-metabolising enzymes. An exploratory study was conducted on 114 CRC samples from Australia. mRNA levels for 17 genes involved in folate and nucleotide metabolism were measured by real-time RT-PCR. CIMP+ was determined by real-time methylation-specific PCR and compared to mRNA expression. Candidate genes showing association with CIMP+ were further investigated in a replication cohort of 150 CRC samples from Japan. In the exploratory study, low expression of gamma-glutamyl hydrolase (GGH) was strongly associated with CIMP+ and CIMP+-related clinicopathological and molecular features. Trends for inverse association between GGH expression and the concentration of folate intermediates were also observed. Analysis of the replication cohort confirmed that GGH expression was significantly lower in CIMP+ CRC. Promoter hypermethylation of GGH was observed in only 5.6% (1 out of 18) CIMP+ tumours and could not account for the low expression level of this gene. CIMP+ CRC is associated with low expression of GGH, suggesting involvement of the folate pathway in the development and/or progression of this phenotype. Further studies of folate metabolism in CIMP+ CRC may help to elucidate the aetiology of these tumours and to predict their response to anti-folates and 5-fluorouracil/leucovorin."

Folate is necessary for hypermethylation as Folate is a vitamin essential for DNA synthesis. Although, excess levels of Folate will likely just be urinated out.

Folate and cancer: how DNA damage, repair and methylation impact on colon carcinogenesis.

"Inappropriate diet may contribute to one third of cancer deaths. Folates, a group of water-soluble B vitamins present in high concentrations in green, leafy vegetables, maintain DNA stability through their ability to donate one-carbon units for cellular metabolism. Folate deficiency has been implicated in the development of several cancers, including cancer of the colorectum, breast, ovary, pancreas, brain, lung and cervix. Generally, data from the majority of human studies suggest that people who habitually consume the highest level of folate, or with the highest blood folate concentrations, have a significantly reduced risk of developing colon polyps or cancer. However, an entirely protective role for folate against carcinogenesis has been questioned, and recent data indicate that an excessive intake of synthetic folic acid (from high-dose supplements or fortified foods) may increase human cancers by accelerating growth of precancerous lesions. Nonetheless, on balance, evidence from the majority of human studies indicates that dietary folate is genoprotective against colon cancer. Suboptimal folate status in humans is widespread. Folate maintains genomic stability by regulating DNA biosynthesis, repair and methylation. Folate deficiency induces and accelerates carcinogenesis by perturbing each of these processes."

Folic Acid (Nature Made Folic Acid Supplement, 400 mcg, 250-Count Tablets (Pack of 3)) as well as Folinic Acid can maximize height growth by protecting against DNA damage. But excess folic or folinic acid are only necessary but not sufficient conditions for DNA Methylation to occur. Every child undergoing growth should ensure that they have appropriate quantities of folic and folinic acid to maximize height growth.

DNA Hypermethylation needs something else to occur and one suggested mechanism is S-Adenosyl methionine.

(*NEW*)

Effect of methotrexate and folinic acid on skeletal growth in mice.

"Four equal groups of Balb/c young male mice (6 animals in each group; mean body weight 11.9 +/- 0.25 g, in their rapid growth phase[3 weeks]) were subjected to the following drug treatment for a period of 3 wk. Group 1 was given intraperitoneal MTX (3.5 mg kg(-1) body weight) every second day. Group 2 received folinic acid (7.0 mg kg(-1) body weight) intraperitoneally every second day. Group 3 was given both drugs (MTX every second day and folinic acid 8 h post-MTX injection). Group 4 was injected with physiological saline every other day to serve as a control group.

Mean lengths of both the tibia and femur of animals were compared in the four treatment groups. A significant decrease in the mean lengths was observed in the group receiving MTX alone. Similarly, there was a significant decrease in the height of the femoral and tibial growth plate in this group when compared with the other groups. The main effect of MTX seemed to be on the hypertrophic proliferative zone of chondrocytes in the growth plate. Furthermore, animals in this MTX-treated group also showed increased levels of MTX in plasma and low levels of erythrocyte folate{thus folate deficiency may play a role in the length decrease}.

Chronic administration of MTX induces suppression of skeletal growth in mice, possibly through the inhibition of the pathway of de novo DNA synthesis{thus other compounds that inhibit new DNA synthesis may also decrease height}. Folinic acid treatment following MTX administration appears to reverse this growth inhibition."

Folinic Acid alone non-significantly increased both femur and tibia length.

A is saline, B is folinic acid, C is MTX, D is MTX + folinic acid. The growth plate quality seems higher in group B.

Same groups as above. Again, GP with highest quality is B. What's interesting to note is that in D the hypertrophic zone seems to be disorganized but the proliferative zone seems expanded. Therefore, Folinic Acid may revert MTX growth inhibition by more an expansion of the proliferative zone rather than reverting all the effects of MTX.

Growth plate height was non-significantly higher in Folinic Acid group than in the other groups by almost 10% for the femur. I think why it was shown as non-significant is that the p value chosen was very low p <0.001.

Folinic Acid increased folate levels in erythrocytes by about 40%.

Prevention of bone growth defects, increased bone resorption and marrow adiposity with folinic acid in rats receiving long-term methotrexate.

"chronic folinic acid supplementation can prevent methotrexate-induced chondrocyte apoptosis and preserve chondrocyte columnar arrangement and number in the growth plate. In the metaphysis, folinic acid supplementation can preserve primary spongiosa heights and secondary spongiosa trabecular volume by preventing osteoblasts from undergoing apoptosis and suppressing methotrexate-induced marrow adiposity and osteoclast formation. Systemically, plasma of folinic acid supplemented rats, in comparison to plasma from rats treated with MTX alone, contained a significantly lower level of IL-1β and suppressed osteoclast formation in vitro in normal bone marrow cells. The importance of IL-1β in supporting plasma-induced osteoclast formation was confirmed as the presence of an anti-IL-1β neutralizing antibody attenuated the ability of the plasma (from MTX-treated rats) in inducing osteoclast formation."

This study did not study Folinic Acid supplementation without MTX.

Are you shorter after you go to the gym?

2013-01-02T15:55:00.003-08:00

If we can maintain our temporary spinal height, we can maintain our morning height into the night.

The effects of upper limb loading on spinal shrinkage during treadmill walking.

"Walking tasks were performed on seven healthy males and motion analysis was used to track four reflective markers at 100 Hz, dividing the spine into three segments. Static data was collected in 5-min intervals over a 30-min period{the height loss probably tapers off after a certain point but the study was too short, however height loss started to taper dramatically between 25 and 30 minutes}.

Total spinal length and lumbar segment decreased with respect to time. Load affected the percentage length change at each spinal segment{so the more time you spend doing the treadmill and the heavier the load on each spinal segment the more (temporary) height you lose}, with the lumbar segment showing greatest height loss at the highest load. The upper and lower thoracic segments showed greater anterior lean with the heavier loads and the lumbar segment showed the opposite trend.

The body adopts less anterior lean with an immediate load-bearing demand, to decrease the necessary extension moment generated by the spinal extensors for spinal stability. Further postural alteration in the same direction is observed with prolonged loading. In combination with lumbar spinal shrinkage, such postural changes are likely to increase the loading on the facet joints and subsequently unload the discs which may be beneficial for those with low back pain."

"The shrinkage that occurs during walking and load carriage is part of the normal diurnal height change where approximately 1 % of total stature loss occurs throughout the day"

"Before each trial participants were required to lie in a supine position for 2 h to standardise the baseline condition of the spine. After 1 h 45 min, the participants changed into black Lycra™ shorts and height and weight were recorded using a standard stadiometer "

"On day 1 they walked unloaded, acting as the control and on days 2 and 3 carrying randomly assigned loads, in a similar fashion to shopping bags, equivalent to 7.5 and 15 % body weight, respectively, equally distributed across both arms. Participants refrained from physical activity for the 48 h preceding each laboratory visit. "

It should be noted that 15% loading actually reduced height loss in the upper and lower thoracic spinal segment and possibly even resulted in height gain in the lower thoracic segment. Although 15% loading dramatically increased height loss.

"The reduction in both anterior lean during instantaneous loading and with time and the straightening of thoracolumbar curvature with load are both suggestive of a spinal shape compensation mechanism to reduce the flexion moment of the load about the lumbar spine."

Stature loss from sustained gentle body loading.

"The effects of low levels of loading on spinal creep have been investigated in nine young men aged between 19 and 24 years. Subjects were measured on a precision stadiometer before and after 25 minutes of free standing and quiet walking, and the resultant stature losses compared with the more substantial losses observed following steady running. All measurements were made following the same presession routine, at exactly the same time on three separate mornings. It was observed that while no appreciable stature loss followed standing (mean = 0.01 mm; SE = 0.65 mm) even quiet walking had a measurable and statistically significant effect (mean = 1.82 mm; SE = 0.49 mm). Compared with these, steady running produced almost 2 1/2 times as great a loss (mean = 4.32 mm; SE = 0.83 mm)."

Standing causes almost no impact so maybe impact is the primary cause of the temporary height loss and not gravity as the height loss following standing was minimal.

Body mass as a factor in stature change

"Twenty volunteers were divided into two equal groups; obese: BMI > 30 kg/m2, non-obese: BMI < 25 kg/m2. Stature was measured at 3 min intervals during a 30 min walking task and a 30 min standing recovery period. Tests were performed on two occasions, once with participants loaded during the walking task (10% body mass) and once unloaded.

In both groups the stature loss was greater in the loaded than unloaded condition (mean (SD)) (6.52 (1.45) mm and 3.55 (0.93) mm non-obese; 8.49 (1.75) mm and 7.02 (1.32) mm obese). The obese presented a greater reduction in stature in both task conditions. The obese group were unable to recover stature regardless of the task condition during the recovery period (loaded: 0.06 (0.3) mm; unloaded: 0.32 (0.6) mm)."

"The non-obese individuals were able to regain approximately 76% of their initial stature during the standing recovery in contrast to the obese group who did not recover from loading. Some of the obese individuals continued to loose stature during the standing recovery period (−0.7% loaded; −4.55% unloaded)."<-So maybe pure loading does have an effect and not just impact. Although it could be due to inflammatory microenvironment caused by obesity that inhibits statural recovery. One way to test this would be to take very heavy and muscular individuals to see if the effect is due to weight or changes in the microenvironment caused by fat.

Relationship between everyday activities and spinal shrinkage

"spinal load was ascertained by stadiometric measurement of the decrease in standing height, “spinal shrinkage”, quantified by the exposure of a 1-h adopted posture or activity. Ten subjects performed five daily life activities: standing, sitting, walking, cycling and lying down.

By doing different activities during 1 h, immediate after getting up in the morning, following average values for shrinkage were measured: standing −7.4 mm (SD 0.5); sitting −5.0 mm (SD 0.6); walking −7.9 mm (SD 0.5); cycling −3.7 mm (SD 0.4) and lying down +0.4 mm (SD 0.5).{interesting that this study got similar measures for standing and walking in contrast to the study which found only a 0.01mm loss after 25 minutes}"

"remaining in bed after a normal night’s rest mostly does not further increase spinal height"

"Spinal shrinkage was correlated with mean IDP[intradiscal pressure] values during these activities, i.e., for lying down 0.11 MPa (ranging from 0.10 MPa for lying prone to 0.12 MPa for lying laterally); relaxed standing 0.50 MPa; sitting 0.38 MPa (ranging from 0.30 MPa for nonchalant sitting to 0.46 MPa for sitting unsupported); walking 0.59 MPa (ranging from 0.53 MPa to 0.65 MPa)."

"spinal shrinkage during sitting on a chair with seat pan moving in the horizontal plane was also lower than in the static sitting situation"

Length of the spine while sitting on a new concept for an office chair.

"Changes in spinal length were used to evaluate a new concept for an office chair. This dynamic chair imparts passive forced motion to the seated subject. The passive forced motion is a rotary movement about an axis, perpendicular to the seat with amplitude of 0.6 degrees and a frequency of 0.08 Hz. Change of stature is assumed to provide a measure for spinal load. Eight subjects were measured in two situations: static (without motion) and dynamic. In both situations the same office tasks were performed and the duration of the sitting period was 1 h. To allow for the normal shrinkage curve the starting time was the same on each of the measurement days. The results indicated a significant difference: when sitting on the dynamic chair the average spinal length increased in comparison to the spinal length in the static chair, where average spinal length decreased."

All but one of the eight subjects increased height in the dynamic versus one individual who lost the same amount of height in both the static and dynamic chair.

"The intervertebral disc is capable of increasing its thickness because of the swelling pressure."

Changes in spine length during and after seated whole-body vibration.

"The authors examined the relation between exposure to seated whole-body vibration (WBV) and an increase in the loss of height of the spine over and above normal diurnal changes. The mean change in body height (diurnal reduction) during two normal days in five men aged 23 to 25 years was 10.6 mm, (SD, 3.2 mm). On the third day, the change in sitting height was measured before and after vertical vibration (5 Hz with a peak-to-peak amplitude of 3 mm, and peak acceleration less than 2 m/s2) and again at the end of the day. The mean reduction in sitting height over the half hour of vibration exposure was 9.0 mm versus less than 1 mm for the control condition. The mean height loss over the third day (the day of 30 minutes of vibration exposure) was only 3.6 mm (compared with 10.6 mm lost over a control day with no vibration exposure). Hence, exposure to vibration increased the creep response in all subjects during exposure but, at the end of the day, there was a recovery in height, such that subjects were taller at the end of the day of vibration exposure. It is hypothesized that this "rebound" effect is due to an inflammatory response in the spine."

So you lose 7 mm less of height with vibration. It'd be interesting if this rebound effect occurred in response to other forms of exercise.