Friday, September 10, 2010

Grow Taller by Increasing Chondrocyte Hypertrophy

Growth via the growth plate mechanisms is primarily determined by the maximum size of growth plate chondrocytes(chondrocyte hypertrophy) and the number of stem cells in the reserve zone(which we attempt to increase by inducing stem cells to differentiate into chondrocytes).

How can we increase the maximum size that chondrocytes achieve during development?

Mammalian limb loading and chondral modeling during ontogeny.

[Ontogeny is the study of an organism's development] 

"The adaptive growth response of cartilage, or chondral modeling, can result in changes in joint and limb proportions during ontogeny and ultimately contribute to the adult form. Here, we characterize the macro- and microanatomical responses of the femoral growth plate, articular cartilage, and bone in 15 juvenile Sus scrofa domestica subjected to different locomotor activity patterns. The exercised animals exhibit thinner cartilage zones, greater cellularity and larger proliferative chondrocyte areas in the growth plate, as well as larger femoral dimensions and a more elongate femoral head compared with sedentary controls. In general, the growth plate demonstrates greater adaptive changes than articular cartilage. \chondrocyte hypertrophy and proliferation were found to be responsive to locomotor loading and thus more important factors in chondral modeling than the extracellular matrix variables that were examined herein. In sum, the underlying mechanisms of adaptive chondrogenesis and bone plasticity are key to informing evolutionary and translational studies regarding determinants of variation in joint form and function. Given the disparity between the predictions of chondral modeling theory and our experimental findings, this suggests a need for further evaluation of chondral modeling responses during ontogeny." 

"The chondral modeling response is posited to include regional or widespread cartilage thickening, changes in cartilage cellular and extracellular matrix (ECM) composition and organization, and potential for increased calcification and ossification. Chondral modeling may result in differential mineralization and ossification of the deepest hyaline cartilage layers (i.e., the calcified layer of articular cartilage, hypertrophic zone of growth plate). As such, it may directly contribute to the form and proportions of bones via influences on subchondral and diaphyseal bone."

"high load-bearing joint cartilage will cease growth yet compensatory growth will occur in adjacent areas to more equally distribute the load."<-this underlines the importance of the LSJL pressure gradient.

"Fifteen castrated male juvenile miniature swine (Sus scrofa domesticus) were used"

"Pigs are not skeletally mature until 5 to 6 years of age, with the femoral proximal growth plate remaining unfused until 3 years old"

"The juvenile pigs began the protocol at 8 months of age and were sacrificed after seventeen weeks of participation in the experiment."

"exercised swine completed treadmill running to exertion limit 5 days a week"

A lot of measurements were taken but there aren't a lot of noticeable trends.  But hypertrophic and proliferative cell count tended to be greater in the exercised groups than the sedentary groups.

"growth plate of exercised swine tended to be thinner, with reductions in average thickness localized in the proliferative zone."

"The proliferative chondrocytes had a larger average cell area in the exercised group as well"

"the exercised cohort had relatively taller epiphyses which created more expansive dorsal subchondral and articular surfaces."

This study actually showed a decrease in ECM production due to exercise.  This may be due to the intensity.

"hypertrophic chondrocytes were smaller than those in the sedentary group, which leaves one to speculate that this is due to an increased turnover rate in the hypertrophic cells of the exercise group."

"elevated loading does not result in a correspondingly larger growth plate, and a larger growth plate is not essential for greater longitudinal bone growth."

Locomotor is similar to dynamic loading.  Loading in motion such as sprinting.  A longer femoral head should result in a taller bone.  Locomotive loading affects chondrocyte hypertrophy and proliferation more than extracellular matrix secretion.  Which means that locomotive loading during development is powerful.

Thyroid hormone-mediated growth and differentiation of growth plate chondrocytes involves IGF-1 modulation of beta-catenin signaling. 

"Thyroid hormone regulates terminal differentiation of growth plate chondrocytes in part through modulation of the Wnt/beta-catenin signaling pathway. Insulin-like growth factor 1 (IGF-1) has been described as a stabilizer of beta-catenin, and thyroid hormone[remember that Lithium has many properties of thyroid hormone, Lithium stabilizes Beta-Catenin as well] is a known stimulator of IGF-1 receptor expression. IGF-1 and the IGF- receptor (IGF1R) stimulate Wnt-4 expression and beta-catenin activation in growth plate chondrocytes. The positive effects of IGF-1/IGF1R on chondrocyte proliferation and terminal differentiation are partially inhibited by the Wnt antagonists sFRP3 and Dkk1. T(3) activates IGF-1/IGF1R signaling and IGF-1-dependent PI3K/Akt/GSK-3beta signaling in growth plate chondrocytes undergoing proliferation and differentiation to prehypertrophy. T(3)-mediated Wnt-4 expression, beta-catenin activation, cell proliferation, and terminal differentiation of growth plate chondrocytes are partially prevented by the IGF1R inhibitor picropodophyllin as well as by the PI3K/Akt signaling inhibitors LY294002 and Akti1/2. The interactions between thyroid hormone and beta-catenin signaling in regulating growth plate chondrocyte proliferation and terminal differentiation are modulated by IGF-1/IGF1R signaling through both the Wnt and PI3K/Akt signaling pathways. While chondrocyte proliferation may be triggered by the IGF-1/IGF1R-mediated PI3K/Akt/GSK3beta pathway, cell hypertrophy is likely due to activation of Wnt/beta-catenin signaling, which is at least in part initiated by IGF-1 signaling or the IGF-1-activated PI3K/Akt signaling pathway." 

Proper cell hypertrophy requires Wnt/beta-catenin signaling which is induced by IGF-1 signaling.  IGF-1 is important in chondrocyte hypertrophy and IGF-1 may able to help increase height in a dose dependent manner up to a point by increasing peak chondrocyte hypertrophy.  Beta-catenin increases chondrocyte hypertrophy and the benefits of IGF-1 may be limited to the amount of beta-catenin for IGF-1 to stabilize.

"GSK-3β is also involved in the IGF-1 signaling pathway. Phosphatidylinositol-3-kinase (PI3K) is an important signal transducer of responses to IGF-1 signaling. Akt is a downstream target of PI3K, and can inactivate GSK-3β by phosphorylation on serine 9"<-Lithium again inhibits GSK-3B again further linking Lithium to thyroid hormone.

"Igf1 is expressed at much lower levels in growth plate chondrocytes than in perichondrium and metaphyseal bone. IGF-1 protein in the in vivo growth plate therefore is considered to derive primarily from surrounding perichondrium and bone than from the chondrocytes themselves."<-You may be able to perform exercises to increase IGF-1 in the periosteum and other regions of the bone to have benefits on growth plate chondrocytes.  This could be why locomotive loading increased chondrocyte proliferation and hypertrophy due to increased levels of IGF-1 and enhanced circulation of IGF-1 to growth plate chondrocytes.  Here's an image from the study that shows how thyroid hormone and in turn possibly Lithium affects height growth.

Thyroid Hormone(and possibly Lithium) can increase height growth up to the number of IGF-1 receptors.  Beta-Catenin over that point may actually decrease height by increasing terminal differentiation without a corresponding increase in proliferation.  IGF-1R is lowered by fasting and IGF-1 itself.  IGF-1R also decreases with age.  How best to enhance IGF-1R expression is unclear.

Ubiquitous overexpression of Hey1 transcription factor leads to osteopenia and chondrocyte hypertrophy in bone. 

"The transcription factor Hey1, a known Notch target gene of the HES family, has recently been described as a target gene of bone morphogenetic protein-2 (BMP-2) during osteoblastic differentiation in vitro. We analyzed bones of mice ubiquitously lacking or overexpressing Hey1. This strategy enabled us to evaluate whether Hey1 modulation in the whole organism could serve as a drug or antibody target for therapy of diseases associated with bone loss. Hey1 deficiency resulted in modest osteopenia in vivo[osteopenia is lowered bone density] and increased number and activity of osteoclasts generated ex vivo. Hey1 overexpression resulted in distinct progressive osteopenia and inhibition of osteoblasts ex vivo, an effect apparently dominant to a mild inhibition of osteoclasts. In both Hey1 deficient and overexpressing mice, males were less affected than females and skeleton was not affected during development. Bone histomorphometry did not reveal major changes in animals at 20 weeks, suggesting that modulation had occurred before. Adult Hey1 transgenics also displayed increased type X collagen expression and an enlarged hypertrophic zone in the growth plate. In vivo Hey1 regulation affects osteoblasts, osteoclasts and chondrocytes. Due to the complex role of Hey1 in bone, inhibition of Hey1 does not appear to be a straightforward therapeutic strategy to increase the bone mass." 

Hey1 may increase chondrocyte hypertrophy.  Hey1 can be induced by BMP-2.

"Hey1 is expressed in calvarial bone, mandibular and maxillary bone, and trabecular osteoblasts of vertebrae. Hey1 co-transfection with Runx2, a major transcription factor regulator of bone formation, completely abolished transcriptional activity of Runx2 in a reporter gene assay. Hey1 down-regulation by siRNA led to increased osteoblast maturation and mineralization"<-Hey1 may be responsible for those kinds of bones unique shape.  Note that they are not long bones.

Hey1 deficiency did not affect bone length.  Hey1 transgenic mice did not affect bone length.

"Hey1 transgenic animals showed a massive overexpression of type X collagen mRNA as compared to wild type littermates"<-Chondrocyte Hypertrophy was increased by Transgenic Hey1

Mice transgenic in Hey1 had a large increase in growth plate height but not an overall increase in bone length.  Either chondrocyte hypertrophy does not increase height in all instances or the loss of bone mineralization and osteoblast maturation also induced by transgenic Hey1 compensates for the increase in hypertrophic cell size resulting in the same net height.

Zfp521 is inhibited by BMP-2 and it represses the activity of Runx2.   Hey1 also inhibits the activity of Runx2.  Zfp521 and Hey1 are closely linked.  An equilibrium quantity of Zinc and BMP-2 may be needed for maximal height growth.  But an increase in chondrocyte hypertrophy may not always increase height.

Locomative methods of increasing chondrocyte hypertrophy shows promise as sprinting during development is something that's easy to do and already recommended.

Here's a study that explains how chondrocytes regulate volume:

Cell volume regulation in chondrocytes.

"Chondrocytes are the cells within cartilage which produce and maintain the extracellular matrix. Volume regulation in these cells is vital to their function and occurs in several different physiological and pathological contexts. Firstly, chondrocytes exist within an environment of changing osmolarity and compressive loads[we try to change osmolarity with LSJL]. Secondly, in osteoarthritic joint failure, cartilage water content changes and there is a notable increase in chondrocyte apoptosis. Thirdly, endochondral ossification requires chondrocyte swelling in association with hypertrophy. Regulatory volume decrease (RVD) and regulatory volume increase (RVI) have both been observed in articular chondrocytes and this review focuses on the mechanisms identified to account for these. There has been evidence so far to suggest TRPV4 is central to RVD; however other elements of the pathway have not yet been identified. Unlike RVD, RVI appears less robust in articular chondrocytes and there have been fewer mechanistic studies; the primary focus being on the Na(+)-K(+)-2Cl(-) co-transporter[manipulating the sodium-potassium pump may be a way to grow taller]. The clinical significance of chondrocyte volume regulation remains unproven. Importantly however, transcript abundances of several ion channels implicated in volume control are changed in chondrocytes from osteoarthritic cartilage. A critical question is whether disturbances of volume regulation mechanisms lead to, result from or are simply coincidental to cartilage damage."

"Cartilage is also greatly hypertonic to most other tissues, typically in excess of 350mOsm[meaning water tends to leave cartilage cells]. This hydration state exists because whilst high concentrations of polyanionic proteoglycan molecules draw water into the tissue by osmosis, water is simultaneously forced out by the tension of interwoven collagen “strands”"

"chondrocytes can recover volume from a 20% increase"<-you have to increase volume by more than 20% to have a permanent change in chondrocyte shape.

"Some report that changing osmolarity with NaCl evokes RVI[Regulatory volume increase]"<-Increase height with sodium chloride?

"NKCC is present at high levels in growth-plate chondrocytes"<-Meaning that the sodium-potassium and chloride pump may be important for height growth.

Chondrocyte cell volume regulation is likely crucial to height growth and therefore NKCC is likely critical as well.

Evidence for lysosomal exocytosis and release of aggrecan-degrading hydrolases from hypertrophic chondrocytes, in vitro and in vivo.

"The abundant proteoglycan, aggrecan, is resorbed from growth plate cartilage during endochondral bone ossification, yet mice with genetically-ablated aggrecan-degrading activity have no defects in bone formation. To account for this apparent anomaly, we propose that lysosomal hydrolases degrade extracellular, hyaluronan-bound aggrecan aggregates in growth plate cartilage, and that lysosomal hydrolases are released from hypertrophic chondrocytes into growth plate cartilage via Ca(2+)-dependent lysosomal exocytosis.  Hypertrophic chondrocytes release hydrolases via lysosomal exocytosis in vitro and we show in vivo evidence for lysosomal exocytosis in hypertrophic chondrocytes during skeletal development. We show that lysosome-associated membrane protein 1 (LAMP1) is detected at the cell surface following in vitro treatment of epiphyseal chondrocytes with the calcium ionophore, ionomycin. Furthermore, we show that in addition to the lysosomal exocytosis markers, cathepsin D and β-hexosaminidase, ionomycin induces release of aggrecan- and hyaluronan-degrading activity from cultured epiphyseal chondrocytes. We identify VAMP-8 and VAMP7 as v-SNARE proteins with potential roles in lysosomal exocytosis in hypertrophic chondrocytes, based on their colocalisation with LAMP1 at the cell surface in secondary ossification centers in mouse tibiae. We propose that resorbing growth plate cartilage involves release of destructive hydrolases from hypertrophic chondrocytes, via lysosomal exocytosis."

"in contrast to adult articular cartilage, resorption of aggrecan in growth plate cartilage does not rely on ADAMTS enzymes."

"Lysosomal exocytosis is a repair mechanism for patching membranes that rupture, for example in response to treatment with pore-forming agents or under conditions of increased biomechanical load. Following membrane disruption, a rapid equilibration of intracellular Ca2+ depolymerises the F-actin network to trigger lysosome accumulation near the plasma membrane and lysosomal fusion with the cell membrane to reseal the perforation"

"LAMP1 was clearly localised at the cell periphery in hypertrophic chondrocytes of the developing secondary centre of ossification"

"membrane damage to mouse epiphyseal chondrocytes leads to a Ca2+-dependent translocation of LAMP1 to the cell membrane."

"lysosomal exocytosis in mouse hypertrophic chondrocytes is potentially mediated, at least in part, by the v-SNARE, VAMP8."

"hypertrophic chondrocytes expand their volume 5–10 fold over a period of only 2 days and are therefore under stretch-induced load, 2) hypertrophic chondrocytes have a requirement for membrane expansion in order to achieve this increase in cell volume that occurs within a few days, 3) lysosomal hydrolases are an alternative source of aggrecan-degrading activity in growth plates of mice with gene mutations targetting aggrecanolysis"

"hypertrophic chondrocytes release their lysosomal contents in response to membrane disruption and high extracellular Ca2+ in vitro, and more importantly, that this process occurs in vivo in normal, unstimulated conditions"

Hypertrophy of growth plate chondrocytes in vivo is accompanied by modulations in the activity state and surface area of their cytoplasmic organelles.

"Using rats at different stages of growth, the surface areas and volumes of the three organellar systems  [rough endoplasmic reticulum(collagen fibril synthesis), Golgi apparatus(GAG synthesis) and mitochondria(energy source)] were quantified in epiphyseal plate chondrocytes at the onset and termination of hypertrophy. Matrix synthesis during the same span was assessed by monitoring the production of its principal components, namely, fibrillar collagen and glycosaminoglycans. Each organelle adapts to increases (21- to 35-day-old rats) and decreases (35- to 80-day-old rats) in growth rate by its own individual combination of the two alternative mechanisms, but modulations in the level of activity predominate over alterations in the surface area or volume of their membranes."

"In each age group, the matrix volume per chondrocyte more than doubled between the end of the proliferative phase and that of the hypertrophic one"

"dilution partially contributed to these increases in most instances, the extent to which this occurred being age dependent"

Density of fibril collagen decreased in 21 day old rats but remained constant in 35 and 80 year old rats.

"The density of glycosaminoglycans decreased during the course of hypertrophy in both 21- and 35-day-old rats (from 51.0 to 33.8 and from 40.3 to 27.0, respectively), but increased in 80-day-old ones (from 7.4 to 9.3)."

"the final density of glycosaminoglycans achieved at the end of hypertrophy decreased with age."

"The production of matrix glycosaminoglycans per cell per hour [increased from 21 to 35 but decreased from 35 to 80]"

"During growth acceleration (21 to 35 days), relative activity changes in cell and matrix volume increases were +11% and +59%, respectively, whereas during the deceleration phase (35 to 80 days), they were –45% and +17%, respectively"

"The combined changes in cell and matrix volume per hour and per unit volume of mitochondria were
+70% during the growth acceleration phase (21 to 35 days) and –28% during the deceleration one (35 to 80 days)"

"Total mitochondrial volume per cell was found not to change significantly during growth acceleration
and, during deceleration, it even decreased (–35%). Hence, mitochondria coped with increased or decreased energy demands within the bounds of the physiological range investigated by modulating
their rate of activity."

"Chondrocyte hypertrophy is characterised by very rapid increases in cell and matrix volumes. The major extracellular components produced are the fibrillar collagens (ca 50% dry weight) and proteoglycans (ca 40% dry weight), and, of the latter, glycosaminoglycans represent the principal constituent (>85% of the proteoglycan molecules)."  Xlkd1 a gene involved in GAG catabolism is upregulated in LSJL.  Glce a gene involved in GAG biosynthesis is also upregulated.  Hexa a gene involved in the GAG metabolic process is downregulated.

"the concentration of fibrillar collagen in 80-day-old animals was higher, and that of glycosaminoglycans significantly lower, than in the two younger age groups (21 and 35 days)."<-Since 80 days is the deceleration phase maybe, increasing the ratio of GAG to fibrillar collagen production in hypertrophic chondrocytes is a way to increase height?

"The 60% reduction in glycosaminoglycan synthesis occurring during growth deceleration (between 35 and 80 days) was achieved by a decrease in activity per unit area (–32%) or per unit volume (–23%) of Golgi membranes per hour"

"Increases in collagen productivity effected during growth acceleration (21 to 35 days; +73%) and deceleration (35 to 80 days; +6%) were accompanied by RER[Rough Endoplasmic Reticulum] activity
increases per unit area of membrane and per hour of 71% and 53%, respectively, and that effected between 21 and 80 days by one of 261%. The tremendous increase in the latter case was accounted for by the smaller total amount of RER membrane present in the hypertrophic chondrocytes of 80-day-old animals, despite an increase in RER membrane surface density"

Endoplasmic Reticulum Genes upregulated in LSJL:


LSJL has a tendency to downregulate endoplasmic reticulum related proteins than to upregulate.  Since the increase in endoplasmic reticulum activity was inversely related to the number of endoplasmic reticulum this could be a result of a larger total number of cells as a result of LSJL.  The upregulation of collagen was huge in LSJL so total endoplasmic reticulum activity could not have decreased, therefore there must be more cells to compensate.

Downregulation of Carbonic Anhydrase IX Promotes Col10a1 Expression in Chondrocytes.

"Carbonic anhydrase (CA) IX is a transmembrane isozyme of CAs that catalyzes reversible hydration of CO(2). CA IX is distributed in human embryonic chondrocytes. Car9 mRNA and CA IX [are] expressed in proliferating but not hypertrophic chondrocytes. Next, we examined the role of CA IX in the expression of marker genes of chondrocyte differentiation in vitro. Introduction of Car9 siRNA to mouse primary chondrocytes obtained from costal cartilage induced the mRNA expressions of Col10a1, the gene for type X collagen α-1 chain, and Epas1, the gene for hypoxia-responsible factor-2α (HIF-2α), both of which are known to be characteristically expressed in hypertrophic chondrocytes. On the other hand, forced expression of CA IX had no effect of the proliferation of chondrocytes or the transcription of Col10a1 and Epas1, while the transcription of Col2a1 and Acan were up-regulated. Although HIF-2α has been reported to be a potent activator of Col10a1 transcription, Epas1 siRNA did not suppress Car9 siRNA-induced increment in Col10a1 expression, indicating that down-regulation of CA IX induces the expression of Col10a1 in chondrocytes in a HIF-2α-independent manner. On the other hand, cellular cAMP content was lowered by Car9 siRNA. Furthermore, the expression of Col10a1 mRNA after Car9 silencing was augmented by an inhibitor of protein kinase A, and suppressed by an inhibitor for phosphodiesterase as well as a brominated analog of cAMP. While these results suggest a possible involvement of cAMP-dependent pathway, at least in part, in induction of Col10a1 expression by down-regulation of Car9, more detailed study is required to clarify the role of CA IX in regulation of Col10a1 expression in chondrocytes."

So Car9 can promote chondroinduction and inhibition can promote chondrocyte hypertrophy.

Acan expression but Col2a1 expression was reduced by Car9 siRNA.

Multiple phases of chondrocyte enlargement underlie differences in skeletal proportions.

"The largest contribution to the lengthening of a skeletal element, and to the differential elongation of elements, comes from a dramatic increase in the volume of hypertrophic chondrocytes in the growth plate as they undergo terminal differentiation. Mammalian chondrocytes undergo three distinct phases of volume increase, including a phase of massive cell swelling in which the cellular dry mass is significantly diluted. In light of the tight fluid regulatory mechanisms known to control volume in many cell types, this is a remarkable mechanism for increasing cell size and regulating growth rate. It is, however, the duration of the final phase of volume enlargement by proportional dry mass increase at low density that varies most between rapidly and slowly elongating growth plates. This third phase is locally regulated through a mechanism dependent on insulin-like growth factor."

"In phase 1, an initial increase of about threefold from approximately 600 fl to 2,000 fl is characterized by true hypertrophy—a proportionate increase in dry mass production and fluid uptake thus maintaining the normal dry mass density at 0.183 pg fl−1. In phase 2, a fourfold enlargement from about 2,000 fl to 8,000 fl is characterized by cell swelling. Volume increases at a rate disproportionate to the continuing rate of dry mass production resulting in a dramatic dilution of dry mass density to approximately 0.07 pg fl−1. In phase 3, at volumes larger than 8,000 fl, the dry mass density once again stabilizes, and cells continue to enlarge another twofold to about 14,000 fl by proportionately increasing dry mass and fluid volume at this lower density. Swelling in phase 2 allows cells to reach volumes two to three times greater in phase 3 than if they relied entirely on the proportionate increase in dry mass at high density"

"the largest cells reduce their dry mass density by approximately 60%, and moreover indicates that dry mass is low throughout the cytoplasm with a slightly higher density ring around the nucleus"

"Igf1-deficient mice are 35% smaller than controls but have the same number of hypertrophic chondrocytes, although each cell is 30% shorter in the direction of elongation, a finding we confirmed in mice where the floxed Igf1tm1Dlr allele was conditionally deleted from the hindlimb using Hoxb6-Cre"

The scientists found that the three phases was not due to osmotic stress.

No comments:

Post a Comment