Monday, April 29, 2013

Flurbiprofen(Grow Taller by manipulating Lymphocyte count)

 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.

Monday, April 22, 2013

Bone lengthening in response to stress?

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."<-Note that the mature periosteum can form cartilage and not just the developmental perichondrium.

"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."

"Deviatoric (distortional or shear) stresses cause material distortions with no change in volume. Dilatational (hydrostatic) 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

"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. [Examination] 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 mechanical stimulation and hyperemia{increase in blood flow} of the constantly strained extremity."

"Significant difference in ulnar length between the two arms in the tennis players [ranging] 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.

Tuesday, April 16, 2013

Increase your Stature with Plastic Deformation

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."


Full-size image (18 K)
"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.

Thursday, April 11, 2013

Do fats and sugars affect your height gain?

 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,

Friday, April 5, 2013

Micro-Growth Plates by LSJL

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. 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. 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, 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"

thumbnail
"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.

Here's some evidence that micro growth plates can undergo endochondral ossification:

Delayed endochondral ossification in early medial coronoid disease (MCD): A morphological and immunohistochemical evaluation in growing Labrador retrievers.

"Early micromorphological changes [occur] in articular cartilage and to describe the postnatal development of the medial coronoid process (MCP) before MCD develops. Three litters of MCD-prone young Labrador retrievers were purpose-bred from a dam and two sires with MCD. Comparisons of the micromorphological appearance of the MCP in MCD-negative and MCD-positive joints demonstrated that MCD was initially associated with a disturbance of endochondral ossification, namely a delay in the calcification of the calcifying zone, without concurrent abnormalities in the superficial layers of the joint cartilage. Cartilage canals containing patent blood vessels were only detected in dogs <12weeks old. Retained hyaline cartilage might ossify as the disease progresses, but weak areas can develop into cracks between the retained cartilage and the subchondral bone, leading to cleft formation and fragmentation of the MCP."

That scattered growth plates could undergo endochondral ossification bodes well that micro growth plates could undergo endochondral ossification.

"Osteochondrosis is characterized by a disturbance of endochondral ossification that leads to an area of retained cartilage"<-osteochondrosis could be key to our understanding of microgrowth plates.

The arrows are pointing to cartilage canals

Full-size image (90 K)Cartilage canals dissappeared at 12 weeks of age.

Pathogenesis of epiphyseal osteochondrosis.

"Osteochondrosis (OC) of the articular epiphyseal cartilage complex (AECC) is a developmental disease that is present in the first weeks of life. It is characterized by focal chondronecrosis and retention of growth cartilage due to failure of endochondral ossification. Fissures may extend from the lesion through the overlying articular cartilage to create a cartilage flap and an osteochondral fragment. This articular form is known as osteochondritis dissecans (OCD). There have been many hypotheses about the etiopathogenesis of OC of the AECC including, amongst others, ischemia of growth cartilage or altered cartilage type II collagen metabolism. The ischemia theory proposes that necrosis of the vessels in the cartilage canals of the sub-articular growth cartilage leads to necrosis of chondrocytes and retention of necrotic cartilage{so re-establishing these vessels in the cartilage canals could be critical to forming new growth plates}. Several studies have measured biomarkers in serum and synovial fluid to demonstrate a consistent increase in type II collagen synthesis in young animals of different species. Although these changes could represent lesion reparative events, there is no comparable increase in the synthesis of cartilage matrix proteoglycan molecule. It is therefore speculated that an altered type II collagen metabolism may be involved in the early changes associated with OC. Further studies of OC susceptible animals in utero and the first weeks of life are required to elucidate the cause of vessel necrosis and the exact role of type II collagen structure and metabolism in OC."

"The bones of diarthrodial joints develop from mesenchymal cells that condense and differentiate into chondrocytes to form a cartilage template. Following formation of the primary ossification center in the diaphysis, the chondrocytes of the central area of the epiphysis proliferate, hypertrophy and undergo cell death. A complex sequence of molecular signals orchestrates these events. The hypertrophic chondrocytes themselves secrete type X collagen and alkaline phosphatase that contribute to calcification of the adjacent matrix. The mineralized cartilaginous septae, between the cell lacunae, undergo proteolytic digestion by septoclasts . Proteinases, such as matrix metalloproteinases (MMPs), cathepsins and gelatinases, degrade the mineralized type II-collagen-rich network to allow blood vessel invasion. The invading vessels are a source of mesenchymal stem cells and bone progenitors that differentiate into osteoblasts, secrete osteoid and promote ossification of the secondary center of ossification in the epiphysis" <-Our goal is to induce the mimicing of these events via LSJL to form new growth plates.

Osteochondrosis of the proximal phalanx of the hallux in adolescent footballers.

"We report two cases with radiographic appearances of osteochondrosis in the proximal phalanx of the big toe in adolescent footballers. The radiological findings were those of initial fragmentation with subsequent healing of the epiphysis. This is the first report of osteochondrosis at this site. Local pain was accompanied by swelling with restriction of dorsiflexion of the metatarsophalangeal joint of the big toe. The condition was self-healing over a 2-4-year period. It needs to be included in the differential diagnosis of painful hallux in adolescent footballers."

Unfortunately I could not get this full study.  It'd be interesting to see the healing of the epiphysis.

Thursday, April 4, 2013

How they treat short stature in medicine

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.

Tuesday, April 2, 2013

CTGF(CCN2), height increase target gene

 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 MMP3Cyclic tension increased CTGF expression.

CCN2/CTGF is required for matrix organization and to protect growth plate chondrocytes from cellular stress.

"The loss of CCN2 leads to perinatal lethality resulting from a severe chondrodysplasia. Upon closer inspection of Ccn2 mutant mice, we observed defects in extracellular matrix (ECM) organization and hypothesized that the severe chondrodysplasia caused by loss of CCN2 might be associated with defective chondrocyte survival. Ccn2 mutant growth plate chondrocytes exhibited enlarged endoplasmic reticula (ER), suggesting cellular stress. Immunofluorescence analysis confirmed elevated stress in Ccn2 mutants, with reduced stress observed in Ccn2 overexpressing transgenic mice. In vitro studies revealed that Ccn2 is a stress responsive gene in chondrocytes. The elevated stress observed in Ccn2-/- chondrocytes is direct and mediated in part through integrin α5. The expression of the survival marker NFκB and components of the autophagy pathway were decreased in Ccn2 mutant growth plates, suggesting that CCN2 may be involved in mediating chondrocyte survival. Absence of a matricellular protein can result in increased cellular stress and highlight a novel protective role for CCN2 in chondrocyte survival. The severe chondrodysplasia caused by the loss of CCN2 may be due to increased chondrocyte stress and defective activation of autophagy pathways, leading to decreased cellular survival. These effects may be mediated through nuclear factor κB (NFκB) as part of a CCN2/integrin/NFκB signaling cascade."


"ER enlargement is a hallmark of defective protein folding and cellular stress. ER and other forms of cellular stress activate the Unfolded Protein Response (UPR), an adaptive mechanism to restore cell homeostasis and viability"


"ER stress activates NFκB via tumor necrosis factor-α (TNF-α) receptor associated factor 2 (TRAF2) and inositol requiring enzyme 1 (IRE1) in vitro"


"CCN2 induces NFκB activity in ATDC5 chondrocytic cells through integrin αvβ3-mediated mechanisms to enhance migration"

In this study CCN2 overexpression did not seem to result in increased height in rats. But the structure of the skeletons is different. The growth plate height in CCN2 mice was lower.  The scientists did mention progressive overgrowth of cartilage elements.

CCN2: a master regulator of the genesis of bone and cartilage.

" CCN2 promotes the proliferation and differentiation of growth-plate chondrocytes"

" the over-expression of CCN2 in cartilage stimulated the proliferation and differentiation of growth-plate chondrocytes, resulting in the promotion of endochondral ossification."

Expression of CCN2 can be induced by TGF-Beta.

In this study mice overexpressing CCN2 had longer bones on postnatal day 1.

" In in vitro short-term cultures of chondrocytes prepared from the cartilage of ccn2-over-expressing mice, the expression of col2a1, aggrecan and ccn2 was substantially enhanced; and in long-term cultures the expression levels of these genes were further enhanced"

"IGF-I and IGF-II mRNA levels were elevated in the transgenic chondrocytes, and treatment of non-transgenic chondrocytes with CCN2 stimulated the expression of these mRNAs"

"phosphorylation of ERK1/2 by CCN2 is involved in chondrocyte proliferation, CCN2-BMP-2 treatment might promote chondrocyte differentiation by suppressing chondrocyte proliferation via decreased ERK1/2 phosphorylation."