Why do people with marfan syndrome grow taller?

The most famous individual with Marfan's Syndrome is the late Vincent Schiavelli who was a towering 6'6".  Marfan's Syndrome is associated with tall stature and marfan's syndrome deals with connective tissue.  The connective tissue gene COL2A1 likely playing a large role in height.  Why do people with Marfan's Syndrome grow taller and can we use this information to grow taller ourselves?

Microenvironmental regulation by fibrillin-1.

"Fibrillin-1 is a ubiquitous extracellular matrix molecule that sequesters latent growth factor complexes.  Fibrillin-1 provides extracellular control of growth factor signaling. Mutations in FBN1 are mainly responsible for the Marfan syndrome (MFS), recognized by its pleiotropic clinical features including tall stature and arachnodactyly, aortic dilatation and dissection, and ectopia lentis. Each of the many different mutations in FBN1 known to cause MFS must lead to similar clinical features through common mechanisms, proceeding principally through the activation of TGFβ signaling[LSJL upregulates TGF-Beta expression]. Here we show that a novel FBN1 mutation in a family with Weill-Marchesani syndrome (WMS) causes thick skin, short stature, and brachydactyly when replicated in mice. WMS mice confirm that this mutation does not cause MFS. The mutation deletes three domains in fibrillin-1, abolishing a binding site utilized by ADAMTSLIKE-2, -3, -6, and papilin. Our results place these ADAMTSLIKE proteins in a molecular pathway involving fibrillin-1 and ADAMTS-10. Investigations of microfibril ultrastructure in WMS humans and mice demonstrate that modulation of the fibrillin microfibril scaffold can influence local tissue microenvironments and link fibrillin-1 function to skin homeostasis and the regulation of dermal collagen production. Hence, pathogenetic mechanisms caused by dysregulated WMS microenvironments diverge from Marfan pathogenetic mechanisms, which lead to broad activation of TGFβ signaling in multiple tissues[WMS causes short stature rather than tall stature and WMS messes where TGF-Beta signaling occurs(more in the skin and less in the cartilage for example) thereby linking TGF-Beta signaling to one of the reasons that people with Marfan's Syndrome grow taller]. We conclude that local tissue-specific microenvironments, affected in WMS, are maintained by a fibrillin-1 microfibril scaffold, modulated by ADAMTSLIKE proteins in concert with ADAMTS enzymes."

"More than a thousand different mutations in FBN1, the gene for fibrillin-1, are known to cause MFS, suggesting that the same general pathogenetic mechanisms are initiated by each of these distinct mutations."<-So many mutations in FBN1 are likely to cause tall stature Marfan's Syndrome but relatively few are likely to cause short stature.

"fibrillin-1 targets and sequesters the large latent Transforming Growth Factor β (TGFβ) complex as well as multiple Bone Morphogenetic Proteins (BMPs) and Growth and Differentiation Factor-5 (GDF-5)"<-all of these proteins have been associated with height growth.

According to "A new mouse model for marfan syndrome presents phenotypic variability associated with the genetic background and overall levels of Fbn1 expression.",  knockout of FBN1 can be lethal and no evidence of larger stature was shown.  Thus, Fibrillin-1 is likely not a purely inhibitive compound on height growth and specific mutations must occur to increase height growth(numerous combinations of mutations work however).

In vivo studies of mutant fibrillin-1 microfibrils.

"While most of the known mutations in fibrillin-1 cause Marfan syndrome, a number of other mutations lead to clinical features unrelated to Marfan syndrome. Pathogenesis of Marfan syndrome is currently thought to be driven by mechanisms due to haploinsufficiency of wild-type fibrillin-1[haploinsufficiency means a partial knockout of the gene]. However, haploinsufficiency-driven mechanisms cannot explain the distinct phenotypes found in other fibrillinopathies. To test the hypothesis that mutations in fibrillin-1 cause disorders through primary effects on microfibril structure[if marfan's syndrom primarily acts on microfibers than perhaps microfibrils play a role in height growth], two different mutations were generated in Fbn1 in mice. One mutation leads to a truncated fibrillin-1 molecule that is tagged with green fluorescent protein, allowing visualization of mutant fibrillin-1 incorporated into microfibrils. In heterozygosity, these mutant mice demonstrate progressive fragmentation of the aortic elastic lamellae and also display fragmentation of microfibrils in other tissues. Fibrillin-2 epitopes are also progressively revealed in these mice, suggesting that fibrillin-2 immunoreactivity can serve as a marker for microfibril degradation. In contrast, a second mutation (in-frame deletion of the first hybrid domain) in fibrillin-1 results in stable microfibrils, demonstrating that fibrillin-1 molecules are not required to be in perfect register for microfibril structure and function and that the first hybrid domain is dispensable for microfibril assembly. Taken together, these results suggest that perturbation of microfibril structure may underlie one of the major features of the Marfan syndrome: fragmentation of aortic elastic lamellae."

"Overexpression of mutant human FBN1 showed incorporation of the human fibrillin-1 into microfibril structures, using human specific fibrillin-1 antibodies, but no apparent effects of the mutant human fibrillin-1 were found on microfibril structure or function in these transgenic mice"<-So merely having higher levels of fibrillin-1 doesn't do anything.

"In one line, fibrillin-1 is truncated[so the gene does not attain it's proper form] and tagged with enhanced green fluorescent protein (eGFP), allowing us to conclusively demonstrate that mutant fibrillin-1 is incorporated into microfibrils. In heterozygosity, these mice develop features of Marfan syndrome and, in homozygosity, do not survive past the early postnatal period. In a second line of mutant mice, exon 7 is deleted, resulting in the in-frame deletion of the first hybrid domain in fibrillin-1. To our surprise, these mice survived normally, even in homozygosity, and manifested no apparent defects in microfibril structure or function"<-So when you have non-functional fibrillin-1 incorporated into microbrils then you get the tall stature of Marfan's syndrome.    However, if you just have lower levels of fibrillin-1, no change is noted at all.  So perhaps when non-functional fibrillin-1 is incorporated into microfibrils it serves some purpose that makes you grow taller.

"Although wild-type fibroblasts assembled abundant fibrillin-1 fibrils, heterozygous GT-8 fibroblasts assembled fewer fibrillin-1 fibrils[mice with some of the fibrillin-1 fibrils non-functional and some functional], and homozygous GT-8 fibroblasts failed to assemble a fibrillin-1 fibril matrix[all the fibrillin-1 fibrils were non-functional]. eGFP-tagged and -truncated fibrillin-1 was assembled into fibrils by heterozygous GT-8 fibroblasts but not by homozygous GT-8 fibroblasts"<-So the mice with marfan's syndrome(and taller stature) assumed fewer fibrillin-1 fibrils.  Thus, fibrillin-1 fibrils may play an inhibitory role on height but you need some quantity of fibrillin-1 fibrils to grow taller and live.  Fewer microfibrils may be more elastic thus allowing for more height growth.

It's possible that the malfunction of the fibroblasts due to the incorporation of truncated fibrillin-1 fibrils interferes with TGF-Beta signaling which results in height growth.

In the first study, we saw that also disorders in FBN1 can cause short stature.  With the malfunctioning microfibril hypothesis in the last study, we can theorize that perhaps a specific truncation of fibrillin-1 results in short stature

What we can conclude is that microfibrils may play a role in height growth and that having less of them in the growth plate or possibly even the bone may be beneficial for height.  Therefore, we might want to come up with ways to reduce the number of microfibrils(we need some though).

Immunolocalisation of fibrillin microfibrils in the calf metacarpal and vertebral growth plate.

"Overgrowth of limbs and spinal deformities are typical clinical manifestations of Marfan syndrome (MFS) and congenital contractural arachnodactyly (CCA){both diseases cause overgrowth}, caused by mutations of the genes encoding fibrillin-1 (FBN1) and fibrillin-2 (FBN2), respectively. FBN1 mutations are also associated with acromicric (AD) and geleophysic dysplasias (GD), and with Weill-Marchesani syndrome (WMS), which is characterised by short stature.Here we investigated the organisation of fibrillin microfibrils in the growth plate of the long bone and vertebra immunohistochemically. Fibrillin-1 was dual-immunostained with elastin, with fibrillin-2 or with collagen X.  Fibrillin microfibrils are distributed throughout all regions of the growth plate, and that fibrillin-1 and fibrillin-2 were differentially organised. Fibrillin-1 was more abundant in the extracellular matrix of the resting and proliferative zones of the growth plate than in the hypertrophic zone. More fibrillin-2 was found in the calcified region than in the other regions. No elastin fibres were observed in either the proliferative or hypertrophic zones. This study indicates that, as fibrillin microfibrils are involved in growth factor binding and may play a mechanical role, they could be directly involved in regulating bone growth."

"Fibrillin microfibrils are associated with the elastic fibre network, which in general consists of an elastin core surrounded by a network of fibrillin microfibrils. An extensive elastic network is distributed in the ECM of many tissues"

"the network of microfibrils could play a functional role in the long bone and spinal growth. Microfibrils, as part of the elastic fibre system, are commonly thought to play a mechanical role"

Contractile Forces by Chondrocytes and Height Increase

Previously, we found that cells have a rounding force when they enter mitosis.  And, it is known that when muscles and heart cells need to generate more force they hypertrophy.  If enochondral ossification were a purely transformative process then you wouldn't grow taller at all.  Your cartilage would just turn into bone and you'd stay the same size.  Since hypertrophy is linked to force generation in muscle organs it follows too that it might be the case that hypertrophy may be important for force generation for chondrocytes.  Chondrocytes also have contractile ability which means they are capable of generating force.  If we knew how much force was required to elongate bone during endochondral ossification then we could mimic this force and generate height via other means.


Contractile forces generated by articular chondrocytes in collagen-glycosaminoglycan matrices.


"The objective of the study was to directly measure the force of contraction of adult articular chondrocytes and to examine their contractile behavior in collagen-glycosaminoglycan analogs of extracellular matrix[this is for articular rather than growth plate cartilage but force generation and contractile behavior should be similar between articular carilage and growth plate cartilage]. The contractile forces generated by passages 2 and 3 adult canine articular chondrocytes were measured using a cell force monitor. Passage 2 cells seeded in a collagen-glycosaminoglycan scaffold were capable of generating a force of 0.3 nN/cell. Chondrocytes subcultured through a third passage generated a force twice that level, paralleling the increase in the alpha-smoothmuscle actin (SMA) content of the cells. Treatment of passage 3 cells with staurosporine reduced the force of contraction by approximately one-half, reflecting the effects of this agent in reducing the SMA content of the cells and disrupting the microfilaments. These values compare with 1 nN previously reported for lapine dermal fibroblasts of passage 5-7[dermal fibroblasts are a type of skin cell that are capable of stretching and elongating], using the same apparatus. Direct live cell imaging documented the contractile behavior of the articular chondrocytes in the collagen-glycosaminoglycan matrix in the time frame in which the force was directly measured in the cell force monitor. the cells acted individually and in unison to buckle the collagen struts[so the chondrocytes could act in unison in the growth plate to make bone longer] making up the matrix. Adult articular chondrocytes are capable of generating a SMA-enabled force of contraction sufficient to model extracellular matrix molecules."

"The force of contraction recorded in the present study for canine chondrocytes is approximately twice the value previously measured for second passage adult human articular chondrocytes in the same apparatus"<-But humans are taller than dogs despite dags having larger contraction forces.  Maybe human chondrocytes are better organized or humans have more chondrocytes than dogs making up for the reduction in force generation.

Growth Factor Regulation of Smooth Muscle Actin Expression and Contraction of Human Articular Chondrocytes and Meniscal Cells in a Collagen–GAG Matrix

"human articular chondrocytes can express the gene for a contractile actin isoform, α-smooth muscle actin (SMA), in vivo. The objective of the present study was to evaluate the effects of two growth factors, transforming growth factor (TGF)-β1 and platelet-derived growth factor (PDGF)-BB, on the SMA content of these cells and their contraction of a collagen–glycosaminoglycan (GAG) analog of extracellular matrix in vitro. TGF-β1 was found to markedly increase SMA content of the cells and PDGF-BB decreased SMA expression, with both findings achieving statistical significance. A notable finding was the increased contraction of the collagen–GAG matrix induced by TGF-β1 and the decrease in contraction resulting from PDGF-BB treatment, indicating a causal relationship between expression of SMA and the contractility of the cells[So TGF-Beta1 may increase height by increasing the contractility of the cells.  However, TGF-Beta1 has not been shown to necessarily increase height only in inducing chondrogenesis(TGF-Beta1 has other effects as well) and dogs are not taller than humans despite having more SMA(a contractile actin)]. A novel cell force monitor, employed to estimate the force exerted per cell, demonstrated a higher force exerted by the TGF-β1-treated cells. The findings demonstrate that the expression of SMA by articular chondrocytes and meniscal cells and their associated contractile behavior can be regulated by selected growth factors."

BMP-2 causing Smad 1/5/8 phosphorylation was found to accelerate mineralization and terminal differentiation whereas TGF-Beta1 causing Smad 2/3 phosphorylation was found to deccelerate terminal differentiation.  Whether either gene inactivation can increase height is unknown.  ESL-1 inhibits TGF-Beta and when ESL-1 knockout mice were generated height decrease resulted.  So when TGF-Beta1 can't be inhibited height increase results.

According to the study "Bone Morphogenetic Protein-2-Induced Signaling and Osteogenesis Is Regulated by Cell Shape, RhoA/ROCK, and Cytoskeletal Tension", "BMP triggered rapid and sustained RhoA/Rho-associated protein kinase (ROCK) activity and contractile tension only in spread cells".  Now chondrocyte growth plate cells tend not to be spread whereas osteoblast cells tend to be spread.  So this indicates possibly that BMP doesn't generate contractile force in unspread cells(like chondrocytes).  "BMP-induced osteogenesis is progressively antagonized with decreased cell spreading".  So BMP is more likely to induce chondrogenesis when the cells are closer perhaps this is linked to the contractile forces.

So it's still unclear which is better for height growth TGF-Beta1 and BMP-2.  LSJL upregulates TGF-Beta1 and BMP-2.  If BMP-2 can't induce contractile forces in unspread cells then that may be a reason why TGF-Beta1 and BMP-2 would have different effects on adult height.  If BMP-2 does increase adult height more than TGF-Beta1 then that would be an indication that chondrocyte cell forces are not the driving force behind bone lengthening.  BMP-2 driven chondrocytes being less able to generate contractile forces than TGF-Beta1 chondrocytes might result in less efficiently being able to model the matrix thus more time to proliferate before hypertrophy.  BMP-2 chondrocytes have less type X collagen(hyptrophic collagen) and more Type II collagen.  Since BMP-2 chondrocytes can't generate any force from chondrocyte hypertrophy they have to generate it all by mitotic cell rounding thus resulting in more chondrocyte proliferation.

So, contractile forces may still be the force behind bone lengthening but with BMP-2 chondrocytes it's primarily chondrocyte proliferation driven whereas with TGF-Beta1 chondrocytes it's chondrocyte proliferation driven.

Smad6/Smurf1 overexpression in cartilage delays chondrocyte hypertrophy and causes dwarfism with osteopenia.

"Biochemical experiments have shown that Smad6 and Smad ubiquitin regulatory factor 1 (Smurf1) block the signal transduction of bone morphogenetic proteins (BMPs). However, their in vivo functions are largely unknown. Here, we generated transgenic mice overexpressing Smad6 in chondrocytes. Smad6 transgenic mice showed postnatal dwarfism with osteopenia and inhibition of Smad1/5/8 phosphorylation in chondrocytes. "

LSJL alteration of either Smurf1 or 2 is predicted and downregulates Smad1.

"Smad ubiquitin regulatory factor 1 (Smurf1) and Smurf2 induce the ubiquitination and degradation of Smad1 and Smad5"<-We need to know the proportion of how much LSJL upregulates Smad1 relative to the Smurf's.

"Smurf1 and Smurf2 interact with nuclear Smad7 and induce the nuclear export of Smad7. Smurf–Smad7 complexes then associate with type I receptor for TGF-β and enhance its turnover "<-LSJL had no effect on Smad7 but LSJL did upregulate TGF-Beta Receptor I.

"Smurf1 binds to BMP type I receptors via Smad6 and Smad7, and that it induces the ubiquitination and degradation of these receptors. Thus, Smad6 and Smurf1 cooperatively down-regulate BMP signals by degradation of R-Smads as well as BMP receptors."<-LSJL induces BMP RII rather than BMP RI which is consistent with the elevated Smurf levels.

"Exogenous rhBMP2 added to Smurf1 transgenic cartilage in organ cultures of metatarsal rudiments at 15.0 d.p.c. caused proliferative cartilage outgrowth and hypertrophic center formation as in normal mice. "<-So maybe the Smurf1 upregulation doesn't inhibit the height growth inducing elements of LSJL.

"Smad6 appears to block BMP signaling, whereas Smad7 blocks that of both TGF-β and BMP"  rhBMP2 is what phosphorylates Smad1/5/8.

So BMP-2 may be better for height growth than TGF-Beta1 and this may be linked to BMP-2 chondrocytes lower contractile ability which results in slower terminal differentiation and mineralization.

In Bone Marrow Chondrogenesis of Mesenchymal Stem Cells

The goal of Lateral Synovial Joint Loading is to use lateral loads to induce hydrostatic pressure in the epiphyseal bone marrow.  This hydrostatic pressure results in chondrogenic differentiation.  Unlike with osteoblasts, chondrocytes are both hydrophillic(water loving) and have their hypertrophy and cellular proliferation coupled in columns.  It is likely some combination of these two properties that allow chondrocytes to be much more effective at lengthening bone than osteoblasts.  Thus, studies of in vivo(within the body) studies of chondrogenic differentiation would be very useful in providing insights to the LSJL method.  As they could both tell us how best to induce chondrogenic differentiation and whether the chondrocytes generated by LSJL could increase height.

In vivo chondrogenesis of adult bone-marrow-derived autologous mesenchymal stem cells.

"[What is the effect of] the normal joint cavity environment on chondrocytic differentiation of bone-marrow-derived mesenchymal stem cells (MSCs)[not the epiphyseal bone marrow which is our target area for inducing MSC chondrogenic differentiation]? Autologous bone marrow was aspirated from the iliac crest of male sheep. Labeled MSCs were then grown on a three-dimensional porous scaffold of poly (L-lactic-co-glycolic acid) in vitro and implanted into the joint cavity by a surgical procedure. At 4 or 8 weeks after implantation, the implants were removed for histochemical and immunohistochemical analysis. The [MSCs] in the implants expressed type II collagen and synthesized sulfated proteoglycans. However, the osteoblast-specific marker, osteocalcin, was not detected by immunohistochemistry indicating that the implanted MSCs had not differentiated into osteoblasts by being directly exposed to the normal joint cavity. To investigate the possible factors involved in chondrocytic differentiation of MSCs further, we co-cultured sheep MSCs with the main components of the normal joint cavity, viz., synovial fluid or synovial cells, in vitro. After 1 or 2 weeks of co-culture, the MSCs in both co-culture systems expressed markers of chondrogenesis. These results suggest that synovial fluid and synovium from normal joint cavity are important for the chondrocytic differentiation of adult bone-marrow-derived MSCs[the interstitial fluid flow from LSJL may help synovial fluid get to the epiphysis and synovial tissue might be able to be accessed via the periosteum]."

"in vivo experiments have demonstrated that MSCs implanted into surgically created, osteochondral defects in rabbits are able to differentiate into chondrocytes and improve the quality of cartilaginous repair"<-But not yet whether MSCs in intact epiphyseal bone marrow can differentiate into chondrocytes. Osteochondral defects give more access to various fluids and nutrients. Bone is well vascularized so it may be able to provide many of the nutrients present in osteochondral defects.

Since LSJL involves synovial joint loading it seems likely that loading the synovial joint plays some role in inducing the chondrogenesis than just increasing the hydrostatic pressure in the bone marrow. Loading the synovial joint cavity may enhance the expression of genes that result in the induction of chondrogenesis. Loading the synovial cavity may also create a pressure gradient that increases in hydrostatic pressure in the epiphyseal bone marrow but direct effects on gene expression through the loading of the synovial cavity may play a role as well. To study this, you'd have to perform LSJL on rats, after removing their joint capsule, and then compare the results to the rats in the other study.

The LSJL scientists studied the effects of surgical holes on bone formation but not chondrogenesis.  But it's likely that any chondrogenesis in the bone would've increased the bone measurements like cortical area and cortical thickness.  Thus, the increase in intramedullary pressure may be the main determinant for both chondrogenic and osteogenic differentiation but synovial fluid transport from the joint cavity may enhance chondrogenesis.  Whereas synovial fluid transport from the joint cavity does not encourage osteogenic differentiation.

Here's a study about what happens to extra MSCs that come into the body:

The dynamic in vivo distribution of bone marrow-derived mesenchymal stem cells after infusion.

"Rat marrow-derived MSCs were ex vivo culture-expanded and infused into syngeneic rats via intra-artery (i.a.), intravenous (i.v.) and intraperitoneal cavity (i.p.) infusions[so put directly into the blood stream basically]. In addition, for i.a. and i.v. infusions, a vasodilator, sodium nitroprusside, was administered prior to the cell infusion and examined for its effect on MSC circulation. The dynamic distribution of infused MSCs was monitored by real-time imaging using a gamma camera immediately after infusion and at 48 h postinfusion. After 48 h, radioactivity in long bones, was measured in a gamma well counter and expressed as a percentage of injected doses. After both i.a. and i.v. infusion, radioactivity associated with MSCs was detected in other organs[including long bones. the homing of labeled MSCs to the marrow of long bones was significantly increased by the pretreatment with vasodilator[Vasodilators widen the blood vessels like Viagra]. These results indicate multiple homing sites for injected MSCs and that the distribution of MSCs can be influenced by administration of vasodilator."

"Vascular cell adhesion molecule-1 (VCAM-1) is expressed by the marrow stromal cells and may play an important role in the interchange of cells between bone marrow and blood."<-So areas with bone marrow already like the long bones express VCAM-1. The more vascularized the blood the more likely it is for MSCs in the blood to bind to the bone marrow.

So to get more MSCs to the epiphysis you'd want more epiphyseal bone marrow to be there and to take a vasodilator.

Induction of chondrogenesis in muscle, skin, bone marrow, and periodontal ligament bydemineralized dentin and bone matrix in vivo and in vitro.

"Induction of chondrogenesis in vivo by rolls of demineralized dentin[dentin is a part of your tooth] implanted in muscle, subcutaneous connective tissue of skin, medullary cavity of femur[not the epiphysis though], and periodontal ligament of rat was investigated. Specimens were examined at various times up to 21 days after implantation. Induction of cartilage occurred most quickly in muscle, followed by subcutaneous connective tissue of skin and medullary cavity of femur, and most slowly in periodontal ligament. Significantly more cartilage was found in muscle than in subcutaneous connective tissue of skin and medullary cavity of femur at the times examined, and least of all in periodontal ligament. Outgrowth of cells from rat muscle, dermis and subcutaneous tissue, bone marrow and periodontal ligament cultured in vitro on demineralized bone matrix for up to 35 days produced similar results."

"Implant in medullary cavity of femur. Wounds were made. A skin incision, approximately 3.0 cm in length,
was made along the lateral aspect of the right thigh, and the femoral muscles were retracted to expose the lateral aspect of the femur. A defect approximately 1.4 mm in diameter was cut in the mid-region of the lateral aspect of the femoral diaphysis, by means of a #3 round bur mounted in a slow-running dental handpiece cooled with phosphate-buffered saline, pH 7.0. The underlying bone marrow was excised with a dental excavator, and debris was removed by being washed with phosphate-buffered saline. One dentin roll was implanted in the medullary cavity proximal to the wound, and parallel to the long axis of the bone. The muscle was sutured with 3.0 silk and the skin with 4.0 silk."

It took 10 days for cartilage to form in the bone.  The cartilage formed directly next to the dentin roll.  No chondrogenesis was evident after day 21.

"demineralized bone matrix is mitogenic for mesenchymal cells; and second, that demineralized bone matrix binds fibronectin, an event that is necessary for induction of cartilage"<-It's possible that hydrostatic pressure demineralizes bone matrix which allows for new cartilage growth to occur.  Acid is also a way to dimeralize bone matrix.

In this study cartilage only formed directly next to the dimineralized dentin roll.  Thus dimineralized matrix is likely necessary for cartilage growth.  Thus for LSJL to be more effective we may need to dimineralize the bone matrix.  Hydrostatic pressure may already contribute to demineralization of the bone as bone tissue is hydrophobic.

Lactic Acid is naturally produced in response to exercise.  Ascorbic acid(Vitamin C), which can induce chondrogenesis, is naturally occurring.  The main minerals that give bone it's stiffness are Vitamin D, phosphorus, and Vitamin C.  However, people with osteomalacia are not growing taller.  In fact people with osteomalacia have stunted height.  So, we want only partial bone demineralization only on the surface area of the bone to allow for chondrogenesis to occur on it.  Hydrostatic pressure may demineralize the surface area of the bone without affecting the mineralization of the interior of the bone providing a safe way to increase height.

The longitudinal ends of the bones may provide a demineralized bone portion for mesenchymal stem cells to differentiate into chondrocytes in the joint capsule.  Osteoarthritis in fact involves changes in subchondral bone mineralization which may affect the stem cells ability to undergo chondrogenesis.

Osteoclasts operate by using an electron transport pump to acidify the surface of the bone.  So osteoclasts would also be a way to dimineralize a bone surface to allow for chondrogenesis.

Surgically, you could insert a roll of demineralized dentin into your growth plate and then take stem cells followed by a vasodilator.

Compressive force promotes sox9, type II collagen and aggrecan and inhibits IL-1beta expression resulting in chondrogenesis in mouse embryonic limb bud mesenchymal cells.

"Biomechanical forces may be one of the major epigenetic factors that determine the form and differentiation of skeletal tissues. In order to test the hypothesis that static compressive forces are transduced into molecular signals during early chondrogenesis, we have developed a unique three-dimensional collagen gel cell culture system which is permissive for the proliferation and differentiation of chondrocytes. Mouse embryonic day 10 (E10) limb buds were microdissected and dissociated into cells which were then cultured within a collagen gel matrix and maintained for up to 10 days. Static compressive forces were exerted onto these cultures. The time course for expression pattern and level for cartilage specific markers, type II collagen and aggrecan, and regulators of chondrogenesis, Sox9 and IL-1beta, were analyzed and compared with non-compressed control cultures. Under compressive conditions, histological evaluation showed an apparent acceleration in the rate and extent of chondrogenesis. Quantitatively, there was a significant 2- to 3-fold increase in type II collagen and aggrecan expression beginning at day 5 of culture and the difference was maintained through 10 days of cultures. Compressive force also causes an elevated level of Sox9, a transcriptional activator of type II collagen. In contrast, the expression and accumulation of IL-1beta, a transcriptional repressor of type II collagen was down-regulated. We conclude that static compressive forces promote chondrogenesis in embryonic limb bud mesenchyme, and propose that the signal transduction from a biomechanical stimuli can be mediated by a combination of positive and negative effectors of cartilage specific extracellular matrix macromolecules."

"During chondrogenesis, it was proposed that the osmotic changes during hyaluronic acid synthesis and degradation could produce sufficient swelling and deswelling to regulate the balance between cell-cell versus cell-ECM contacts"

"[mesenchymal] condensation is promoted by a regulated balance between cell-ECM and cell-cell adhesion involving type I and type III collagen, tenascin, fibronectin, cellular receptors for these matrix proteins such as integrins, and other non-integrin adhesion molecules, such as cell-cell adhesion molecules N-CAM and N-cadherin"

"As matrix molecules are deposited between cells, cell-cell contacts are lost"

"Static compressive forces were applied by the use of a weight placed on the lid of the culture plate exerted through a well insert which fitted on top of the collagen gel"

"1, 1.5, and 2 kPa was empirically determined to yield a 20-30% deformation of the gel"<-Can LSJL induce a 20-30% deformation of the epiphyseal bone marrow?

"When 1.5 and 2 kPa of force was loaded, type II collagen mRNA increased 1.5- and 3.8-fold, respectively, over transcript levels detected in samples subjected to 1 kPa of force magnitude"

"A collagen gel matrix under compressive force is likely to become more compact when compared with non-compressed matrix. Compaction may lead to changes in gel density and cell number per unit volume."<-The study also mentions that high cell density promotes chondrogenesis.

"Possible mechanisms of how compressive stimuli are translated into biochemical signals might involve integrin or non-integrin ECM receptors, cell-cell adhesion molecules or alteration of cytoskeletal architecture mediated by MAP kinase, rho kinase or focal adhesion associated tyrosine kinase signal transduction pathways "

Here's a diagram of the bone marrow of the intramedullary cavity, the epiphyseal bone marrow cavity should be similar from the study(Hierarchical organization and regulation of the hematopoietic stem cell osteoblastic niche):

Schematic of osteoblastic niche in bone marrow. Osteoblastic niche locates in trabecular region of bone marrow. Active osteoblats (OB), bone-lining cells (BLC), and osteoclasts (OC) constitutively line its bony boundary. Hematopoietic stem cell (HSC)–mesenchymal stem cell (MSC) pairings share common niches. Hierarchical regulators from immune, humoral, and neural systems are engaged in the osteoblastic niche and are represented by FoxP3 regulatory T (Treg), parathyroid hormone (PTH), and β-adrenergic nerve cell (β-ANC) with ensheathing glial cells (GC) respectively. Abbreviations: MK, megakaryocytes; OM, endosteal macrophages; EDL, endothelium; PPR, PTH/PTHRP receptor; brown dot ()—Ca²⁺; blue dot ()—PTH; —PPR.

What's important to note is that the ratio of MSCs to other cells is very small so work will be needed to achieve mesenchymal condensation to induce chondrogenesis.

Other facts of note from the paper: "BMMSCs preferentially locate in developing sinusoids[a blood vessel], their own microvascular niche, which is a primary producer of angiopoietin-1, a vital molecule for HSCs vascular niche remodeling"

"increased osteoclastogenesis decreases bone trabecular acreage, as well as hematopoietic stem and progenitor cell (HSPC) pools"<-So osteoclastgenesis may be helpful for inducing chondrogenesis by increasing the number of potentially chondrogenic cells.

Regeneration of cartilage and bone by defined subsets of mesenchymal stromal cells--potential and pitfalls.

"the triple positive subset (CD56+, CD271+, TNAP+) displayed a pronounced chondrogenic potential"

Inferred Functions of "Novel" Genes Identified in Fibroblasts Chondroinduced by Demineralized Bone

" several functional classes of genes (cytoskeletal and matrix elements, cell adhesion proteins, peptide growth factors, and signal transduction proteins) were found to be altered in human dermal fibroblasts (hDFs) cultured in porous collagen sponges with chondroinductive demineralized bone powder (DBP) for 3 days. In addition, a number of "novel" sequences were identified. In this study, molecular techniques were combined with computational methods to characterize those sequences. Gene expression of all 10 novel sequences tested was found in hDFs by RT-PCR. The sequences were compared to the human genome, and their cellular functions were inferred from genes that mapped to the same chromosomal coordinates. Only one of the novel sequences contained a protein-coding region (kinesin superfamily protein 26B){up in LSJL}. The others contained 3′ untranslated (osteonectin, alpha-V integrin, RAP2B) or other untranslated regions (PTPN21, GAS6) of mRNAs."

"DBP induces osteogenesis by an endochondral mechanism.  Mesenchymal cells are attracted to DBP, differentiate into chondroblasts, and secrete a cartilage matrix. The cartilage matrix is then calcified, resorbed, and replaced by bone and hematopoietic bone marrow"

Demineralized bone alters expression of Wnt network components during chondroinduction of post-natal fibroblasts.

"Human dermal fibroblasts (hDFs) were cultured in porous, three-dimensional (3D) collagen sponges with or without chondroinductive DBP. In some experiments, lithium chloride (LiCl), an agonist of the Wnt/beta-catenin signaling pathway, was added to the culture media. Sponges were cultured for intervals (0.5-21 days) before processing for molecular, histologic, and biochemical analyses. Expression of wnt, fzd, and sfrp genes was characterized by semi-quantitative RT-PCR. Fibroblasts' contacts with DBP were documented by histology. Accumulation of proteoglycan in extracellular matrix was evaluated by histology (metachromasia in toluidine blue-stained sections) and quantitative immunoassay (chondroitin 4-sulfate ELISA).

Expression of 15 wnt, fzd, and sfrp family members was detected in hDFs by RT-PCR. A subset of those genes (wnt2b, wnt5b, wnt10b, fzd6, fzd7) showed altered expression in hDFs exposed to DBP for 3 days. wnt and fzd gene expression was not altered before hDFs contacted the DBP within the collagen sponge. Human DFs cultured in plain collagen sponges and treated with LiCl accumulated significantly more metachromatic matrix than NaCl-treated controls on day 10, and showed a trend towards increased matrix chondroitin-4 sulfate content.

These data suggest that changes in Wnt signaling contribute to chondroinduction of post-natal fibroblasts by DBP. This is the first evidence that Wnt components, which are essential regulators of pre-natal chondrocyte differentiation, may also influence post-natal chondrocyte differentiation induced by DBP."

Lithium chloride is an activator of the Wnt pathway.

"hDFs cultured in LiCl-treated sponges were surrounded by significantly more metachromatix matrix"

"In the chick, chondrogenesis in embryonic mesenchymal cells is enhanced by ectopic expression of wnt5b and inhibited by fzd7"

"human bone marrow stromal cells induced with TGF-β [to chondrogenesis] show decreased fzd2{up} and increased wnt11 mRNAs"

"In human bone marrow stromal cells, LiCl-treatment increases the chondrogenic response to TGF-β. In the embryonic mouse limb, chondrogenic cells do not express Dkk-1, an inhibitor of the Wnt/β-catenin pathway. In the murine mesenchymal cell line, C3H10T1/2, overexpression of wnt3a increases nuclear β-catenin levels and these cells’ chondrogenic response to bone morphogenetic protein 2 (BMP-2). Curiously, exposure of C3H10T1/2 cells to LiCl inhibits BMP-2-induced chondrogenesis. Chick embryonic limb mesenchymal cells exposed to LiCl also show reduced chondrogenesis. Finally, β-catenin expression inversely correlates with expression of the cartilage matrix protein, collagen type II, during in vitro chondrogenesis of chick embryonic mesenchymal cells"

"During mouse limb development, loss of calcium-pathway-signaling Wnt5a increases β-catenin and inhibits chondrogenesis"

In vitro model of mesenchymal condensation during chondrogenic development.

"The extracellular matrix (ECM) secreted by cells are macromolecules with nanofibrillar features. The ECM plays a crucial role in tissue development, by providing a natural ‘scaffold’ to guide cell responses, in terms of regulation of cell anchorage, migration, organization and triggering signals that govern cell survival, cell cycle progression and expression of differentiated phenotypes during development. Basement membranes (BM) are 50-100 nm thick nano-fibrous sheet-like ECM architectures that underpin epithelial and endothelial cells. BM is also found supporting several mesenchymal cell types, such as smooth muscle cells, adipocytes- the cells which are derived from the same progenitor cells as chondrocytes."

"Chondrocytes are terminally differentiated cells originating from pluripotent mesenchymal stem cells. Mature human Chondrocytes are not in direct contact with BM in cartilage."

"individual BM components [are present] in adult cartilage matrix, such as laminin, perlecan, nidogen-2  and collagen type IV"

"With progression of age this extensive ECM layer gradually becomes less and less distinct. In the cartilage of newborn mice, BMs are widespread in surrounding matrices, whereas in mature cartilage of adult mice BMs are localized mainly to a narrow pericellular zone"

"on the 30 cm matrix (malleable nanofibrous matrix) cells initially assumed a spread morphology but after 3-4 days started to assume a rounded morphology. After 8-10 days of culture in the presence of chondrogenic media, cells migrated and formed aggregates on the 30 cm mats."

"As H9 ES cells (passage 35) were seeded on the 30 cm mat in aggregated form, thus no significant modulation in cell migration or aggregate size was noticed over 10 days of culture. TEM images of the ES cell clusters showed that initially all the ES cells were in close contact with each other, maintaining a rounded cell shape typical of ES cells. After 10 days within the aggregates individual cells were separated by putative ECM, which may be simulating “chondron”-like morphology. The prominent presence of lipid vacuoles in the cytoplasm was also noticed in the ES aggregates, as reported earlier for chondrogenic differentiation of mouse ES cells"

"The level of chondrogenesis regulating transcripts, such as collagen-II, Sox-9, aggrecan and fibronectin were significantly lower in nanofibrillar matrices with higher stiffness, where cells maintained their spread morphology and hence, poor tissue forming ability, compared to the cells grown on nanofibrillar matrices with relatively reduced stiffness"

Gene-Induced Chondrogenesis of Mesenchymal Stem Cells: A Comparative Analysis of Candidate cDNAs

[This is not a full study but just a paper]

"Gene transfer to MSCs can be used to achieve sustained expression of specific protein factors capable of inducing chondrogenic differentiation. In this study, using adenoviral mediated gene transfer and bone marrow derived MSCs in an aggregate culture system, we compared the relative condroinductive capacity of several cDNAs whose protein products have been associated with the induction or maintenance of the articular cartilage phenotype. First generation, E1, E3 deleted, serotype 5 adenoviral vectors carrying the complete cDNAs for TGF-b1, bone morphogenetic protein (BMP) -2, BMP -4 , BMP- 7, Sox 9, indian hedgehog (IHH), and insulin-like growth factor-1 (IGF-1) were constructed using cre-lox recombination. As a source of MSCs, adherent, colony forming cells from the bone marrow of 3-week-old calves were isolated, cultured and used at low passage. Individual flasks of MSCs were infected with Ad.TGF-b1, Ad.BMP-2, Ad.BMP-4, Ad.BMP-7 and Ad.IGF-1 at doses sufficient to provide low (<10 ng/ml) , medium (10-100 ng/ml) and high (>100 ng/ml) amounts of transgene expression per 24 hrs. Delivery of Sox-9 and Ad.IHH was performed over a wide range of viral doses. At 24 hours post infection, 2.0 x 10^5 cells of each culture were pelleted by centrifugation. The aggregates were cultured for 3 weeks in conical tubes in a defined, serum-free medium containing ascorbate 2-phosphate and dexamethasone. Chondrogenesis was determined using histologic staining for glycosaminoglycans and by immunohistochemistry for collagen types I and II. By this method, gene transfer and expression of BMP-2 and BMP-4 showed the greatest chondrogenic potential. This was followed to a lesser extent by TGF-b1 and then Sox-9 and IGF-1. Delivery of IHH or BMP-7 was met with only minimal chondrogenic activity which was only slightly greater than uninfected control cells. The potent activity of the secreted factors BMPs-2, -4 and TGF-b1 were expected and are consistent with the literature. The capacity to induce chondrogenesis through delivery of the Sox-9 transcription factor offers a promising intracellular alternative to the overexpression of secretable pleiotropic growth factors. The relative lack of chondroinduction following overexpression of IGF-1 and IHH is perhaps not surprising because these products thought to be more important for maintenance than induction of the chondrocytic phenotype."

Differentiation of mesenchymal stem cells to osteoblasts and chondrocytes: a focus on adenosine receptors.

Adenosine receptors signal via cAMP.

"if β-catenin is activated, it upregulates Runx2 expression and MSCs differentiate to osteoblasts, rather than chondrocytes "

"In the prechondrogenic mesenchyme, Runx2 is expressed along with Sox9 [but with Sox9 expression dominant]" "Nkx3.2 inhibits Runx2 transcription, enforcing the differentiation to chondrocytes"

"The expression of Sox9, along with the activation of cAMP response element binding protein (CREB) and c-Fos, maintains the chondrocytes in a proliferative state. In order for the chondrocytes to exit the cell cycle and become hypertrophic, Runx2 must be upregulated and Sox9 suppressed. The exit from the cell cycle and apoptosis is necessary for the eventual invasion by osteoblasts and ossification of the bone matrix "

Hoxa2 inhibits Runx2.

"high expression of the A2AAR and A2BAR receptors [adenosine receptors] can be found in cartilage"

"In human MSCs, A2BAR activation increased osteoblast differentiation, as determined by an increase in alkaline phosphatase activity"

"caffeine impairs new endochondral bone formation by inhibiting the proliferation and differentiation of chondroprogenitor cells."

JunB promotes osteoblast differentiation.

"TNF-α [promotes] Runx2 protein degradation in C2C12 and 2T3 osteoblast precursor cells by upregulating the E3 ligases Smurf1 and Smurf2 "

"High extracellular concentrations of adenosine can be achieved during cell injury or stress"

PKA stimulates Sox9 activation of Col2a1.

Mesenchymal stromal cell-derived extracellular matrix influences gene expression of chondrocytes.

"the ability of adult human mesenchymal stem cell (hMSC)-derived ECM to rescue the phenotype of osteoarthritic (OA) chondrocytes and to further stimulate the differentiation of healthy (HL) chondrocytes was evaluated. ECMs were prepared by decellularizing hMSCs cultured in basic medium (BM) and chondrogenic medium (CM). The obtained ECM was then combined with a polymeric solution of Poly (ε-caprolactone) (PCL) dissolved in 1, 1, 1, 3, 3, 3-hexafluoro-2-propanol (HFIP) and electrospun meshes were fabricated. Electrospun ECM scaffolds were characterized using scanning electron microscopy (SEM) and picrosirius red staining was used to confirm the presence of collagen. OA and HL chondrocytes were cultured on scaffolds containing hMSC ECM in BM or CM and compared to PCL electrospun scaffolds without ECM. Metabolic activity and chondrogenic gene expression were assessed by Alamar blue assay and quantitative PCR (qPCR) analysis, respectively. The ECM presence resulted in a significant difference in chondrocyte metabolic activity compared to PCL scaffolds alone. HL chondrocytes cultured for 21 days in chondrogenic medium on electrospun scaffolds containing hMSC ECM from BM showed a significant increase in collagen II and aggrecan expression compared to hMSC ECM from CM and PCL scaffolds without ECM incorporation. No significant influence of hMSC ECM presence on the chondrogenic signature of OA chondrocytes was found."

" hMSCs (female, 75 years old) were isolated from the bone marrow"

Aggrecan, Col2a1, Col1a1 were upregulated by healthy chondrocytes in MSC ECM.  Type X Collagen was only upregulated in the presence of chondrogenic medium.  The other three genes were upregulated in basal media but more significantly in chondrogenic medium.

Mechanical Signals As a Non-Invasive Means to Influence Mesenchymal Stem Cell Fate, Promoting Bone and Suppressing the Fat Phenotype.

"MSCs are ideally positioned as mechanosensitive elements central to musculoskeletal adaptation, but that the signals needn't be large to be influential."

The study states that mechanical loading suppresses the adipose phenotype but does not state much of enhancing the chondrogenic phenotype.  Mechanical stimulation also increases the number of progenitor cells which makes mesenchymal condensation easier to occur.

Biophysical Regulation of Stem Cell Differentiation.

"Bone marrow-derived mesenchymal stem cells-the most broadly studied source of osteoblastic progenitors-undergo osteoblastic differentiation in vitro in response to biophysical signals, including hydrostatic pressure, fluid flow and accompanying shear stress, substrate strain and stiffness, substrate topography, and electromagnetic fields. Furthermore, stem cells may be subject to indirect regulation by mechano-sensing osteocytes positioned to more readily detect these same loading-induced signals within the bone matrix. Such paracrine and juxtacrine regulation of differentiation by osteocytes occurs in vitro"<-perhaps these mechano-sensing osteocytes can induce stem cell chondrogenesis as well.

"Membrane-bound stretch-activated or voltage-activated cation channels also open as a consequence of membrane deformation, electromagnetic field stimulation, or fluid streaming potentials, initiating an influx of Ca2+. MSCs may also transduce membrane strains directly by the Piezo family of cation-selective mechano-sensitive channels" <-the primary cilium can also detect mechanical stimuli.

"In osteoporotic bone, ImP greatly increases, approaching arterial blood pressure in children with paralytic osteoporosis. Adipose tissue in the marrow was found to increase with age and disease, leading to decreased marrow viscosity and proportional decreases in fluid shear stresses"<-Osteoperosis can be a way to study increases in Intramedullary Pressure and the converse can be studied with increases in adipose tissue.

"Oscillatory strains compressing MSC-seeded fibrin scaffolds by 15 % produced chondrogenic differentiation, but inhibition of ERK1/2 abolished this chondrogenic response in favor of osteogenesis"

New insights into osteogenic and chondrogenic differentiation of human bone marrow mesenchymal stem cells and their potential clinical applications for bone regeneration in pediatric orthopaedics.

" trabecular bone[has] been identified as [a] potential [source] of MSCs"<-very promising for LSJL.  What would be even better if cortical bone was identified as a source for MSCs.

According to the book Stem Cells and Bone Tissue, there is a MSC population within cortical bone.  Trabecular bone MSCs are similar to bone marrow MSCs except they do not have the markers for Hematopoetic stem cells which is actually good as HSCs are not a component of the growth plate.

The study identifies the following transcription factors of regulating chondrogenic differentiation: Sox 5/6/9, Slug,  TSRP1, and GDF5.

"SOX9 specifically interacts with two binding sites for HMG-domain proteins and activates elements in the promoters of Col2a1, Col9a1, Col11a2, and aggrecan. Removal of SOX9 before mesenchymal condensation results in mice without cartilage development, while loss of SOX9 after condensation causes the arrest of chondrocytes differentiation"

"Procedures devoted to recruit stem cells from BM by penetration of the subchondral bone have been widely used to treat localized cartilage defects"

Why does hydrostatic pressure induce chondrocyte growth?

We hear a lot in biology that structure equals function.  The structure of a compound correlates to the purpose.  The induction of stimulus to stem cells that a chondrocyte is better to counteract than the other cell types that a stem cell can differentiate into is likely to induce stem cells to differentiate into chondrocytes.  Stem Cells have been found to differentiate into chondrocytes as a result of hydrostatic pressure and microgravity.  Note that going into a pool actually reduces hydrostatic pressure by counteracting the internal hydrostatic pressure generated from the blood in the body.

The fate of mechanically induced cartilage in an unloaded environment.

"Persistent intermittent mechanical stimulation is required to maintain differentiated cartilage. In three groups of rats, regenerating mesenchymal tissue was submitted to different loading conditions in bone chambers{epiphyseal bone marrow is like a bone chamber and also contains mesenchymal tissue so it is similar to the terms of this study}. Two groups were immediately killed after loading periods of 3 or 6 weeks (the 3-group and the 6-group). The third group was loaded for 3 weeks and then kept unloaded for another 3 weeks (the (3 + 3)-group). Cartilage was found in all loaded groups{so loading mesenchymal tissue resulted in chondrogenic differentiation of MSCs}. Without loading, cartilage does not appear in this model[remember that in a random position machine(in which stem cells differentiated into chondrocytes in response to microgravity), the cartilage does not undergo complete unloading but only constant stimulation to gravity from different directions while getting a rest in others]. In the 3-group there was no clear ongoing endochondral ossification, the 6-group showed ossification in 2 out of 5 cartilage containing specimens, and in the (3 + 3)-group all cartilage was undergoing ossification.  cartilage [has a tendency] to be maintained under unloaded conditions until it is reached by bone that can replace it through endochondral ossification{once the stem cells have been differentiated they stay chondrocytes upon unloading}.Additional measurements showed less amount of new bone in the loaded specimens. In most of the loaded specimens in the 3-group, necrotic bone fragments were seen embedded in the fibrous tissue layer close to the loading piston, indicating that bone tissue had been resorbed due to the hydrostatic compressive load{hydrostatic pressure can resorb bone helping to induce a more youthful state}. In some specimens, a continuous cartilage layer covered the end of the specimen and seemed to protect the underlying bone from pressure-induced resorption{cartilage protects the bone from hydrostatic pressure-induced resorption which is why stem cells under hydrostatic pressure differentiate into chondrocytes to protect bone from resorption, these new chondrocytes can new form growth plates which will result in you growing taller!}. one of the functions of the cartilage forming in the compressive loaded parts of a bone callus is to protect the surrounding bone callus from pressure-induced fluid flow leading to resorption."

"male Sprague–Dawley rats were used (365–425 g)"

"The bone conduction chamber consists of a threaded titanium cylinder, formed out of two half cylinders held together by a hexagonal closed screw cap. One end of the implant is screwed into the bone. The interior of the chamber has a diameter of 2 mm, and is 7 mm long. There are two bone ingrowth openings at one end where tissue can grow in from the cortical and the subcortical cancellous bone but not from extracortical soft tissue. At 3 weeks after the implantation, the specimens within the chamber usually contain 3 different zones of ingrown tissue as described previously. At the bottom there is a zone with cancellous bone with a marrow cavity, followed higher up by more immature woven bone formed by membranous ossification occurring as an advancing ossification frontier. Above this frontier there is vascularized fibrous tissue. At the fourth and sixth week the marrow cavity has expanded, the cancellous bone is found higher up, and the fibrous zone is thinner."

You can see in b that it is very similar to an apparatus used to generate hydrostatic pressure.

"The load chamber consists of the same two half cylinders with its two ingrowth openings. The hexagonal screw cap is replaced by a cap equipped with a 1.8 mm diameter piston protruding into the chamber, from the subcutaneous end towards the intraosseous end. By applying a known force on the top of the piston, a mechanical load can be transmitted to the tissue within the chamber. When loading is interrupted, the piston returns to its original position by means of a spring and no further mechanical stimuli act upon the tissue within the chamber. The inside diameter of the LC is the same as in the BCC (2 mm), the distance between the chamber bottom with its ingrowth openings and the piston is 5 mm when the chamber is unloaded, and 1.5 mm when the piston is in its most downward position. The top, with its mobile parts, is covered with a rubber coat to prevent overlying tissues from interfering with the moving parts. Originally we aimed at evaluating the ingrowth distance of new bone and used an unloaded control. In the first 3-group, bilateral chambers were thus used, a BCC on one side and an LC on the other side. Since it became more interesting to merely show the presence of cartilage, the unloaded control BCC was left out in the later six-series and the (3+3)-series."

"The loading device consisted of a metal rod, a spring and a metal cylinder that could glide over the rod, at the same time compressing the spring"<-this is very similar to a clamp except the clamp is applied laterally rather than axially.

After 6 weeks of loading the mean cartilage area in millimeters squared was 0.017 with the highest being 0.065.  There were non-responders so there definitely could be a genetic component in not responding to hydrostatic pressure.

The scientists reported tissue ingrowth but no mention of tissue outgrowth is made(which is what causes height growth).  The study is definitely indicative however of the ability for hydrostatic pressure to form new cartilagenous regions in bone marrow.

"Intermittent hydrostatic compressive stress and/or low oxygen tension will stimulate mesenchymal tissue to form cartilage"<-still don't know what purpose low oxygen tension stimulating chondrocyte differentiation does yet.

"High shear stresses were hypothesized to be associated with formation of fibrous tissue, whereas high hydrostatic compressive stresses seemed to guide the cellular differentiation into the chondrogenic pathway"<-which is why you have to load the epiphysis in LSJL.  The area that is clamped will experience hydrostatic pressure but the other areas of the bone will mostly experience shear strain.

The scientists in the study used a bone conduction chamber to generate hydrostatic pressure.  Then there's a piston used to push the fluid in the bone.

"As cortical bone is stiffer than the newly formed membranous trabecular bone within the bone chamber, the deformations of cortical bone are less than the strain threshold for cartilage to form. This indicates that bone resorption can be initiated by hydrostatic stresses, as low as the lowest stresses that induce cartilage.In conclusion, mechanically induced cartilage can be maintained in unloaded conditions."<-So hydrostatic pressure is possible to cause resorption of the cortical bone allowing for more stem cell growth and thus a more youthful organ.

"In a few specimens, a continuous cartilaginous layer had developed adjacent to the bone frontier, which had reached far into the chamber. No signs of bone resorption or necrotic trabeculae were found in these cases. It seems that this cartilage, which covered the whole end of the specimen, protected the underlying bone from resorption"<-cartilage is the best adaptive mechanism to hydrostatic pressure so the body responds to hydrostatic pressure by encouraging chondrogenesis.  "Cartilage could play a protective role against hydrostatic stress-induced resorption of the underlying bone, possibly by hindering massive fluid flow through the bone"

"(a) Chamber tissue specimen after 3 weeks of loading. At the bottom next to the ingrowth openings a marrow cavity is formed. Above, the frontier of ingrowing bone is covered by a layer of cartilage extending across the whole end of the specimen. No signs of bone resorption or necrosis are seen. (Hematoxylin Eosin×20.) (b) Detail of (a) with the cartilaginous tissue at the top. Beneath, the cartilage has been ossified to form a subchondral bone plate."

"(a) Chamber tissue specimen from another animal also after 3 weeks loading. A layer of cartilaginous cells is seen in the middle of the picture. The bone layer under the cartilage has been resorbed and only sparse necrotic bone trabeculas are present at the bottom. (Hematoxylin Eosin×20.) (b) Detail of (A) Cartilaginous cells at the top and a necrotic bone trabecula at the lower end."

The formation of chondrocytes may be an adaptive mechanism in response to hydrostatic pressure.  In the study, 40% of the cartilage containing groups underwent endochondral ossification.  However, it's possible that more of the groups could undergo endochondral ossification after the 6 week period.

So like muscle adapts to microdamage by growing bigger(over simplified), bone responds to hydrostatic pressure by forming cartilagenous possible growth plate like regions.

Hydrostatic pressure and the actomyosin cortex drive mitotic cell rounding.

"During mitosis, adherent animal cells undergo a drastic shape change, from essentially flat to round[chondrocyte cells tend to be round]. Mitotic cell rounding is thought to facilitate organization within the mitotic cell and be necessary for the geometric requirements of division.  We show that cells have an outward rounding force, which increases as cells enter mitosis. Mitotic rounding force depends both on the actomyosin cytoskeleton and the cells' ability to regulate osmolarity[so LSJL requires both a sensitive actin cytoskeleton and sufficient hydrostatic pressure]. The rounding force itself is generated by an osmotic pressure.  the actomyosin cortex is required to maintain this rounding force against external impediments[the actin cytoskeleton is need to prevent chondrogenic differentiation]. Instantaneous disruption of the actomyosin cortex leads to volume increase, and stimulation of actomyosin contraction leads to volume decrease. osmotic pressure is balanced by inwardly directed actomyosin cortex contraction[osmotic pressure is required to generate the stimulus to a chondrogenic phenotype but the actin cytoskeleton is required to respond to the stimulus]. Thus, by locally modulating actomyosin-cortex-dependent surface tension and globally regulating osmotic pressure, cells can control their volume, shape and mechanical properties."

"If the osmolarity is higher inside the cell than outside, water will flow into the cell and generate a hydrostatic pressure"<-LSJL alters solute concentration by means of compression.

"Introduction of hypotonic medium (−Δ100 mosM l⁻¹) led to an immediate increase in the volume of metaphase cells (40 ± 6%; n = 9), indicating that water entered the cells. This was accompanied by a concurrent increase in the measured rounding pressure (76 ± 20%; n = 9), presumably because the intracellular pressure increased. Within 3 min of the osmolarity changing, the cell volume and rounding pressure returned to close to their original values. This is probably because, in response to increased osmotic pressure, regulatory volume decrease causes cells to release ions"<-so pressure may have to be sustained longer for LSJL to be active to prevent dedifferentiation or perhaps LSJL needs to be applied in 3 minute intervals.

"The exchange of a proton with a Na⁺ ion increases the intracellular osmolarity because pH is strongly buffered in the cytoplasm; thus, a Na⁺ ion has a greater effect on osmolarity than a proton"<-High Sodium intake may help with LSJL?

The physics of tissue formation with mesenchymal stem cells.

"Mesenchymal stem cells (MSCs) consistently proliferate or differentiate under cues from hydrostatic pressure, diffusive mass transport, shear stress, surface chemistry, mechanotransduction, and molecular kinetics"

"HP refers to the pressure change in a fluid caused by an external force in a closed geometry. HP is expressed as HP=ρgh, where g is gravity, ρ is density, and h is height, assuming the fluid is incompressible. The external force (ΔPex), which can be that exerted by compression, tension, or ultrasound, increases HP, as HP=ΔPex + ρgh. The term ‘ρgh’ is negligible, because it corresponds to an increase in HP of approximately 100 Pa per cm of height. Thus, external stimulation is the main cause of pressure changes."

"physiological loadings from 0.1 to 10 MPa have a chondrogenic effect on MSCs"<-lower pressures tend to be more osteogenic.

"For chondrogenesis of MSCs, although HP >0.1 MPa induces proteoglycans expression, HP >1 MPa consistently stimulates the expression of a broader range of healthy cartilage markers"

"30–200 mW/cm2, F=1–1.5 MHz"<-for ultrasound are found to be chondrogenic.

"high intensities (30–200 mW/cm2) [of ultrasound] induce chondrogenic differentiation"

"sound waves may induce HP more homogeneously than might compression or tension, enhancing MSC viability and preventing MSC death."

"the geometry of a construct determines the distribution of forces and the amount of pressure that each MSC experiences, hence the type of differentiation. For example, forces applied on meniscus-like structures yield cartilage"

SMITH&NEPHEW  EXOGEN 2000+   LOW INTENSITY ULTRASOUND FRACTURE HEALING SYSTEM is mentioned as being 30 mW/cm2

Grow with triterpenoids?

Synthetic triterpenoids, CDDO-Imidazolide and CDDO-Ethyl amide, induce chondrogenesis.

"Here we show that the synthetic oleanane triterpenoids, CDDO-Imidazolide (CDDO-Im) and CDDO-Ethyl amide (CDDO-EA), at concentrations as low as 200 nM, induce chondrogenesis in organ cultures of newborn mouse calvaria. The cartilage phenotype was measured histologically with metachromatic toluidine blue staining for proteoglycans and by immunohistochemical staining for type II collagen. Furthermore, real-time polymerase chain reaction (PCR) analysis using mRNA from calvaria after 7-day treatment with CDDO-Im and CDDO-EA showed up-regulation of the chondrocyte markers SOX9 and type II collagen (alpha1). In addition, TGF-β; BMPs 2 and 4; Smads 3, 4, 6, and 7; and TIMPs-1 and -2 were increased[LSJL upregulated BMP-2 and TIMP-1]. In contrast, MMP-9 was strongly down-regulated. Treatment of human bone marrow-derived mesenchymal stem cells with CDDO-Im and CDDO-EA (100 nM) induced expression of SOX9, collagen IIα1, and aggrecan, as well as BMP-2 and phospho-Smad5, confirming that the above triterpenoids induce chondrogenic differentiation."

BMP vs. TGF-Beta chondrogenesis

Gene expression profile of multipotent mesenchymal stromal cells: Identification of pathways common to TGFbeta3/BMP2-induced chondrogenesis.

Human stem cells were used.

"The genes belonging to class 1 correspond to genes barely but continuously expressed during chondrogenesis and involved in chondrocyte proliferation and cell cycle regulation. This class is characterized by the expression of Notch3, insulinlike growth factor binding protein 1 (IGFBP1), interleukin 1 receptor-like 1 (IL1RL1), Wingless 5a (Wnt5a), endothelial growth factor-related protein (PGF), preprourokinase/urokinase-type plasminogen activator receptor (PLAU/PLAUR), matrilin 2 (MATN2), laminin alpha 2 (LAMA2), phorbol-12-myristate-13-acetate-induced protein 1 (PMAIP1)"<-LSJL upregulates IL1RL1 and MATN2.

"The second class corresponds to 17 factors that are up-regulated within the first 3 days of chondrogenesis and then downregulated at the end of chondrogenesis. This class includes the 5 integrin (ITGA5), as well as tissue factor pathway inhibitor-2 (TFPI2), regulator of G-protein signaling 2, (RGS2), B-cell lymphoma 6 (BCL6), spermidinespermine N1-acetyltransferase (SAT1), N-myc downstream regulated (NDRG1), nuclear factor, interleukin 3 regulated (NFIL3){downregulated by LSJL}, adipose differentiation-related protein (ADFP), Max-interacting protein 1 (MXI1){downregulated by LSJL}, hypoxia-inducible protein 2 (HIG2) and B-cell translocation gene 1 (BTG1). These genes are implicated in cell attachment and apoptosis prevention."

"Class 3 consists of 15 genes that are expressed in the late phase of chondrogenesis. These genes included factors involved in osteogenesis inhibition: Dickkopf 1 (Dkk1), apolipoprotein E and D (APOE/APOD){APOD is upregulated in LSJL}, serine proteinase inhibitor (SERPINF1), tissue inhibitor of metalloproteinase 4 (TIMP4), and nidogen (enactin, NID)."<-LSJL decreases Dkk expression.

"Classes 4 and 5 are characterized by genes that are either downregulated (class 4) or upregulated (class 5) as soon as day 1 of chondral differentiation, among which are connective tissue growth factor (CTGF), cysteine-rich angiogenic inducer 61 (CYR61), and homolog of chicken slug zing-finger protein (SLUG)."

Both TGF-Beta3 and BMP-2 had low effect on Class 1 genes.  

Shared pathways between TGF-Beta3 and BMP-2: Wnt5a, CHI3L1, VEGF, PLAUR, PGAR, ITGalpha5,  NID2, DPT{Upregulated by LSJL}, DKK1, SPARCL1.

"Phase 1. Cell attachment and apoptosis induction. This phase is characterized by the transitory upregulation of the class 2 genes whose function is associated with transcription regulation, cell attachment (ITGA5, TFPI2), and hypoxia (HIG2). The regulation of transcription factors as BCL6, BTG1, NFIL3{Upregulated by LSJL}, MXI1{Downregulated by LSJL} suggest a pro-apoptotic effect at this early stage. In parallel, CTGF and CYR61{upregulated by LSJL}, belonging to the class 4 of genes that are involved in skeletal tissue development, are down-regulated during this phase." 

"Phase 2. Differentiation induced by Wnt, Notch, and IGF signaling. This phase is characterized by the class 1 genes that are expressed during the whole period of differentiation and are members of Wnt/ catenin signaling, IGF signaling (as IGFBP, Foxo3A), and Notch signaling regulating cellular growth and cell death (SGK, Notch3)."

"Phase 3. Wnt signaling inhibition and hypertrophy. At the late stage of chondral differentiation, among upregulated genes belonging to class 3 we identified genes associated with hypertrophy, including TIMP4 and SERPINF1 or involved in lipid metabolism, indicating the formation of adipocytes inside the pellet culture. We also identified DKK1, a strong Wnt inhibitor recently shown to play a major role in the prevention of osteogenesis."

sFrp1 is also upregulated during chondrogenesis.

Height with Notch?

There are four Notch's: Notch 1, Notch 2, Notch 3, and Notch4.

Immunolocalization of notch signaling protein molecules in a maxillary chondrosarcoma and its recurrent tumor.

"Both primary and recurrent tumors were histopathologically diagnosed as conventional hyaline chondrosarcoma (WHO Grade I). Hypercellular tumor areas strongly expressed Notch3 and Jagged1 in spindle and pleomorphic cells suggesting up-regulation of these protein molecules at sites of tumor proliferation. Expression patterns were distinct with some overlap. Differentiated malignant and atypical chondrocytes demonstrated variable expression levels of Jagged1, and weak to absent staining for Notch1, 4 and Delta1. Protein immunolocalization was largely membranous and cytoplasmic, sometimes outlining the lacunae of malignant chondrocytes. Hyaline cartilage demonstrated a diffuse or granular precipitation of Jagged1 suggesting presence of soluble Jagged1 activity at sites of abnormal chondrogenesis. No immunoreactivity for the other Notch members was observed. Calcified cartilage was consistently Notch-negative indicating down-regulation of Notch with cartilage maturation[upregulate Notch's to decrease cartilage maturation and to grow taller for longer?].
Results indicate that Notch signaling pathway may participate in cellular differentiation and proliferation in chondrosarcoma. Findings implicate Notch3 and Jagged1 as key molecules that influence the differentiation and maturation of cells of chondrogenic lineage."

"Notch receptors are structurally homologous transmembrane proteins with distinct differences in their extracellular and intracellular domains (ICD)."

"Binding of Notch ligands to their receptors on neighboring cells induce proteolytic cleavages, releasing Notch ICD which translocates to the nucleus to interact with DNA-bound proteins. This in turn activates the transcription of selected target genes such as Hes1{Upregulated by LSJL}, Hes5{Upregulated by LSJL}, and Hes7"

"Jagged1mediated Notch signaling in human bone marrow stem cells is necessary to initiate chondrogenesis but must be switched off for chondrogenesis to proceed"<-So ideally Notch signaling would be induced while performing LSJL and then downregulated when LSJL ceases.

According to Notch receptor and Notch ligand expression in developing avian cartilage., "abolition of Notch signalling is needed to allow full chondrogenesis to occur in human bone marrow stromal cells demonstrating that Notch signalling is not conserved between species."

Cartilage, SOX9 and Notch signals in chondrogenesis.

"Cell rounding has been shown to help restore the chondrocyte phenotype"

"TFP medium contains transforming growth factor β1 (TGFβ1), fibroblast growth factor 2 (FGF-2) and platelet-derived growth factor BB (PDGF-BB), with 10% fetal bovine serum (FBS). In the presence of these three growth factors the chondrocytes divide more rapidly, they become spindle-shaped and demonstrate dramatically reduced contact inhibition.  After culture in TFP the chondrocytes showed increased multipotency with some cells able to differentiate into osteoblasts and adipocytes as well as regaining a chondrocyte phenotype. Once transduced the cells expressed 10-fold higher levels of SOX9 and this level remained high as the cells were further passaged."<-so this study will show us whether elevated levels of Sox9 will help increase height regardless of how Sox9 degrades Beta-Catenin.

"the cells had an increased potential to respond to chondrogenic signals after they were transduced with SOX9"<-Maybe lack of Sox9 in epiphyseal bone marrow can lead to a reduced response to methods intended to induce epiphyseal chondrogenesis.

"In lowered oxygen conditions there is increased COL2A1 gene expression when chondrocytes are grown in 3D culture systems and that this involves increases in endogenous SOX9 expression"

In the study FGF2 increased expression of the Sox trio so FGF2 may be a potential way to induce Sox9 as a precursor to inducing chondrogenesis.

"Notch-2 receptor [is] broadly distributed throughout the epiphyseal growth plate"

According to Histone deacetylase inhibitors upregulate Notch-1 and inhibit growth in pheochromocytoma cells., valproic acid (VPA) and suberoyl bis-hydroxamic acid (SBHA) can activate Notch.  So possibly use those chemicals to induce chondrogenesis and then wait for serum levels to decrease until Notch signaling is abolished to allow chondrogenesis to continue.

Beta-Catenin may help regulate Notch Signaling.

Beta-catenin modulates the level and transcriptional activity of Notch1/NICD through its direct interaction.

"Wnt and Notch1 signaling pathways play an important role in a variety of biological processes including embryonic induction, the polarity of cell division, cell fate, and cell growth. This report shows that beta-catenin can regulate the level and transcriptional activity of the Notch1 and Notch1 intracellular domain (NICD). The in vivo and in vitro results demonstrate that beta-catenin binds with Notch1 and NICD, for which its Armadillo repeat domain is essential. It was further demonstrated that beta-catenin could upregulate the level of Notch1 and NICD[interesting since Sox9 degrades beta-catenin which would decrease Notch1 and Notch1 is needed to induce chondrogenesis], possibly by competing the common ubiquitin-dependent degradation machinery. In addition, beta-catenin enhanced the transcriptional activity of NICD on the hairy and enhancer of split 1 (HES1) and CSL through its C-terminal transactivation domain. This effect of cooperative regulation by beta-catenin could also be observed in bone morphogenetic protein 2 (BMP2) induced osteogenic differentiation of C2C12 cells. beta-catenin coexpression with NICD enhanced the alkaline phosphatase (ALP) activity in C2C12 cells compared with either beta-catenin or NICD expression alone. Culturing C2C12 cells on Delta-1 coated dishes together with Wnt3-conditioned media induced noticeable increases in ALP staining, verifying that employed physiological levels of NICD and beta-catenin are sufficient to induce ALP activation. Furthermore, effects of beta-catenin on Notch1 were dramatically diminished by overexpressed LEF1[Maybe Sox9 degrades the Beta-Catenin earmarked for the TCF/LEF1 complex rather than NICD]. Overall, our data suggest that beta-catenin can act as a switching molecule between the classical TCF/LEF1 mediated pathway and NICD mediated pathway."

"In the presence of Wnt signaling, β-catenin is relieved from the destruction complex including APC/Axin/GSK3β, and the degradation pathway of β-catenin by the ubiquitination machinery is inhibited."

"In this condition, β-catenin accumulates in the cytoplasm and the nucleus where it interacts with the LEF1/TCF family transcription factors (LEF1, TCF-1, -3 and -4)"

"NICD has been reported to interact with LEF1, a transcriptional component of Wnt signaling, and has been suggested to function as a coactivator for LEF1. Presenilin and GSK3β are other proteins acting to meet Notch signaling and Wnt signaling at the cytoplasmic level. Presenilin has the activity of Notch cleavage into NICD, and is involved in the stability of β-catenin. Likewise, GSK-3β is involved in Wnt/β-catenin signaling and Notch signaling. β-catenin can be phosphorylated by GSK3β and regulated by the proteosome-dependent degradation machinery, and Notch2 is also associated with GSK3β, which inhibits the Notch2 activity"

"Notch1 and NICD directly bind to the arm repeat domain of β-catenin, resulting in reciprocal increases in the levels of Notch1 and β-catenin."

Repression of chondrogenesis through binding of notch signaling proteins HES-1 and HEY-1 to N-box domains in the COL2A1 enhancer site.

"Notch signaling is implicated in the repression of mesenchymal stem cell (MSC) chondrogenic differentiation.
Notch intracellular domain (NICD) protein levels were monitored via Western blotting throughout chondrogenic differentiation of human MSCs in pellet cultures. Overexpression of Notch signaling components and their effect on chondrogenesis was achieved by transfecting plasmids coding for NICD, HES-1, and HERP-2/HEY-1. COL2A1 and AGGRECAN expression was monitored via quantitative polymerase chain reaction analysis. Chromatin immunoprecipitation (ChIP) was used to test whether HES-1 and HEY-1 bind putative N-box domains in intron 1 of COL2A1.
High levels of NICD proteins were reduced during chondrogenesis of human MSCs, and this was mediated by transforming growth factor beta3 (TGFbeta3). COL2A1 gene expression was repressed following overexpression of NICD (2-fold) and HES-1 (3-fold) and was markedly repressed by overexpression of HEY-1 (80-fold). HEY-1 repressed AGGRECAN expression 10-fold, while NICD and HES-1 had no effect. We identified 2 putative N-box domains adjacent to, and part of, the SOX9 enhancer binding site located in intron 1 of COL2A1. ChIP studies showed that endogenous HES-1 and HEY-1 bound to these sites. Transducin-like enhancer, the HES-1 corepressor protein, was displaced during chondrogenic differentiation and following TGFbeta3 treatment.
Notch signaling proteins act on the SOX9 binding site in the COL2A1 enhancer and prevent SOX9-mediated transcriptional activation of COL2A1 and, thus, chondrogenic differentiation."

"Hes-1{upregulated in LSJL} and Hey-1 bind N-box domains in the enhancer region of Col2a1. Since this site is required for Sox9 mediated transcription of Col2a1, we propose that bHLH proteins negatively compete with Sox9 binding to the enhancer site and prevent Sox9-mediated transcriptional activation of Col2a1 and thus chondrogenic differentiation."<-So it's likely a bad thing that Hes-1 increased in expression however Sox9 increased in expression 0.5 fold higher than Hes-1.

Notch gain of function inhibits chondrocyte differentiation via Rbpj-dependent suppression of Sox9.

"Sustained Notch activation in cartilage leads to chondrodysplasia[short stature]. Genetic evidence indicates that Notch regulates limb bud mesenchymal stem cell differentiation into chondrocytes via an Rbpj-dependent Notch pathway. Cartilage specific Notch gain-of-function (GOF) mutant mice display chondrodysplasia accompanied by loss of Sox9 expression in vertebrae. To evaluate the contribution of an Rbpj-dependent Notch signaling to this phenotype, we deleted Rbpj on the Notch GOF background. These mice showed persistent spine abnormalities characterized by "butterfly" vertebrae suggesting that removal of Rbpj does not fully rescue the axial skeleton deformities caused by Notch GOF. However, Sox9 protein level was restored in Rbpj deficient Notch GOF mice compared to Notch GOF mutants, demonstrating that regulation of Sox9 expression is canonical or Rbpj-dependent. The association of the Rbpj/NICD complex with the Sox9 promoter is associated with transcriptional repression of Sox9 in a cellular model of chondrocyte differentiation. Notch negatively regulates chondrocyte differentiation in the axial skeleton by suppressing Sox9 transcription, and Rbpj-independent Notch signaling mechanisms may also contribute to axial skeletogenesis."

"The signaling from Notch ligands to Notch receptors is initiated by the release of the active Notch intracellular domain (NICD) through proteolytic events mediated by Presenilin1/2 within the gamma-secretase complex."  

"NICD forms a complex with Rbpj to activate the transcription of downstream targets including the Hes and Hey family of genes"   

"loss of function (LOF) in DLL3 or JAGGED1, both Notch ligands, or loss of function in HES7, LFNG or MESP2 [can result in short stature]"

"Over-expression of the Notch ligand DLL1 in chick limb buds blocked differentiation from pre-hypertrophic to hypertrophic chondrocytes and resulted in shortened skeletal elements with decreased expression of Col2a1 and Col10a1."

"increased expression of Ihh and Col10a1 with an elongated hypertrophic zone [occurred] when Presenilin1/ 2 [was] deleted in limb bud mesenchymal progenitor cells"

"an elongated hypertrophic zone resulted from deletion of Notch1/2 receptors by Prx1-cre"

"In the earlier progenitor pool, Notch signaling promotes chondrocyte proliferation. In committed chondrocytes Notch inhibits chondrocyte differentiation and maturation."

"canonical Notch targets Hey1 and Hey2{upregulated with LSJL} were up-regulated in the GOF mice"

"Notch receptor physically interacts with activated Beta-catenin to constrain the accumulation of Beta-catenin in stem or progenitor cells"

Notch Suppresses Nuclear Factor of Activated T Cells (Nfat) Transactivation and Nfatc1 Expression in Chondrocytes.

"Notch1 to Notch4 transmembrane receptors determine cell fate, and release of the Notch intracellular domain (NICD) in the cytoplasm induces gene expression. Notch regulates endochondral ossification, . Nuclear factor of activated T cells (Nfatc) transcription factors regulate chondrogenesis, and we asked whether Notch modifies Nfat signaling in chondrocytes. Notch was induced in teratocarcinoma ATDC5 chondrogenic cells infected with a retroviral vector, where the cytomegalovirus (CMV) promoter directs NICD expression. NICD suppressed chondrocyte differentiation and inhibited Nfat transactivation and Nfatc1 expression. Notch was activated in chondrocytes from Rosa(Notch) mice, where the Rosa26 promoter is upstream of a loxP-flanked STOP cassette and NICD. To excise the STOP cassette and express NICD, Rosa(Notch) chondrocytes were infected with an adenoviral vector where the CMV promoter directs Cre expression (Ad-CMV-Cre). Notch1 and Notch2 mediate the effects of Notch in skeletal cells, and to inhibit Notch signaling, chondrocytes from mice homozygous for Notch1 and Notch2 alleles targeted with loxP sites were infected with Ad-CMV-Cre. NICD suppressed chondrogenic nodules formation and expression of selected chondrocyte gene markers, induced Col10a1{up} and Mmp13, and suppressed Nfat transactivation and Nfatc1 expression, whereas inactivation of Notch1 and Notch2 did not affect chondrocyte differentiation. To investigate Nfatc1 function in chondrocytes, Nfatc1 was induced in Rosa(Notch) chondrocytes overexpressing NICD or controls. Nfatc1 suppressed chondrocyte differentiation and opposed Col10a1 induction by Notch. Notch suppresses Nfat transactivation in chondrocytes and Notch and Nfatc1 regulate chondrocyte differentiation."

"In the Notch canonical signaling pathway,NICD translocates to the nucleus and interacts with Epstein-Barr virus latencyC promoter binding factor 1, Suppressor of hairless and Lag-1 (Csl), also known as Rbpj in mice, a DNA-binding protein that suppresses gene expression by recruiting repressors of transcription."

"Overexpression of NICD in vitro suppresses the differentiation of chondrogenic cells, and Notch signaling inhibition in limb bud cell cultures enhances chondrogenesis"

"NICD overexpression in mesenchymal cells suppresses chondrogenesis"

"inactivation of Csl in chondrocytes causes elongation of the hypertrophic zone"

"The phosphatase calcineurin induces Nfat transactivation by dephosphorylating specific serine residues in the SRR and SPXXrepeat motifs of the regulatory domain of Nfat"

"Dephosphorylated Nfat translocates to the nucleus and induces expression of Nfat target genes, such as the isoform of Regulator of calcineurin 1 transcribed from the promoter region upstream of exon 4 (Rcan1.4).  Phosphorylation of the SRR and SPXX repeat motifs prevents association to DNA and induces Nfat nuclear export, resulting in inhibition of Nfat transactivation.  Nfatc1 to Nfatc4 are expressed in murine chondrocytes, and Nfatc2-null mice display ectopic chondrogenesis and joint abnormalities due to hypertrophic differentiation of articular chondrocytes"

"a constitutively active Nfatc3{down} mutant promoted chondrogenic differentiation of mesenchymal cells in vitro"

"activation of Nfatc2 during early chondrogenesis of limb bud cell cultures leads to enhanced chondrocyte differentiation, whereas Nfatc2 activation in more mature cells prevented further differentiation"

In ATDC5 cells:  "NICD suppressed Col10a1, Mmp13, and Vegf transcripts" but it did not inhibit early markers like Sox9 and Col2a1 significantly but it did affect early markers Sox9 and Ihh non-significantly but in a noticeable way.  NICD also upregulated Hey2{up} and Hes1{up}.


Notch signaling in chondrocytes modulates endochondral ossification and osteoarthritis development.

"Intracellular domains of Notch1 and -2 were translocated into the nucleus of chondrocytes with their differentiation in mouse limb cartilage and in mouse and human OA articular cartilage. A tissue-specific inactivation of the Notch transcriptional effector recombination signal binding protein for Ig kappa J (RBPjκ) in chondroprogenitor cells of SRY-box containing gene 9 (Sox9)-Cre;Rbpj(fl/fl) mouse embryos caused an impaired terminal stage of endochondral ossification in the limb cartilage. The RBPjκ inactivation in adult articular cartilage after normal skeletal growth using type II collagen (Col2a1)-Cre(ERT);Rbpj(fl/fl) mice by tamoxifen injection caused resistance to OA development in the knee joint. Notch intracellular domain with the effector RBPjκ stimulated endochondral ossification through induction of the target gene Hes1{up} in chondrocytes. Among the Notch ligands, Jagged1 was strongly induced during OA development. Finally, intraarticular injection of N-[N-(3,5-diflurophenylacetate)-l-alanyl]-(S)-phenylglycine t-butyl ester (DAPT), a small compound Notch inhibitor, to the mouse knee joint prevented OA development. The RBPjκ-dependent Notch signaling in chondrocytes modulates the terminal stage of endochondral ossification."

"Although the conditional knockout (Sox9-Cre;Rbpjfl/fl) mice died shortly after birth, they showed a slight dwarfism during the embryonic periods compared with the Rbpjfl/fl littermates: the limbs and vertebrae were about 5–10% shorter in Sox9-Cre;Rbpjfl/fl mice than in these littermates"

"the percentage of the hypertrophic zone relative to the limb length was much increased, whereas that of the bone area was considerably decreased in the Sox9-Cre;Rbpjfl/fl limbs, indicating that the RBPjκ knockout impaired the terminal differentiation stage in such aspects as matrix degradation and vascular invasion"

"Mmp13 and Vegfa, as well as Hes1, were decreased by the RBPjκ knockout"

"Under the surgical OA induction, however, the cartilage degradation as well as expressions of Mmp13, Vegfa, and Hes1 were suppressed in the Col2a1-CreERT;Rbpjfl/fl knee joints, compared with the Rbpjfl/fl joints, confirming the resistance to OA development by the RBPjκ insufficiency in adult articular cartilage."<-interesting this indicates that osteoarthritis may be due to endochondral ossification of articular cartilage than due to degeneration of chondrocytes into fibroblasts.

"In the cultures of articular chondrocytes, treatment with DAPT inhibited Mmp13, Vegfa, and Hes1 expressions and also alkaline phosphatase and Alizarin red stainings"