Lubricin Protects the Temporomandibular Joint Surfaces from Degeneration

The temporomandibular joint (TMJ) is a specialized synovial joint essential for the mobility and function of the mammalian jaw. The TMJ is composed of the mandibular condyle, the glenoid fossa of the temporal bone, and a fibrocartilagenous disc interposed between these bones. A fibrous capsule, lined on the luminal surface by the synovial membrane, links these bones and retains synovial fluid within the cavity. The major component of synovial fluid is lubricin, a glycoprotein encoded by the gene proteoglycan 4 (Prg4), which is synthesized by chondrocytes at the surface of the articular cartilage and by synovial lining cells. We previously showed that in the knee joint, Prg4 is crucial for maintenance of cartilage surfaces and for regulating proliferation of the intimal cells in the synovium. Consequently, the objective of this study was to determine the role of lubricin in the maintenance of the TMJ. We found that mice lacking lubricin have a normal TMJ at birth, but develop degeneration resembling TMJ osteoarthritis by 2 months, increasing in severity over time. Disease progression in Prg4 ^−/− mice results in synovial hyperplasia, deterioration of cartilage in the condyle, disc and fossa with an increase in chondrocyte number and their redistribution in clusters with loss of superficial zone chondrocytes. All articular surfaces of the joint had a prominent layer of protein deposition. Compared to the knee joint, the osteoarthritis-like phenotype was more severe and manifested earlier in the TMJ. Taken together, the lack of lubricin in the TMJ causes osteoarthritis-like degeneration that affects the articular cartilage as well as the integrity of multiple joint tissues. Our results provide the first molecular evidence of the role of lubricin in the TMJ and suggest that Prg4 ^−/− mice might provide a valuable new animal model for the study of the early events of TMJ osteoarthritis.     

Shox2-deficiency leads to dysplasia and ankylosis of the temporomandibular joint in mice

The temporomandibular joint (TMJ) is a unique synovial joint whose development differs from the formation of other synovial joints. Mutations have been associated with the developmental defects of the TMJ only in a few genes. In this study, we report the expression of the homeobox gene Shox2 in the cranial neural crest derived mesenchymal cells of the maxilla-mandibular junction and later in the progenitor cells and undifferentiated chondrocytes of the condyle as well as the glenoid fossa of the developing TMJ. A conditional inactivation of Shox2 in the cranial neural crest-derived cells causes developmental abnormalities in the TMJ, including dysplasia of the condyle and glenoid fossa. The articulating disc forms but fuses with the fibrous layers of the condyle and glenoid fossa, clinically known as TMJ ankylosis. Histological examination indicates a delay in development in the mutant TMJ, accompanied by a significantly reduced rate of cell proliferation. In situ hybridization further demonstrates an altered expression of several key osteogenic genes and a delayed expression of the osteogenic differentiation markers. Shox2 appears to regulate the expression of osteogenic genes and is essential for the development and function of the TMJ. The Shox2 conditional mutant thus provides a unique animal model of TMJ ankylosis.     

Osteophyte formation and matrix mineralization in a TMJ osteoarthritis mouse model are associated with ectopic hedgehog signaling

The temporomandibular joint (TMJ) is a diarthrodial joint that relies on lubricants for frictionless movement and long-term function. It remains unclear what temporal and causal relationships may exist between compromised lubrication and onset and progression of TMJ disease. Here we report that Proteoglycan 4 (Prg4)-null TMJs exhibit irreversible osteoarthritis-like changes over time and are linked to formation of ectopic mineralized tissues and osteophytes in articular disc, mandibular condyle and glenoid fossa. In the presumptive layer of mutant glenoid fossa's articulating surface, numerous chondrogenic cells and/or chondrocytes emerged ectopically within the type I collagen-expressing cell population, underwent endochondral bone formation accompanied by enhanced Ihh expression, became entrapped into temporal bone mineralized matrix, and thereby elicited excessive chondroid bone formation. As the osteophytes grew, the roof of the glenoid fossa/eminence became significantly thicker and flatter, resulting in loss of its characteristic concave shape for accommodation of condyle and disc. Concurrently, the condyles became flatter and larger and exhibited ectopic bone along their neck, likely supporting the enlarged condylar heads. Articular discs lost their concave configuration, and ectopic cartilage developed and articulated with osteophytes. In glenoid fossa cells in culture, hedgehog signaling stimulated chondrocyte maturation and mineralization including alkaline phosphatase, while treatment with hedgehog inhibitor HhAntag prevented such maturation process. In sum, our data indicate that Prg4 is needed for TMJ integrity and long-term postnatal function. In its absence, progenitor cells near presumptive articular layer and disc undergo ectopic chondrogenesis and generate ectopic cartilage, possibly driven by aberrant activation of Hh signaling. The data suggest also that the Prg4-null mice represent a useful model to study TMJ osteoarthritis-like degeneration and clarify its pathogenesis.     

A distinct cohort of progenitor cells participates in synovial joint and articular cartilage formation during mouse limb skeletogenesis

The origin, roles and fate of progenitor cells forming synovial joints during limb skeletogenesis remain largely unclear. Here we produced prenatal and postnatal genetic cell fate-maps by mating ROSA-LacZ-reporter mice with mice expressing Cre-recombinase at prospective joint sites under the control of Gdf5 regulatory sequences (Gdf5-Cre). Reporter-expressing cells initially constituted the interzone, a compact mesenchymal structure representing the first overt sign of joint formation, and displayed a gradient-like distribution along the ventral-to-dorsal axis. The cells expressed genes such as Wnt9a, Erg and collagen IIA, remained predominant in the joint-forming sites over time, gave rise to articular cartilage, synovial lining and other joint tissues, but contributed little if any to underlying growth plate cartilage and shaft. To study their developmental properties more directly, we isolated the joint-forming cells from prospective autopod joint sites using a novel microsurgical procedure and tested them in vitro. The cells displayed a propensity to undergo chondrogenesis that was enhanced by treatment with exogenous rGdf5 but blocked by Wnt9a over-expression. To test roles for such Wnt-mediated anti-chondrogenic capacity in vivo, we created conditional mutants deficient in Wnt/β-catenin signaling using Col2-Cre or Gdf5-Cre. Synovial joints did form in both mutants; however, the joints displayed a defective flat cell layer normally abutting the synovial cavity and expressed markedly reduced levels of lubricin. In sum, our data indicate that cells present at prospective joint sites and expressing Gdf5 constitute a distinct cohort of progenitor cells responsible for limb joint formation. The cells appear to be patterned along specific limb symmetry axes and rely on local signaling tools to make distinct contributions to joint formation.     

The role of TGF-beta signaling in regulating chondrogenesis and osteogenesis during mandibular development

During craniofacial development, Meckel's cartilage and the mandible bone derive from the first branchial arch, and their development depends upon the contribution of cranial neural crest (CNC) cells. We previously demonstrated that conditional inactivation of Tgfbr2 in the neural crest of mice (Tgfbr2^fl/fl;Wnt1-Cre) results in severe defects in mandibular development, although the specific cellular and molecular mechanisms by which TGF-β signaling regulates the fate of CNC cells during mandibular development remain unknown. We show here that loss of Tgfbr2 does not affect the migration of CNC cells during mandibular development. TGF-β signaling is specifically required for cell proliferation in Meckel's cartilage and the mandibular anlagen and for the formation of the coronoid, condyle and angular processes. TGF-β-mediated connective tissue growth factor (CTGF) signaling is critical for CNC cell proliferation. Exogenous CTGF rescues the cell proliferation defect in Meckel's cartilage of Tgfbr2^fl/fl;Wnt1-Cre mutants, demonstrating the biological significance of this signaling cascade in chondrogenesis during mandibular development. Furthermore, TGF-β signaling controls Msx1 expression to regulate mandibular osteogenesis as Msx1 expression is significantly reduced in Tgfbr2^fl/fl;Wnt1-Cre mutants. Collectively, our data suggest that there are differential signal cascades in response to TGF-β to control chondrogenesis and osteogenesis during mandibular development.     

Canonical Wnt signaling activity during synovial joint development

Wnt signaling plays important roles in skeletal development. However, the activation and function of canonical Wnt signaling in joint development remains unclear. We analyzed the lineage identity and developmental changes of the Wnt-responsive cells during synovial joint formation as well as adulthood in the Wnt signaling reporter TOPgal transgenic mice. At embryonic day (E) 12.5, we found that the TOPgal was inactivated in the presumptive joint forming interzone, but it was intensively activated in the cartilage anlage of developing long bones and digits. At E14.5, the TOPgal activity was found in a subgroup of the articular chondrocyte lineage cells, which were co-immunolabeled with Doublecortin intensively and with Vinculin weakly. At E18.5, the TOPgal/Doublecortin co-immunolabeled cells were found in the superficial layer of the developing articular cartilage. During postnatal development, the TOPgal(+) articular chondrocytes were abundant at P7 and decreased from P10. A small number of TOPgal(+) articular chondrocytes were also found in adult joints. Our study suggests an age- and lineage-specific role of canonical Wnt signaling in joint development and maintenance.     

Replacing Shox2 with human SHOX leads to congenital disc degeneration of the temporomandibular joint in mice

The temporomandibular joint (TMJ) consists in the glenoid fossa arising from the otic capsule through intramembranous ossification, the fibrocartilaginous disc and the condyle, which is derived from the secondary cartilage by endochondral ossification. We have reported previously that cranial neural-crest-specific inactivation of the homeobox gene Shox2, which is expressed in the mesenchymal cells of the maxilla-mandibular junction and later in the progenitor cells and perichondrium of the developing chondyle, leads to dysplasia and ankylosis of the TMJ and that replacement of the mouse Shox2 with the human SHOX gene rescues the dysplastic and ankylosis phenotypes but results in a prematurely worn out articular disc. In this study, we investigate the molecular and cellular bases for the prematurely worn out articular disc in the TMJ of mice carrying the human SHOX replacement allele in the Shox2 locus (termed Shox2 ^SHOX-KI/KI). We find that the developmental process and expression of several key genes in the TMJ of Shox2 ^SHOX-KI/KI mice are similar to that of controls. However, the disc of the Shox2 ^SHOX-KI/KI TMJ exhibits a reduced level of Collagen I and Aggrecan, accompanied by increased activities of matrix metalloproteinases and a down-regulation of Ihh expression. Dramatically increased cell apoptosis in the disc was also observed. These combinatory cellular and molecular defects appear to contribute to the observed disc phenotype, suggesting that, although human SHOX can exert similar functions to mouse Shox2 in regulating early TMJ development, it apparently has a distinct function in the regulation of those molecules that are involved in tissue homeostasis.     

Development of synovial joints

Synovial joints arise through two main processes. In long bone elements, cartilaginous differentiation occurs across the locations of the prospective joints that then segment secondarily. This process occurs through the development of a noncartilaginous region known as the interzone. The interzone becomes an important signaling center to the opposing elements, which can regulate growth through such factors as GDF-5. The interzone also expresses bone morphogenetic proteins (BMPs) and their antagonists, such as noggin. Overexpression of BMPs, or the loss of noggin leads to joint fusions. The interzone also expresses Wnt-14, which appears to be specific for this region in the developing anlagen, and regulates its nonchondrogenic nature. Cavitation of the joint follows, driven by selective high-level synthesis of hyaluronan by interzone cells and presumptive synovial cells. In addition, as the interzone disperses during cavity enlargement, data are now accruing that suggest that both the synovium and articular cartilage develop from this population. Finally, the development of articular cartilage progresses through appositional growth driven by a progenitor/stem cell subpopulation that resides in the articular surface. The individual elements of the skeleton are connected together at regions termed joints or articulations. Classically, there are three broad categories of joints: immovable joints (syntharthroses); mixed articulations, in which the range of movement is limited (amphiarthroses); and the movable, or synovial, joints (diarthroses). This review concentrates on the development of the synovial joints.     

Noncanonical Transforming Growth Factor β (TGFβ) Signaling in Cranial Neural Crest Cells Causes Tongue Muscle Developmental Defects

Microglossia is a congenital birth defect in humans and adversely impacts quality of life. In vertebrates, tongue muscle derives from the cranial mesoderm, whereas tendons and connective tissues in the craniofacial region originate from cranial neural crest (CNC) cells. Loss of transforming growth factor β (TGFβ) type II receptor in CNC cells in mice (Tgfbr2^fl/fl;Wnt1-Cre) causes microglossia due to a failure of cell-cell communication between cranial mesoderm and CNC cells during tongue development. However, it is still unclear how TGFβ signaling in CNC cells regulates the fate of mesoderm-derived myoblasts during tongue development. Here we show that activation of the cytoplasmic and nuclear tyrosine kinase 1 (ABL1) cascade in Tgfbr2^fl/fl;Wnt1-Cre mice results in a failure of CNC-derived cell differentiation followed by a disruption of TGFβ-mediated induction of growth factors and reduction of myogenic cell proliferation and differentiation activities. Among the affected growth factors, the addition of fibroblast growth factor 4 (FGF4) and neutralizing antibody for follistatin (FST; an antagonist of bone morphogenetic protein (BMP)) could most efficiently restore cell proliferation, differentiation, and organization of muscle cells in the tongue of Tgfbr2^fl/fl;Wnt1-Cre mice. Thus, our data indicate that CNC-derived fibroblasts regulate the fate of mesoderm-derived myoblasts through TGFβ-mediated regulation of FGF and BMP signaling during tongue development.     

Augmented BMPRIA-Mediated BMP Signaling in Cranial Neural Crest Lineage Leads to Cleft Palate Formation and Delayed Tooth Differentiation

The importance of BMP receptor Ia (BMPRIa) mediated signaling in the development of craniofacial organs, including the tooth and palate, has been well illuminated in several mouse models of loss of function, and by its mutations associated with juvenile polyposis syndrome and facial defects in humans. In this study, we took a gain-of-function approach to further address the role of BMPR-IA-mediated signaling in the mesenchymal compartment during tooth and palate development. We generated transgenic mice expressing a constitutively active form of BmprIa (caBmprIa) in cranial neural crest (CNC) cells that contributes to the dental and palatal mesenchyme. Mice bearing enhanced BMPRIa-mediated signaling in CNC cells exhibit complete cleft palate and delayed odontogenic differentiation. We showed that the cleft palate defect in the transgenic animals is attributed to an altered cell proliferation rate in the anterior palatal mesenchyme and to the delayed palatal elevation in the posterior portion associated with ectopic cartilage formation. Despite enhanced activity of BMP signaling in the dental mesenchyme, tooth development and patterning in transgenic mice appeared normal except delayed odontogenic differentiation. These data support the hypothesis that a finely tuned level of BMPRIa-mediated signaling is essential for normal palate and tooth development.     

Development of the temporomandibular joint in miniature pig embryos

The temporomandibular joint (TMJ) is a synovial joint involved in sliding and hinge movements of lower jaw in mammals. Studies on TMJ development in embryos have been mainly performed using rodents. However, the TMJ structure in rodents differs in several aspects from that in humans. There are few studies on the embryonic development of TMJ in large mammals. In the present study, we investigated the embryonic developmental characteristics of the TMJ in pigs histologically. Embryonic day 35 (E35), E45, E55, E75, E90, and postnatal day 1(P1) embryos/fetuses from the pigs were used for the study. The results showed condensation of mesenchymal cells on E35. The inferior articular cavity was formed on E45, together with a narrow crack in the superior articular cavity region. The superior and inferior articular cavities and articular disc of the TMJ were completely formed on E55. On E75, the condyle showed an obvious conical shape and the superior and inferior joint cavities were enlarged. Furthermore, the mandibular ramus and mandibular body under the neck of the condyle were ossified from E75 to P1 day. The chondrocyte layer of the condyle was significantly thinner from E75 to P1. It is speculated that the spatiotemporal development of the TMJ in miniature pig embryos is similar to that in humans. Embryonic development of the pig TMJ is an important bridge for translating the results of rodent research to medical applications.     

Collagen I and II mRNA distribution in the rat temporomandibular joint region during growth

The distribution of type I and II collagen synthesis in the temporomandibular joint (TMJ) area of 1- to 28-day-old rats was studied after hybridization with probes to pro alpha1(I) and pro alpha1(II) collagen mRNA, and stain intensity through the various cartilaginous zones of the mandibular condyle and other areas of TMJ was assessed. The pro alpha(I) collagen mRNA was detected in the perichondrium/periosteum, in the fibrous and undifferentiated cell layers of the mandibular condyle, in the articular disc, and in all bone structures and muscles. The pro alpha1(II) collagen mRNA was found in the condylar cartilage and the articular fossa. Intensity in the condyle was highest in the chondroblastic layer and decreased towards the lower hypertrophic layer. In the condylar cartilage of the 21- to 28-day-old rats the chondroblastic cell zone was relatively narrow compared with the younger animals, whereas the reverse seems to be the case in the cartilage of the articular fossa. Changes in the pro alpha1(II) collagen mRNA were observed in the osseochondral junction area of the primary spongiosa, in that at the age of 5 days intense staining was found, whereas no staining was observed by 14 days. In the mineralizing zone, however, the majority of osteoblastic cells gave a positive signal with the pro alpha1(I) collagen probe. In conclusion, type II collagen synthesis of the mandibular condyle is restricted to its upper area. This differs from the long bone epiphyseal plate, where this type of collagen is produced virtually throughout the cartilage. Type II collagen synthesis of the fossal cartilage seems to increase as a function of age.     

Trps1 is necessary for normal temporomandibular joint development

Mutation of the human TRPS1 gene leads to trichorhinophalangeal syndrome (TRPS), which is characterized by an abnormal development of various organs including the craniofacial skeleton. Trps1 has recently been shown to be expressed in the jaw joints of zebrafish; however, whether Trps1 is expressed in the mammalian temporomandibular joint (TMJ), or whether it is necessary for TMJ development is unknown. We have analyzed (1) the expression pattern of Trps1 during TMJ development in mice and (2) TMJ development in Trps1 knockout animals. Trps1 is expressed in the maxillo-mandibular junction at embryonic day (E) 11.5. At E15.5, expression is restricted to the developing condylar cartilage and to the surrounding joint disc progenitor cells. In Trps1 knockout mice, the glenoid fossa of the temporal bone forms relatively normally but the condylar process is extremely small and the joint disc and cavities do not develop. The initiation of condyle formation is slightly delayed in the mutants at E14.5; however, at E18.5, the flattened chondrocyte layer is narrowed and most of the condylar chondrocytes exhibit precocious chondrocyte maturation. Expression of Runx2 and its target genes is expanded toward the condylar apex in the mutants. These observations underscore the indispensable role played by Trps1 in normal TMJ development in supporting the differentiation of disc and synoviocyte progenitor cells and in coordinating condylar chondrocyte differentiation.     

TGF-beta mediated Dlx5 signaling plays a crucial role in osteo-chondroprogenitor cell lineage determination during mandible development

Transforming growth factor-β (TGF-β) signaling is crucial for mandible development. During its development, the majority of the mandible is formed through intramembranous ossification whereas the proximal region of the mandible undergoes endochondral ossification. Our previous work has shown that TGF-β signaling is required for the proliferation of cranial neural crest (CNC)-derived ectomesenchyme in the mandibular primordium where intramembranous ossification takes place. Here we show that conditional inactivation of Tgfbr2 in CNC cells results in accelerated osteoprogenitor differentiation and perturbed chondrogenesis in the proximal region of the mandible. Specifically, the appearance of chondrocytes in Tgfbr2^fl/fl;Wnt1-Cre mice is delayed and they are smaller in size in the condylar process and completely missing in the angular process. TGF-β signaling controls Sox9 expression in the proximal region, because Sox9 expression is delayed in condylar processes and missing in angular process in Tgfbr2^fl/fl;Wnt1-Cre mice. Moreover, exogenous TGF-β can induce Sox9 expression in the mandibular arch. In the angular processes of Tgfbr2^fl/fl;Wnt1-Cre mice, osteoblast differentiation is accelerated and Dlx5 expression is elevated. Significantly, deletion of Dlx5 in Tgfbr2^fl/fl;Wnt1-Cre mice results in the rescue of cartilage formation in the angular processes. Finally, TGF-β signaling-mediated Scleraxis expression is required for tendonogenesis in the developing skeletal muscle. Thus, CNC-derived cells in the proximal region of mandible have a cell intrinsic requirement for TGF-β signaling.     

Biomechanical properties of murine TMJ articular disc and condyle cartilage via AFM-nanoindentation

This study aims to quantify the biomechanical properties of murine temporomandibular joint (TMJ) articular disc and condyle cartilage using AFM-nanoindentation. For skeletally mature, 3-month old mice, the surface of condyle cartilage was found to be significantly stiffer (306 ± 84 kPa, mean ± 95% CI) than those of the superior (85 ± 23 kPa) and inferior (45 ± 12 kPa) sides of the articular disc. On the disc surface, significant heterogeneity was also detected across multiple anatomical sites, with the posterior end being the stiffest and central region being the softest. Using SEM, this study also found that the surfaces of disc are composed of anteroposteriorly oriented collagen fibers, which are sporadically covered by thinner random fibrils. Such fibrous nature results in both an F-D^3/2 indentation response, which is a typical Hertzian response for soft continuum tissue under a spherical tip, and a linear F-D response, which is typical for fibrous tissues, further signifying the high degree of tissue heterogeneity. In comparison, the surface of condyle cartilage is dominated by thinner, randomly oriented collagen fibrils, leading to Hertzian-dominated indentation responses. As the first biomechanical study of murine TMJ, this work will provide a basis for future investigations of TMJ tissue development and osteoarthritis in various murine TMJ models.     

新石器时代人骨颞下颌关节的研究

对宝鸡、华县新石器时代人骨22个成年个体的颞下颌关节进行研究。发现该时期人类中23％者存在髁状突和关节结节骨质的严重磨损、不对称磨损和不规则增生,认为这是由于强大的咀嚼压力所造成的继发性关节退行性变。颞下颌关节的测量结果表明,与现生人类相比较,新石器时代人类的髁状突较大,关节结节较高,关节凹较深,关节结节后斜面斜度较小,反映了人类进化过程中由于生活方式和口腔功能的改变颞下颌关节的变小和关节承压结构的减弱。     

Deficiency of Thrombospondin-4 in Mice Does Not Affect Skeletal Growth or Bone Mass Acquisition,but Causes a Transient Reduction of Articular Cartilage Thickness

Although articular cartilage degeneration represents a major public health problem, the underlying molecular mechanisms are still poorly characterized. We have previously utilized genome-wide expression analysis to identify specific markers of porcine articular cartilage, one of them being Thrombospondin-4 (Thbs4). In the present study we analyzed Thbs4 expression in mice, thereby confirming its predominant expression in articular cartilage, but also identifying expression in other tissues, including bone. To study the role of Thbs4 in skeletal development and integrity we took advantage of a Thbs4-deficient mouse model that was analyzed by undecalcified bone histology. We found that Thbs4-deficient mice do not display phenotypic differences towards wildtype littermates in terms of skeletal growth or bone mass acquisition. Since Thbs4 has previously been found over-expressed in bones of Phex-deficient Hyp mice, we additionally generated Thbs4-deficient Hyp mice, but failed to detect phenotypic differences towards Hyp littermates. With respect to articular cartilage we found that Thbs4-deficient mice display transient thinning of articular cartilage, suggesting a protective role of Thbs4 for joint integrity. Gene expression analysis using porcine primary cells revealed that Thbs4 is not expressed by synovial fibroblasts and that it represents the only member of the Thbs gene family with specific expression in articular, but not in growth plate chondrocytes. In an attempt to identify specific molecular effects of Thbs4 we treated porcine articular chondrocytes with human THBS4 in the absence or presence of conditioned medium from porcine synovial fibroblasts. Here we did not observe a significant influence of THBS4 on proliferation, metabolic activity, apoptosis or gene expression, suggesting that it does not act as a signaling molecule. Taken together, our data demonstrate that Thbs4 is highly expressed in articular chondrocytes, where its presence in the extracellular matrix is required for articular cartilage integrity.     

Mouse limb skeletal growth and synovial joint development are coordinately enhanced by Kartogenin

Limb development requires the coordinated growth of several tissues and structures including long bones, joints and tendons, but the underlying mechanisms are not wholly clear. Recently, we identified a small drug-like molecule – we named Kartogenin (KGN) – that greatly stimulates chondrogenesis in marrow-derived mesenchymal stem cells (MSCs) and enhances cartilage repair in mouse osteoarthritis (OA) models. To determine whether limb developmental processes are regulated by KGN, we tested its activity on committed preskeletal mesenchymal cells from mouse embryo limb buds and whole limb explants. KGN did stimulate cartilage nodule formation and more strikingly, boosted digit cartilaginous anlaga elongation, synovial joint formation and interzone compaction, tendon maturation as monitored by ScxGFP, and interdigit invagination. To identify mechanisms, we carried out gene expression analyses and found that several genes, including those encoding key signaling proteins, were up-regulated by KGN. Amongst highly up-regulated genes were those encoding hedgehog and TGFβ superfamily members, particularly TFGβ1. The former response was verified by increases in Gli1-LacZ activity and Gli1 mRNA expression. Exogenous TGFβ1 stimulated cartilage nodule formation to levels similar to KGN, and KGN and TGFβ1 both greatly enhanced expression of lubricin/Prg4 in articular superficial zone cells. KGN also strongly increased the cellular levels of phospho-Smads that mediate canonical TGFβ and BMP signaling. Thus, limb development is potently and harmoniously stimulated by KGN. The growth effects of KGN appear to result from its ability to boost several key signaling pathways and in particular TGFβ signaling, working in addition to and/or in concert with the filamin A/CBFβ/RUNX1 pathway we identified previously to orchestrate overall limb development. KGN may thus represent a very powerful tool not only for OA therapy, but also limb regeneration and tissue repair strategies.     

Versican expression during synovial joint morphogenesis

The extracellular matrix (ECM) plays a critical role in governing cell behavior and phenotype during limb skeletogenesis. Chondroitin sulfate proteoglycans (Cspgs) are highly expressed in the ECM of precartilage mesenchymal condensations and are important to limb chondrogenesis and cartilage structure, but little is known regarding their involvement in formation of synovial joints in the embryonic limb. Matrix versican Cspg expression has previously been reported in the epiphysis of developing long bones and presumptive joint; however, detailed analysis has not yet been conducted. In the present study we immunolocalized versican and aggrecan Cspgs during chick elbow joint morphogenesis between HH st25-41 of development. In this study we show that versican and aggrecan expression initially overlapped in the incipient cartilage model of long bones in the wing, but versican was also highly expressed in the perichondrium and presumptive joint interzone during early stages of morphogenesis (HH st25-34). By HH st36-41 versican localization was restricted to the future articular surfaces of the developing joint and surrounding joint capsule while aggrecan localized in an immediately adjacent and predominately non-overlapping region of chondrogenic cells at the epiphyses. These results suggest a potential role for versican proteoglycan in development and maintenance of the synovial joint interzone.