Ectopic chondro-ossification with LSJL

Here's a reply to an email by Dan Huey: Co-Author of the excellent paper "Unlike Bone, Cartilage Regeneration Remains Elusive".

"While MSCs derived from bone marrow have shown the ability to differentiate down the chondrocytic lineage both in vitro and in vivo the efficiency and completeness of this process hinders the formation of stable hyaline tissue {although we don't care about the stableness of this as we want transient endochondral ossification of cartilage to make us taller}. Ectopic differentiation of MSCs into chondrocytes does not occur in the marrow cavity due to a lack of the appropriate signals (both mechanical and biochemical) {and we try to induce the appropriate mechanical signals via LSJL}. These MSCs are tuned by their environment to contribute to the natural bone remodeling process {we need to alter that tuning}. However, even when these cells are introduced into a cartilage defect via microfracture, complete chondrocyte differentiation does not occur, as evidenced by the formation of fibrous tissue{Likely due to the levels of shear strain and fluid pressure not being in the equilibrium range}. For these cell to undergo complete chondrogenesis the proper combination of mechanical and biochemical cues must be provided. As the clot formed in microfracture is quite soft the cells within the clot will not receive the appropriate level of mechanical forces for chondrogenesis {Mechanical forces can be altered by LSJL}. With regards to the biochemical signals, a cartilage stimulating growth factor analagous to BMP's effect for osteogenic differenetiation has not been identified {TGF-Beta may qualify as such a signal}. With respect to the term microfracture, in cartilage and bone it means two different things. For cartilage microfracture is a surgical procedure that involves creating holes in the bone underlying a cartilage defect to allow stem cells to enact a healing response. With regards to bone, microfractures are the very small breaks in bone that occur during strenuous activity.  does not occur in cartilage as it does in bone. In bone, microfracture occurs during strenuous activity and heals."

His bias towards microfracture induced healing of cartilage versus our attempts to induce ectopic growth plates in cartilage can be seen.  However, his statements of belief that chondrogenic differentiation can occur in vivo in MSCs if the proper mechanical and biochemical stimulation can occur, provides weight towards LSJL theory if LSJL does in fact provide those signals.  Some of these signals can be observed by the upregulation of Cyr61, Sox9, and FGF2 in the gene expression study and signs of mesenchymal condensation in LSJL histology slides.

Another important insight from the statements is that the microfracture clot is too soft to induce chondrogenesis.  Maybe LSJL can be used as a stimulus to encourage cartilage development in existing microfractures and co-creation of microfractures along with doing LSJL will enhance LSJL results.  Sprinting could be one such mechanism of inducing microfractures.

LSJL upregulates Biglycan 2.057 fold.  Ectopic ossification is a form of hetertropic ossification that is not invasive meaning it is (usually) within the bone like a growth plate.  Our goal is ectopic chondro-ossification within the epiphyseal bone marrow.

Ectopic chondro-ossification and erroneous extracellular matrix deposition in a tendon window injury model.

"The acquisition of chondro-osteogenic phenotypes and erroneous matrix deposition may account for poor tissue quality after acute tendon injury. We investigated the presence of chondrocyte phenotype, ossification, and the changes in the expression of major collagens and proteoglycans in the window wound in a rat patellar tendon window injury model using histology, von Kossa staining and immunohistochemistry of Sox 9, major collagens, and proteoglycans. The repair tissue did not restore to normal after acute injury. Ectopic chondrogenesis was observed in 33% of samples inside wound at week 4 while ectopic ossification surrounded by chondrocyte-like cells were observed in the window wound in 50% of samples at week 12. There was sustained expression of biglycan and reduced expression of aggrecan and decorin in the tendon matrix in the repair tissue. The erroneous deposition of extracellular matrix and ectopic chondro-ossification in the repair tissue, both might influence each other, might account for the poor tissue quality after acute injury. Higher expression of biglycan and aggrecan were observed in the ectopic chondro-ossification sites in the repair tissue, suggesting that they might have roles in ectopic chondro-osteogenesis{expression of both biglycan and aggrecan was elevated during LSJL which provides further evidence that LSJL can induce ectopic chondrogenesis}."

"Knock-down of biglycan in a mouse model resulted in low bone mass and biglycan was essential for bone formation while the knock-down of decorin in a mouse model resulted in normal bone mass."

"We observed earlier expression of Sox 9 and collagen type II in healing tendon fibroblasts and this preceded their expression in the chondrocyte-like cells and ossified area. "<-So in LSJL the expression of Sox9 and COL2A1 might have proceeded the actual presence of exogenous growth plates.

"There was rapid upregulation of expression of proteoglycans during chondrogenic differentiation of bone marrow mesenchymal stem cells (BMSCs). The expression of collagen types II and X occurred only at the late stage of the process, suggesting the importance of proteoglycans in modulating stem cell differentiation"<-Col10A1 was upregulated by LSJL which would seem to be a knock on LSJL.

Our hypothesis was that of the four samples only the first sample was from a growth plate region and the rest were from diaphyseal regions and the expression of Agc1, Sox9, and COL2A1 were consistent with this. MATN3 was not with 1,3,4 showing an increase while 2 decreased.  The expression of Pax 1 is consistent with the hypothesis of 1 being from a growth plate as 2,3, and 4 increased in expression whereas 1 decreased.  COL10A1 increased in 2,3,4 where it decreased in 1.

What we'd expect is for COL10A1 to increase in expression in 2,3,4 but decrease in 1 as 1 is the hypothetical growth plate and COL10A1 is typically expressed in mature chondrocytes.  However, 49 hours may have been enough time for chondrocytes to reach enough maturity to express more COL10A1.  Biglycan increased in all four samples.

"The differentiation of marrow stromal cells [depends] on the expression of biglycan"

Direct conversion of tenocytes into chondrocytes by Sox9.

"Sox9 is a high-mobility group box-containing transcription factor that functions as a key regulator of chondrogenesis. Sox9 mediates the direct conversion of tenocytes to chondrocytes through an intermediate state in which both differentiation programs are active. Sox9 is abundantly expressed in cartilage but is undetectable in limb tendons that express Scleraxis (Scx) and Tenomodulin (Tnmd){LSJL upregulates both Scx and Tnmd; Tnmd by over 6-fold; the increase was universal across all four samples}, tendon-specific early and late molecular markers, respectively. Upon forced expression of Sox9 in the chick forelimb, ectopic cartilage formation is preferentially observed in fibrous tissues including the tendons, ligaments, perichondrium/periosteum, dermis, and muscle connective tissues. Tnmd expression in tenocytes isolated from leg tendons was markedly upregulated by forced expression of basic helix-loop-helix (b-HLH) activators including Scx, Paraxis, Twist1 and Twist2. In contrast, the overexpression of Sox9 in monolayer tenocytes resulted in the downregulation of Tnmd and Scx expressions during passaging in culture, and the induction of cartilage molecular markers such as type II collagen (Col2a1) and Chondromodulin-I (ChM-I). This Sox9-driven switching from a tenocytic to a chondrocytic gene expression profile was associated with a dramatic change from a spindle to a polygonal cellular morphology. The extracellular accumulation of cartilage-characteristic proteoglycans [occurred].  Tenocytes have a strong potential for conversion into chondrocytes through the activities of Sox9 both in vitro and in vivo."

Maybe tenocytes are an intermediary which then undergo chondrogenic differentiation within the epiphyseal bone marrow.

"Progenitor cells for the tendons, ligaments, cartilage, and bone are all derived from the same origins including the sclerotome, lateral plate mesoderm, and neural crest"

"Scx was also detected in cultured chondrocytes expressing ChM-I and Sox9 at a high level"

Conversion of human bone marrow-derived mesenchymal stem cells into tendon progenitor cells by ectopic expression of scleraxis.

"During embryonic development, the tendon-specific cells descend from a sub-set of mesenchymal progenitors condensed in the syndetome, a dorsolateral domain of the sclerotome. These cells are defined by the expression of the transcription factor scleraxis (Scx), which regulates tendon formation and several other characteristic genes, such as collagen type I, decorin, fibromodulin, and tenomodulin (Tnmd).  We investigated whether the expression of Scx can lead to the direct commitment of mesenchymal stem cells (MSCs) into tendon progenitors. First, MSC derived from human bone marrow (hMSC) were lentivirally transduced with FLAG-Scx cDNA to establish 2 clonal cell lines, hMSC-Scx and hMSC-Mock. Subsequent to Scx transduction, hMSC underwent cell morphology change and had significantly reduced proliferation and clonogenicity. Collagen type I and several T/L-related proteoglycans were upregulated in hMSC-Scx cells. When stimulated toward 3 different mesenchymal lineages, hMSC-Scx cells failed to differentiate into chondrocytes and osteoblasts, whereas adipogenic differentiation still occurred. Lastly, we detected a remarkable upregulation of the T/L differentiation gene Tnmd in hMSC-Scx. Scx delivery results in the direct programming of hMSC into tendon progenitors and that the newly generated hMSC-Scx cell line can be a powerful and useful tool in T/L research."

"On Scx ectopic expression in hMSC, we observed almost a complete loss of Sox9 expression."<-Which did not happen in LSJL.

"Scx and E47 can directly cooperate with Sox9 and regulate its transcription"

Correlation of COL10A1 induction during chondrogenesis of mesenchymal stem cells with demethylation of two CpG sites in the COL10A1 promoter.

"Human articular chondrocytes do not express COL10A1 and do not undergo hypertrophy except in close vicinity to subchondral bone. Chondrocytes produced in vitro from mesenchymal stem cells (MSCs) show premature COL10A1 expression{this is what we see under LSJL: premature COL10A1 expression} and cannot form stable ectopic cartilage transplants, which indicates that they may be phenotypically unstable and not suitable for treatment of articular cartilage lesions. CpG methylation established during natural development may play a role in suppression of COL10A1 expression and hypertrophy in human articular chondrocytes. This study was undertaken to compare gene methylation patterns and expression of COL10A1 and COL2A1 in chondrocyte and MSC populations, in order to determine whether failed genomic methylation patterns correlate with an unstable chondrocyte phenotype after chondrogenesis of MSCs.
COL10A1 and COL2A1 regulatory gene regions were computationally searched for CpG-rich regions. CpG methylation of genomic DNA from human articular chondrocytes, MSCs, and MSC-derived chondrocytes was analyzed by Combined Bisulfite Restriction Analysis and by sequencing of polymerase chain reaction fragments amplified from bisulfite-treated genomic DNA.
The CpG island around the transcription start site of COL2A1 was unmethylated in all cell groups independent of COL2A1 expression, while 9 tested CpG sites in the sparse CpG promoter of COL10A1 were consistently methylated in human articular chondrocytes. Induction of COL10A1 expression during chondrogenesis of MSCs correlated with demethylation of 2 CpG sites in the COL10A1 promoter.
Methylation-based COL10A1 gene silencing is established in cartilage tissue and human articular chondrocytes. Altered methylation levels at 2 CpG sites of COL10A1 in MSCs and their demethylation during chondrogenesis may facilitate induction of COL10A1 as observed during in vitro chondrogenesis of MSCs{there are a couple of genes upregulated by LSJL that are associated with methylation}."

"one of the [demethylated] CpG sites [on COL10A1] is part of a Pax8 and a N-myc transcription factor DNA binding motif. The analysis of the methylation status of the chondrocyte-specific gene chondromodulin has shown that methylation of one CpG site coinciding with a binding site of Sp-1 and Sp-3 correlated with chondromodulin gene expression and that methylation affected binding of Sp-3 to this site"

Misexpression of Sox9 in mouse limb bud mesenchyme induces polydactyly and rescues hypodactyly mice.

"We first generated mutant mice in which Sox9 was misexpressed in the limb bud mesenchyme. The mutant mouse embryos exhibited polydactyly in limb buds in association with ectopic expression of Sox5 and Sox6 although markers for the different axes of limb bud development showed a normal pattern of expression. Misexpression of Sox9 stimulated cell proliferation in limb bud mesenchyme, suggesting that Sox9 has a role in recruiting mesenchymal cells to mesenchymal condensation. Second, despite the facts that misexpression of Sonic hedgehog (Shh) induces polydactyly in a number of mutant mice and Shh-null mutants have severely defective cartilage elements in limb buds, misexpression of Sox9 did not restore limb bud phenotypes in Shh-null mutants. Rather, there was no expression of Sox9 in digit I of Hoxa13Hd mutant embryos, and Sox9 partially rescued hypodactyly in Hoxa13Hd mutant embryos. Sox9 induces ectopic chondrogenesis in mesenchymal cells and its expression may be regulated by Hox genes{LSJL alters the expression of several Hox genes} during limb bud development."

"Expression of Hoxd genes during limb bud development consists first in uniform activation of Hoxd9 and Hoxd10 {upregulated 2.612 fold by LSJL} and subsequently Hoxd11, Hoxd12, and Hoxd13, which are activated sequentially at the posterior border of the limb bud. Hoxa gene activation proceeds from Hoxa9 and Hoxa10 to Hoxa11 through Hoxa13. Activation of Hoxa13 occurs at the posterior and distal tip of the limb bud after Hoxd13 activation"

Arginase II was downregulated 0.36 fold by LSJL and it's a marker of chondrocyte differentiation but apparently lack of ArgII does not inhibit chondrogenesis.

Identification and characterization of arginase II as a chondrocyte phenotype-specific gene.

"Activation of extracellular signal-regulated protein kinase-1 and -2 (ERK1/2) causes chondrocyte dedifferentiation. We identified genes involved in the ERK1/2 regulation of chondrocyte dedifferentiation. Several genes were identified by subtractive hybridization, and, of these, arginase II was selected for further functional characterization. Similar to the pattern of type II collagen expression, which is a hallmark of chondrocyte differentiation, arginase II expression was increased during chondrogenesis of mesenchymal cells. The high expression level of arginase II was decreased during dedifferentiation of chondrocytes, whereas its expression was restored during redifferentiation of the dedifferentiated chondrocytes. Inhibition of ERK1/2 signaling in chondrocytes enhanced type II collagen expression with a concomitant increase in expression and activity of arginase II. However, ectopic expression of arginase II or inhibition of its activity did not affect chondrocyte differentiation {in LSJL we had downregulation}."

"both down-regulation of ERK1/2 and induction of p38 kinase activities are required for chondrogenic differentiation of mesenchymal cells"

"inhibition of ERK1/2 with PD98059 caused a significant increase in type II collagen expression. ERK1/2 inhibition caused increased expression and activity of arginase II"

"increased expression and activity of arginase II in differentiated chondrocytes ensures the availability of proline for the synthesis of a large amount of collagen and therefore contributes to the maintenance of the differentiated phenotypes of articular chondrocytes."<-there were several proteins related to proline that had expression altered due to LSJL.

Enchondromatosis revisited: New classification with molecular basis.

""enchondromatoses" are skeletal disorders defined by the presence of ectopic cartilaginous tissue within bone tissue {What we're trying to within LSJL in the epiphyseal bone marrow}. The clinical and radiographic features of the different enchondromatoses are distinct, and grouping them does not reflect a common pathogenesis but simply a similar radiographic appearance. [Different] molecular and cellular bases confirm the heterogeneous nature of the different enchondromatoses. Some, like Ollier disease, Maffucci disease, metaphyseal chondromatosis with hydroxyglutaric aciduria, and metachondromatosis are produced by a dysregulation of chondrocyte proliferation, while others (such as spondyloenchondrodysplasia or dysspondyloenchondromatosis) are caused by defects in structure or metabolism of cartilage or bone matrix. In other forms (e.g., the dominantly inherited genochondromatoses), the basic defect remains to be determined. The classification was based on the radiographic appearance, the anatomic sites involved, and the mode of inheritance. The new classification proposed here integrates the molecular genetic advances and delineates phenotypic families based on the molecular defects."

"An island, or nodule, of cartilagineous tissue enclosed in bone tissue is called an “enchondroma.” On conventional radiographs, enchondromas appear as radiolucent lesions in more radiodense osseous tissue. Enchondromas may arise from the abnormal proliferation of chondrocytes, as seen in Ollier disease (OD), or from misdirected chondrocyte growth such as the exophytic chondromas seen in metachondromatosis. Enchondromas may also be the result of failure of reabsorption of cartilage at sites of enchondral ossification {enchondrodysplasia, such as that caused by the deficiency of acid phosphatase in spondyloenchondrodysplasia (SPENCD)}. "<-So do people with encohondrodysplasia grow taller?

"[In some enchondromas] a dominant effect [occurs] that trans-specifies the enzyme protein and confers the ability to convert isocitrate to d-2-hydroxyglutarate rather than to alpha-ketoglutarate. The hyperproduction of d-2-hydroxyglutarate combined with the depletion of alpha-ketoglutarate has several consequences, including an activation of the HIF-1alpha pathway and a change in the methylation pattern of several genes. Deregulation of the HIF-1alpha pathway is a plausible mechanism [for formation of chondroma] since it is essential for chondrocytes in the growth plate"

Solitary epiphyseal enchondromas.

"Typically, multiple small cartilaginous nodules are formed; these nodules tend to coalesce and to become interspersed with areas of normal marrow fat. Furthermore, these islands often develop enchondral ossification, which is the basis for the radiographic "arcs and rings" pattern."

"A mutation in the type-I PTHrP receptor constitutively activates Ihh signaling in vitro and has been demonstrated to cause enchondroma development in transgenic mice"  " It is probable that a similar disturbance in humans results in the failure of physeal chondrocyte apoptosis and in continued proliferation as the mutant chondrocytes migrate from the growth plate with continued physeal growth."

Unilateral Mosaic Cutaneous Vascular Lesions, Enchondroma, Multiple Soft Tissue Chondromas and Congenital Fibrosarcoma— A Variant of Maffucci Syndrome?

Enchondromas that can cause overgrowth: Klippel-Trenaunay syndrome and Proteus Syndrome.

Regeneration of Growth Plate Cartilage Induced in the Neonatal Rat Hindlimb by Reamputation

"Following primary hindlimb amputations dividing the lower femur or the central tibiofibula, the neonatal[new born] rat innately regenerates the distal growth plate(s) with a frequency of about 20-30%. One or two reamputation procedures were performed in an effort to increase the frequency of physeal regeneration, noting that such procedures, and related forms of tissue stimulation, have been repeatedly shown to induce regenerative growth at limb amputation sites of some amphibians that display little innate regenerative capacity{amputation is the removal of a limb}. The present reamputation sequences divided the skeletal stump through the cartilaginous mass arising at its distal end. Following first reamputation an approximate three fold increase in the frequency of growth plate cartilage regeneration was observed at transfemoral and transtibiofibular sites. Only after second reamputation, however, did tibiofibular physeal cartlage regeneration equal in frequency that observed after first reamputation through the lower femur. Ectopic growth plate cell architecture was identified in cartilaginous extensions arising from the side of the distal femoral shaft, and also within the regrown secondary cartilage body, which unites the lower tibia and fibula in the shank of the rat. Moreover, among 3 of 11 femoral amputees that had sustained reamputations, regrowth of the distal femoral condylar mass and profile were achieved to varying degrees. A regimen of reamputation, known to induce regenerative growth in the amphibian limb, also induces skeletal regeneration in the mammalian limb, and leads to the appearance of ectopic growth plate cell architecture at adjacent sites."

"growth plate regeneration is a relatively uncommon occurrence, evident histologically among only 20-30% of hindlimb amputees, where new cartilage is often laid down as an incomplete hemiphysis restricted to one side of the shaft of the femur, or is applied to the cut surface of one, but not both, bones of the tibiofibula. Furthermore, innate physeal regrowth does not occur following distal humeral amputations"

"In the adult mouse that exposure of digital amputation sites to a repeated regimen of skin removal followed by surgical disruption of subdermal soft tissues could impart to the stump the appearance of an early amphibian limb regenerate"

"In most instances closing the amputation wound, either by suturing or with a skin graft, prevents the regeneration of the limb"

"Human finger tips regrow readily after covering the amputation surface with a full-thickness skin graft"

Podoplanin a marker of ectopic chondrogenesis is upregulated by LSJL 3.5 fold.

Expression of podoplanin in human bone and bone tumors: New marker of osteogenic and chondrogenic bone tumors.

"Podoplanin mRNA was expressed at a high level in bone marrow tissue and cartilage, and was upregulated with differentiation to osteoblasts in bone marrow cells. Strong podoplanin expression was seen in osteocytes, chondrocytes, and osteoblasts on immunohistochemistry. Podoplanin mRNA was expressed at a high level in several osteosarcoma and chondrosarcoma cell lines, whereas podoplanin was expressed at a low level in a Ewing's/primitive neuroectodermal tumor cell line. In the clinical samples, osteosarcomas (22/26) expressed podoplanin at various levels. In small cell osteosarcomas (2/2), podoplanin was expressed strongly, although the tissue samples included few diagnostic osteoids. Chondrosarcomas (10/10) expressed podoplanin strongly, and chondroblastomas (5/5) expressed podoplanin moderately, while podoplanin was absent or expressed at low levels in Ewing's sarcomas (0/5), chordomas (0/6) and giant cell tumors of bone (1/7)."

However, normal osteoblasts and osteocytes do express podoplanin.

Podoplanin is expressed by a sub-population of human foetal rib and knee joint rudiment chondrocytes.

"Podoplanin was immunolocalised in first trimester human foetal rib and knee joint rudiments to a sub-population of chondrocytes deep in the rib rudiments, tibial and femoral growth plates and cells associated with the cartilage canals of the foetal knee joint rudiments. Lymphatic vessels in the loose stromal tissues surrounding the developing rudiments were also demonstrated on the same histology slides using antipodoplanin (MAb D2-40) and anti-LYVE-1 and differentiated from CD-31 positive blood vessels confirming the discriminative capability of the antibody preparations used. The D2-40 positive rib and knee rudiment chondrocytes were not stained with antibodies to LYVE-1, CD-31 or CD-34 however perlecan was a prominent pericellular proteoglycan around these cells confirming their chondrogenic phenotype. Discernable differences were evident between the surface and deep rudiment chondrocytes in terms of their antigen reactivities detected with MAb D2-40 or antiperlecan antibodies. Binding of the cytoplasmic tail of PDPN to the ERM proteins ezrin, radixin and moeisin may result in changes in cytoskeletal organisation which alter the phenotype of this central population of rudiment cells. This may contribute to morphological changes in the rudiment cartilages which lead to establishment of the primary ossification centres and is consistent with their roles as transient developmental scaffolds during tissue development."

PDPN may be a sign of formation of new primary ossification centers.

"PDPN is an early osteoblast marker protein"

Arterial injury promotes medial chondrogenesis in Sm22 knockout mice.

"Expression of SM22 (also known as SM22alpha and transgelin){LSJL upregulates Transgelin}, a vascular smooth muscle cells (VSMCs) marker, is down-regulated in arterial diseases involving medial osteochondrogenesis. We investigated the effect of SM22 deficiency in a mouse artery injury model to determine the role of SM22 in arterial chondrogenesis.

Sm22 knockout (Sm22(-/-)) mice developed prominent medial chondrogenesis 2 weeks after carotid denudation as evidenced by the enhanced expression of chondrogenic markers including type II collagen, aggrecan, osteopontin, bone morphogenetic protein 2, and SRY-box containing gene 9 (SOX9){all of these are upregulated by LSJL}. This was concomitant with suppression of VSMC key transcription factor myocardin and of VSMC markers such as SM α-actin and myosin heavy chain. The conversion tendency from myogenesis to chondrogenesis was also observed in primary Sm22(-/-) VSMCs and in a VSMC line after Sm22 knockdown: SM22 deficiency altered VSMC morphology with compromised stress fibre formation and increased actin dynamics. Meanwhile, the expression level of Sox9 mRNA was up-regulated while the mRNA levels of myocardin and VSMC markers were down-regulated, indicating a pro-chondrogenic transcriptional switch in SM22-deficient VSMCs. Furthermore, the increased expression of SOX9 was mediated by enhanced reactive oxygen species production and nuclear factor-κB pathway activation."

Maybe the upregulation of transgelin plays a role in reduced adaptation to LSJL stimulus over time.

Acta2 which is downregulated in Transgelin knockout was upregulated in LSJL.

"the up-regulation of Sox9 might be initiated by ROS increase after Sm22 knockdown in PAC1 cells. Indeed, we recently showed that disruption of SM22 expression by Sm22 knockdown in PAC1 cells boosted ROS production."

"NF-κB{which participates in Sox9 expression and chondrogenesis} pathway is activated after Sm22 knockdown in PAC1 cells and is associated with boosted ROS production."

"After inhibition of the NF-κB pathway during Sm22 knockdown in PAC1 cells using NF-κB inhibitors, Bay-11–7082 or IMD-0354, transcriptional activation of Sox9 was significantly reduced"

"VSMCs derive from mesenchymal cells and disruption of actin cytoskeleton with increased actin dynamics in mesenchymal cells leads to chondrogenesis."

Chondrocytes isolated from tibial dyschondroplasia lesions and articular cartilage revert to a growth plate-like phenotype when cultured in vitro.

"We had analyzed the electrolytes and amino acid levels in the extracellular fluid of avian growth plate chondrocytes. Using these data, we constructed a culture medium (DATP5) in which growth plate cells essentially recapitulate their normal behavior in vivo. Here, we used DATP5 to examine the behavior of chondrocytes isolated from lesions of tibial dyschondroplasia (TD). Once isolated from lesion and grown in this supportive medium, dysplasic chondrocytes behaved essentially like normal growth plate cells. The cause of TD is local factors operating in vivo to prevent these cells from developing normally. With respect to articular chondrocytes, our data indicate that they more closely retain normal protein and proteoglycan synthesis when grown in serum-free media. These cells readily induced mineral formation in vitro, both in the presence and absence of serum. However, in serum-containing media, mineralization was significantly enhanced when the cells were exposed to retinoic acid (RA) or osteogenic protein-1 (OP-1). autocrine factors [are present that are] produced by articular chondrocytes in vivo that prevent mineralization and preserve matrix integrity. The lack of inhibitory factors and the presence of supporting factors are likely reasons for the induction of mineralization by articular chondrocytes in vitro."

"While mineral deposition was evident in both normal and TD cells by day 24 of culture, levels of Ca2+ and Pi in TD cultures were only about half that in normal growth plate cultures. However, by day 35, mineral deposition increased significantly and both normal and TD cultures had similar levels of Ca2+ and Pi in the matrix/cell layer."

"By day 6, both normal and TD chondrocyte populations had increased in number (2,720 vs. 1,530 large cells/mm2, respectively), maintaining a rounded shape. By day 12, both normal and TD cultures had attained confluence, and due to cell–cell interaction, assumed a polygonal morphology. By day 15, while cells from normal tissue remained confluent and polygonal in shape (i.e., attached to the surface of the culture dish), the TD chondrocytes became rounded and partially detached from the culture surface. But by day 17, both normal and TD cells assumed rounded morphology and were densely distributed in the cultures. In some chondrocytes from normal tissue, numerous small vesicular structures about 0.5 µm in diameter were present on the cell surface. The first visible (opaque) mineral deposits were seen on day 21 in both normal and TD cultures; by day 27 and 35 mineral deposition had expanded significantly in both normal and TD cultures."

"In TD, there is no lack of Ca2+ or Pi in the circulating fluid, yet there is no calcification."

"With regard to articular cartilage, it is important to realize that less than 1% of the tissue is actually occupied by cells"

"Articular chondrocytes expressed little ALP activity when grown in serum-containing DATP5 medium, but showed substantial AP activity when grown in serum-free HL-1 medium. Since mineralization of the cultured articular chondrocytes was supported by both DATP5 and HL-1 media, this indicates that mineral formation in articular cartilage must not be directly related to ALP activity. On the other hand, the effects just described for articular chondrocytes are opposite those seen with growth plate chondrocytes. Growth plate cells, when grown in DATP5, express high levels of ALP activity, while in serum-free HL-1, the levels of ALP are much lower "

"It is noteworthy that RA (50 nM) was inhibitory to proteoglycan synthesis by articular chondrocytes under all culture conditions, reducing levels to about 50% of the control from day 28 onward. This finding is similar to that previously seen with cultured growth plate and sternum chondrocytes. Since RA is carried in blood plasma bound to proteins that cannot readily diffuse through the dense articular cartilage matrix, it is probable that the levels of RA in articular cartilage are low. Bioassays of retinoids in 8.5–10-day chick embryonic cartilage (metaphyseal–diaphyseal portion of the humerus) reveal levels of ∼3 nM in the perichondrial region, with lower levels in the core of the cartilaginous anlagen. Since RA is required for differentiation of chondrocytes to the hypertrophic state, exclusion of RA from articular chondrocytes may well be essential for maintenance of a normal cartilage matrix."

So retinoic acid is key to inducing hypertrophy in ectopic cartilage structures.

Post growth plate fusion the marrow is a vascular tissue and arteries are also a vascular tissue.  Chondrocyte is an avascular tissue so in what conditions can chondrocyte differentiation occur in vascular tissues?

Arterial Calcification Is Driven by RAGE in Enpp1–/– Mice

"Ectopic osteochondral differentiation, driven by ENPP1-catalyzed generation of the chondrogenesis and calcification inhibitor inorganic pyrophosphate (PPi), promotes generalized arterial calcification of infancy. The multiligand receptor for advanced glycation end-products (RAGE), which promotes atherosclerosis and diabetic cardiovascular and renal complications, also mediates chondrocyte differentiation in response to RAGE ligand calgranulins such as S100A11. Here, we tested RAGE involvement in ENPP1 deficiency-associated arterial calcification.

Because ectopic artery calcification in Enpp1–/– mice is Pi-dependent and mediated by PPi deficiency, in vitro studies on effects of S100A11 and RAGE on mouse aortic explants were conducted using exogenous Pi, as well as alkaline phosphatase to hydrolyze ambient PPi.

S100A11 induced cartilage-specific collagen IX/XI expression and calcification dependent on RAGE in mouse aortic explants that was inhibited by the endogenous RAGE signaling inhibitor soluble RAGE (sRAGE). Enpp1–/– aortic explants demonstrated decreased Pi-stimulated release of sRAGE, and increased calcification and type IX/XI collagen expression that were suppressed by exogenous sRAGE and by Rage knockout. Last, Rage knockout suppressed spontaneous aortic calcification in situ in Enpp1–/– mice.

Cultured Enpp1–/– aortic explants have decreased Pi-stimulated release of sRAGE, and RAGE promotes ectopic chondrogenic differentiation and arterial calcification in Enpp1–/– mice."

"Arterial calcification appears to be actively initiated and organized by osteochondral differentiation of intra-arterial stem cells, pericytes, SMCs, and adventitial myofibroblasts. Deficiency of physiologic inhibitors of chondro-osseous differentiation such as the BMP-2 inhibitor matrix GLA protein (MGP) can be compounded by lesion excess of BMP-2, and other inducers of chondro-osseous commitment and maturation"

"artery calcification in Enpp1–/– mice is associated with intra-arterial chondrogenic differentiation, and cultured ENPP1-deficient artery SMCs undergo accelerated chondrogenic trans-differentiation upon provision of a source of Pi"

Chondro/osteoblastic and cardiovascular gene modulation in human artery smooth muscle cells that calcify in the presence of phosphate and calcitriol or paricalcitol.

"Vitamin D sterol administration, a traditional treatment for secondary hyperparathyroidism, may increase serum calcium and phosphorus, and has been associated with increased vascular calcification (VC). In the presence of uremic concentrations of phosphorus, vitamin D sterols regulate gene expression associated with trans-differentiation of smooth muscle cells (SMCs) to a chondro/osteoblastic cell type. This study examined effects of vitamin D sterols on gene expression profiles associated with phosphate-enhanced human coronary artery SMC (CASMC) calcification. Cultured CASMCs were exposed to phosphate-containing differentiation medium (DM) with and without calcitriol, paricalcitol, or the calcimimetic R-568 (10(-11)-10(-7) M) for 7 days. Calcification of CASMCs, determined using colorimetry following acid extraction, was dose dependently increased (1.6- to 1.9-fold) by vitamin D sterols + DM. In contrast, R-568 did not increase calcification. Compared with DM, calcitriol (10(-8) M) + DM or paricalcitol (10(-8) M) + DM similarly and significantly regulated genes of various pathways including: metabolism, CYP24A1; mineralization, ENPP1; apoptosis, GIP3; osteo/chondrogenesis, OPG, TGFB2, Dkk1, BMP4, BMP6; cardiovascular, HGF, DSP1, TNC; cell cycle, MAPK13; and ion channels, SLC22A3 KCNK3. R-568 had no effect on CASMC gene expression. Thus, SMC calcification observed in response to vitamin D sterol + DM may be partially mediated through targeting mineralization, apoptotic, osteo/chondrocytic, and cardiovascular pathway genes, although some gene changes may protect against calcification."

"genes related to mineralization were altered in vitamin D sterol-treated CASMC resulting in a gene expression pattern indicative of a shift to a mineralizing osteoblast-like cell phenotype. Such a shift is represented by concurrent increases in pro-mineralization gene expression (e.g., ALPL, TGFB2, BMP4 and 6) and decreases in anti-mineralization gene expression (ENPP1) along with changes in IBSP, OPG and the chondrocyte genes, ANXA3, CILP, TNC, ITGA8, and cytokine IL-6."

Chondroinduction in vascular tissue can occur without injury:

Overexpression of transforming growth factor beta1 in arterial endothelium causes hyperplasia, apoptosis, and cartilaginous metaplasia.

"Uninjured rat arteries transduced with an adenoviral vector expressing an active form of transforming growth factor beta1 (TGF-beta1) developed a cellular and matrix-rich neointima, with cartilaginous metaplasia of the vascular media. Explant cultures of transduced arteries showed that secretion of active TGF-beta1 ceased by 4 weeks, the time of maximal intimal thickening. Between 4 and 8 weeks, the cartilaginous metaplasia resolved and the intimal lesions regressed almost completely, in large part because of massive apoptosis. Thus, locally expressed TGF-beta1 promotes intimal growth and appears to cause transdifferentiation of vascular smooth muscle cells into chondrocytes. Moreover, TGF-beta1 withdrawal is associated with regression of vascular lesions."

"Arteries [with positive expression of type II collagen] revealed rounded cells with a high nuclear/cytoplasmic ratio, surrounded by lacunae. A loose extracellular matrix was present, appearing more cartilaginous than vascular, with collagen fibers, abundant proteoglycans, and little elastin"

"The appearance of chondrocytes in the arterial wall was not caused by migration of cells from cartilage "

"TGF-β1 expression leads to increased focal vascular cell proliferation"

Unlike bone, cartilage regeneration remains elusive.

"bone marrow MSCs or resident chondroprogenitor cells [cannot] generate hyaline ECM"  Highlighting the importance of hyaluronic acid supplementation.

"Microfracture involves subchondral bone penetration to release bone marrow that forms a stem cell–rich clot. "

"Chondro-differentiation of MSCs results in an unnatural differentiation pathway that is unlike either endochondral ossification or permanent cartilage formation in that markers of hyaline cartilage (collagen type II{up} and SOX-9), hypertrophy (collagen type X{up} and MMP13), and bone (osteopontin{up} and bone sialoprotein{up}) are expressed concurrently"

"Cartilage-to-cartilage integration is exceedingly difficult to achieve, because cartilage displays low metabolism and contains dense, anti-adhesive ECM. For example, proteins transcribed from the PRG4 gene, contributors to cartilage’s low friction, and GAGs have been shown to directly inhibit cell adhesion"

Premature induction of hypertrophy during in vitro chondrogenesis of human mesenchymal stem cells correlates with calcification and vascular invasion after ectopic transplantation in SCID mice.

"Functional suitability and phenotypic stability of ectopic transplants are crucial factors in the clinical application of mesenchymal stem cells (MSCs) for articular cartilage repair, and might require a stringent control of chondrogenic differentiation. This study evaluated whether human bone marrow-derived MSCs adopt natural differentiation stages during induction of chondrogenesis in vitro, and whether they can form ectopic stable cartilage that is resistant to vascular invasion and calcification in vivo.

During in vitro chondrogenesis of MSCs, the expression of 44 cartilage-, stem cell-, and bone-related genes and the deposition of aggrecan and types II and X collagen were determined. Similarly treated, expanded articular chondrocytes served as controls. MSC pellets were allowed to differentiate in chondrogenic medium for 3-7 weeks, after which the chondrocytes were implanted subcutaneously into SCID mice; after 4 weeks in vivo, samples were evaluated by histology.

The 3-stage chondrogenic differentiation cascade initiated in MSCs was primarily characterized by sequential up-regulation of common cartilage genes. Premature induction of hypertrophy-related molecules (type X collagen and matrix metalloproteinase 13) occurred before production of type II collagen and was followed by up-regulation of alkaline phosphatase activity. In contrast, hypertrophy-associated genes were not induced in chondrocyte controls. Whereas control chondrocyte pellets resisted calcification and vascular invasion in vivo, most MSC pellets mineralized, in spite of persisting proteoglycan and type II collagen content.

An unnatural pathway of differentiation to chondrocyte-like cells was induced in MSCs by common in vitro protocols. MSC pellets transplanted to ectopic sites in SCID mice underwent alterations related to endochondral ossification rather than adopting a stable chondrogenic phenotype. Further studies are needed to evaluate whether a more stringent control of MSC differentiation to chondrocytes can be achieved during cartilage repair in a natural joint environment."

"dedifferentiated late-passage chondrocytes lose their ability to form ectopic cartilage and generate only fibrous-like tissue after transplantation"<-Maybe something similar happens physiologically to inhibit new growth plate formation?

In vitro differentiation of MSCs:

Up in LSJL:

Bgn

Lum

Col10a1

Col11a1

Col2a1

Col1a1

Spp1(as Osteopontin)

The appearance of Col10a1 before Col2a1 is abnormal.

"human adult MSCs derived from bone marrow can be programmed to produce ectopic fibrocartilage rich in proteoglycans and types I, II, and X collagen, and to undergo calcification and vascular invasion consistent with a program related to endochondral ossification. Remarkably, this sequence occurred in the absence of a 3-dimensional carrier and a growth factor depot, and without genetic manipulation of the cells."

The medium to induce chondrogenic differentiation of MSCs included TGFB3 and was pellet culture.  The ectopic cultures were implanted on the backs of 8 week old mice.  No hydrostatic pressure, tensile strain stimulation, or dynamic compression was used which could help LSJL from more appropriate endochondral ossification cartilage than present in this study.

Sequence of development of innately regenerated growth-plate cartilage in the hindlimb of the neonatal rat

"the neonatal rat can regenerate the distal femoral growth-plate. The age of the rodent and level and angle of amputation as significant modifiers of the regeneration process. Examination of these issues constitutes the objective of the present report. Fifty-four male, outbred albino rats sustained low femoral (48 rats) or midtibiofibular (six rats) hind-limb amputations when ten to eleven days old. They were killed after 0, 1, 2, 4, 7, 14, 22 or 29 postoperative days; and their amputation stumps were sectioned longitudinally. Twenty-four hr after amputation, the distal femoral periosteum was thickened and metachromatic regions were observed forming within it. Intraperiosteal cartilage was observed by the end of the second postoperative day in four of six limb stumps and, during the following week, expanded considerably in volume. Regenerated growth-plate cell architecture was recognized within the enlarging cartilage mass by the end of the second week; and, by the end of the fourth postoperative week, the regenerating growth-plate region had achieved considerable architectural maturity."

Rats lose the ability to regenerate growth plates after 5 months of age and are only able to regenerate growth plates in the lower but not upper extremities.

"Among rats killed two or four days after surgery, clots were present at the amputation surface and had begun to infiltrate between the nonskeleta1 tissues of the limb stumps, appearing in intermuscular spaces and along fascia1 planes."

"[48 hours post operation] Four of six femurs displayed small, irregular bodies of hyaline cartilage forming within the distal periosteum"

"[96 hours post operation] All seven amputation stumps displayed expanding cartilage bodies applied to their femoral termini. This cartilage formed a cuff surrounding the skeletal terminus, and within it ossification was proceeding as indicated by the appearance of delicate trabeculae of newly formed endochondral bone"

"[3 weeks post operation] new bone extended beyond the original amputation plane for a considerable
length, but in no specimen was evidence of distinct growth-plate regeneration observed."

Signs of a new growth plate appeared 4 weeks post operation.  In this type of growth plate regeneration the new growth plates appear de novo and not from tissues of the same type.

"the regenerated physis is formed via proliferation of periosteal cells which rapidly transform into chondrocytes"

"Unlike the amphibian limb, the regeneration of the growth-plate in the mammalian hindlimb occurs even after closure of the skin with structures"

Ectopic osteogenesis and chondrogenesis of bone marrow stromal stem cells in alginate system

"The present study sought to determine the ectopic osteogenic and chondrogenic ability of BMSSCs in combination with a scaffolding material made from alginate gel. After isolation from the bone marrow of BALB/C [8 week old male] mice, BMSSCs were expanded in vitro and induced to chondrogenesis or osteogenesis for 14 days, respectively. Subsequently, these induced cells were seeded into alginate gel, and the constructs implanted into BALB/C nude mice subcutaneously for up to 8 weeks. In the histological analysis, the transmission electron microscopy of the retrieved specimens at various intervals showed obvious trends of ectopic cartilage or bone formation along with the alteration of the cellular phenotype. Simultaneously, the results of the immunohistochemical staining and RT-PCR both confirmed the expression of specific extracellular matrix (ECM) markers for cartilaginous tissue, such as collagen type II (Col-II), SOX9, and aggrecan, or alternatively, markers for osteoid tissue, such as osteopontin (OPN), osteocalcin (OCN), and collagen type I (Col-I). During subcutaneous implantation, the elevating production of ECM and the initiation of the characteristic structure were closely correlated with the increase of time. In contrast, there was an apparent degradation and resorption of the scaffolding material in blank controls, but with no newly formed tissues. Finally, the constructs that were made of non-induced BMSSCs nearly disappeared during the 8 weeks after implantation. Therefore, it is suggested that alginate gel, which is combined with BMSSCs undergoing differentiation into skeletal lineages, may represent a useful strategy for the clinical reconstruction of bone and cartilage defects."

"After placed into the chondrogenic medium, BMSSCs were modulated from an elongated fibroblastic appearance to a smaller polygonal or round shape. The immunocytostaining of Col-II was a strong positive in 80% of the cells. When the chondrogenic induced BMSSCs were seeded into alginate, the scanning electronic microscopy results show the cells to be well attached to the scaffolds. Subsequently, the BMSSCs/alginate was implanted into 6-week-old female BALB/C nude micesubcutaneously."

"As shown in the immunostaining, Col-II was positive at 8 weeks after implantation"

"Col-II, aggrecan, and SOX9 were observed in differentiated monolayer cells and engineered constructs that were not observed in the controls "

In the osteogenic medium "With HE staining, the experimental implants showed surrounding fibrous tissue, and a mild infiltration of inflammatory cells and fibroblasts at 4 weeks. The resorption of alginate and endochondral ossification appeared to some extent, but there was no obvious initiation of bone formation at this point"<-indicating that chondroinduction can occur in an osteogenic medium too.

" in the osseous constructs, the phenomenon of endochondral ossification occurred at 4 weeks, and the structure of bone trabecula containing a large number of cells and collagenous ECM formed in another 4 weeks. "

"Although differentiated BMSSCs were not actual chondrocytes or osteoblasts, their chondrogenic and osteogenic nature was confirmed with not only morphological features but also immunohistochemical staining of the marker proteins in cartilage and bone."<-maybe mechanical stimulation could cement their phenotype.

"In the neo-cartilage, the intensity of mRNA encoding aggrecan, SOX9, and Col-II maintained stably at high levels as shown in the RT-PCR results. Correspondingly, there was a significant accumulation of ECM components in the pericellular matrix with increasing time. The same trend of Col-I, OCN, and OPN production was also found in the new bone, which promoted the confluence of ECM and the development of a bony structure."

"Morphological and gross observation of cartilage tissues. Two weeks after chondrogenic induction, the BMSSCs indicated a smaller polygonal or round shape as is in chondrocytes. The immunocytostaining of Col-II was a strong positive in 80% of the cells, the arrow points to the positive cell (A). When the chondrogenic induced BMSSCs were seeded into alginate, the scanning electronic microscopy results show the cells to be well attached with the scaffolds (B). The engineered chondroid constructs formed white spheroid aggregates and were capsuled by connective tissues. The ultramicro cellular observation indicated the specific markers of cartilage formation, such as the presence of abundant ribosome and rough endoplasmic reticulum, and pericellular collagen fibrils and proteoglycan granules at 4 weeks. The arrow points to the collagen fibers (C). HE staining indicated the initial cartilage formation after 4 weeks of implantation (D) and neo-cartilage becoming confluent and mature at 8 weeks (E). The 8 weeks constructs positively stained with the antibodies against Col-II proteins, the arrow points to the lacunae (F)."

"Morphological and gross observation of osteoid tissues. After culture in an osteogenic medium for two weeks, the BMSSCs became a cuboidal shape and were surrounded with an abundant matrix, when confluent, the monolayer cells aggregated to form nodules with differentiated cells trapped in an abundant matrix, which was positive in alizarin red S. The arrow points to the calcified nodule (A). The retrieved osteoid constructs exhibited white, rigid constitution and the alginate content decreased stepwise. When the osteogenic induced BMSSCs were seeded into alginate, the scanning electronic microscopy results show the cells to be well attached with the scaffolds, the arrow points to the cells attached on the scaffold (B). The transmission electron microscopy indicated an abundance of ribosome and rough endoplasmic reticulum, and pericellular paralleling collagen fibrils at 4 weeks, and there were many vacuoles in cytoplasm and calcification in some areas, the arrow points to the calcification area (C). HE staining indicated the resorption of alginate and endochondral ossification in constructs after 4 weeks of implantation with the surrounding fibrous tissue and a mild infiltration of inflammatory cells and fibroblasts (D). After 8 weeks, remnants of a little alginate were distributed in the partially mineralized ECM and new bone formation appeared in a closely knitted arrangement throughout the implanted constructs (E). In an OCN immunohistochemical assay, which is a marker of osteoblasts, the extensively regenerated bone was confirmed and the structure of bone trabecula contained an abundance of cells and collagenous ECM (F). There was no detectable signal of bone formation in the blank controls."

So ectopic chondrossification is theoretically possible.

Transcriptional mechanisms in osteoblast differentiation and bone formation

"Osx-null cells acquire a chondrocyte phenotype implies that Osx is a negative regulator of Sox9 and of the chondrocyte phenotype."

" if Sox9 is inactivated after the establishment of mesenchymal condensations, Runx2 expression and osteoblast differentiation takes place."

Perichondrium phenotype and border function are regulated by Ext1 and heparan sulfate in developing long bones: A mechanism likely deranged in Hereditary Multiple Exostoses

"During limb skeletogenesis the cartilaginous long bone anlagen and their growth plates become delimited by perichondrium with which they interact functionally. Despite being so intimately associated with cartilage, perichondrium acquires and maintains its distinct phenotype and exerts its border function. Because perichondrium becomes deranged and interrupted by cartilaginous outgrowths in Hereditary Multiple Exostoses (HME), a pediatric disorder caused by EXT mutations and consequent heparan sulfate (HS) deficiency, we asked whether EXT genes and HS normally have roles in establishing its phenotype and function. Indeed, conditional Ext1 ablation in perichondrium and lateral chondrocytes flanking the epiphyseal region of mouse embryo long bone anlagen –a region encompassing the groove of Ranvier– caused ectopic cartilage formation. A similar response was observed when HS function was disrupted in long bone anlagen explants by genetic, pharmacological or enzymatic means, a response preceded by ectopic BMP signaling within perichondrium. These treatments also triggered excess chondrogenesis and cartilage nodule formation and overexpression of chondrogenic and matrix genes in limb bud mesenchymal cells in micromass culture. Interestingly, the treatments disrupted the peripheral definition and border of the cartilage nodules in such a way that many nodules overgrew and fused with each other into large amorphous cartilaginous masses. Interference with HS function reduced the physical association and interactions of BMP2 with HS and increased the cell responsiveness to endogenous and exogenous BMP proteins. In sum, Ext genes and HS are needed to establish and maintain perichondrium's phenotype and border function, restrain pro-chondrogenic signaling proteins including BMPs, and restrict chondrogenesis."

So is Ext1 removal can induce ectopic cartilage formation can it also increase height?

"HME is characterized by cartilaginous and bony outgrowths (exostoses) that form next to, but never within, the growth plates."  Apparently HME results in short rather than tall stature(most of the time).

"when we compared the frequency of ectopic cartilage formation with respect to the long bone longitudinal axis, it was clear that the incidence was higher in the epiphyseal than diaphyseal region by a ratio of about 4 to 1"

"the epiphysis needs to enlarge and expand laterally by appositional growth and achieve a much larger diameter compared to the diaphysis"

"it is conceivable and possible that the ectopic cartilage formation occurring in Ext1-deficient long bone anlagen in vivo or explant culture is an amplification of that natural process propelled by ectopic pro-chondrogenic signaling activity, increased availability of “free” chondrogenic factors, and precocious recruitment of progenitor cells into the chondrogenic lineage."

Specific inductive potential of a novel nanocomposite biomimetic biomaterial for osteochondral tissue regeneration.

"Osteochondral lesions require treatment to restore the biology and functionality of the joint. A novel nanostructured biomimetic gradient scaffold was developed to mimic the biochemical and biophysical properties of the different layers of native osteochondral structure. The scaffold presents important physicochemical characteristics and can support the growth and differentiation of mesenchymal stromal cells (h-MSCs), which adhere and penetrate into the cartilaginous and bony layers. H-MSCs grown in chondrogenic or osteogenic medium decreased their proliferation during days 14-52 on both scaffold layers and in medium without inducing factors used as controls. Both chondrogenic and osteogenic differentiation of h-MSCs occurred from day 28 and were increased on day 52, but not in the control medium. Safranin O staining and collagen type II and proteoglycans immunostaining confirmed that chondrogenic differentiation was specifically induced only in the cartilaginous layer{finding why chondrogenic differentiation only occurred in the cartilaginous layer could be key to finding methods to induce ectopic chondrogenesis}. Conversely, von Kossa staining, osteocalcin and osteopontin immunostaining confirmed that osteogenic differentiation occurred on both layers."

Control chondrogenic medium and chondrogenic medium were the same except TGF-B1 was added to the chondrogenic medium.

So TGFB-1 is key for chondrocyte differentiation.

Cartilage formation in growth plate and arteries: from physiology to pathology

"vascular smooth muscle cells (VSMCs) undergo chondrogenic commitment eventually leading to vascular calcification, by mechanisms similar to those governing ossification in the cartilage growth plate."

"VSMCs express Sox9 and type II and IX collagen"

An artery carries blood away from the blood into the body.  There are arteries in the bone.  If ectopic growth plates can be induced in these arteries than it could be possible to form new growth plates.