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. 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. Our results showed that 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. "
"Sox9 is a high-mobility group box-containing transcription factor that functions as a key regulator of chondrogenesis. We here report that 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 was also observed. 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). In contrast to other mesenchymal progenitors, the genealogy and biology of the tenogenic lineage is not yet fully understood due to the lack of simple and efficient protocols enabling generation of progenitors in vitro. Here, 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. Gene expression analysis demonstrated that 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. From these results, we conclude that 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."
"Interestingly, on Scx ectopic expression in hMSC, we observed almost a complete loss of Sox9 expression."
"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. In contrast, 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.
Our findings indicate that 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. These results provide evidence that Sox9 induces ectopic chondrogenesis in mesenchymal cells and strongly suggest that 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 also 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."
""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 plate}. 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 and thus the need for a differential diagnosis. Recent advances in the understanding of their 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, proposed by Spranger and associates in 1978 and tentatively revised twice, 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)]. "
"[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. The precise mechanisms leading to chondroma formation remain to be determined but deregulation of the HIF-1alpha pathway is a plausible mechanism 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. It is concluded that 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). We found that once isolated from lesion and grown in this supportive medium, dysplasic chondrocytes behaved essentially like normal growth plate cells. These findings suggest that 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"
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.