Increasing Bone Length by increasing Chondrocyte Proliferative Capacity?

The phenomena of catch-up growth is said to be related to the fact that chondrocytes have a finite proliferative capacity. The chondrocytes simply stay inactive until there are appropriate circumstances for endochondral ossification to occur.  Stem cells have an unlimited proliferative capacity but if there are no stem cells in the hyaline cartilage growth plate line no further growth is possible.  It is the infinite ability of stem cells to proliferate that allows tall stature to occur in syndromes like Gigantism.  HGH increases IGF-1 which increases stem cell proliferation.  The reason that HGH on it's own doesn't usually increase height is that in Gigantism cellular homeostasis is altered(i.e. enlarged pituitary gland) whereas just simply injecting HGH will result in the bodies negative feedback mechanisms.

Is there anyway to increase the proliferative capacity of chondrocytes?  What mechanisms control it's proliferative capacity? 

Catch-up growth after glucocorticoid excess: a mechanism intrinsic to the growth plate. 

"In humans and other mammals, the release from growth-inhibiting conditions, such as glucocorticoid excess, leads to supranormal linear growth. The prevailing explanation for this catch-up growth involves a central nervous system mechanism that compares actual body size to an age-appropriate set-point and adjusts growth rate accordingly via a circulating factor. Although such a neuroendocrine "sizostat" was hypothesized more than 30 yr ago, its existence has never been confirmed experimentally. Here we show that suppression of growth within a single growth plate by locally administered glucocorticoid is followed by local catch-up growth that is restricted to the affected growth plate. Thus, the catch-up growth cannot be explained by neuroendocrine mechanism but, rather, must arise from a mechanism intrinsic to the growth plate. To explain this finding, we propose that the normal senescent decline in growth plate function depends not on age per se, but on the cumulative number of stem cell divisions, and that glucocorticoid administration, by suppressing stem cell proliferation, delays senescence, resulting in catch-up growth after the growth-inhibiting agent is removed" 

Now, in this study they propose that the mechanism is based on inhibiting stem cell proliferation.  If it did in fact affect stem cell proliferation then once could still increase bone length by increasing chondrocyte proliferation.  Now we know that myostatin inhibits stem cell proliferation.  Increasing testosterone inhibits myostatin.  Glutocorticoids may actually increase stem cell proliferation.  It makes no sense for catch up growth to be a restriction on stem cell proliferation as then the stem cells would still differentiate into chondrocytes and they would lose the number of stem cell proliferations during the time that the growth slows down.    It makes much more sense for it to be based on chondrocyte proliferative capacity.  

Depletion of resting zone chondrocytes during growth plate senescence. 

"With age, the growth plate undergoes senescent changes that cause linear bone growth to slow and finally cease. Based on previous indirect evidence, we hypothesized that this senescent decline occurs because growth plate stem-like cells, located in the resting zone, have a finite proliferative capacity that is gradually depleted. Consistent with this hypothesis, we found that the proliferation rate in rabbit resting zone chondrocytes (assessed by continuous 5-bromo-2'-deoxy-uridine labeling) decreases with age, as does the number of resting zone chondrocytes per area of growth plate. Glucocorticoid excess slows growth plate senescence. To explain this effect, we hypothesized that glucocorticoid inhibits resting zone chondrocyte proliferation, thus conserving their proliferative capacity. Consistent with this hypothesis, we found that dexamethasone treatment decreased the proliferation rate of rabbit resting zone chondrocytes and slowed the numerical depletion of these cells. Estrogen is known to accelerate growth plate senescence. However, we found that estradiol cypionate treatment slowed resting zone chondrocyte proliferation. Our findings support the hypotheses that growth plate senescence is caused by qualitative and quantitative depletion of stem-like cells in the resting zone and that growth-inhibiting conditions, such as glucocorticoid excess, slow senescence by slowing resting zone chondrocyte proliferation and slowing the numerical depletion of these cells, thereby conserving the proliferative capacity of the growth plate. We speculate that estrogen might accelerate senescence by a proliferation-independent mechanism, or by increasing the loss of proliferative capacity per cell cycle."

The basis for Estrogen inhibition is that Estrogen might decrease the proliferative capacity of chondrocytes per cell cycle.  This might explain the sex related height differences.  However, there are other possible explanations like testosterone inhibiting myostatin thus increasing stem cell proliferation or different genetic expression between males and females.  A lot of height seekers take aromatase inhibitors.  It's the basis for Height FX or Estrogenex Aromatase Inhibitor by Hi-Tech Pharmaceuticals - 90 Tablets.  More studies need to be done as to whether estrogen actually affects the proliferative capacity of chondrocytes.

"We have recently shown evidence that the resting zone chondrocytes serve as a pool of stem-like cells that generate columnar clones of proliferative zone chondrocytes[these resting zone stem like cells are basically stem cells but with a rounder shape which can be attained by hydrostatic pressure]. We have also found evidence suggesting that growth plate senescence is not a function of time per se but rather of the cumulative number of divisions that the chondrocytes have undergone[doesn't explain Gigantism though which seems to take place at a systematic level rather than at local growth plate levels]. Taken together, these previous findings suggest the following model: 1) stem-like cells in the resting zone have a finite proliferative capacity which is gradually exhausted[based on DNA-methylation and telomere length]; 2) as this proliferative capacity is exhausted, the proliferation rate of the proliferative zone chondrocytes (which are derived from the stem-like cells) decreases, causing longitudinal bone growth to slow with age and eventually cease."

"Although we attribute the decrease in proliferation with age to a decrease in the proliferative capacity, this decreased capacity is not necessarily cell autonomous and irreversible. The decrease in proliferative capacity could be context-dependent, and reversible[methylation status can be restored and telomeres can be lengthened, also you can get new stem cells to differentiate into chondrocytes in the epiphysis]. Previous studies suggest that the decrease is not due to hormonal or other systemic factors[But, again, what of Gigantism?], but the decrease could reflect a local change in concentrations of paracrine growth factors[possibly Ihh, Pth, etc.]."

"We speculate that estrogen might increase the loss of proliferative capacity that occurs with each cell cycle, or that estrogen might cause loss of proliferative capacity by a cell-cycle-independent mechanism[not that there seems to be a need for an equilibrium value for estrogen(not too high or too low)]. For example, if senescence is caused by epigenetic changes such as loss of DNA methylation with each cell cycle, then estrogen might act by decreasing expression of maintenance methylases, causing greater loss of methylation with each cell replication. The effects of estrogen on the resting zone chondrocytes could be mediated by the estrogen receptor- or -ß, both of which are expressed by resting zone chondrocytes in humans, rabbits, and rats. The combination of a decreased rate of growth (in most mammals) and an increased rate of senescence seems to be an effect specific to estrogen[again too little estrogen has been shown to have growth detrimental effects as well]. Other growth-inhibiting conditions, such as glucocorticoid excess and hypothyroidism, decelerate growth plate senescence. The mechanism responsible for this paradoxical effect of estrogen remains to be determined."

"The reserve region contains pairs of flattened cells[hydrostatic pressure may act to flatten stem cells making them look like reserve growth plate chondrocytes] that give the appearance of nascent proliferative columns. Finally, the concept is supported by functional studies; after surgical ablation, the reserve region can apparently be regenerated by epiphyseal chondrocytes[if you get new chondrocytes to the growth then you can grow taller], and the proliferative zone can apparently be generated by reserve chondrocytes"

Growth plate senescence is associated with loss of DNA methylation.

"The overall body size of vertebrates is primarily determined by longitudinal bone growth at the growth plate. With age, the growth plate undergoes programmed senescence, causing longitudinal bone growth to slow and eventually cease. Indirect evidence suggests that growth plate senescence occurs because stem-like cells in the growth plate resting zone have a finite proliferative capacity that is gradually exhausted. Similar limits on replication have been observed when many types of animal cells are placed in cell culture, an effect known as the Hayflick phenomenon. However, we found that the number of population doublings of rabbit resting zone chondrocytes in culture did not depend on the age of the animal from which the cells were harvested, suggesting that the mechanisms limiting replicative capacity of growth plate chondrocytes in vivo are distinct from those in vitro. We also observed that the level of DNA methylation in resting zone chondrocytes decreased with age in vivo. This loss of methylation appeared to occur specifically with the slow proliferation of resting zone chondrocytes in vivo and was not observed with the rapid proliferation of proliferative zone chondrocytes in vivo (i.e. the level of DNA methylation did not change from the resting zone to the hypertrophic zone), with proliferation of chondrocytes in vitro, or with growth of the liver in vivo. Thus, the overall level of DNA methylation decreases during growth plate senescence. This finding is consistent with the hypothesis that the mechanism limiting replication of growth plate chondrocytes in vivo involves loss of DNA methylation and, thus, loss of DNA methylation might be a fundamental biological mechanism that limits longitudinal bone growth in mammals, thereby determining the overall adult size of the organism."

So to increase chondrocyte proliferative capacity we want to increase levels of DNA methylation.  DNA methylation refers to the addition of the methyl group to DNA.  Of course, DNA methylation may be a necessary but not sufficient condition for chondrocyte replication so more pieces of the puzzle may be needed(telomere length is another example).

"growth plate senescence, appears to be a function, not of time per se, but rather of the number of replications that the growth plate chondrocytes have undergone"<-so just make more growth plate chondrocytes

"Some CG sequences in mammalian genomic DNA are methylated on the cytosine moiety. When DNA is replicated, the new strand is initially not methylated. However, DNA methyltransferase 1, a maintenance methylase, recognizes the hemimethylated CGs and adds the missing methyl groups"

"growth plate chondrocytes cultured at high density and exposed to a demethylating agent (5-azacytidine), undergo hypertrophic differentiation"<-so the signal to undergo chondrogenic differentiation doesn't involve hormonal control but rather due to cell concentration and methylation status

"Chondrocytes in primary culture were small, had a round or polygonal shape, and stained for alcian blue and alkaline phosphatase activity, but not for senescence related ß-galactosidase . In contrast, chondrocytes that were becoming senescent were larger, only stained faintly or not at all for alcian blue and alkaline phosphatase activity, but showed an increased senescence-related ß-galactosidase activity"<-Terminal differentiation depends less on alkaline phosphatase levels but rather more on B-galactosidase levels.

"For growth plate chondrocytes in vivo, three lines of evidence suggest that telomere shortening is not the primary cell-division counter causing replicative senescence. First, telomere length varies widely among different strains of mice with similar skeletal sizes. Indeed, some strains of mice have far greater telomere lengths than humans. Thus, telomere length does not correlate with skeletal size. Secondly, functional ablation of telomerase in mice leads to progressive shortening of telomere length in subsequent generations of the mice but has little effect on skeletal size. Thirdly, telomere length does not decrease significantly with growth plate senescence in mice"<-Telomere length may still affect the ability of mesenchymal stem cells to undergo chondrogenesis


Rescuing loading induced bone formation at senescence.

"We present an agent-based model of real-time Ca(2+)/NFAT signaling amongst bone cells that fully described periosteal bone formation induced by a wide variety of loading stimuli in young and aged animals. The model predicted age-related pathway alterations underlying the diminished bone formation at senescence, and hence identified critical deficits that were promising targets for therapy. Based upon model predictions, we implemented an in vivo intervention and show for the first time that supplementing mechanical stimuli with low-dose Cyclosporin A[ciclosporin(available by prescription)] can completely rescue[restart] loading induced bone formation in the senescent skeleton."

" NFAT activation dynamics (within minutes) is remarkably specific to Ca2+ amplitudes and frequencies known to influence distinct downstream cell functions, including proliferation, differentiation and apoptosis"<-LSJL downregulates NFAT5 and NFATC3.

"the model predicted age-related deficits in the ability of Ca2+ oscillations to dephosphorylate NFAT and in NFAT DNA binding capacity"

"Simulated restoration of age-related deficits in α and NFATnx (that are downstream of Ca2+ signaling) did not differentially influence Ca2+ oscillations in cells around the surface in response to the loading protocol"

"loading bone every 24 hrs vs 48 hrs does not differentially influence bone formation"

"supplementation with low-dose CsA would mitigate predicted deficits in loading (and Ca2+) induced dephosphorylation and nuclear translocation of NFAT (α) in part via CsA suppression of negative regulators such as p38 and/or GSK-3β"

"CsA supplementation could also effectively counteract predicted deficits in NFAT-DNA binding (NFATnx) by enhancing cooperative binding interactions between loading induced translocation of NFAT and CsA enhanced activation of transcription factor families such as AP-1"