Wednesday, January 4, 2012

How do the growth plates make us taller?

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

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

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

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

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

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

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

Stages of endochondral ossification:

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

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

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

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

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

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

The growth plate chondrocyte and endochondral ossification.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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


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

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

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

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

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

"The pores in, for example, the diaphysis at 6 weeks were mainly orientated in the longitudinal direction; their structure was very irregular with many branches that were not perpendicular to each other[meaning that growth from the growth plate is not perfectly structured either]. The pores, i.e. vascular canals being incorporated to form primary osteons, were mainly orientated parallel to the long bone axis"
"Mechanical loading is not considered to be the direct stimulus for bone remodelling but rather the stimulus that determines the expression of biochemical signalling molecules. The local concentration of this biochemical factor is subsequently compared with a set-point value at the bone surface. Upon exceeding a certain threshold, local bone apposition occurs."<-so there may be a conditioning effect that reduces the concentration of the biochemical factors in response to the same loads.
  
Skeletal dysplasias and the growth plate.

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

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

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

Histology of epiphyseal cartilage calcification and endochondral ossification

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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


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

"Before the appearance of the ossification centers epiphyseal growth rests exclusively on chondrocytes proliferation (interstitial growth), without any detectable differentiated cellular organization"<-If LSJL induces new growth plate formation then it's expected for their to not be much cellular organization initially.

This study mentions provides more information about two stages:

"CaPO4 deposition on the matrix interposed between hypertrophic chondrocytes"

"remodeling of trabeculae from primary to secondary (in other words, only lamellar without a core of calcified cartilage)."

"The diaphyseal ossification center early in the fetal development is remodelled progressively and substituted by the periosteal lamellar bone. The whole physis is cartilagineous with a differentation toward the diaphysis corresponding to the metaphyseal growth plate."

"The typical cellular organization of the endochondral ossification and the chondrocytes columns are oriented parallel to the bonemajor axis at the top of the ossified diaphysis and below the epiphysis (still completely cartilagineous)."

"From the time of appearance of the ossification center, the physeal growth is governed by the interstitial chondrocytes proliferation and by the endochondral ossification advancement from the center to the periphery.The progressive occupation of the epiphyseal volume by the ossification center showed that the rate of advancement of the endochondral ossification is faster than expansion driven by the cartilage interstitial growth."

"In terms of the growth mechanics of the whole system, the middle compartment (diaphysis) is the stable point, and expansion of the cartilages (longitudinal growth) occurs at the extremities because the vectors of the distal physis are inverted in respect to the proximal."

"the interstitial chondrocytes proliferation at the level of the articular cartilage and the apposition/resorption modeling at the level of the subchondral bone [can affect growth plate growth]"

"if the density of the cells remain evenly distributed, the shape of the model cannot change but only its volume increases: on the contrary, a differentiated topography of chondrocytes proliferation canmodulate shape variations."

"the bone development [of] the general length increment [occurs in] two fronts: one facing the metaphyseal growth plate but with the growth vector in the opposite direction, the other facing the joint surface with the vector in the same direction as that of the growth plate."

Suggested fundamental concepts in skeletal physiology

"Each living structural tissue (bone, cartilage, fibrous tissue) can develop microscopic fatigue damage called microdamage.As examples, in bone this includes microcracks and prefailure planes; in cartilage it includes chondromalacia; in fibrous tissues it includes ruptures of a few collagen fibers or bundles"

"Mediator mechanisms in each living tissue can detect and repair its microdamage. Each tissue has a kind of microdamage threshold. The threshold exists in the sense that below it, normal microdamage
repair can keep up with the need, but above it, the repair can become overwhelmed and microdamage
can begin accumulating. These thresholds can be expressed as numbers of strain-destraln cycles at a given peak strain.
Expressed as a strain, each tissue's microdamage threshold is only a minor fraction of its ultimate fracture or rupture strain. In bone, this threshold is about one eighth its fracture strain.
When microdamage overwhelms its repair, it can accumulate and cause fatigue failures. Examples of such failures include stress fractures and loosening of orthopedic and dental implants in bone; chondromalacia and arthroses in cartilage and joints; cracks in teeth; and spontaneous ruptures in tendon, ligament (especially the spinal annulus fibrosis), and fascia.
Three things can overwhelm microdamage repair: creating too much, hindering its repair, or a combination. As examples, impaired repair in bone in osteomalacia causes pseudofractures. In fibrous tissue it causes many spontaneous tendon ruptures. In articular cartilage it causes chondromalacia. Excessive microdamage production can happen in athletes, special forces trainees,
and in the bone embedding many load-bearing orthopedic and dental implants.
During growth, skeletal architecture probably adapts to increasing total loads (and their gradients) in ways that keep each tissue's strains below its microdamage threshold. To repeat, this would apply to bones, joints, cartilage, fibrous tissue, ligaments, tendons, and fascia"

"To keep strains below the microdamage thresholds of articular cartilage and subchondral spongiosa, a growing, increasingly heavily loaded joint enlarges its bearing area. It does that by increasing its diameter, by changing its shape (including reducing the curvature of its surfaces), and by adjusting the stiffness of its articular cartilage and the spongiosa supporting it. Growth at the perichondral ring can increase joint diameter, and chondral modeling can change joint shape and reduce surface curvature (stiffness is discussed shortly)"<-It could possibly do this after the growing period as well.

"Joint tissues always deform (strain) elastically under loads in ways that tend to increase the MLA[momentarily loaded area] carrying a load at any moment"

"Chondrocytes control articular cartilage stiffness, whereas bone modeling drifts, and remodeling BMUs control the subchondral spongiosa's stiffness. This elasticity lets the MLA of a joint under large loads usually exceed that under small ones. This tends to lower the peak unit loads on (and strains of) those tissues under large loads. Reducing a growing joint's curvature should make this mechanism more effective and may partly explain why joint curvatures do decrease during normal
growth."

"The ability to change articular size and shape to adapt to increasing loads depends mainly on chondral growth."

"Whether directly or indirectly, excess growth hormone may decrease the set points that control how chondral growth and modeling, and bone modeling drifts and remodeling BMUs, respond to mechanical usage. As a result, joints would become bigger and bones longer, wider, and denser than needed for their usual mechanical usage."

"If so, removing the pituitary adenoma should let those set points return towards normal, where upon the skeleton would "sense" more bone than needed and typical disuse responses should begin. These responses include reducing or stopping longitudinal bone growth and the diametric growth
of joints, and also the modeling drifts that increase outside bone diameter. Meanwhile, BMU-based remodeling should begin removing bone next to marrow (trabecular and cortical=endosteal bone) to enlarge the marrow cavity and cause a relative trabecular osteopenia. That is exactly what
happens in human gigantism and acromegaly after removal of the pituitary adenoma. Yet, if the hormone worked by directly stimulating chondroblasts and osteoblasts, as many believe, after removing the pituitary adenoma, the then existing bone mass should simply persist instead of reducing."

8 comments:

  1. Dear Tyler,

    at the beginning of the article you mention that hydrostatic pressure causes chondrogenic differentiation of stem cells. Where did you read this? is there any reference about that? In addition, where are the stem cells -that will be differentiated in chondrocites- located in the bone?

    Thanks

    ReplyDelete
  2. @Anonymous

    Here's a brief explanation:

    http://hakker.betaboard.net/t19-thinking-out-aloud

    I suggest you read Minigolfers other posts to find out more information...

    Ummmm, Tyler? I was thinking maybe you could check out our recent research regarding CNP and some other stuff?

    I have my own forum since the GTF board wasn't keeping up with me. (Lag, post limits, spam, server errors etc)

    Do you mind helping us out? (Me, MYFP, Altitude, getter, and some other researchers)

    Thanks.

    ReplyDelete
  3. CNP is interesting as it does seem to inhibit FGFR3 which causes dwarfism. I don't know if this will increase height in bone without actively proliferating chondrocytes.

    ReplyDelete
  4. TYLER!

    I think I've figured it out! I'm struggling on my own. No-one, not even you are helping me with my research... Please bro?

    Is there any way we can get in touch? I have a present for you! Trust me - this present is nothing like you've ever received/will receive from anyone. (hint - Book courtesy of MYFP)

    I have a solution(s) for LSJL's flaws and you can correct my flaws, together we'll be able to finish the quest for height once and for all!

    ReplyDelete
  5. so do you recommend using ipriflavone for the estrogen inhibiting proliferation problems outlined in this post?

    or do you not suggest that?

    ReplyDelete
  6. ipriflavone also inhibits osteoclast resorption which is another coause of endochrondral ossification besides vegf growth plate vascularization.

    increase nitric oxide and cgmp (arginine, icariin),etc to boosts cnp
    boost nitric oxide to inhibit vegf,
    take letrozole to lower estrogen
    and take ipriflavone to mediate osteoclast resorption and increase chondrocyte proliferation

    ReplyDelete
  7. any thoughts on this?
    http://www.ncbi.nlm.nih.gov/pmc/articles/PMC126613/

    ReplyDelete
  8. tyler can you find anything any methods any substances that upregulate
    (increase sentivity) of tgf, fgf, and igf-1 receptors without antagonizing them?

    ReplyDelete