Understanding growth plates and growth plate mechanics by helping us try to form new ones. LSJL attempts to form new growth plates by increasing the hydrostatic pressure in the bone marrow to induce chondrogenic differentiation. Is this enough to form new growth plates? What can a growth plate model teach us about what's needed for growth plate formation?
A mathematical model of epiphyseal development: hypothesis of growth pattern of the secondary ossification centre.
"This paper introduces a 'hypothesis about the growth pattern of the secondary ossification centre (SOC)', whereby two phases are assumed. First, the formation of cartilage canals as an event essential for the development of the SOC[
Cartilage canals are formed by MMPs and VEGF;
LSJL does upregulate MMP expression(specifically MMP-3),
VEGF is regulated by Estrogen which is why you need an equilibrium of Estrogen or to inhibit the bad effects of Estrogen like
ER-alpha]. Second, once the canals are merged in the central zone of the epiphysis,
molecular factors are released (primarily Runx2 and MMP9) spreading and causing hypertrophy of adjacent cells[Low concentrations of diosigen increase Runx2 and BMP-2 and
ultrasound increases Runx2 expression as well; MMP-9 can be increased by tensile strain(stretching)]. In addition, there are two important molecular factors in the epiphysis: PTHrP and Ihh. The first one inhibits chondrocyte hypertrophy and the second helps the cell proliferation. Between these factors, there is negative feedback, which generates a highly localised and stable pattern over time. From a mathematical point of view, this pattern is similar to the patterns of Turing. The spread of Runx2 hypertrophies the cells from the centre to the periphery of the epiphysis until found with high levels of PTHrP to inhibit hypertrophy. This mechanism produces the epiphyseal bone-plate. Moreover,
the hypertrophy is inhibited when the cells sense low shear stress and high pressure levels that maintain the articular cartilage structure[LSJL induces high pressure in the epiphyseal bone marrow thus inhibiting hypertrophy]."
Note that the secondary ossification center is actually within the epiphysis rather than in the metaphyseal growth plate. Thus, LSJL may form growth plates within the epiphysis itself thus increasing the size of the epiphysis. Tweaking may be needed to get LSJL effective for the metaphyseal area.
"
The mechanical loads induce deformation of the cartilage matrix, causing alterations in interstitial fluid hydrostatic pressure and changes in ionic and osmotic composition of both the tissue and interstitial fluid. The chondrocytes perceive these variations and generate specific responses to these stimuli. Besides, they can coordinate changes in expression and synthesis of matrix proteins and finally, in the biomechanical performance of the tissue"<-Now in adults there is no cartilage matrix, however the bone marrow is still deformed and there is still a change in hydrostatic pressure that can induce chondrogenic differentiation.
"The high octahedral stresses (or shear) accelerate the hypertrophy and high pressure levels maintain the cartilaginous structure, that is, the mechanical environment is of vital importance in epiphyseal growth. Mechanical loads play a relevant role in endochondral development. During bone growth, cartilage is particularly sensitive to mechanical factors for its deformation facility to sustain loads over time. The octahedral stresses play an important role in the growth of cartilage canals towards the centre of the epiphysis, where they release, especially Runx2 and MMP9s."<-shear stress helps form cartilage canals whereas hydrostatic pressure helps maintain the cartilage structure. Both shear stress and hydrostatic pressure are likely needed in sufficient quantities to form new growth plates.
"This stress is the mechanical factor influencing the growth of cartilage canals towards the central area. The results of the formation and growth of the canals in the random sites of perichondrium are shown"<-shear stress did result in the formation of new cartilage canals from the perichondrium(immature periosteum). Can shear stress be used to get cartilage canals to form from the periosteum into the metaphyseal area?
An equilibrium quantity of shear stress versus hydrostatic pressure may be needed for optimal growth plate formation?
Developmental regulation of the growth plate
"Bone formation begins when mesenchymal cells form condensations — clusters of cells that adhere through the expression of adhesion molecules. In a few areas, most notably
the flat bones of the skull, the cells of these condensations differentiate directly into bone-forming osteoblasts[thus osteoblasts can increase height directly in the flat bone of the skull]. These cells lay down a matrix particularly rich in type I collagen in a process named intramembranous bone formation. But this straightforward process is the exception. In most condensations, the cells become chondrocytes, the
primary cell type of cartilage; cells at the border of condensations form a perichondrium. Chondrocytes have a characteristic shape, secrete a matrix rich in type II collagen and the proteoglycan aggrecan, and, more generally, express a characteristic genetic program driven by SOX9"<-the goal of LSJL driven hydrostatic pressure is to get stem cells to adopt that chondrogenic cell shape.
"The hypertrophic chondrocyte, simply through its size, is the principal engine of bone growth"<-so most of the increase in size in bones is due to hypertrophic chondrocytes. However, osteoblasts can hypertrophy but do not result in taller bones(except for intramembranous ossification).
"While hypertrophic chondrocytes perform multiple tasks in the centre of the cartilage mould, the mould enlarges further through continued proliferation of chondrocyte"<-hypertrophic chondrocytes are the regulators while proliferating chondrocytes provide the growth. Osteoblasts do not have coupled hypertrophy and proliferating cells which is likely why osteoblasts do not make you taller.
""As bones enlarge further, so-called secondary ossification centres are established when chondrocytes in
characteristic, new locations stop proliferating, hypertrophy, and attract vascular invasion along with osteoblasts. In the long bones of the limb, growth chondrocytes continue to proliferate between regions of bone of the primary and secondary ossification centre"<-So if you could keep those growth plate chondrocytes proliferating in the primary ossification centre you would grow in baby-like proportions.
"The proliferative effect of Ihh is likely to be a direct action on chondrocytes, because the expression of Ptc-1 and of other general targets of hedgehog signalling is stimulated by Ihh in chondrocytes"<-so increase serum levels of Ihh increase chondrocyte proliferation and grow taller.
"PTHrP acts primarily to keep proliferating chondrocytes in the proliferative pool"<-so you can also possibly grow taller and keep growth plates open by increasing levels of PTHrP.
"When chondrocytes are no longer sufficiently stimulated by PTHrP, they stop proliferating and synthesize Ihh. Ihh can then, by mechanisms that are still unknown, stimulate the production of PTHrP at the ends of bone"<-so you need specific ratios of PTHrP and Ihh to optimize height growth.
"Knockout of Fgfr3 in mice leads to an increased rate of proliferation of chondrocytes and an expansion of
the length of chondrocyte columns"<-Inhibitors of FGFR3 would be a great way to grow taller. Transgenic expression of FGFR3 causes dwarfism.
"FGF signalling through FGFR3 inhibits proliferation; this inhibition is at least partly through activation of the Janus kinase–signal transducer and activator of transcription-1 (JAK–STAT1) pathway"<-so inhibiting parts of the JAK-STAT1 pathway could serve to increase height as well.
"knockout of Fgf18 leads to an increase in chondrocyte proliferation that closely resembles the effect of Fgfr3"<-inhibiting FGF18 is another potential height increase therapeutic option.
"knockout of the Fgfr3 gene increases Ihh expression"<-thus increasing Ihh expression may be one of the ways that knocking out FGFR3 therefore increasing Ihh expression like knocking out FGFR3 may be a way to grow taller.
"Addition of BMPs to bone explants increases proliferation of chondrocyte"<-thus BMPs are likely a very effective way to grow taller especially BMPs 2,4, and 7.
"BMP signalling increases the expression of Ihh by prehypertrophic chondrocyte"<-so BMP may too increase height by an Ihh mechanism so it may be best to directly increase Ihh levels.
"SOX9 is expressed in cells of mesenchymal condensations and in proliferating chondrocytes, but not in hypertrophic chondrocytes."<-if we can stimulate SOX9 expression in mesenchymal condensations(the bone marrow) then we can stimulate new growth plate information and height growth.
According to the study "
TNF-alpha upregulates expression of BMP-2 and BMP-3 genes in the rat dental follicle--implications for tooth eruption", TNF-alpha can upregulate BMP-2 and BMP-3. TNF-alpha is an inflammatory cytokine so it has negative effects as well but it's possible it might help you grow taller if it can upregulate those genes. TNF-alpha is upregulated by exercise.
Follistatin is a BMP agonist but
Follistatin may have beneficial effects. Too much BMP-3 can cause weaker bones for example.
A mechanobiological model of epiphysis structures formation
"Developing bone consists of epiphysis, metaphysis and diaphysis. The secondary ossification centre (SOC) appears and grows within the epiphysis, involving two histological stages. Firstly, cartilage canals appear; they carry hypertrophy factors towards the central area of the epiphysis. Canal growth and expansion is modulated by stress on the epiphysis. Secondly, the diffusion of hypertrophy factors causes SOC growth. Hypertrophy is regulated by biological and mechanical factors present within the epiphysis. Cartilage canal spatial-temporal growth patterns were obtained as well as the SOC formation pattern."
"Cartilage canal formation is due to invagination developing at specific sites on the perichondrium. The canals carry nutrients, blood vessels, growth factors and hormones to the central area of the epiphysis"<-If we can form new cartilage canals we can get new height growth.
"These canals expand in response to the mechanical stress supported by bone due to a load being exerted by the surrounding muscles in motion"<-but there must be a limit or perhaps cartilage canal expansion does not affect adult height?
"The canals release molecular factors in the inner area of the epiphysis triggering the chondrocyte hypertrophy phase. These factors are mainly matrix metalloproteinase-9 (MMP9), also known as gelatinase-B, and the RUNX2 transcription factor. RUNX2 is a positive modulator of hypertrophy and is released by hypertrophic chondrocytes "
"Parathyroid hormone-related protein (PTHrP) and Indian hedgehog (Ihh) protein are found in the epiphysis and are critical for future bone development. PTHrP inhibits Ihh expression whilst Ihh stimulates PTHrP expression; these two molecules interact with each other in a negative loop. Ihh action stimulates proliferation and PTHrP inhibits hypertrophy"<-So Ihh is more powerful than PTHrP which may be why
Ihh genes are more associated with height.
"PTHrP is expressed in the articular perichondrium and Ihh in pre-hypertrophic and hypertrophic chondrocytes"
"Hypertrophy rate of chondrocytes is positively modulated by RUNX2 transcription factor expression and dissemination and is inhibited by the presence of PTHrP"<-also of note that hypertrophic chondrocytes are located at the region of greatest octahedral(shear) stress.
"SOC[Secondary Ossification Center] growth thus initially follows a radial pattern from the centre of the epiphysis where RUNX2 is released from the canals and spreads outwards. Once RUNX2 induces hypertrophy, the hypertrophic cells collaborate in forming RUNX2. PTHrP inhibits hypertrophy and decreases RUNX2 expression"
"cartilage maturation and ossification are inhibited by intermittent hydrostatic compression stress and accelerated by intermittent non-destructive octahedral shear caused by physiological loading"
"hydrostatic pressure increases cartilage tissue expression"
"Cartilage canals appear randomly and become extended due to octahedral mechanical stresses occurring in the epiphysis."<-Thus the body is capable of forming more than two cartilage canals.
"An experiment applied 200 mN cyclical forces at 1 Hz for 12 h. Their results showed average formation of eight cartilage canals and the appearance of SOC."<-Like LSJL.
"The 200 mN mechanical cyclic loading at 1 Hz applied for 12 h at a single firmly attached condyle explant in an organ culture. Shear forces were generated due to the convex curvature of articular surface. High magnification of the formation of a canal correlates with the applied mechanical load."
Epiphyseal growth plate and secondary peripheral chondrosarcoma: the neighbours matter
"Chondrocytes interact with their neighbours through their cartilaginous extracellular matrix (ECM). Chondrocyte–matrix interactions compensate the lack of cell–cell contact and are modulated by proteoglycans and other molecules. The epiphyseal growth plate is a highly organized tissue responsible for long bone elongation. T
he growth plate is regulated by gradients of morphogens that are established by proteoglycans. Morphogens diffuse across the ECM, creating short- and long-range signalling that lead to the formation of a polarized tissue. Mutations affecting genes that modulate cell–matrix interactions are linked to several human disorders. Homozygous mutations of EXT1/EXT2 result in reduced synthesis and shortened heparan sulphate chains on both cell surface and matrix proteoglycans. This disrupts the diffusion gradients of morphogens and signal transduction in the epiphyseal growth plate, contributing to loss of cell polarity and osteochondroma formation. Osteochondromas are cartilage-capped bony projections arising from the metaphyses of endochondral bones adjacent to the growth plate. The osteochondroma cap is formed by cells with homozygous mutation of EXT1/EXT2 and committed stem cells/wild-type chondrocytes. Osteochondroma serves as a niche (a permissive environment), which facilitates the committed stem cells/wild-type chondrocytes to acquire secondary genetic changes to form a secondary peripheral chondrosarcoma."
"Cell–cell interactions and the transcription factor Sox9 regulate the formation of [mesenchymal] condensations"
"N-cadherin and N-CAM, are important in establishing an aggregation centre by recruiting mesenchymal cells from surrounding tissue"
"Down-regulation of N-CAM by the binding of syndecan to fibronectin and activation of homeobox genes (ie Msx-1 and Msx-2) by the presence of BMP-2 and BMP-4 stop condensation growth and initiate pre-chondrocyte differentiation "
" In the resting zone, chondrocytes are non-polarized and irregularly arranged. Resting chondrocytes serve as precursors (committed stem cell pool) for proliferative chondrocytes. In the proliferating zone, cell division of chondrocytes occurs perpendicular to the long axis of the growing bone. Proliferating chondrocytes have to undertake a series of cell movements/rotations (intercalation) and shape changes to align one on top of the other to generate the typical chondrocyte columns of the growth plate. Once acquired, this columnar organization is maintained. Chondrocytes require adhesion to cartilaginous ECM for all types of shape changes. Integrins are an important family of receptors that mediate chondrocyte–matrix adhesion. Integrins regulate centrosome function, the assembly of the mitotic spindle and cytokinesis. In β1-null growth plates, chondrocytes display mitotic figures perpendicular to the long axis, but they stay side-by-side and failed to move over each other and form columns, suggesting that β1 integrins regulate chondrocyte shape and rotation. In the hypertrophic zone, chondrocytes stop proliferating and change their expression profile to synthesize type X collagen and to prepare for mineralization of the surrounding cartilaginous ECM"
"Proteoglycans, such as heparan and chondroitin sulphate, function in concert to establish an Indian hedgehog (Ihh) gradient, either through affecting its diffusion or by protecting it from degradation"
"Planar cell polarity pathway has been demonstrated to regulate chondrocyte polarity in the epiphyseal growth plate. Non-polarized resting chondrocytes, which are committed progenitor cells responsible for the generation of proliferating chondrocytes, become polarized proliferating chondrocytes assuming a precise position and orientation in the epiphyseal growth plate, creating columns of stacked cells. Planar cell polarity pathway comprises molecules such as Wnt5A, the Rho family of GTPases and Gpi-anchored proteins. These molecules are shown to regulate orientated cell division and movements (intercalation) of chondrocytes"
"conditional deletion of Kif3a in the growth plate chondrocytes results in the depletion of cilia and loss of columnar organization"->Will overexpression of Kif3a result in more cilia?
"Areas with a growth plate-like distribution of proteoglycans in gradients possibly contain chondrocytes with functional EXT1/EXT2. Areas with reduced amount of proteoglycans with no gradient formation have probably chondrocytes with homozygous inactivation of EXT1/EXT2."
The skeleton: a multi-functional complex organ: the growth plate chondrocyte and endochondral ossification.
"Chondrocytes secrete factors that regulate the behaviour of the invading bone cells, including vascular endothelial growth factor and receptor activator of NFκB ligand."
"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."
"In the presence of IHH signalling, [Gli] proteins promote transcription of IHH target genes, but when IHH is absent, Gli2 and Gli3{up in LSJL} are subject to proteolytic conversion into transcriptional repressors. 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"
"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 signalling, and for chondrocyte proliferation. IHH signalling, and thus proliferation of growth plate chondrocytes, is also dependent on the presence of an intact primary cilium, 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"
"Phosphorylation of Smads 1 and 5 in chondrocytes is dependent on the presence of neogenin, which is a receptor for the neuronal axon guidance cues netrins and repulsive guidance molecules "
"mice that lack both Shn2 and Shn3 (but not each gene individually) show defects in endochondral ossification including inadequate chondrocyte proliferation, an effect that is likely to be due to failure of BMP signalling"
"FGFR3 is a tyrosine kinase receptor, and its suppression of chondrocyte proliferation in the growth plate appears to be mediated by STAT1. The activity of FGFs, like that of IHH, is modulated by glycosaminoglycans. Mice that lack sulphatase-modifying factor 1, which activates sulphatases (and thus proteoglycan desulphation) show dwarfism, which can be rescued by crossing with FGF18-null mice. Thus, it appears that FGF-induced repression of chondrocyte proliferation is limited by the desulphation of glycosaminoglycans. The relevant glycosaminoglycan is most likely to be heparan sulphate, found in proteoglycans such as perlecan in the cartilage ECM. BMP signalling antagonises the inhibition of chondrocyte proliferation caused by activation of FGFR3"
"TRPS1-null mice demonstrate abnormally low levels of proliferation in growth plate chondrocytes, which can be attributed to a role for TRPS1{up in LSJL} in repression of STAT3 expression, which in turn allows for elevated expression of cyclin D1"
"TRPS1 binds the transcriptional activator form of Gli3"<-So maybe the form of Gli3 is the transcription activator form in LSJL.
"Cartilage proteoglycans, in addition to aggrecan, include the small leucine-rich proteoglycans decorin, biglycan{up} and fibromodulin, as well as the large proteoglycan perlecan. Other non-collagenous proteins found in cartilage ECM include the matrilins and thrombospondin family members, such as thrombospondin-5, also known as cartilage oligomeric matrix protein (COMP)."
"Pseudoachondroplasia in humans is caused by mutations in COMP, and the milder multiple epiphyseal dysplasia can be caused by mutations in the genes encoding COMP, collagen type IX{up} or matrilin-3{up}" "these proteins are at least partly able to substitute for each other"
"Mice in which the gene for hyaluronan synthase 2 (Has2) is inactivated in tissues derived from limb bud mesoderm possess abnormally short limbs"
"SOX9-activated transcription appears to be modulated by epigenetic mechanisms, since it occurs predominantly in hyperacetylated chromatin; 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"
"Overexpression of HDAC1 or 2 in chondrocytes results in down-regulation of expression of Aggrecan and Col2a1"
" aggrecan degradation does not appear to be required. There are no morphological defects in the growth cartilage of mice in which aggrecan is rendered resistant to MMP cleavage"
"the role of hyaluronan may be to initiate hypertrophy-inducing intracellular signalling in chondrocytes emerging from the proliferative phase."
"Hypothyroidism in humans results in slowing of longitudinal bone growth, with abnormally thin growth plates and impaired chondrocyte hypertrophy. Studies in genetically manipulated mice have demonstrated that the receptor responsible for these effects is thyroid hormone receptor α and not thyroid hormone receptor β"
"In chondrocytes in vitro, T3 up-regulates Wnt4 mRNA and protein expression as well as cellular accumulation of β-catenin, and inhibition of WNT signalling by secreted WNT antagonists inhibits T3-induced hypertrophy"
"PTHrP stimulates cyclin D1 expression in chondrocytes, and is unable to down-regulate RUNX2 expression in chondrocytes from cyclin D1-null mice, apparently because cyclin D1 contributes to proteasomal degradation of RUNX2. The transcriptional co-regulator Zfp521, which is induced by PTHrP, has also recently been identified as an effector of PTHrP's actions in the growth plate; the growth cartilage of mice with chondrocyte-specific deletion of Zfp521 resembles that of PTHrP-null mice, and PTHrP is unable to stimulate cyclin D1 expression or inhibit RUNX2 expression in the absence of Zfp521"
"Mice with a heterozygous in-frame deletion of the DNA-binding domain of TRPS1 show elevated expression of RUNX2 in the growth plate; moreover, TRPS1 directly interacts with RUNX2 to inhibit its function. These observations suggest that TRPS1 acts to fine-tune cell cycle exit and progression to the hypertrophic state, by limiting both the antihypertrophic activity of PTH and the pro-hypertrophic activity of RUNX2, while at the same time supporting proliferation in a cyclin D1-dependent manner"
"growth plate mineralisation is not required for normal function of the growth plate itself. The importance of growth plate mineralisation may lie in provision of an appropriate composition for the remnants of cartilage matrix on which bone is deposited in the primary centre of ossification, since bone volume in the newly formed bone of the metaphysis is reduced in the absence of carminerin"
"light chondrocytes appear to disintegrate within their cell membrane and dark chondrocytes progressively extrude their cytoplasm into the extracellular space."
"growth plate cartilage degradation is not dramatically altered by failure to express cathepsin K"
"there is abnormal retention of growth plate cartilage in MMP9-null mice, it appears that osteoclasts depend more on this enzyme than on cathepsin K for their degradation of cartilage matrix"
"blood vessels appear to precede osteoclasts as the first cells that enter the lacunae recently vacated by dying hypertrophic chondrocytes, thus it is not particularly surprising that they are able to invade the growth plate in the absence of osteoclast activity. Another cell type that has recently been described as accompanying the blood vessels at the ossification front is the ‘septoclast’, a perivascular cell that expresses cathepsin B; it has been proposed that septoclasts assist in the degradation of the transverse cartilage septa, thus allowing entry of capillaries"
"High-mobility group box 1 protein (HMGB1) is secreted by hypertrophic chondrocytes, and acts as a chemoattractant for endothelial cells, osteoclasts and osteoblasts; HMGB1-null mice display delayed invasion of the growth plate by cells of the ossification centre"
Regulatory mechanisms for the development of growth plate cartilage.
"RhoA signaling through its main effector ROCK inhibits chondrogenesis by suppressing the expression of Sox9"
"Sox9 suppresses the expression of Runx2 and β-catenin signaling and thereby inhibits the progression from proliferation to prehypertrophy of chondrocytes. Twist-1 is a basic helix-loop-helix-type transcription factor, which represses the expression of Runx2 in the perichondrium. Runx2 enhances the expression of fibroblast growth factor 18 (Fgf18) and exerts an indirect negative effect on chondrocyte maturation. Osterix regulates the calcification and degradation of cartilaginous matrix through MMP13 expression in association with Runx2 "
"Genetic deletion of Mef2c in endochondral cartilage impairs hypertrophic maturation, while the forced expression of a superactivating form of Mef2c resulted in precocious chondrocyte hypertrophy. The activity of Runx2/3 and Mef2c/d is inhibited by the histone deacetylase HDAC4. Other transcription factors, such as Msx2, the AP1 family member Fra2, and FoxA family transcription factors, also positively control chondrocyte hypertrophy"
"chondrocytes express several integrin subunits including fibronectin receptors (α5β1, αnβ3, αnβ5), a laminin receptor (α6β1) and collagen receptors (α1β1, α2β1, α10β1). The importance of β1 integrin-mediated signaling in chondrogenesis was demonstrated by the chondrodysplasia-like phenotype of chondrocyte-specific β1 integrin-knockout mice"
"Inactivation of the α10 integrin gene resulted in growth plate dysfunction, which was associated with an abnormal cell shape and increased apoptosis of chondrocytes" Knockout of alpha1 integrin resulted in no growth plate defects.
"HDAC1 and HDAC2 were shown to repress the expression of some cartilage-specific genes including Col2a1, and the Snail transcription factor was identified as a mediator of the repression. The up-regulation of HDAC7 expression was suggested to contribute to the cartilage degradation by promoting the expression of MMP13"
. It's also unclear what the optimal interaction dosages would be between Cnidium and Icariin.