Are bone marrow cells capable of chondrogenesis?

The goal of LSJL is to induce chondrogenic differentiation of the mesenchymal stem cells in the epiphyseal bone marrow.  The chondrogenic potential of bone marrow cells is therefore of interest to us.  Are adult bone marrow cells capable of differentiating into chondrocytes?

Chondrogenesis of mesenchymal stem cells: role of tissue source and inducing factors.

"the cardio-vascular reparative effects attributed to MSCs appear to be mediated predominantly through the secretion of factors targeting cells at the site of repair"<-so what MSCs differentiate into depends on what factors are secreted at certain sites.  So at the sites where we want to grow taller we want our cells to secrete pro-chondrogenic factors.  LSJL encourages secretion of TGF-Beta which is pro-chondrogenic.

"MSC populations are heterogeneous cell populations whose composition depends on isolation methods and expansion conditions that differ largely among investigators. Nearly 50% of CFU-Fs from BM were tripotent MSCs while the remaining population of cells showed varied phenotypes"<-So the ability of MSCs to undergo chondrogenesis may vary by individual.

"SM[synovial membrane] MSCs [contain] two populations: 30% of cells were tripotent while the remainder displayed only osteo-chondral differentiation potential"<-so all synovial membrane cells have the ability to differentiate into chondrocytes.  However, the synovial membrane lines the articular cartilage and does not necessarily have access to epiphyseal bone marrow.

"embryonic stem (ES) cells can clearly be discriminated from MSCs by specific hypermethylation of numerous genes. The comparison of AT[adipose tissue] and BM MSCs revealed few differences. specific hypermethylation of numerous genes [occurs] in HSCs while the methylation patterns of MSCs from different sources were very similar"<-So DNA Methylation affects the differences between embryonic and adult stem cells much more than methylation differences between individuals.  We need to find out which specific methylations affect height.

" promoter hypomethylation is not predictive for the differentiation potential of cells, while hyper-methylation sets restrictions that define frames for differentiation potentials"<-so we may want to un hyper-methylate some genes.

"two cytosines in the COL10A1(Collagen Type X or terminal differentiation) promoter were consistently hypomethylated in MSCs in comparison with articular chondrocytes, correlating to the inducibility of COL10A1 expression and hypertrophy during in vitro chondrogenesis of MSCs "->so the hypomethylation of the COL10A1 gene in articular chondrocytes could be why articular chondrocytes do not ossify and undergo terminal differentiation.  For LSJL to work properly we need to ensure that the COL10A1 gene is hypomethylated.

"Histone modifications and histone-modifying molecules are regulated, while MSCs enter senescence in vitro and could be involved in the ensuing loss of differentiation potential. They are also actively involved in differentiation. Histone deacetylases, in particular HDAC4, may represent important regulators of chondrogenesis "<-So we need to worry about histones as well for inducing chondrogenesis.

"During embryogenesis the development of cartilage is initiated by a phase of condensation of mesenchymal precursor cells, and the cell-cell contact arising from condensation appears to be crucial for the onset of chondrogenesis. N-cadherin seems to be involved in cell-cell contact in pre-cartilage condensations, and functional N-cadherin was necessary for chondrogenesis of chick limb mesenchymal cells in vitro and in vivo. In human MSCs, N-cadherin is strongly up-regulated during the condensation phase during the first few days of chondrogenic induction in vitro . When MSCs are submitted to chondrogenic conditions in monolayer culture, they begin to condensate in response to the stimulus and form high-density three-dimensional cell aggregates. proper chondrogenic differentiation occurs also for MSCs embedded in gel-like biomaterials that keep cells apart from each other and thus limit direct cell-cell contact. This suggests that, although cell-cell contact facilitates chondrogenic induction of MSCs compared with monolayer culture, it does not represent an absolute requirement for in vitro chondrogenic differentiation of human MSCs in a three-dimensional structure."<-So cells may be able to differentiate into chondrocytes without contact with other MSCs.  This is good for LSJL as we only have to encourage chondrogenesis(and subsequent endochondral ossification) of one cell rather than several.

So evidence supporting chondrogenesis in epiphyseal bone marrow.  We need to learn more about methylation and histones that encourage height growth as we can manipulate those to grow taller.

The Therapeutic Effect of Bone Marrow Derived Stem Cell Implantation Following Epiphyseal Plate Injury is Abrogated by Chondrogenic Pre-Differentiation.

"Chondrogenesis was induced with TGF-Beta1 treatment in high-density monolayer cultures of BMSCs in vitro. The pre-differentiated or undifferentiated BMSCs were either seeded into agarose gels for continued in vitro culture, or injected into growth plate defects via an in situ gelling agarose. Pre-differentiated BMSCs had higher Sox-9, type II collagen, and aggrecan mRNA levels compared to undifferentiated cells after high-density monolayer culture. After transfer to agarose gels, pre-differentiated cells did not produce a cartilaginous matrix{so injecting chondrocytes into the bone may not help you grow taller}, even with continued TGF-Beta1 stimulation, whereas undifferentiated cells produced a cartilaginous matrix in this system. Three-dimensional images of the growth plate showed that delivery of either pre-differentiated or undifferentiated cells to defects resulted in a decrease in mineralized tether formation (fusion) in the growth plate tissue surrounding the defect to normal levels. Limb length discrepancy between injured and control limbs was corrected following treatment with undifferentiated but not pre-differentiated cells."

"delivery of an in situ gelling agarose to centralized growth plate defects in a rat animal model decreased limb length discrepancies, but did not fully restore function to the injured area"

"Growth factor stimulation is required to induce chondrogenesis of stem cells, although the specific treatment protocol depends on the tissue source from which the cells are derived"

"Bone marrow was flushed from diaphyses with alpha-MEM supplemented with 1% antibiotic/antimycotic" Our target is epiphyseal bone marrow but they should be similar to diaphyseal bone marrow stem cells.

"control samples continued to receive 1ng/ml FGF-2 and chondrogenic samples were switched to receiving 10ng/ml TGF-Beta1"

"BMSCs in high density monolayer showed very little proliferation after switching to chondrogenic media in both FGF-2 and TGF-Beta1 treated samples over the culture period"<-Maybe it's better to try to upregulate stem cell proliferation proteins when starting LSJL rather than inducing chondrogenesis?

"The limb length discrepancy was corrected in only the group treated with undifferentiated cells and the defect legs of this group were significantly longer then the empty defect limbs"<-overgrowth

Cultured mesenchymal stem cell transfers in the treatment of partial growth arrest.

"Mesenchymal stem cells were cultured from periosteum harvested from the tibias of New Zealand White (NZW) rabbits. An experimental model for growth arrest was created by excising the medial half of the proximal growth plate of the tibia of 6-week-old NZW rabbits. The cultured mesenchymal stem cells were embedded in agarose and transferred into the growth-plate defect after excision of the bony bridge in established growth arrest. Transfer of agarose alone and a periosteum flap without cells served as the control groups. In cases of transfer of mesenchymal stem cells, growth arrest with angular deformation and loss of length of the tibia was corrected. Transfer of agarose alone and a periosteum flap yielded poor results."

"periosteal derived MSCs delivered in agarose generated a new growth plate and completely corrected angular deformity"

Normal structure, function, and histology of the bone marrow.

Bone marrow accounts for 5% of body weight in humans.

Thus the natural bone marrow MSCs may actually be better than exogenous chondrocytes.

Biological properties of mesenchymal Stem Cells from different sources.

"Bone marrow MSCs (BM-MSCs) are a subpopulation of the stromal cells that line the endosteal surface of the marrow space. Cells in many respects identical to BMMSCs can be isolated from trabecular and compact bone and from non-hematopoietic bone marrow sites, such as the femoral head"

"MSCs have been first isolated from the bone marrow. They have been defined as nonhematopoietic, multipotential cells that support hematopoietic stem cells expansion in vitro and can differentiate into cells of various connective tissues. They are easy to harvest and they are hold in bone marrow in relatively high concentration"

Bone Marrow MSCs are rated as having very high chondrogenic differentiation capability second though to Umbillical Cord, Tendon, and Synovial Stem Cells.

Make Yourself Taller with Chondrogenesis

Note: I am sick so no LSJL experimentation until I get better.  Unfortunately, what's anabolic for growing new tissues is also anabolic for various bacteria and viruses(i.e. like sugar which creates cavities) so I'm going to have to wait until I'm healthy until I perform LSJL again.

In previous articles, I've talked about factors that affect chondrocyte proliferation.  There are tons of genes whose expression we can alter by mechanical loading and cells we can transport to areas that could increase growth(like stem cells or IGF-1) by increasing fluid flow in the bone.  Long bone grows mainly as a result of chondrogenesis.  Are there any other stages of chondrogenesis aside from chondrocyte proliferation or differentiation that we can manipulate to make ourselves taller?


Identification of Five Developmental Processes during Chondrogenic Differentiation of Embryonic Stem Cells.

"ESCs were differentiated into the chondrocyte lineage, forming small cartilaginous aggregates in suspension. Differentiated ESCs showed that chondrogenesis was typically characterized by five overlapping stages. During the first stage, cell condensation and aggregate formation was observed. The second stage was characterized by differentiation into chondrocytes and fibril scaffold formation within spherical aggregates. Deposition of cartilaginous extracellular matrix and cartilage formation were hallmarks of the third stage[Chondrocytes probably deposit this matrix] Apoptosis of chondrocytes, hypertrophy and/or degradation of cartilage occurred during the fourth stage. Finally, during the fifth stage, bone replacement with membranous calcified tissues took place.
ESCs show the chondrogenic differentiation pathway from the pluripotent stem cell to terminal skeletogenesis through these five stages in vitro. During each stage, morphological changes acquired in preceding stages played an important role in further development as a scaffold or template in subsequent stages."

Stage four and five of chondrogenic development are well known as part of endochondral ossifcation.  Could the deposition of extracellular matrix and cartilage formation be important in making yourself taller?  Chondrocytes have to die to be invaded by bone cells and extracellular matrix would inhibit that.  Although a larger hyaline cartilage layer(growth plate resting zone) could be beneficial for growth.  Stem cells tend to take up the characteristics of the surrounding tissues.  Stem cells within the hyaline cartilage growth plate line tend to differentiate into chondrocytes so stages one and two should automatically occur once the stem cells arrive in the hyaline cartilage growth plate line.


The balance of WNT and FGF signaling influences mesenchymal stem cell fate during skeletal development


"Craniosynostosis, a developmental disorder resulting from premature closure of the gaps (sutures) between skull bones, can be caused by excessive intramembranous ossification, a type of bone formation that does not involve formation of a cartilage template (chondrogenesis). Endochondral ossification, a type of bone formation that proceeds through a cartilage intermediate, caused by switching the fate of mesenchymal stem cells to chondrocytes, can also result in craniosynostosis. Simultaneous knockout of Axin2, a negative regulator of the WNT-beta-catenin pathway, and decreased activity of fibroblast growth factor (FGF) receptor 1 (FGFR1) in mice induced ectopic chondrogenesis[upregulating Beta-Catenin and downregulating FGFR1 is an interesting way to induce chondrogenesis], leading to abnormal suture morphogenesis and fusion. Genetic analyses revealed that activation of beta-catenin cooperated with FGFR1 to alter the lineage commitment of mesenchymal stem cells to differentiate into chondrocytes, from which cartilage is formed. We showed that the WNT-beta-catenin pathway directly controlled the stem cell population by regulating its renewal and proliferation, and indirectly modulated lineage specification by setting the balance of the FGF and bone morphogenetic protein pathways. This study identifies endochondral ossification as a mechanism of suture closure during development and implicates this process in craniosynostosis."

You know what this study shows? It shows that endochondral ossification(long bone growth) can occur at the fibrocartilage sutures between the bones of the skull. There are fibroblast cells within your synovial joints as well.

Since articular cartilage is like hyaline cartilage, and like this study shows endochondral ossifcation is possible at a lot of joint spaces. Maybe LSJL could work within the articular cartilage and not just the hyaline cartilage but one difference between the articular cartilage and the sutures in the skull is the synovial fluid. So even if articular cartilage began proliferating and differentiating chondrocytes the synovial fluid should provide enough nutrients to prevent the chondrocytes from dying and being invaded by bone cells.

"WNT–β-catenin pathway directly controls the skeletal precursor population by stimulating its renewal and proliferation, and indirectly influences lineage specification by setting the balance of the FGF and bone morphogenetic protein (BMP) pathways[The WNT/Beta-Catenin pathway determines the lineage of mesenchymal stem cells]. Switching the fate of mesenchymal stem cells to chondrocytes is an etiologic mechanism for craniosynostosis in mice[switching the fate of MSCs to chondrocytes causes the fusion of the sutures in the brain, manipulating the WNT/Beta-Catenin can be used to induce chondrogenesis]."

"loss of FGFR1 activity tips the balance toward chondrogenesis."<-inhibiting FGFR1 activity in the bone may be a way to encourage height growth.

"altering β-catenin and FGF signaling still changed the fate of the bone marrow precursors, suggesting that both β-catenin and FGF signaling are important for chondrocyte development in multiple contexts."

There are mechanisms specific within the articular cartilage that prevent endochondral ossification from happening...


Articular chondrocytes secrete PTHrP and inhibit hypertrophy of mesenchymal stem cells in coculture during chondrogenesis.


"[During cell-based cartilage regeneration the] unwanted upregulation of hypertrophic markers like alkaline phosphatase (ALP) and collagen-type X [occurs] during in vitro chondrogenesis and formation of instable calcifying cartilage at heterotopic sites[i.e. chondrocytes undergoing endochondral ossification]. In contrast, stable non-mineralizing cartilage is obtained from articular chondrocytes. Aim of this study was to address whether coculture with human articular chondrocytes (HAC) has the capacity to suppress undesired hypertrophy in differentiating MSC. 
MSC were differentiated in chondrogenic medium which had or had not been conditioned by parallel chondrocyte pellet cultures, or were mixed in the same pellet with chondrocytes (1:1, 1:2) and cultured for six weeks. Following in vitro differentiation, pellets were transplanted into SCID mice. 
The gene expression ratio of COL10A1/COL2A1 and IHH/COL2A1 was significantly reduced by HAC-conditioned medium and less collagen-type X protein was deposited relative to collagen-type II. ALP-activity was significantly lower (p<0.05) in the conditioned-medium-group and transplants showed significantly reduced calcification in vivo. In mixed HAC/MSC pellets, suppression of ALP was dose-dependent and in vivo calcification was fully inhibited. Chondrocytes secreted PTHrP throughout culture while PTHrP was downregulated in favour of IHH upregulation in control MSC after 2-3 weeks of chondrogenesis. Main inhibitory effects seen with HAC-conditioned medium could be reproduced by PTHrP supplementation of unconditioned medium."

So, in articular cartilage there is a method of inhibiting chondrocytes from undergoing endochondral ossification.

Conclusion: Endochondral ossification can occur at almost any cartilage layer(including fibrocartilage) however there are inhibitors of this occurring such as that within articular cartilage at the synovial joints. If you get stem cells to differentiate into chondrocytes, chondrogenesis and thereby endochondral ossifcation will occur.

Are these stem cell derived-chondrocytes different from "natural" chondrocytes?

Differences Between Chondrocytes and Bone Marrow-Derived Chondrogenic Cells.

"In this study, we analyzed the differences between the iMSCs[artificially induced Mesenchymal Stem Cells] and chondrocytes. Human bone marrow-derived MSCs were collected and induced to exhibit the chondrogenic phenotype by culturing the pelleted MSCs in a chemically defined culture medium supplemented with transforming growth factor-beta 1[note that maybe the marrow derived MSCs were not complete chondrocytes as the TGF-Beta 1 alone was not enough to induce full differentiation]. The molecular biological properties of iMSCs and culture-expanded chondrocytes, including their mRNA profiles and surface proteomics, were analyzed using reverse transcription-polymerase chain reaction (RT-PCR) and flow cytometry, respectively. The biomechanical properties of iMSCs and native chondrocytes, including their surface topology, adhesion force, and membrane stiffness, were analyzed using atomic force microscopy (AFM). Both iMSCs and chondrocytes presented type II collagen and glycosaminoglycan, whereas only chondrocytes presented type X collagen[Type X collagen is consistent with chondrocyte hypertrophy so derived chondrocytes don't hypertrophy.  It's possible though for this to change with other stimulus and the stem cells just differentiated so they may begin expressing type X collagen over time]. Flow cytometric assays showed that the expression of type II collagen and integrin-1 was higher in the chondrocytes than in the iMSCs[integrin-1 mediates attachment so it can be very vital in forming growth plates]. AFM revealed that the MSCs, iMSCs, and chondrocytes greatly differed in their shape. The MSCs were spindle shaped and easily distinguishable from the spherical chondrocytes. The iMSCs appeared round and resembled the spherical chondrocytes; however, the iMSCs were flatter with a central hump of condensed mass and a surrounding thin and broad pleat[so again the iMSCs likely did not completely differentiate into chondrocytes and needed hydrostatic pressure or something to become more like spherical chondrocytes]. The mean adhesion force and mean surface stiffness were significantly lower for the iMSCs (4.54 nN and 0.109 N/m, respectively) than for the chondrocytes (6.86 nN and 0.134 N/m, respectively)."

"Although MSCs are found in various adult tissues, the MSCs from bone marrow have higher potential for chondrogenesis than the MSCs from other sources"<-this since heterotropic ossification can occur in other tissues it is very likely for heterotopic ossification to occur in bone marrow.

It's very likely that the low quality of iMSCs was due to the presence of only one differentiation factor(TGF-Beta1) rather than an inherent malfunction of MSC derived chondrocytes.

JAWS coordinates chondrogenesis and synovial joint positioning.

"We show that mice carrying an insertional mutation in a previously uncharacterized gene, which we have named Jaws (joints abnormal with splitting), die perinatally with striking skeletal defects, including ectopic interphalangeal joints. These ectopic joints develop along the longitudinal axis and persist at birth, suggesting that JAWS is uniquely required for the orientation and consequent positioning of interphalangeal joints within the endochondral skeleton. Jaws mutant mice also exhibit severe chondrodysplasia characterized by delayed and disorganized maturation of growth plate chondrocytes, together with impaired chondroitin sulfation and abnormal metabolism of the chondroitin sulfate proteoglycan aggrecan. Our findings identify JAWS as a key regulator of chondrogenesis and synovial joint positioning required for the restriction of joint formation to discrete stereotyped locations in the embryonic skeleton."

"Jaws−/− chondrocytes lacked the pseudocolumnar organization of their WT counterparts"

"normal short-range but disrupted long-range Ihh signaling in the Jaws−/− growth plate"

Would overexpression of JAWS increase height or is JAWS only needed for proper endochondral ossification?

Effects of overexpression of membrane-bound transferrin-like protein (MTf) on chondrogenic differentiation in Vitro.

"Membrane-bound transferrin-like protein (MTf) is expressed in parallel with the expression of cartilage-characteristic genes during differentiation of chondrocytes, and the MTf level is much higher in cartilage than in other tissues. To investigate the role of MTf in cartilage, we examined the effects of growth factors on MTf expression in mouse prechondrogenic ATDC5 cells and the effect of MTf overexpression on differentiation of ATDC5 and mouse pluripotent mesenchymal C3H10T1/2 cells. In ATDC5 cultures, bone morphogenetic protein-2 and transforming growth factor-beta as well as insulin induced MTf mRNA expression when these peptides induced chondrogenic differentiation. Forced expression of rabbit MTf in ATDC5 cells induced aggrecan, type II collagen, matrilin-1, type X collagen mRNAs, and cell-shape changes from fibroblastic cells to spherical chondrocytes. Accordingly, the synthesis and accumulation of proteoglycans were higher in MTf-expressing cultures than in control cultures. These effects of MTf overexpression correlated with the MTf protein level on the cell surface and decreased in the presence of anti-MTf antibody. However, the aggrecan mRNA level in the ATDC5 cells overexpressing MTf was lower than that in wild type ATDC5 cells exposed to 10 microg/ml insulin. MTf overexpression in C3H10T1/2 cells also induced aggrecan and/or type II collagen mRNA but not the spherical phenotype[so Mtf could be chondroinductive]."

"BMP-2 elicited the greatest stimulation of MTf "

Chondrogenic differentiation of human bone marrow-derived mesenchymal stem cells in self-gelling alginate discs reveals novel chondrogenic signature gene clusters.

"We have used a disc-shaped self-gelling alginate hydrogel as a scaffold for in vitro chondrogenic differentiation of human bone marrow-derived mesenchymal stem cells. The comparison of monolayer cells and alginate embedded cells with or without differentiation medium allowed us to perform a detailed kinetic study of the expression of a range of genes and proteins known to be involved in chondrogenesis, using real-time polymerase chain reaction, fluorescence immunohistochemistry, and glycosaminoglycan measurement in the supernatant. mRNA encoding type II collagen (COL2), COL10, aggrecan, and SOX5, 6, and 9 were greatly elevated already at day 7, whereas COL1 and versican mRNA were gradually reduced[LSJl gene expression was taken at 48 hours]. COL2 and aggrecan were dispersed throughout the extracellular matrix at day 21, whereas COL10 distribution was mainly intra/pericellular. COL1 seemed to be produced by only some of the cells. SOX proteins were predominantly localized in the nuclei. Then, using microarray analysis, we identified a signature cluster of extracellular matrix and transcription factor genes upregulated during chondrogenesis similar to COL2A1, and clusters of genes involved in immune responses, blood vessel development, and cell adhesion downregulated similar to the chemokine CXCL12. "

"A number of genes encoding proteins with a modifying effect on chondrogenesis were upregulated, including epiphycan (EPYC){upregulated over 6 fold in LSJL}, matrilin 3 (MATN3){upregulated in LSJL}, fibromodulin (FMOD), TIMP metalloproteinase inhibitor 4 (TIMP4), chondroadherin (CHAD), and dermatopontin (DPT){upregulated 3 fold by LSJL}"

Divide, accumulate, differentiate: cell condensation in skeletal development revisited.

"The pre-condensation phase is characterized by expression of Hox genes, growth factors (TGF-beta and BMP-2) and the cell surface proteoglycan receptor, syndecan-1"

"Other molecules, such as versican, syndecan-3 and tenascin, present in low concentrations before condensation, are up-regulated during condensation. Yet other molecules--Hox genes, transcription factors, growth factors (activin, BMP-4 and -5, GDF-5), cell adhesion molecules and proteoglycans--are only expressed during the condensation phase, while the transcription factor Pax-1, fibronectin, hyaluronan and hyaladherin are expressed both during and after condensation"

So this furthers the hypothesis that LSJL induces pre-chondrogenic mesenchymal condensation.

"The formation of a condensation is associated with reduction in hyaluronan (hyaluronate, hyaluronic acid) and increase in chondroitin sulphate. Hyaluronan blocks chondrogenesis; removal of hyaluronan permits chondrogenesis;"<-this is contrary to other evidence and LSJL increases both chondroitin and hyaluronan synthesis.

Identification and characterization of chondrogenic progenitor cells in the fascia of postnatal skeletal muscle.

"Intramuscular injection of bone morphogenetic proteins (BMPs) has been shown to induce ectopic bone formation. A chondrogenic phase is typically observed in this process, which suggests that there may exist a chondrogenic subpopulation of cells residing in skeletal muscle. Two prospective cell populations were isolated from rat skeletal muscle: fascia-derived cells (FDCs), extracted from gluteus maximus muscle fascia (epimysium) and muscle-derived cells (MDCs) isolated from the muscle body. Both populations were investigated for their cell surface marker profiles (flowcytometry analysis), proliferation rates as well as their myogenic and chondrogenic potentials. The majority of FDCs expressed mesenchymal stromal cell markers but not endothelial cell markers. FDCs underwent chondrogenic differentiation after BMP4 treatment in vitro. Although MDCs showed chondrogenic potential, they expressed the myogenic cell marker desmin and readily underwent myogenic differentiation in vitro; however, the chondrogenic potential of the MDCs is confounded by the presence of FDC-like cells residing in the muscle perimysium and endomysium. To clarify the role of the muscle-derived myogenic cells in chondrogenesis, mixed pellets with varying ratios of FDCs and L6 myoblasts were formed and studied for chondrogenic potential. the chondrogenic potential of the mixed pellets decreased with the increased ratio of myogenic cells to FDCs supporting the role of FDCs in chondrogenesis."

" fascia tissue is also rich in blood vessels, which implies that these chondrogenic progenitor cells might reside in the ‘stem cell niche’ surrounding the blood vasculature. The blood cell walls may harbor a dormant reserve of chondrogenic progenitors that could be recruited when they receive a BMP4 stimulation signal."

"connective tissue-derived cells (epimysium, perimysium and endomysium) [may be] required for chondrogenesis"

High expression of vimentin(LSJL upregulates Vim) and low expression of desmin may be a more chondrogenic environment.


Metabolic labeling of human bone marrow mesenchymal stem cells for the quantitative analysis of their chondrogenic differentiation.

"The average age of the patients was 70 years (range 65-74 years). The study has been approved by the Ethics Committee of Galicia, and all patients gave their informed consent. Cells were isolated from trabecular bone marrow samples by washing the femoral heads"<-We're trying to induce chondrogenic differentiation in the trabecular bone marrow of the femoral epiphysis!

This study proves that adult epiphyseal bone marrow cells are capable of chondrogenesis.

"chondrogenesis [involves] protein synthesis and turnover, cytoskeleton organization and chaperones/stress response"

"the cell pellet was resuspended in DMEM medium with 20% FBS and then plated at a seeding density of 1x105 cells/cm2 and incubated at 37ºC in a 5% CO2 atmosphere. After 48 hours, the medium was replaced to remove non-adherent hematopoietic cells."<-Can LSJL induce a similar environment?  It can increase the density by laterally compressing the epiphysis.

Genes upregulated at 14 days of chondrogenic differentiation versus day 2 of chondrogenic differentiation of HMBMSC's that are also altered during LSJL:

PTRF

ARPC4(down in LSJL)

VIM

COL6A2(partially up and down in LSJL)

COL6A3

LMNA

Tenascin(LSJL upregulates Tenascin W as TNN which is found in developing bone)

Downregulated:

Increase your Height with Sox9

In the study where the recreated the growth plate in hyaline cartilage using mesenchymal chondrosarcoma, they used Sox9 as a key marker for chondrogenesis.  If there's no Sox9 expression in the growth plate, then delivering mesenchymal stem cells there by trabecular microfracture followed by an increase in interstitial fluid flow won't recreate the growth plate(the chondrocytes won't proliferate and differentiate).  Of course the cells have to die too or otherwise the osteoblasts won't invade the dead chondrocytes.  How can we control Sox9 genetic expression in the growth plate and can that expression be increased by foods or exercise?

Anterior cruciate ligament-derived cells have high chondrogenic potential. 

"Anterior cruciate ligament (ACL)-derived cells have a character different from medial collateral ligament (MCL)-derived cells. However, the critical difference between ACL and MCL is still unclear in their healing potential and cellular response. The objective of this study was to investigate the mesenchymal differentiation property of each ligament-derived cell. Both ligament-derived cells differentiated into adipogenic, osteogenic, and chondrogenic lineages. In chondrogenesis, ACL-derived cells had the higher chondrogenic property than MCL-derived cells. The chondrogenic marker genes, Sox9 and alpha1(II) collagen (Col2a1), were induced faster in ACL-derived pellets than in MCL-derived pellets. Sox9 expression preceded the increase of Col2a1 in both pellet-cultured cells. However, the expression level of Sox9 and a ligament/tendon transcription factor Scleraxis[Scx] did not parallel the increase of Col2a1 expression along with chondrogenic induction." 

"ACL includes more chondrocytic cells, identified as fusiform, ovoid, and spheroid cells, compared with MCL. Fusiform and spheroid cells packaged in the ligament-to-bone interface can produce a cartilage specific extracellular matrix (ECM), type II collagen"

"Repair-associated molecules including transforming growth factor (TGF)-β and type III collagen do not increase in rabbit ACL injury, unlike MCL injury"

"TGF-β signal Smad3 promotes the early chondrogenesis through the activation of Sox9"<-Smad3 was not upregulated by LSJL.

"TGF-β-regulated Smad3 also activates the Sox9-dependent transcription on chromatin without influencing Sox9 expression itself"

"Scx expression is transiently increased in the early stage of embryonic stem cell-derived chondrogenesis. We have previously demonstrated that Scx stimulates the Sox9-dependent transcription by forming transcriptional complex with E47 and p300 in early chondrogenesis"

So maybe it is important to load the ligaments in Lateral Synovial Joint Loading.  Anterior is the outer part of the knee so perhaps outer knee loading is better for LSJL.  

Enhanced chondrogenic differentiation of human bone marrow-derived mesenchymal stem cells in low oxygen environment micropellet cultures. 

"Chondrogenesis of mesenchymal stem cells (MSCs) is typically induced when they are condensed into a single aggregate and exposed to transforming growth factor-beta (TGF-beta). Hypoxia, like aggregation and TGF-beta delivery, may be crucial for complete chondrogenesis. However, the pellet dimensions and associated self-induced oxygen gradients of current chondrogenic methods may limit the effectiveness of in vitro differentiation and subsequent therapeutic uses. Here we describe the use of embryoid body-forming technology to produce microscopic aggregates of human bone marrow MSCs (BM-MSCs) for chondrogenesis. The use of micropellets reduces the formation of gradients within the aggregates, resulting in a more homogeneous and controlled microenvironment. These micropellet cultures (approximately 170 cells/micropellet) as well as conventional pellet cultures (approximately 2 x 10(5) cells/pellet) were chondrogenically induced under 20% and 2% oxygen environments for 14 days. Compared to conventional pellets under both environments, micropellets differentiated under 2% O(2) showed significantly increased sulfated glycosaminoglycan (sGAG) production and more homogeneous distribution of proteoglycans and collagen II. Aggrecan and collagen II gene expressions were increased in pellet cultures differentiated under 2% O(2) relative to 20% O(2) pellets but 2% O(2) micropellets showed even greater increases in these genes, as well as increased SOX9. These results suggest a more advanced stage of chondrogenesis in the micropellets accompanied by more homogeneous differentiation. Thus, we present a new method for enhancing MSC chondrogenesis that reveals a unique relationship between oxygen tension and aggregate size." 

Hypoxia is something we can induce by exercise.  Just run until you're out of breath.  Something to experiment with LSJL if results slow.  Do jumping jacks until you are out of breath and then apply the dumbell to the epiphysis of the bone.  Hypoxia enhances stem cell proliferation too. 

Sox9 family members negatively regulate maturation and calcification of chondrocytes through up-regulation of parathyroid hormone-related protein. 

"Sox9 is a transcription factor that plays an essential role in chondrogenesis and has been proposed to inhibit the late stages of endochondral ossification. We found that overexpression of Sox9 alone or Sox9 together with Sox5 and Sox6 (Sox5/6/9) inhibited the maturation and calcification of murine primary chondrocytes and up-regulated parathyroid hormone-related protein (PTHrP) expression in primary chondrocytes and the mesenchymal cell line C3H10T1/2. Sox5/6/9 stimulated the early stages of chondrocyte proliferation and development. In contrast, Sox5/6/9 inhibited maturation and calcification of chondrocytes in organ culture. The inhibitory effects of Sox5/6/9 were rescued by treating with anti-PTHrP antibody. Moreover, Sox5/6/9 bound to the promoter region of the PTHrP gene and up-regulated PTHrP gene promoter activity. Interestingly, we also found that the Sox9 family members functionally collaborated with Ihh/Gli2 signaling to regulate PTHrP expression and chondrocyte differentiation. Our results provide novel evidence that Sox9 family members mediate endochondral ossification by up-regulating PTHrP expression in association with Ihh/Gli2 signaling." 

Parathyroid Hormone is pretty important for growth.  Not only does it increase osteoblast activity(which increases height in the spine) but it is involved in chondrocyte differentiation as well. 

p38 MAPK mediated in compressive stress-induced chondrogenesis of rat bone marrow MSCs in 3D alginate scaffolds. 

"Mesenchymal stem cells (MSCs) are well known to have the capability to form bone and cartilage, and chondrogenesis derived from MSCs is reported to be affected by mechanical stimuli. This research was aimed to study the effects of cyclic compressive stress on the chondrogenic differentiation of rat bone marrow-derived MSCs (BMSCs) which were encapsulated in alginate scaffolds and cultured with or without chondrogenic medium, and to investigate the role of p38 MAPK phospho-relay cascade in this process. The results show that the gene expression of chondrocyte-specific markers of Col2alpha1, aggrecan, Sox9, Runx2, and Ihh was upregulated by dynamic compressive stress introduced at the 8th day of chondrogenic differentiation in vitro. The p38 MAPK was activated by chondrogenic cytokines in a slow and lagged way, but activated by cyclic compressive stimulation in a rapid and transient manner. And inhibition of p38 activity with SB203580 suppressed gene expression of chondrocyte-specific genes stimulated by chondrogenic medium and (or) cyclic compressive stress. These findings suggest that p38 MAPK signal acts as an essential mediator in the mechano-biochemical transduction and subsequent transcriptional regulation in the process of chondrogenesis." 

MAPK's can be affected by the hormone leptin(positively).  Cyclic compressive stress is something we can induct with exercise in methods like you guess it LSJL.  A foam roller could induce compressive stress too(it's pretty awkward to use on your limbs though when you're turned on your side) . 

This research seems to suggest that both aerobic(induces hypoxia) and heavy compressive lifting(cyclic compressive stimulation) would be good for increasing height growth during development.  They could also help the factors involved in Lateral Synovial Joint Loading as well. 

Here's a study involving R. Bollock on Sox9:

Sox9 directs hypertrophic maturation and blocks osteoblast differentiation of growth plate chondrocytes.

"The transcription factor Sox9 is necessary for early chondrogenesis. Using a doxycycline-inducible Cre transgene and Sox9 conditional null alleles in the mouse, we show that Sox9 is required to maintain chondrocyte columnar proliferation and generate cell hypertrophy, two key features of functional growth plates. Sox9 keeps Runx2 expression and β-catenin signaling in check and thereby inhibits not only progression from proliferation to prehypertrophy, but also subsequent acquisition of an osteoblastic phenotype. Sox9 protein outlives Sox9 RNA in upper hypertrophic chondrocytes, where it contributes with Mef2c to directly activate the major marker of these cells, Col10a1."

"Typical markers include Col2a1 (collagen 2) and Acan (aggrecan) for early chondrocytes; Fgfr3 (fibroblast growth factor receptor 3) for columnar cells; Ppr (parathyroid hormone-related protein receptor), Ihh (Indian hedgehog), and Col10a1 (collagen 10) for prehypertrophic cells; and Col10a1 only for hypertrophic cells. Terminal chondrocytes express Mmp13 (matrix metalloproteinase 13) and Bsp (bone sialoprotein), and mineralize the extracellular matrix, as do mature osteoblasts, whereas early osteoblasts express Osx (Osterix) and Col1a1 (collagen 1)"

"The three Sox proteins[Sox5, 6, and 9] are needed and sufficient for early chondrogenesis, and thus referred to as the chondrogenic trio"

"We previously showed that an Acan (aggrecan) upstream enhancer was sufficient to activate the Col2a1 promoter in differentiated chondrocytes in transgenic mice"

"Sox9 may delay prehypertrophy by downregulating Runx2 and Mef2c, but must control hypertrophy differently"

"Sox9 protein is present in cells activating Col10a1."

"Sox9 and Mef2c transactivate Col10a1, and act additively."

"The Sox trio is required for Col2a1 transcription"

MLTK may be involved with the Sox trio.

The protein kinase MLTK regulates chondrogenesis by inducing the transcription factor Sox6.

"Sox9 acts together with Sox5 or Sox6 as a master regulator for chondrogenesis. The protein kinase MLTK plays an essential role in the onset of chondrogenesis through triggering the induction of Sox6 expression by Sox9. We find that knockdown of MLTK in Xenopus embryos results in drastic loss of craniofacial cartilages without defects in neural crest development. Sox6 is specifically induced during the onset of chondrogenesis, and Sox6 induction is inhibited by MLTK knockdown. Sox6 knockdown phenocopies MLTK knockdown. Ectopic expression of MLTK induces Sox6 expression in a Sox9-dependent manner. p38 and JNK pathways function downstream of MLTK during chondrogenesis."

"Sox5 and Sox6 bind to the enhancers of cartilage matrix genes and enhance the activity of Sox9"

"MLTK-alpha and MLTK-beta form homo- and heterodimers, and can be activated by osmotic stress"<-LSJL can cause osmotic stress. This may be part of the mechanism as to how hydrostatic pressure induces chondrogenic differentiation.

"Activated MLTK-alpha and MLTK-beta activate the p38 and JNK pathways through MKK3/6 and MKK4/7 phosphorylation"

"overexpression of xSox9 alone did not induce a substantial increase in xSox6 expression, co-expression of xMLTK-alpha and xMLTK-beta with xSox9 induced marked increases in xSox6 expression in a dose-dependent manner"

"p38 signaling can increase the transcriptional activity of Sox9, and transgenic mice in which p38 signaling is constitutively activated in chondrocytes, show phenotypes similar to those of mice that overexpress Sox9 in chondrocytes"<-increasing p38 signaling might be a way to grow taller.

Disruption of a Sox9 - β-catenin circuit by mutant Fgfr3 in Thanatophoric Dysplasia Type II.

"Using a mouse model of Thanatophoric Dysplasia Type II (TDII) in which FGFR3(K650E) expression was directed to the appendicular skeleton, we show that the mutant receptor caused a block in chondrocyte differentiation specifically at the prehypertrophic stage. The differentiation block led to a severe reduction in hypertrophic chondrocytes that normally produce vascular endothelial growth factor, which in turn was associated with poor vascularization of primary ossification centers and disrupted endochondral ossification. We show that the differentiation block and defects in joint formation are associated with persistent expression of the chondrogenic factor Sox9[so maybe FGFR3 can increase height in the right circumstances] and downregulation of β-catenin levels and activity in growth plate chondrocytes. Consistent with these in vivo results, FGFR3(K650E) expression was found to increase Sox9 and decrease β-catenin levels and transcriptional activity in cultured mesenchymal cells. Coexpression of Fgfr3(K650E) and Sox9 in cells resulted in very high levels of Sox9 and cooperative suppression of β-catenin-dependent transcription. Fgfr3(K650E) had opposing effects on Sox9 and β-catenin protein stability with it promoting Sox9 stabilization and β-catenin degradation. Both Sox9 overexpression and β-catenin deletion independently blocks hypertrophic differentiation of chondrocytes and cause chondrodysplasias similar to those caused by mutations in FGFR3."

"Sox9 is expressed in progenitor or “resting” chondrocytes, proliferating chondrocytes and chondrocytes in the initial stage of differentiation (prehypertrophy), but is then abruptly downregulated as these cells differentiate into non-proliferative and terminally differentiated hypertrophic chondrocytes"

"Sox9 downregulation in hypertrophic chondrocytes is thought to play a critical function in endochondral growth and ossification since its forced expression in growth plate chondrocytes suppressed the production of hypertrophic cells and Vegfa expression, resulting in compromised vascularization at the ossification front and a severe chondrodysplasia"

"Sox9 can also suppress β-catenin transcriptional activity independent of β-catenin degradation, possibly by interfering with β-catenin - Tcf/Lef interactions"

"Sox9 protein levels are negatively regulated by Beta-catenin"

"strong correlation between mutant Fgfr3 expression, decreased Beta-catenin expression and activity and a failure to downregulate Sox9."

"Addition of FGF2 repressed β-catenin activity alone and augmented Fgfr3K650E driven repression"

"deletion of Sox9 in growth plate chondrocyte was recently found to induce apoptosis, suppress

Col10a1 expression and prevent proper hypertrophic differentiation"

"Beta-catenin stability is regulated by a multiprotein complex that includes of Beta-catenin, Axin, APC, and Gsk-3Beta. According to canonical regulation of Beta-catenin degradation, Gsk-3Beta phosphorylates primed Beta-catenin within this complex, which marks Beta-catenin for ubiquitylation and subsequent proteasome degradation in the cytoplas. Sox9 promotes nuclear translocation of Beta-catenin together with components of the Beta-catenin destruction complex, which leads to enhanced phosphorylation and degradation of Beta-catenin in the nucleus. Fgfr3K650E promoted both upregulation of Sox9 and Beta-catenin phosphorylation at Gsk-3Beta sites"

Unraveling the transcriptional regulatory machinery in chondrogenesis.

"The catalytic subunit of cyclic AMP-dependent protein kinase A (PKA) and the Rho-associated coiled coil-forming kinase (ROCK) interacted with Sox9 and directly phosphorylate it at serine 181. This modification caused nuclear accumulation of Sox9, increased the efficiency of Sox9 binding to DNA, and/or increased transcriptional activity. It occurred principally in the prehypertrophic zone of the growth plate, which is the major site of expression of the parathyroid hormone-related peptide (PTHrP) receptor, suggesting that PTHrP may inhibit conversion of proliferating cells into prehypertrophic cells, at least in part, through activating PKA and thereby the activity of Sox9. Histone acetyl transferase Tip60 increased Sox9/Sox5-dependent transcription in association with acetylation of Sox9 at lysine 61, 253, and 398. Tip60, together with Sox9 and Sox5, was also present in the chromatin of the Col2a1 enhancer. The Sox9 protein is also modified by protein inhibitor of activated STAT (PIAS)1-mediated SUMOylation. PIAS1 [may repress] or [stimulate] Sox9 activity. "

"P54nrb, 54-kDa nuclear RNA-binding protein, which physically interacted with Sox9 to increase transactivation of Col2a1 and promoted splicing of the Col2a1 mRNA"<-Grow taller with P54nrb?

"Trap230 (Med12) [is] a coactivator of Sox9"

Chondrogenesis enhanced by overexpression of sox9 gene in mouse bone marrow-derived mesenchymal stem cells.

"a high density micromass culture may compensate for the necessity of co-factors, L-sox5 and sox6."

The study states that overexpression of Sox9 enhanced chondrogenesis.

The postnatal role of Sox9 in cartilage

"The postnatal inactivation of Sox9 led to stunted growth characterized by decreased proliferation, increased cell death, and de‐differentiation of growth plate chondrocytes."  Loss of Sox9 resulted in compression and degeneration in other cartilagenous areas too such as the discs.  So ensuring an equilibrium quantity of Sox9 in those areas may help increase height.

"Upon postnatal Sox9 inactivation in the articular cartilage, the sulfated proteoglycan and aggrecan content of the uncalcified cartilage were rapidly depleted and the degradation of aggrecan was accompanied by higher ADAMTS5 immunostaining and increased detection of the aggrecan neoepitope, NITEGE."

"the loss of Sox9 in the IVD decreased the expression of cytokines, cell surface receptors, and ion channels"

Genes downregulated by loss of Sox9:
MATN3(up in LSJL)
UCMA
Col2a1(up in LSJL)
Sfrp5
Grem1
Rspo4
Cytl1
Clec3a
Fxyd2(up in LSJL)
Cilp2
Col27a1
THBS1
CHAD
HAPLN1(up)
COL11A2
COMP
VCAN(up)
CILP
ACAN(up)
BGN(up)
Lect1
Frzb
IGFBP5
LOXL2,3,4
PAPSS2
FAM38B
PIEZO2
PKD1
FGFR3
TRPV4
GPR126

Upregulated genes:
Dbh
Ddc
Syt1
Syt4
Ucp1
Mpo
CXCR2

"Sox9 is almost exclusively localized to the articular chondrocytes of the uncalcified cartilage, with very few Sox9 positive cells in the region of calcified cartilage"

"the polycystin gene (pkd1) that encodes for an integral membrane protein that has been shown to be a mechanical flow “sensor” in the renal epithelium was downregulated. The homozygous pkd1-null mice display skeletal abnormalities, including stunted growth and kyphosis of the spine"

Sox9 sustains chondrocyte survival and hypertrophy in part through Pik3ca-Akt pathways.

"During endochondral bone formation, Sox9 expression starts in mesenchymal progenitors, continues in the round and flat chondrocyte stages at high levels, and ceases just prior to the hypertrophic chondrocyte stage. Sox9 is important in mesenchymal progenitors for their differentiation into chondrocytes, but its functions post-differentiation have not been determined. To investigate Sox9 function in chondrocytes, we deleted mouse Sox9 at two different steps after chondrocyte differentiation. Sox9 inactivation in round chondrocytes resulted in a loss of Col2a1 expression and in apoptosis. Sox9 inactivation in flat chondrocytes caused immediate terminal maturation without hypertrophy and with excessive apoptosis. Inactivation of Sox9 in the last few cell layers resulted in the absence of Col10a1 expression, suggesting that continued expression of Sox9 just prior to hypertrophy is necessary for chondrocyte hypertrophy. SOX9 knockdown also caused apoptosis of human chondrosarcoma SW1353 cells. These phenotypes were associated with reduced Akt phosphorylation {LSJL upregulates Sox9 and increases p-Akt}. Forced phosphorylation of Akt by Pten inactivation partially restored Col10a1 expression and cell survival in Sox9(floxdel/floxdel) mouse chondrocytes, suggesting that phosphorylated Akt mediates chondrocyte survival and hypertrophy induced by Sox9. When the molecular mechanism of Sox9-induced Akt phosphorylation was examined, we found that expression of the PI3K subunit Pik3ca (p110α) was decreased in Sox9(floxdel/floxdel) mouse chondrocytes. Sox9 binds to the promoter and enhances the transcriptional activities of Pik3ca. Thus, continued expression of Sox9 in differentiated chondrocytes is essential for subsequent hypertrophy and sustains chondrocyte-specific survival mechanisms by binding to the Pik3ca promoter, inducing Akt phosphorylation."

"mesenchymal condensation and subsequent cartilage formation are absent in the limbs of Prx1-Cre"

"misexpression of Sox9 in hypertrophic chondrocytes results in a lack of bone marrow, and that Sox9 is a major negative regulator of cartilage vascularization"

" In 11Enh-Cre transgenic mice, Cre recombinase activities are controlled by the Col11a2 promoter and enhancer and begin during the round chondrocyte stage. 11Enh-Cre directs recombination at a later stage (both developmental and within the chondrocyte differentiation pathway) than Col2a1-Cre"

"11Enh-Cre initiated the direct recombination of floxed Sox9 genes in round chondrocytes"

"chondrocytes lacking Sox9 enter into terminal maturation without hypertrophy"

" the deletion of Pten results in the accumulation of PtdIns(3,4,5)P3, leading to forced activation of Akt."

"most differentiated cell types, including osteoblasts and adipocytes, do not express Sox9 and survive."<-thus if osteoblasts were expressing Sox9 in LSJL then there should have been apoptosis markers whereas in LSJL many apoptosis markers were downregulated.

Regulation of bone and cartilage development by network between BMP signalling and transcription factors

"Sox9 has been shown to directly regulate chondrogenic genes, such as Col2a1, Col11a2 and aggrecan"

"BMP2 markedly stimulated Sox9 expression in mouse limb bud cells"

"p54nrb, a component of para-speckled body, associates with Sox9 and stimulates its transcriptional activity"

"Znf219 and Arid5a, which physically interact with each other, also associate and co-localize with Sox9"<-These two genes are involved in histone related alterations in chondrogenesis.

"Sox9 associates with Gli2, a mediator of Ihh, and Sox9 and Gli2 co-operatively stimulate PTHrP expression and PTHrP gene promoter activity"

"Overexpression of Msx2 also consistently stimulates maturation of chondrocytes. In addition, cartilage development seems to be reduced in Msx2 knockout mice"

"Runx2 stimulates and Sox9 inhibits maturation of chondrocytes."

"Osterix is expressed in the prehypertrophic zones of growth plates and that, in global and conditional Osterix knockout mice, chondrogenesis was totally blocked at the hypertrophic stage and there was no evidence of calcification of chondrogenic matrices or formation of matrix vesicles"

"When cells experience ER stress, several ER sensors, including IRE1, ATF6, Perk, Oasis{up in LSJL as CREB3l1} and BBF2H7, all of which are anchored in the membrane of the ER, are released by membrane truncation, and subsequently respond to the ER stress. Interestingly, truncated forms of Oasis and BBF2H7 translocate to nuclei and function as transcription factors."

"Sec23 is a major target of BBF2H7 in chondrocytes. As expected, introduction of Sec23, an important chaperon protein for protein folding, rescued chondrocyte differentiation in BBF2H7-deficient cells"

Retinoids regulate stem cell differentiation.

"All-trans retinoic acid (RA) and other active retinoids are generated from vitamin A (retinol), but key aspects of the signaling pathways required to produce active retinoids remain unclear. Retinoids generated by one cell type can affect nearby cells, so retinoids also function in intercellular communication. RA induces differentiation primarily by binding to RARs, transcription factors that associate with RXRs and bind RAREs in the nucleus. Binding of RA: (1) initiates changes in interactions of RAR/RXRs with co-repressor and co-activator proteins, activating transcription of primary target genes; (2) alters interactions with proteins that induce epigenetic changes; (3) induces transcription of genes encoding transcription factors and signaling proteins that further modify gene expression (e.g., FOX03A, Hoxa1, Sox9, TRAIL, UBE2D3); and (4) results in alterations in estrogen receptor α signaling. Proteins that bind at or near RAREs include Sin3a, N-CoR1, PRAME, Trim24, NRIP1, Ajuba, Zfp423, and MN1/TEL. Interactions among retinoids, RARs/RXRs, and these proteins explain in part the powerful effects of retinoids on stem cell differentiation."

"high Stra6 expression is suggestive of a requirement for the actions of retinol."

"Inside the cell, RA is transported to the nucleus bound to CRABP2{up in LSJL}"

"Once in the nucleus, RA binds to RARα, β, or γ. These RARs can bind to one of the RXRs (RXRα, β, or γ)."<-RXRb is downregulated in LSJL.

"The Rex1 (Zfp42) gene is expressed at high levels in embryonic stem cells, and is transcriptionally inhibited by RA. The mechanism of inhibition involves loss of binding of the positively acting transcription factor Oct4 to an Oct4 site in the Rex1 promoter in response to RA. Interestingly, transcriptional repression by RA in embryonic stem cells is often mediated by a different mechanism, an increase in the expression of the orphan nuclear receptor GCNF (germ cell nuclear receptor) (Nr6a1), which then represses pluripotency genes such as Sox2, Nanog, or Oct4"

"Sox9 was shown to inhibit proliferation by activating the transcription factor Hes1{up in LSJL}. Sox9 [is transcriptionally activated] by RA"

Control of chondrogenesis by the transcription factor Sox9

"In chondrocytes, Sox9 binds as a homodimer to a pair of the consensus sequences of Col2a1{up}, Col9a1{up}, Col27a1, or Matrilin-1, and this binding is mediated by a dimerization domain located closer to the N-terminus than the HMG domain"

"Sox9-null cells do not express chondrogenic marker genes, such as Col2a1, Col9a2, Col11a2, and aggrecan{up}."

"fibroblast growth factor (FGF), insulin-like growth factor I (IGF-I), human cartilage glycoprotein 39, transient receptor potential vanilloid 4 (TRPV4), RAR agonists, and Src inhibitor increase Sox9 expression"

"CCAAT-binding factor, Sp1, CREB, Sonic hedgehog, and hypoxia-inducible factor 1α directly transactivate the Sox9 proximal promoter"

"transcription factors including Pax1{up}, Pax9, Nkx3.1, Nkx3.2, and Barx2{up} control the level of Sox9"

"Sox9 [inhibits] hypertrophic conversion by phosphorylation of S64 and S211 of Sox9 by protein kinase A (PKA), a downstream intracellular signaling molecule of parathyroid hormone-related peptide (PTHrP)/PTHrP receptor"

"Translocation of Sox9 into the nucleus is controlled by Sox9-calmodulin interaction through the nuclear localization signal of the HMG domain within a consensus calmodulin-binding region"

"cGMP-dependent protein kinase type II{PRKG2 is up in LSJL} reduces the activity of Sox9 by inhibiting the nuclear import of Sox9, a process that is mediated by phosphorylation of S181. Sox9 activity is also regulated by protein inhibitor of activated STAT1 (PIAS1)-mediated sumoylation. PIAS1 enhances the sumoylation at K398 and represses Sox9 activity. The stability and degradation of Sox9 protein are determined by the ubiquitin-proteosome pathway"

"Sox9 uses a cAMP response element-binding protein-binding protein (CBP)/p300 to exert its effects, which is mediated by histone acetylation"

"Smad3, a signaling molecule of transforming growth factor-β,(TGF-β) stimulates the Sox9-dependent transcriptional activation by modulating the interaction between Sox9 and CBP/p300"

"peroxisome proliferation-activated receptor-gamma co-activator 1-α (PGC1-α) is a coactivator of Sox9 during chondrogenesis"

"Sox9 interacts with a component of the mediator complex, the thyroid hormone receptor-associated protein (Trap) 230/Med12"

Analysis of post transcriptional regulation of SOX9 mRNA during in vitro chondrogenesis.

"SOX9 mRNA half life exhibited an inverse correlation with total SOX9 mRNA levels in both dedifferentiating human articular chondrocytes and chondrogenic pellet cultures."

"SOX9 mRNA exhibits a moderately short half life in human articular chondrocytes (HACs) which can be extended if the cells are exposed to environmental stresses such as cycloheximide treatment or hyperosmolarity."

"as levels of the SOX9 mRNA were falling, its half life was increased"

"The BMSCs exhibit a rapid turnover of SOX9 in both their undifferentiated state in monolayer culture and as they differentiate into chondrocytic cells in pellet culture"

"fine tuning of SOX9 mRNA decay rates, through some form of feedback mechanism, keeps overall levels of SOX9 mRNA within a certain threshold, even as transcriptional rates change."

Trans-activation of the mouse cartilage-derived retinoic acid-sensitive protein gene by Sox9.

"To determine whether Sox9 is an inducer of the chondrocyte phenotype, we investigated the role of Sox9 in transcription of another cartilage gene encoding the cartilage-derived retinoic acid-sensitive protein (CD-RAP)[also known as MIA]. CD-RAP is specifically expressed during chondrogenesis. We show here that Sox9 protein is able to bind to a SOX consensus sequence in the CD-RAP promoter. Mutation of the SOX motif led to decreased transcription of a CD-RAP promoter construct in chondrocytes. Overexpression of SOX9 resulted in a dose-dependent increased activity of CD-RAP promoter-driven reporter gene in both chondrocytes and nonchondrogenic cells. A truncated SOX9, which contains a binding domain but no trans-activation function, inhibited CD-RAP promoter activity. Overexpression of SOX9 increased the level of endogenous CD-RAP mRNA in chondrocytes, but was unable to induce endogenous gene expression in 10T1/2 mesenchymal cells or BALB/c-3T3 fibroblasts. These results suggest that Sox9 is a general transcriptional regulator of cartilage-specific genes. However, Sox9 does not appear to be able to induce the chondrocyte phenotype in nonchondrogenic cells{but TGFB1 and BMP-2 which are involved in LSJL may be able to}, implying that other factors are involved in chondrogenesis."

"CD-RAP/MIA is down-regulated coordinately by retinoic acid with type II collagen, the most abundant extracellular protein made by chondrocytes and generally considered to be characteristic of cartilage."

"SOX9 enhanced the CD-RAP promoter activity in both chondrocytes and nonchondrogenic cells, including BALB/3T3, MC3T3, and 10T1/2 cells"

"The CD-RAP promoter activity was modulated by bone morphogenetic proteins, parathyroid hormone-related peptide, transforming growth factor β, fibroblast growth factor, interleukin-1 and interleukin-8, epidermal growth factor, and retinoic acid"

"Sox9 is unable to activate endogenous Col2a1 in nonchondrogenic cells."

"a 48-bp enhancer element in the first intron of type II collagen (which includes a Sox9 motif) is able to generate chondrocyte-specific expression in vivo. However, mutation in each of three HMG-like domains in this fragment abolished the cartilage-specific expression in vitro and in vivo, suggesting that additional HMG-like DNA domains other than the Sox9 binding site are also critical for the cartilage-specific expression."

SOX9 is a major negative regulator of cartilage vascularization, bone marrow formation and endochondral ossification.

"Sox9 is highly expressed in chondrocytes of the proliferating and prehypertrophic zone but declines abruptly in the hypertrophic zone, suggesting that Sox9 downregulation in hypertrophic chondrocytes might be a necessary step to initiate cartilage-bone transition in the growth plate. In order to test this hypothesis, we generated transgenic mice misexpressing Sox9 in hypertrophic chondrocytes under the control of a BAC-Col10a1 promoter. The transgenic offspring showed an almost complete lack of bone marrow in newborns, owing to strongly retarded vascular invasion into hypertrophic cartilage and impaired cartilage resorption, resulting in delayed endochondral bone formation associated with reduced bone growth. In situ hybridization analysis revealed high levels of Sox9 misexpression in hypertrophic chondrocytes but deficiencies of Vegfa, Mmp13, RANKL and osteopontin expression in the non-resorbed hypertrophic cartilage, indicating that Sox9 misexpression in hypertrophic chondrocytes inhibits their terminal differentiation. Searching for the molecular mechanism of SOX9-induced inhibition of cartilage vascularization, we discovered that SOX9 is able to directly suppress Vegfa expression by binding to SRY sites in the Vegfa gene. Postnatally, bone marrow formation and cartilage resorption in transgenic offspring are resumed by massive invasion of capillaries through the cortical bone shaft, similar to secondary ossification. These findings imply that downregulation of Sox9 in the hypertrophic zone of the normal growth plate is essential for allowing vascular invasion, bone marrow formation and endochondral ossification."

Sox9 is good for height growth in all stages aside from the hypertrophic stage.

"Differentiation of proliferating cells into prehypertrophic and hypertrophic chondrocytes is marked by an up to ten-fold increase in cell volume and development of a granular cell surface with numerous microvilli, which release matrix vesicles required for cartilage mineralization. This step is associated with substantial matrix remodelling: the hyaline cartilage matrix, comprising aggrecan, type II, VI and XI collagen made by resting and proliferating chondrocytes, is substituted by a calcifiying matrix deposited by hypertrophic chondrocytes that produce type X collagen and alkaline phosphatase"

"Maturation of chondrocytes in the lower hypertrophic zone to terminally differentiated chondrocytes is marked by upregulation of osteopontin, Vegfa, and Mmp13"

"RUNX2 plays a key role in endochondral ossification as it promotes not only expression of type X collagen[and MMP13] and maturation to hypertrophic chondrocytes, but also induces Vegfa in hypertrophic chondrocytes"

"Sox9 is expressed in all chondroprogenitor cells; it is essential for the formation of cartilage blastema of the limb mesenchyme, for proliferation and differentiation of chondrocytes in the foetal growth plate and for regulation of cartilage-specific genes including Col2a1, Col9a1, Col11a1, aggrecan and others. In the foetal and juvenile growth plates, Sox9 is expressed in resting and proliferating chondrocytes, with a maximum of expression in prehypertropic chondrocytes, but disappears completely from the hypertrophic zone"

"misexpression of Sox9 in hypertrophic chondrocytes significantly impaired postnatal skeletal growth and bone length[20% length decrease]. "

SOX9 transduction of a human chondrocytic cell line identifies novel genes regulated in primary human chondrocytes and in osteoarthritis.

"To identify novel genes regulated by SOX9 we investigated changes in gene expression by microarray analysis following retroviral transduction with SOX9 of a human chondrocytic cell line (SW1353). From the results the expression of a group of genes (SRPX, S100A1, APOD, RGC32, CRTL1, MYBPH, CRLF1 and SPINT1) was evaluated further in human articular chondrocytes (HACs). First, the same genes were investigated in primary cultures of HACs following SOX9 transduction, and four were found to be similarly regulated (SRPX, APOD, CRTL1 and S100A1). Second, during dedifferentiation of HACs by passage in monolayer cell culture, during which the expression of SOX9 progressively decreased, four of the genes (S100A1, RGC32, CRTL1 and SPINT1) also decreased in their expression. Third, in samples of osteoarthritic (OA) cartilage, which had decreased SOX9 expression compared with age-matched controls, there was decreased expression of SRPX, APOD, RGC32, CRTL1 and SPINT1. The results showed that a group of genes identified as being upregulated by SOX9 in the initial SW1353 screen were also regulated in expression in healthy and OA cartilage. Other genes initially identified were differently expressed in isolated OA chondrocytes and their expression was unrelated to changes in SOX9. The results thus identified some genes whose expression appeared to be linked to SOX9 expression in isolated chondrocytes and were also altered during cartilage degeneration in osteoarthritis."

"[SW1353] cells showed increased gene expression of SOX6 (up to 14-fold) and COL2A1 (up to 13-fold), but aggrecan expression was low and was unchanged by SOX9. The SW1353 cells expressed high levels of COL1A1 and this was reduced 6-fold by SOX9 transduction."

Genes upregulated by Sox9 also upregulated by LSJL:

Apod

Crlf1

Genes downregulated:

Spint1

Sox9 directly promotes Bapx1 gene expression to repress Runx2 in chondrocytes.

Bapx1 is also known as Nkx-3.2

"The suppression mechanism by Sox9 on late-stage chondrogenesis partially results from the inhibition of Runt-related transcription factor 2 (Runx2), the main activator of hypertrophic chondrocyte differentiation.  The transcriptional repressor vertebrate homolog of Drosophila bagpipe (Bapx1) was found to be a direct target of Sox9 for repression of Runx2 expression in chondrocytes.  Sox9 physically bound to [a] region of the Bapx1 promoter. Consistent with the notion that Bapx1 and Sox9 act as negative regulators of chondrocyte hypertrophy by regulating Runx2 expression, transient knockdown of Sox9 or Bapx1 expression by shRNA in chondrocytes increased Runx2 expression, as well as expression of the late chondrogenesis marker, Col10a1. Furthermore, while over-expression of Sox9 decreased Runx2 and Col10a1 expressions, simultaneous transient knockdown of Bapx1 diminished that Sox9 over-expressing effect. "

"Paired box gene 1 (Pax1){up}, Pax9, and mesenchyme homeobox 1 (Meox1) proteins regulate Bapx1 expression by directly activating its promoter"

"Pax1/Pax9 and Meox1 expression is maintained in chondrogenic mesenchymal cells until they reach the pre-chondrocyte stage"

"Pax1/9 and Meox1 may regulate initial and/or early induction of Bapx1, and Sox9 contributes to maintain Bapx1 expression during subsequent chondrogenic differentiation. "

"S100A1 and S100B were direct Sox9 targets, and that they suppress hypertrophic chondrocyte differentiation"

Sox9 modulates cell survival and adipogenic differentiation of multipotent adult rat mesenchymal stem cells.

"Sox9 activity level affect the expression of the key transcription factor in adipogenic differentiation, C/EBPß, and moreover, cyclin D1 mediated, the expression of osteogenic marker osteocalcin in undifferentiated adult bone marrow derived rat mesenchymal stem cells (rMSC).  Introducing a stable Sox9 knockdown in undifferentiated rMSC resulted in a marked decrease in proliferation rate and an increase in apoptotic activity. This was linked to a profound upregulation of p21 and cyclin D1 gene and protein expression accompanied with an induction of caspase 3/7 activity and an inhibition of Bcl-2.   Sox9 silencing provoked a delayed S-phase progression and an increased nuclear localization of p21. The protein stability of cyclin D1 was induced in the absence of Sox9 presumably as a function of altered p38 signalling.In addition, the major transcription factor for adipogenic differentiation, C/EBPß, was repressed after silencing Sox9. The nearly complete absence of C/EBPß protein due to increased destabilization of the C/EBPß mRNA and the impact on osteocalcin gene expression and protein synthesis, suggests that a delicate balance of Sox9 level is not only imperative for proper chondrogenic differentiation of progenitor cells, but also affects the adipogenic and most likely osteogenic differentiation pathways of MSC."

"In a rat chondrocytic cell line (CFK2), overexpression of Sox9 induces alteration of cell cycle progression. Stable Sox9 transfected cells accumulate an increased proportion of the cells in the G0/G1 and S-phase of the cell cycle with a reduced number of cells in the G2/M phase"

"As a consequence of using a specific inhibitor for p38 (SB203580), Sox9 expression was repressed in rMSC during in vitro chondrogenesis"

"On average, after knockdown Sox9 specific mRNA copies were reduced 10-fold compared to control cells. Overexpression of Sox9 resulted in an average 100-fold increase in gene expression compared to controls"

"The number of control cells increased 8-fold during 7 days (doubling time2.3days), -SOX9 cells increased 4-fold (doubling time 3.5days) whereas +SOX9 cells showed a 15-fold (doubling time 1.8days) increase in cell number."<-So Sox9 transgenic had the greatest number of cells and the shortest doubling time.

"10% increased S-phase population after overexpressing Sox9 (+SOX9) compared to control cells"

"Overexpression of Sox9 resulted in decreased expression of p21 and cyclin D1 on mRNA and
on protein level"

"reduced activity of Sox9 induces apoptosis in rMSC (increased caspase3/7activity) and reduces expression of the pro-survival protein Bcl-2 while overexpression of Sox9 induces expression of Bcl-2 and reduces activity of caspase3/7 resulting in less apoptosis. In this line, it was reported that inhibition of Bcl-2 resulted in reduction of Sox9 gene expression and consequently in Sox9-dependent chondrogenic gene expression"