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After high fractures of the mandibular condyle, the insufficient blood supply to the condyle often leads to poor bone and cartilage repair ability and poor clinical outcome. Parathyroid hormone (PTH) can promote the bone formation and mineralization of mandibular fracture, but its effects on cartilage healing after the free reduction and internal fixation of high fractures of the mandibular condyle are unknown. In this study, a rabbit model of free reduction and internal fixation of high fractures of the mandibular condyle was established, and the effects and mechanisms of PTH on condylar cartilage healing were explored. Forty-eight specific-pathogen-free (SPF) grade rabbits were randomly divided into two groups. In the experimental group, PTH was injected subcutaneously at 20 µg/kg (PTH (1-34)) every other day, and in the control group, PTH was replaced with 1 ml saline. The healing cartilages were assessed at postoperative days 7, 14, 21, and 28. Observation of gross specimens, hematoxylin eosin staining and Safranin O/fast green staining found that every-other-day subcutaneous injection of PTH at 20 µg/kg promoted healing of condylar cartilage and subchondral osteogenesis in the fracture site. Immunohistochemistry and polymerase chain reaction showed that PTH significantly upregulated the chondrogenic genes Sox9 and Col2a1 in the cartilage fracture site within 7-21 postoperative days in the experimental group than those in the control group, while it downregulated the cartilage inflammation gene matrix metalloproteinase-13 and chondrocyte terminal differentiation gene ColX. In summary, exogenous PTH can stimulate the formation of cartilage matrix by triggering Sox9 expression at the early stage of cartilage healing, and it provides a potential therapeutic protocol for high fractures of the mandibular condyle.

In childhood, skeletal growth is driven by transient expansion of cartilage in the growth plate. The common belief is that energy production in this hypoxic tissue mainly relies on anaerobic glycolysis and not on mitochondrial respiratory chain (RC) activity. However, children with mitochondrial diseases causing RC dysfunction often present with short stature, which indicates that RC activity may be essential for cartilage-mediated skeletal growth. To elucidate the role of the mitochondrial RC in cartilage growth and pathology, we generated mice with impaired RC function in cartilage. These mice develop normally until birth, but their later growth is retarded. A detailed molecular analysis revealed that metabolic signaling and extracellular matrix formation is disturbed and induces cell death at the cartilage-bone junction to cause a chondrodysplasia-like phenotype. Hence, the results demonstrate the overall importance of the metabolic switch from fetal glycolysis to postnatal RC activation in growth plate cartilage and explain why RC dysfunction can cause short stature in children with mitochondrial diseases., (© 2019 Holzer et al.)

Objective: Heterozygous pathogenic variants in the COL2A1 gene result in several clinical features including impaired skeletal growth, ocular and otolaryngological abnormalities. Missense mutations in the triple helical region of the COL2A1 protein have been associated with lethal spondyloepiphyseal dysplasia (SED). In this study, we aimed to identify the underlying cause of a case of SED congenita (SEDC) in a 27-month-old child. Materials and Methods: A patient who was diagnosed initially with osteochondrodysplasia underwent a detailed clinical and radiological examination to obtain a conclusive diagnosis. The patient did not show any clinical features of hypochondrogenesis. Whole exome sequencing of the COL2A1 gene was carried out to identify the underlying genetic cause of the disorder. Results: Variant annotation and filtration detected a heterozygous missense mutation c.1357G>A (p.G453S) in the exon 21 of the COL2A1 gene of the proband which was confirmed by Sanger sequencing. Neither parent carried the mvariant suggesting this was a new mutation. Conclusion: The COL2A1 mutation (c.1357G>A), identified in this case, results in more mild phenotype than other missense mutations in exon 21 which are known to cause lethal hypochondrogenesis. We showed, for the first time, that a missense mutation (p.G453S) in the triple helical region of the alpha 1 (II) chain of the COL2A1 protein underlies SEDC and is not always lethal.

Objective: Hyaline cartilage degenerative pathologies induce morphologic and biomechanical changes resulting in cartilage tissue damage. In pursuit of therapeutic options, electrical and mechanical stimulation have been proposed for improving tissue engineering approaches for cartilage repair. The purpose of this review was to highlight the effect of electrical stimulation and mechanical stimuli in chondrocyte behavior., Design: Different information sources and the MEDLINE database were systematically revised to summarize the different contributions for the past 40 years., Results: It has been shown that electric stimulation may increase cell proliferation and stimulate the synthesis of molecules associated with the extracellular matrix of the articular cartilage, such as collagen type II, aggrecan and glycosaminoglycans, while mechanical loads trigger anabolic and catabolic responses in chondrocytes., Conclusion: The biophysical stimuli can increase cell proliferation and stimulate molecules associated with hyaline cartilage extracellular matrix maintenance.

Objective:: To characterize the morphology of chondrocytes and the expression and secretion of active collagen II by these cells cultured within a regenerated silk fibroin film. Silk fibroin film cytocompatibility and the effect of silk fibroin on chondrocytes in vitro were also evaluated., Methods:: Chondrocytes were transfected with a lentivirus containing a green fluorescent protein marker and cultured within a regenerated silk fibroin film. Effects on chondrocyte adhesion, growth, and expression of functional collagen II were assessed in vitro by analysis with immunofluorescent histochemistry and laser scanning confocal microscopy., Results:: The results of this study showed that the regenerated silk fibroin film had no cytotoxic effect on chondrocytes. The regenerated silk fibroin film facilitated the adhesion of chondrocytes with typical morphology. Chondrocytes cultured within silk fibroin films exhibited the expression of collagen II in vitro., Conclusion:: Regenerated silk fibroin film was found to be an excellent biomaterial with good cytocompatibility for chondrocytes, because these cells remained functional and maintained normal cell morphology when cultured in silk-based biomaterials. These results suggest that silk-based chondrocyte biomaterial complexes may provide a feasible and functional biomaterial for repairing clinical cartilage defects.

Human life expectancy has been steadily increasing at a rapid rate, but this increasing life span also brings about increases in diseases, dementia, and disability. A global burden of disease 2010 study revealed that hip and knee osteoarthritis ranked the 11th highest in terms of years lived with disability. Wear and tear can greatly influence the quality of life during ageing. In particular, wear and tear of the articular cartilage have adverse effects on joints and result in osteoarthritis. The articular cartilage uses longevity of type II collagen as the foundation around which turnover of proteoglycans and the homeostatic activity of chondrocytes play central roles thereby maintaining the function of articular cartilage in the ageing. The longevity of type II collagen involves a complex interaction of the scaffolding needs of the cartilage and its biochemical, structural and mechanical characteristics. The covalent cross-linking of heterotypic polymers of collagens type II, type IX and type XI hold together cartilage, allowing it to withstand ageing stresses. Discerning the biological clues in the armamentarium for preserving cartilage appears to be collagen cross-linking. Therapeutic methods to crosslink in in-vivo are non-existent. However intra-articular injections of polyphenols in vivo stabilize the cartilage and make it resistant to degradation, opening a new therapeutic possibility for prevention and intervention of cartilage degradation in osteoarthritis of aging., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The present study aimed to investigate the therapeutic effect of injections of local bone marrow mesenchymal stem cells (BMSCs) on osteoarthritis (OA) of the temporomandibular joint (TMJ) and to explore the role of stromal cell-derived factor 1 (SDF-1) and regulated on activation, normal T-cell expressed and secreted (RANTES) in this effect. Fundamentally, OA of the TMJ was induced by unilateral anterior crossbite in mice. Exogenous green fluorescent protein-labeled BMSCs (GFP-BMSCs) were weekly injected into the TMJ region for 4, 8, and 12 wk. The reparative effects of exogenous GFP-BMSCs were investigated by morphological observation and micro-computed tomography. The differentiation of GFP-BMSCs in the cartilage was examined by double immunofluorescence of GFPs with type II collagen, and the expression of related factors in the condylar cartilage was quantified by real-time polymerase chain reaction. The role of RANTES and SDF-1 in the therapeutic effect of exogenous BMSCs was examined by both in vitro and in vivo studies. The OA cartilage of the TMJ displays a synchronous increase in SDF-1 and RANTES expression and a higher capability of attracting the migration of GFP-BMSCs. The implanted GFP-BMSCs differentiated into type II collagen-positive cells and reversed cartilage degradation and subchondral bone loss in mice with OA of the TMJ. The migration of GFP-BMSCs towards OA cartilage and the rescuing effect of GFP-BMSC injections were impaired by the inhibitors of C-X-C chemokine receptor type 4 (CXCR4) and C-C chemokine receptor type 1 (CCR1), which are the receptors of SDF-1 and RANTES, respectively. Our data indicated that SDF-1/CXCR4 and RANTES/CCR1 signals are pivotal and function synergistically in the recruitment of GFP-BMSCs towards degraded cartilage in mice OA of the TMJ., (© International & American Associations for Dental Research 2015.)

Objective: In healthy joints, a zone of calcified cartilage (ZCC) provides the mechanical integration between articular cartilage and subchondral bone. Recapitulation of this architectural feature should serve to resist the constant shear force from the movement of the joint and prevent the delamination of tissue-engineered cartilage. Previous approaches to create the ZCC at the cartilage-substrate interface have relied on strategic use of exogenous scaffolds and adhesives, which are susceptible to failure by degradation and wear. In contrast, we report a successful scaffold-free engineering of ZCC to integrate tissue-engineered cartilage and a porous biodegradable bone substitute, using sheep bone marrow stromal cells (BMSCs) as the cell source for both cartilaginous zones., Design: BMSCs were predifferentiated to chondrocytes, harvested and then grown on a porous calcium polyphosphate substrate in the presence of triiodothyronine (T3). T3 was withdrawn, and additional predifferentiated chondrocytes were placed on top of the construct and grown for 21 days., Results: This protocol yielded two distinct zones: hyaline cartilage that accumulated proteoglycans and collagen type II, and calcified cartilage adjacent to the substrate that additionally accumulated mineral and collagen type X. Constructs with the calcified interface had comparable compressive strength to native sheep osteochondral tissue and higher interfacial shear strength compared to control without a calcified zone., Conclusion: This protocol improves on the existing scaffold-free approaches to cartilage tissue engineering by incorporating a calcified zone. Since this protocol employs no xenogeneic material, it will be appropriate for use in preclinical large-animal studies., (Copyright © 2015 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.)

Aims: To investigate the association between type II collagen fragments and the presence of knee osteoarthritis (OA) in the Tunisian population and to determine whether this biomarker can predict X-ray progression of this disease., Methods: Type II collagen C-telopeptide (uCTX-II) and helical peptide (sHelix-II) were assessed in 125 female patients with knee osteoarthritis aged 54 ± 8 years over 2 years and 57 female age-matched controls. The markers were measured at baseline, 1 and 2 yrs' follow-up corresponding to x-ray time points., Results: Only urinary CTX-II values were significantly 48% higher in knee OA patients compared with controls (p=0.001). The longitudinal changes over 2 yrs in Helix-II were also significantly associated with Joint Space Narrowing: JSN (p=0.03). Over the 2-yr study period average CTX-II levels were not significantly higher in progressor compared with non-progressor (339.96 vs 256.00; NS)., Conclusion: The data presented here suggest that CTX-II may be useful to identify patients with knee OA. These results demonstrate significantly association between progression of this disease and alterations levels of Helix-II.

The objective of this study was to determine the effects of hydrostatic pressure (HP) on the biochemical properties and gene expression of mesenchymal stem cells (MSCs) on scaffolds for cartilage tissue engineering composed of poly(caprolactone) (PCL) poly(vinyl alcohol) (PVA) gelatin (GEL) semi interpenetrating polymer network (semi-IPN). The MSCs were cultured on PCL-PVA-GEL semi-IPN scaffolds in two groups (A and B) for 7 and 21 days, respectively, and then loaded with hydrostatic pressure (5 MPa, 0.5 Hz) for 2 h per day for the period of 7 days and compared with two non-loaded groups (C and D) as controls. DMMB and real-time PCR analysis for assaying cartilage-specific extracellular matrix (ECM) gene markers were carried out. According to the results, there were no significant differences in GAG amounts between the loaded and non-loaded constructs were observed after 14 days. However, significant and considerable increases in the expression amount of type II collagen mRNA levels in group A ( from 2.43 × 10-4 ± 5.32 × 10-5 to 2.09 × 10-3 ± 1.07 × 10-4 time), and in group B (from 3.04 × 10-4 ± 4.31 × 10-5 to 2.08 × 10-3 ± 1.59 × 10-4 time) in comparison with non-loaded groups (C and D) were observed, respectively. Results showed the beneficial role of hydrostatic pressure on the increase of type II collagen mRNA levels in articular cartilage tissue engineering.

Objective: Currently, our ability to treat intervertebral disc (IVD) degeneration is hampered by an incomplete understanding of disc development and aging. The specific function of matricellular proteins, including CCN2, during these processes remains an enigma. The aim of this study was to determine the tissue-specific localization of CCN proteins and to characterize their role in IVD tissues during embryonic development and age-related degeneration by using a mouse model of notochord-specific CCN2 deletion., Methods: Expression of CCN proteins was assessed in IVD tissues from wild-type mice beginning on embryonic day 15.5 to 17 months of age. Given the enrichment of CCN2 in notochord-derived tissues, we generated notochord-specific CCN2-null mice to assess the impact on the IVD structure and extracellular matrix composition. Using a combination of histologic evaluation and magnetic resonance imaging (MRI), IVD health was assessed., Results: Loss of the CCN2 gene in notochord-derived cells disrupted the formation of IVDs in embryonic and newborn mice, resulting in decreased levels of aggrecan and type II collagen and concomitantly increased levels of type I collagen within the nucleus pulposus. CCN2-knockout mice also had altered expression of CCN1 (Cyr61) and CCN3 (Nov). Mirroring its role during early development, notochord-specific CCN2 deletion accelerated age-associated degeneration of IVDs., Conclusion: Using a notochord-specific gene targeting strategy, this study demonstrates that CCN2 expression by nucleus pulposus cells is essential to the regulation of IVD development and age-associated tissue maintenance. The ability of CCN2 to regulate the composition of the intervertebral disc suggests that it may represent an intriguing clinical target for the treatment of disc degeneration., (Copyright © 2013 by the American College of Rheumatology.)

Objective: Osteoarthritis (OA) is a degenerative disease resulting in severe joint cartilage destruction and disability. While the mechanisms underlying the development and progression of OA are poorly understood, gene mutations have been identified within cartilage-related signaling molecules, implicating impaired cell signaling in OA and joint disease. The Notch pathway has recently been identified as a crucial regulator of growth plate cartilage development, and components are expressed in joint tissue. This study was undertaken to investigate a novel role for Notch signaling in joint cartilage development, maintenance, and the pathogenesis of joint disease in a mouse model., Methods: We performed the first mouse gene study in which the core Notch signaling component, RBP-Jκ, was tissue specifically deleted within joints. The Prx1Cre transgene removed Rbpjk loxP-flanked alleles in mesenchymal joint precursor cells, while the Col2Cre(ERT2) transgene specifically deleted Rbpjk in postnatal chondrocytes. Murine articular chondrocyte cultures were also used to examine Notch regulation of gene expression., Results: Loss of Notch signaling in mesenchymal joint precursor cells did not affect embryonic joint development in mice, but rather, resulted in an early, progressive OA-like pathology. Additionally, partial loss of Notch signaling in murine postnatal cartilage resulted in progressive joint cartilage degeneration and an age-related OA-like pathology. Inhibition of Notch signaling altered the expression of the extracellular matrix (ECM)-related factors type II collagen (COL2A1), proteoglycan 4, COL10A1, matrix metalloproteinase 13, and ADAMTS., Conclusion: Our findings indicate that the RBP-Jκ-dependent Notch pathway is a novel pathway involved in joint maintenance and articular cartilage homeostasis, a critical regulator of articular cartilage ECM-related molecules, and a potentially important therapeutic target for OA-like joint disease., (Copyright © 2013 by the American College of Rheumatology.)

Transgenic overexpression of the Notch1 intracellular domain inhibits osteoblast differentiation and causes osteopenia, and inactivation of Notch1 and Notch2 increases bone volume transiently and induces osteoblastic differentiation. However, the biology of Notch is cell-context-dependent, and consequences of Notch activation in cells of the osteoblastic lineage at various stages of differentiation and in osteocytes have not been defined. For this purpose, Rosa(Notch) mice, where a loxP-flanked STOP cassette placed between the Rosa26 promoter and the NICD coding sequence, were crossed with transgenics expressing the Cre recombinase under the control of the Osterix (Osx), Osteocalcin (Oc), Collagen 1a1 (Col2.3), or Dentin matrix protein1 (Dmp1) promoters. At 1 month, Osx-Cre;Rosa(Notch) and Oc-Cre;Rosa(Notch) mice exhibited osteopenia due to impaired bone formation. In contrast, Col2.3-Cre;Rosa(Notch) and Dmp1-Cre;Rosa(Notch) exhibited increased femoral trabecular bone volume due to a decrease in osteoclast number and eroded surface. In the four lines studied, cortical bone was either not present, was porous, or had the appearance of trabecular bone. Oc-Cre;Rosa(Notch) and Col2.3-Cre;Rosa(Notch) mice exhibited early lethality so that their adult phenotype was not established. At 3 months, Osx-Cre;Rosa(Notch) and Dmp1-Cre;Rosa(Notch) mice displayed increased bone volume, and increased osteoblasts although calcein-demeclocycline labels were diffuse and fragmented, indicating abnormal bone formation. In conclusion, Notch effects in the skeleton are cell-context-dependent. When expressed in immature osteoblasts, Notch arrests their differentiation, causing osteopenia, and when expressed in osteocytes, it causes an initial suppression of bone resorption and increased bone volume, a phenotype that evolves as the mice mature.

Recent analyses of Col2a1-Cre; ROSA26R reporter mice showed that synovial fibroblasts in 7-day mice were LacZ positive, due to a history of Col2a1-Cre expression conferred by their origin in the interzone of the developing joint. We have examined LacZ staining in adult Col2a1-Cre(+/0); ROSA26R(LacZ) mice, with and without inflammatory arthritis, and found that synovial fibroblasts in normal and inflamed synovium are LacZ positive, but Cre negative. Our results suggest that Cre-mediated recombination in joint interzone cells during development endure in adult synovial cells despite the absence of ongoing Cre expression. These findings have important implications and applications for the study of synovial inflammation in models of experimental arthritis., (Crown Copyright © 2012. Published by Elsevier Ltd. All rights reserved.)

Objective: Increasing evidence points to a strong genetic component to osteoarthritis (OA) and that certain changes that occur in osteoarthritic cartilage recapitulate the developmental process of endochondral ossification. As zebrafish are a well validated model for genetic studies and developmental biology, our objective was to establish the spatiotemporal expression pattern of a number of OA susceptibility genes in the larval zebrafish providing a platform for functional studies into the role of these genes in OA., Design: We identified the zebrafish homologues for Mcf2l, Gdf5, PthrP/Pthlh, Col9a2, and Col10a1 from the Ensembl genome browser. Labelled probes were generated for these genes and in situ hybridisations were performed on wild type zebrafish larvae. In addition, we generated transgenic reporter lines by modification of bacterial artificial chromosomes (BACs) containing full length promoters for col2a1 and col10a1., Results: For the first time, we show the spatiotemporal expression pattern of Mcf2l. Furthermore, we show that all six putative OA genes are dynamically expressed during zebrafish larval development, and that all are expressed in the developing skeletal system. Furthermore, we demonstrate that the transgenic reporters we have generated for col2a1 and col10a1 can be used to visualise chondrocyte hypertrophy in vivo., Conclusion: In this study we describe the expression pattern of six OA susceptibility genes in zebrafish larvae and the generation of two new transgenic lines marking chondrocytes at different stages of maturation. Moreover, the tools used demonstrate the utility of the zebrafish model for functional studies on genes identified as playing a role in OA., (Copyright © 2012 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.)

The role of the renin-angiotensin system (RAS) in vasoregulation is well established, but a localized RAS exists in multiple tissues and exerts diverse functions including autonomic control and thermogenesis. The role of the RAS in the maintenance and function of skeletal muscle is not well understood, especially the role of angiotensin peptides, which appear to contribute to muscle atrophy. We tested the hypothesis that mice lacking the angiotensin type 1A receptor (AT(1A)(-/-)) would exhibit enhanced whole body and skeletal muscle function and improved regeneration after severe injury. Despite 18- to 20-wk-old AT(1A)(-/-) mice exhibiting reduced muscle mass compared with controls (P < 0.05), the tibialis anterior (TA) muscles produced a 25% higher maximum specific (normalized) force (P < 0.05). Average fiber cross-sectional area (CSA) and fiber oxidative capacity was not different between groups, but TA muscles from AT(1A)(-/-) mice had a reduced number of muscle fibers as well as a higher proportion of type IIx/b fibers and a lower proportion of type IIa fibers (P < 0.05). Measures of whole body function (grip strength, rotarod performance, locomotor activity) were all improved in AT(1A)(-/-) mice (P < 0.05). Surprisingly, the recovery of muscle mass and fiber CSA following myotoxic injury was impaired in AT(1A)(-/-) mice, in part by impaired myoblast fusion, prolonged collagen infiltration and inflammation, and delayed expression of myogenic regulatory factors. The findings support the therapeutic potential of RAS inhibition for enhancing whole body and skeletal muscle function, but they also reveal the importance of RAS signaling in the maintenance of muscle mass and for normal fiber repair after injury.

The present chronological investigation assessed the distribution of type II collagen expression in the developing mouse mandibular condyle using immunohistochemical staining with respect to the anatomy of the anlage of the mandibular condyle, the histological characteristics of which were disclosed in our previous investigation. We analyzed fetuses, obtained by cross breeding of ICR strain mice, between 14.0 and 19.0 days post-conception (dpc) and pups on 1, 3, and 5 days post-natal (dpn) using immunohistochemical staining with 2 anti-type II collagen antibodies. The expression of type II collagen was first detected at 15.0 dpc in the lower part of the hypertrophic chondrocyte zone; thereafter, this type II collagen-positive layer was expanded and intensified (P1 layer). At 17.0 dpc, we identified a type II collagen-negative layer (N layer) around the P1 layer and we also identified another newly formed type II collagen-positive layer (P, layer) on the outer surface of the N layer. The most typical and conspicuous 3-layered distribution was observed at 1 dpn; thereafter, there was a reduction in the intensity of expression, and with it, the demarcation between the layers was weakened by 5 dpn. The P1 layer was derived from the central region of the core cell aggregate of the anlage of the mandibular condyle and participated in endochondral bone formation. The N layer was derived from the fringe of the core cell aggregate of the anlage, formed the bone collar at the side of the condyle by intramembranous bone formation, and showed a high level of proliferative activity at the vault. The P2 layer was formed from the outgrowth of the N layer, and could be considered as the secondary cartilage. The intensive expression of type II collagen from 17.0 dpc to 3 dpn was detected in the fibrous sheath covering the condylar head, which is derived from the peripheral cell aggregate of the anlage. Since its expression in the fibrous sheath was not detected in the neighboring section in the absence of hyaluronidase digestion, some changes in the extracellular matrix of the fibrous sheath appear to participate in the generation of the lower joint space. The results of the present investigation indicate that further studies are required to fully characterize the development of the mouse mandibular condyle.

Aiming at regeneration of articular cartilage, we have established stable lines of mouse chondrogenic ATDC5 cells expressing green fluorescent protein under the control of type II collagen promoter fused with four repeats of a SOX9 enhancer (COL2A1-GFP) , as a monitoring system for chondrogenic differentiation. A screening of natural and synthetic compound libraries using the system identified some novel compounds. Combined with cell-sheet technology, a novel small compound was applied to the treatment of full-thickness knee cartilage defects in murine and canine models.

To identify a group of transcription factors required for chondrogenesis, several researchers tried to detect a chondrocyte-specific enhancer element of Col2a1 gene. Benoit de Crombrugghe's group finally found out 48bp in the first intron of Col2a1 gene as a chondrocyte-specific enhancer element, and moreover they also concluded that binding of homodimer of Sox9 and homo-or heterodimer of Sox5÷Sox6 to this element is indispensable for Col2a1 transcription in chodrocytes. Furthermore, mouse genetic approaches revealed that Sox9, Sox5 and Sox6 are required for chondrogenesis, leading to conclusion that these Sox transcription factors are master regulators in chondrogenesis. Recent studies showed that p300÷CBP, Trap230 (med12) , Wwp2, and Med25 are components of transcriptional machinery of Sox9 in chondrogenesis.

The skeleton of zebrafish fins consists of lepidotrichia and actinotrichia. Actinotrichia are fibrils located at the tip of each lepidotrichia and play a morphogenetic role in fin formation. Actinotrichia are formed by collagens associated with non-collagen components. The non-collagen components of actinotrichia (actinodins) have been shown to play a critical role in fin to limb transition. The present study has focused on the collagens that form actinotrichia and their role in fin formation. We have found actinotrichia are formed by Collagen I plus a novel form of Collagen II, encoded by the col2a1b gene. This second copy of the collagen II gene is only found in fishes and is the only Collagen type II expressed in fins. Both col1a1a and col2a1b were found in actinotrichia forming cells. Significantly, they also expressed the lysyl hydroxylase 1 (lh1) gene, which encodes an enzyme involved in the post-translational processing of collagens. Morpholino knockdown in zebrafish embryos demonstrated that the two collagens and lh1 are essential for actinotrichia and fin fold morphogenesis. The col1a1 dominant mutant chihuahua showed aberrant phenotypes in both actinotrichia and lepidotrichia during fin development and regeneration. These pieces of evidences support that actinotrichia are composed of Collagens I and II, which are post-translationally processed by Lh1, and that the correct expression and assembling of these collagens is essential for fin formation. The unique collagen composition of actinotrichia may play a role in fin skeleton morphogenesis., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Antibodies against citrullinated proteins (ACPAs) are highly specific for RA. Since the discovery of these antibodies, several of studies that focused on the presence and identity of citrullinated proteins in the joints of RA patients have been carried out. The best-known antigens that bind ACPAs are citrullinated filaggrin, Type II collagen (CII), α-enolase, fibrinogen and vimentin. This review compares citrullinated filaggrin, CII, α-enolase and fibrinogen with vimentin in their contribution to ACPA triggering, and gives an overview of the literature in which the role of citrullinated and non-citrullinated vimentin in the onset of ACPA production and the pathogenesis of RA is discussed.

Aims: We previously reported that Lactobacillus casei (L. casei) has beneficial effects in experimental rheumatoid arthritis (RA) by suppressing inflammatory immune responses. The major purpose of this study was to evaluate therapeutic effects of L. casei on pathological responses in experimental rodent model of osteoarthritis (OA)., Main Methods: Experimental OA was induced by intra-articular injection of monosodium iodoacetate (MIA) in Wistar rats. L. casei alone or together with type II collagen (CII) and glucosamine (Gln) was orally administered into OA rats. The pathophysiological aspects of OA were investigated by analyzing mechanical hyperalgesia and histology of articular tissues. Expression of inflammatory molecules was analyzed in CD4(+) T cells, synovial fibroblasts, and chondrocytes by quantitative real-time PCR., Key Findings: Oral administration of L. casei together with CII and Gln more effectively reduced pain, cartilage destruction, and lymphocyte infiltration than the treatment of Gln or L. casei alone. This co-administration also decreased expression of various pro-inflammatory cytokines (interleukin-1β (IL-1β), IL-2, IL-6, IL-12, IL-17, tumor necrosis factor-α (TNF-α), and interferon-γ (IFN-γ)) and matrix metalloproteinases (MMP1, MMP3, and MMP13), while up-regulating anti-inflammatory cytokines (IL-4 and IL-10). These results are concomitant with reduced translocation of NF-κB into the nucleus and increased expression of the tissue inhibitor of MMP1 (TIMP1) and CII in chondrocytes., Significance: Our study provides evidence that L. casei could act as a potent nutraceutical modulator for OA treatment by reducing pain, inflammatory responses, and articular cartilage degradation., (Copyright © 2010 Elsevier Inc. All rights reserved.)

We investigated the role of transforming growth factor-beta activated kinase 1 (TAK1) in collagen II signaling in primary human chondrocytes (PHCs). We asked whether TAK1 acts as a modulator of collagen II signaling with respect to collagen-II-dependent induction of cyclooxigenase-2 (COX-2) in PHCs and release of PGE2 from PHCs. Therefore, PHCs were incubated with collagen II, and cells were then analyzed by RT-PCR for the expression of COX-2. ELISA was used to quantify PGE2 release. To examine the influence of TAK1 on these events, TAK1 gene silencing was performed by RNAi in PHCs prior to collagen II treatment. Results indicated that COX-2 gene expression and PGE2 release are specific outcomes of collagen II signaling and that both depend on TAK1 mediation. These findings are promising in that therapeutic inhibition of TAK1 might be used to reduce pain and relieve inflammatory symptoms that are common in osteoarthritis.

After extensively expanding in monolayer culture, the cultured chondrocytes become quiescent. The aim of this study was to establish the hypothesis that the phenotypic function of extensively expanded primary chondrocytes may be restored with extracellular matrix (ECM) compositions with or without growth factors. The restoring effects of these microenvironmental factors on the near quiescent passage 9 (P9) chondrocyte were investigated. The data showed that exogenous type I collagen and type II collagen at 1:1 ratio stimulate cell proliferation greatly while type II collagen alone was enough to revive most of cartilaginous functions of near quiescent P9 chondrocytes. Exogenous type II collagen by itself was more effective in restoring cell proliferation rate, elevating glycosaminoglycan (GAG) accumulation and promoting the re-expression of type II collagen mRNAs in the near quiescent chondrocytes. The supplement of P9 chondrocytes with type II collagen plus TGF-beta1 and IGF-I appeared essential for the re-expression of aggrecan and type II collagen mRNA in monolayer culture. In 3D type II collagen construct, P9 chondrocytes appeared healthy as chondrocytes and showed clear lacuna. However, in 3D type I collagen matrix, only some P9 chondrocytes exhibited lacuna. The cartilaginous microenvironments are crucial to restoring chondrocyte-phenotypic features of the quiescent or 'dedifferentiated' chondrocytes, implicating the potential of expanding a scarcity of healthy chondrocytes for cartilage repair or regeneration.

Genetic and developmental alterations associated with the evolution of amphibian direct development remain largely unexplored. Specifically, little is known of the underlying expression of skeletal regulatory genes, which may reveal early modifications to cranial ontogeny in direct-developing species. We describe expression patterns of three key skeletal regulators (runx2, sox9, and bmp4) along with the cartilage-dominant collagen 2alpha1 gene (col2a1) during cranial development in the direct-developing anuran, Eleutherodactylus coqui. Expression patterns of these regulators reveal transient skeletogenic anlagen that correspond to larval cartilages, but which never fully form in E. coqui. Suprarostral anlagen in the frontonasal processes are detected through runx2, sox9, and bmp4 expression. Previous studies have described these cartilages as missing from Eleutherodactylus cranial ontogeny. These transcriptionally active suprarostral anlagen fuse to the more posterior cranial trabeculae before they are detectable with col2a1 staining or with the staining techniques used in earlier studies. Additionally, expression of sox9 fails to reveal an early anterior connection between the palatoquadrate and the neurocranium, which is detectable through sox9 staining in Xenopus laevis embryos (a metamorphosing species). Absence of this connection validates an instance of developmental repatterning, where the larval quadratocranial commissure cartilage is lost in E. coqui. Expression of runx2 reveals dermal-bone precursors several developmental stages before their detection with alizarin red. This early expression of runx2 correlates with the accelerated embryonic onset of bone formation characteristic of E. coqui and other direct-developing anurans, but which differs from the postembryonic bone formation of most metamorphosing species. Together these results provide an earlier depiction of cranial patterning in E. coqui by using earlier markers of skeletogenic cell differentiation. These data both validate and modify previously reported instances of larval recapitulation and developmental repatterning associated with the evolution of anuran direct development.

Notch signalling, via its downstream mediators HES1 and HES5, regulates development of several different tissues. In vitro studies suggest that these genes are also involved in chondrogenesis and endochondral bone formation. In order to investigate the importance of HES1 and HES5 for these developmental processes, mice lacking chondrogenic expression of HES1 and HES5 were constructed by interbreeding HES5(-/-) mice homozygous for the floxed HES1 allele (HES1(flox/flox)) with COL2A1-Cre transgenic mice, creating conditional HES1;HES5 double mutant mice. The formation of cartilage and endochondral bone was studied in these mice using histological and immunohistochemical stainings, including Alcian Blue van Gieson, Safranin-O, modified Mallory Aniline Blue, tartrate-resistant acid phosphatase and collagen type II stainings. The mice were also studied using several different morphometrical analyses and the differentiation potential of the chondrocytes was evaluated in vitro. Unexpectedly, the conditional HES1;HES5 double mutant mice did not display impaired development of cartilage or endochondral bone. Lack of altered phenotype in the conditional HES1;HES5 double mutant mice can be explained either by the HES1 and HES5 genes not being involved in cartilage and endochondral bone development or by functional redundancy between the genes belonging to the family of HES genes: that is, disruption of one gene could be compensated for by the activity of another. Our results further shed light on the compensatory reserves available during the developing cartilage and bone., (Copyright 2010 S. Karger AG, Basel.)

Although the importance of knowing the magnitude of residual stress (RS) and its functional significance are widely recognized, there is still disagreement and confusion regarding the nature of physical mechanisms giving rise to RS in tissues and organs. Here an attempt is made to examine the various mechanisms which may be involved in producing RS, and to estimate their roles and significance based on previously published experimental observations. Two concepts are introduced. The first establishes a hierarchy of different possible RS producing mechanisms from the micro (local) level of the tissue space, through the meso-level of the whole tissue, to the macro (organ) one. Whereas micro-level RS seem to be present in all soft tissues, the existence of macro- and meso-level mechanisms are tissue and organ specific. The second concept introduced highlights the significance of tissue swelling as an RS producing mechanism in the local micro-level. The implications of RS mechanism hierarchy are discussed regarding the interpretations of commonly used experimental methods aimed to study RS or to estimate its magnitude. Of the three categories of RS mechanisms, the local micro-RS is the least understood. It is analyzed here in terms of the tissue's multiconstituent structure, in the framework of mixture theory. It is shown that the micro-RS can stem either from interactions between the solid tissue constituents or between its solids and its fluidlike matrix. The latter mode is associated with osmotic-driven tissue swelling. The feasibility of these two mechanisms is analyzed based on published observations and measured data. The analysis suggests that under conditions not too remote from the in vivo homeostatic one, osmotic-driven tissue swelling is a predominant RS producing mechanism. The analysis also suggests that a true stress-free configuration can be obtained only if all RS producing mechanisms are relieved, and outlines a manner by which this may be achieved.

Background: Articular cartilage is exposed to high mechanical loads under normal physiological conditions and articular chondrocytes regulate the composition of cartilaginous matrix, in response to mechanical signals. However, the intracellular pathways involved in mechanotransduction are still being defined. Using the well-characterized chondrocyte/agarose model system and dynamic compression, we report protocols for preparing and characterizing constructs of murine chondrocytes and agarose, and analyzing the effect of compression on steady-state level of mRNA by RT-PCR, gene transcription by gene reporter assay, and phosphorylation state of signalling molecules by Western-blotting. The mouse model is of particular interest because of the availability of a large choice of bio-molecular tools suitable to study it, as well as genetically modified mice., Results: Chondrocytes cultured in agarose for one week were surrounded by a newly synthesized pericellular matrix, as revealed by immunohistochemistry prior to compression experiments. This observation indicates that this model system is suitable to study the role of matrix molecules and trans-membrane receptors in cellular responsiveness to mechanical stress. The chondrocyte/agarose constructs were then submitted to dynamic compression with FX-4000C Flexercell Compression Plus System (Flexcell). After clearing proteins off agarose, Western-blotting analysis showed transient activation of Mitogen-activated protein kinases (MAPK) in response to dynamic compression. After assessment by capillary electrophoresis of the quality of RNA extracted from agarose, steady-state levels of mRNA expression was measured by real time PCR. We observed an up-regulation of cFos and cJun mRNA levels as a response to compression, in accordance with the mechanosensitive character observed for these two genes in other studies using cartilage explants submitted to compression. To explore further the biological response of mouse chondrocytes to the dynamic compression at the transcriptional level, we also developed an approach for monitoring changes in gene transcription in agarose culture by using reporter promoter constructs. A decrease in promoter activity of the gene coding for type II procollagen, the most abundant protein in cartilage, was observed in response to dynamic loading., Conclusion: The protocols developed here offer the possibility to perform an integrated analysis of the molecular mechanisms of mechanotransduction in chondrocytes, at the gene and protein level.

Study Design: Immunohistochemical analyses on the axial skeleton from wild type mice., Objective: In the clinic, we have previously observed cervical spine defects associated with deviations in the posterior part of the occipital bone and with morphologic and functional variations in the craniofacial skeleton. As examples, cervical spine fusions occurred frequently in patients with mandibular overjet and even more frequently and more caudally in the cervical spine in patients with sleep apnoea. The aims of the present study were to elucidate this association between the spine and the cranium by comparing gene expression domains of important developmental genes known to be involved in vertebral column formation with gene expression in the craniofacial region., Summary of Background Data: This is the first study looking specifically on gene expression in the basilar part of the occipital bone that is formed around the cranial part of the notochord, thus connecting the spine and the craniofacial skeleton., Methods: The material consisted of 4 mouse embryos p.c. day 13.5, NMRI wild-type mice, from the same litter. The body axis, the cranial base, and the craniofacial area were studied by immunohistochemical analyses using Collagen II, Pax9, Pax1, and Noggin antibodies., Results: Pax1 expression was highly similar in the posterior part of the occipital bone and in the vertebral column, indicating that the basilar part of the occipital bone from a developmental standpoint can be considered the uppermost vertebra. Pax9 and Noggin expression domains were in accordance with those described previously., Conclusion: The present study supports that the basilar part of the occipital bone may be regulated by similar developmental mechanisms as the vertebral column and may thus be regarded the uppermost vertebra. Thus, the clinically observed association between the cervical column and the craniofacial area has been proved by immunohistochemical methods.

The growing field of skeletal developmental biology provides new molecular markers for the cellular precursors of cartilage and bone. These markers enable resolution of early features of skeletal development that are otherwise undetectable through conventional staining techniques. This study investigates mRNA distributions of skeletal regulators runx2 and sox9 along with the cartilage-dominant collagen 2(alpha)1 (col2a1) in embryonic limbs of the direct-developing anuran, Eleutherodactylus coqui. To date, distributions of these genes in the limb have only been examined in studies of the two primary amniote models, mouse and chicken. In E. coqui, expression of transcription factors runx2 and sox9 precedes that of col2a1 by 0.5-1 developmental stage (approximately 12-24 h). Limb buds of E. coqui contain unique distal populations of both runx2- and sox9-expressing cells, which appear before formation of the primary limb axis and do not express col2a1. The subsequent distribution of col2a1 reveals a primary limb axis similar to that described for Xenopus laevis. Precocious expression of both runx2 and sox9 in the distal limb bud represents a departure from the conserved pattern of proximodistal formation of the limb skeleton that is central to prevailing models of vertebrate limb morphogenesis. Additionally, runx2 is expressed in the early joint capsule perichondria of the autopod and in the perichondria of long bones well before periosteum formation. The respective distributions of sox9 and col2a1 do not reveal the joint perichondria but instead are expressed in the fibrocartilage that fills each presumptive joint capsule. These distinct patterns of runx2- and sox9-expressing cells reveal precursors of chondrocyte and osteoblast lineages well before the appearance of mature cartilage and bone.

The coracoacromial ligament forms part of the coracoacromial arch and is implicated in impingement syndrome and acromial spur formation. Here, we describe its structure and the composition of its extracellular matrix. Ligaments were obtained from 15 cadavers, nine from older people (average age 74.7 years) and six from younger individuals (average age 24.2 years). Cryosections of methanol-fixed tissue were cut and sections were immunolabelled with monoclonal antibodies against collagens, glycosaminoglycans, proteoglycans, matrix proteins and neurofilament proteins. Both ligament entheses were highly fibrocartilaginous and immunolabelled strongly for type II collagen, aggrecan and link protein. The area of labelling was more extensive in older people. However, fibrocartilage also characterized the ligament midsubstance, particularly with increased age. Signs of fibrocartilage degeneration were more common in older people. Ligament fat (containing blood vessels and nerve fibers) was conspicuous in both age groups, especially between fiber bundles at the entheses. We conclude that fibrocartilage is a normal feature but becomes more pronounced with age. It is not necessarily pathological, for it simply indicates that the ligament is subject to compression and/or shear. Nevertheless, the prominence of fibrocartilage at the acromial enthesis may relate to the frequency with which enthesophytes develop.

Objective: To determine whether engineered cartilage generated by nasal chondrocytes (ECN) is responsive to different regimens of loading associated with joint kinematics and previously shown to be stimulatory of engineered cartilage generated by articular chondrocytes (ECA)., Methods: Human nasal and articular chondrocytes, harvested from 5 individuals, were expanded and cultured for 2 weeks into porous polymeric scaffolds. The resulting ECN and ECA were then maintained under static conditions or exposed to the following loading regimens: regimen 1, single application of cyclic deformation for 30 minutes; regimen 2, intermittent application of cyclic deformation for a total of 10 days, followed by static culture for 2 weeks; regimen 3, application of surface motion for a total of 10 days., Results: Prior to loading, ECN constructs contained significantly higher amounts of glycosaminoglycan (GAG) and type II collagen compared with ECA constructs. ECN responded to regimen 1 by increasing collagen and proteoglycan synthesis, to regimen 2 by increasing the accumulation of GAG and type II collagen as well as the dynamic modulus, and to regimen 3 by increasing the expression of superficial zone protein, at the messenger RNA level and the protein level, as well as the release of hyaluronan. ECA constructs were overall less responsive to all loading regimens, likely due to the lower extracellular matrix content., Conclusion: Human ECN is responsive to physical forces resembling joint loading and can up-regulate molecules typically involved in joint lubrication. These findings should prompt future in vivo studies exploring the possibility of using nasal chondrocytes as a cell source for articular cartilage repair.

Matrix vesicles (MVs) are involved in de novo mineral formation by nearly all vertebrate tissues. The driving force for MV mineralization is a nucleational core composed of three principal constituents: (i) amorphous calcium phosphate (ACP), complexed in part with phosphatidylserine (PS) to form (ii) calcium-phosphate-lipid complexes (CPLX), and (iii) annexin A5 (AnxA5), the principal lipid-dependent Ca(2+)-binding protein in MVs. We describe methods for reconstituting the nucleational core using a biomimetic approach and for analyzing the kinetics of its induction of mineral formation. The method is based on light scattering by the nascent crystallites at 340 nm and monitors mineral formation at regular intervals without disturbing the system using an automated plate reader. It yields precise replicate values that typically agree within less than 5%. As with MVs, mineral formation by the synthetic complex follows a sigmoidal pattern; following a quiescent induction period, rapid formation ensues for a limited time, followed by a distinct decline in rate that continues to slow, ultimately reaching a maximal asymptotic value. Key to quantization of mineral formation is the use of first-derivative analysis, which defines the induction time, the rate and the amount of initial mineral formation. Furthermore, using a five-parameter logistic curve-fitting algorithm, the maximal amount of mineral formation can be predicted accurately. Using these methods, we document the dramatic finding that AnxA5 synergistically activates PS-CPLX, transforming it from a very weak nucleator of mineral formation to a potent one. The methods presented should enable systematic study of the effects of numerous other factors thought to contribute to mineral formation.

Neutral sphingomyelinase SMPD3 (nSMase2), a sphingomyelin phosphodiesterase, resides in the Golgi apparatus and is ubiquitously expressed. Gene ablation of smpd3 causes a generalized prolongation of the cell cycle that leads to late embryonic and juvenile hypoplasia because of the SMPD3 deficiency in hypothalamic neurosecretory neurons. We show here that this novel form of combined pituitary hormone deficiency is characterized by the perturbation of the hypothalamus-pituitary growth axis, associated with retarded chondrocyte development and enchondral ossification in the epiphyseal growth plate. To study the contribution by combined pituitary hormone deficiency and by the local SMPD3 deficiency in the epiphyseal growth plate to the skeletal phenotype, we introduced the full-length smpd3 cDNA transgene under the control of the chondrocyte-specific promoter Col2a1. A complete rescue of the smpd3(-/-) mouse from severe short-limbed skeletal dysplasia was achieved. The smpd3(-/-) mouse shares its dwarf and chondrodysplasia phenotype with the most common form of human achondrodysplasia, linked to the fibroblast-growth-factor receptor 3 locus, not linked to deficits in the hypothalamic-pituitary epiphyseal growth plate axis. The rescue of smpd3 in vivo has implications for future research into dwarfism and, particularly, growth and development of the skeletal system and for current screening and future treatment of combined dwarfism and chondrodysplasia.

Objective: Interleukin-1beta (IL-1beta) stimulates collagenase-1 (Matrix Metalloproteinase-1 (MMP-1)) expression in articular chondrocytes, leading to cleavage of type II collagen and irreversible cartilage degradation. The nuclear factor-kappa B (NF-kappaB) pathway is potently activated in IL-1beta-stimulated cells and has been implicated as an intermediate in MMP-1 gene expression. However, the roles of individual NF-kappaB family members during IL-1beta-induced MMP-1 gene expression have not been defined., Results: To address the relationship between the NF-kappaB pathway and MMP-1 gene activation in chondrocytes, primary cultured human articular chondrocyte cultures (HAC) and SW-1353 cells were stimulated with IL-1beta over a 24-h time course and MMP-1, NF-kappaB1, NF-kappaB2 and RelA gene expression was assayed. IL-1beta-induced MMP-1 expression was comparable in HAC and SW-1353 cells both temporally and quantitatively. MMP-1 gene expression was mirrored by increases in NF-kappaB gene expression, and inhibition of NF-kappaB nuclear translocation with dominant-negative IkappaBalpha reduced IL-1beta-dependent MMP-1 gene expression. IL-1beta activated the NF-kappaB pathway in chondrocytes, both through phosphorylation and transient degradation of IkappaBalpha, as well as through sustained phosphorylation of RelA. Small inhibitory RNAs (siRNA) specific for RelA resulted in significant reduction of MMP-1 mRNA, whereas siRNA for NF-kappaB1 and NF-kappaB2 augmented IL-1beta-induced MMP-1 expression., Conclusions: Our data demonstrate that IL-1beta activation of the NF-kappaB pathway is required for IL-1beta induction of MMP-1 in chondrocytes and that RelA can work independently of NF-kappaB1 or NF-kappaB2 to activate this gene expression program.

Resveratrol is a polyphenolic phytoalexin that is present in various fruits, in the skin of red grapes and peanuts. Recent studies have shown that resveratrol exhibits potent antioxidant properties and is able to exert anti-inflammatory and anti-catabolic properties in several cell types. The pro-inflammatory cytokine interleukin-1beta (IL-1beta) plays a pivotal role in the pathogenesis of osteoarthritis (OA) in humans and animals. In this article we investigated whether resveratrol is able to block the effects of IL-1beta, specifically the activation of caspase-3 and subsequent cleavage of poly (ADP-ribose) polymerase (PARP) in human articular chondrocytes. Cultures of human chondrocytes were prestimulated with 10 ng/mL IL-1beta for 1, 12, and 24 h before being co-treated with IL-1beta and 100 microM resveratrol or 50 microM of the caspase inhibitor Z-DEVD-FMK for 1, 12, and 24 h, respectively in vitro. Resveratrol significantly reduced the IL-1beta-induced inhibition of expression of cartilage-specific collagen type II and signal transduction receptor beta1-integrin in a time-dependent manner. Incubation of chondrocytes with IL-1beta resulted in the activation of caspase-3 and PARP cleavage. These effects were abolished through co-treatment with resveratrol. Furthermore, co-treatment of IL-1beta-stimulated cells with the caspase inhibitor Z-DEVD-FMK blocked activation of caspase-3 and PARP cleavage, suggesting that this process is a caspase-dependent pathway. In summary, our results confirm that resveratrol is an effective inhibitor of chondrocyte apoptosis in vitro. These findings suggest that this dietary polyphenolic compound may have future applications in the nutraceutical-based therapy of human and animal OA.

The molecular mechanism triggering the organization of endothelial cells (ECs) in multicellular tubules is mechanistically still poorly understood. We demonstrate that cell-autonomous endothelial functions of the AP-1 subunit JunB are required for proper endothelial morphogenesis both in vivo in mouse embryos with endothelial-specific ablation of JunB and in in vitro angiogenesis models. By cDNA microarray analysis, we identified core-binding factor beta (CBFbeta), which together with the Runx proteins forms the heterodimeric core-binding transcription complex CBF, as a novel JunB target gene. In line with our findings, expression of the CBF target MMP-13 was impaired in JunB-deficient ECs. Reintroduction of CBFbeta into JunB-deficient ECs rescued the tube formation defect and MMP-13 expression, indicating an important role for CBFbeta in EC morphogenesis.

We investigated the molecular mechanisms underlying canonical Wnt-mediated regulation of chondrocyte hypertrophy using chick upper sternal chondrocytes. Replication competent avian sarcoma (RCAS) viral over-expression of Wnt8c and Wnt9a, upregulated type X collagen (col10a1) and Runx2 mRNA expression thereby inducing chondrocyte hypertrophy. Wnt8c and Wnt9a strongly inhibited mRNA levels of Sox9 and type II collagen (col2a1). Wnt8c further enhanced canonical bone morphogenetic proteins (BMP-2)-induced expression of Runx2 and col10a1 while Wnt8c and Wnt9a inhibited TGF-beta-induced expression of Sox9 and col2a1. Over-expression of beta-catenin mimics the effect of Wnt8c and Wnt9a by upregulating Runx2, col10a1, and alkaline phosphatase (AP) mRNA levels while it inhibits col2a1 transcription. Western blot analysis shows that Wnt8c and beta-catenin also induces Runx2 protein levels in chondrocytes. Thus, our results indicate that activation of the canonical beta-catenin Wnt signaling pathway induces chondrocyte hypertrophy and maturation. We further investigated the effects of beta-catenin-TCF/Lef on Runx2 promoter. Co-transfection of lymphoid enhancer factor (Lef1) and beta-catenin in chicken upper sternal chondrocytes together with deletion constructs of the Runx2 promoter shows that the proximal region spanning the first 128 base pairs of this promoter is responsible for the Wnt-mediated induction of Runx2. Mutation of the TCF/Lef binding site in the -128 fragment of the Runx2 promoter resulted in loss of its responsiveness to beta-catenin. Additionally, gel-shift assay analyses determined the DNA/protein interaction of the TCF/Lef binding sites on the Runx2 promoter. Finally, our site-directed mutagenesis data demonstrated that the Runx2 site on type X collagen promoter is required for canonical Wnt induction of col10a1. Altogether we demonstrate that Wnt/beta-catenin signaling is regulated by TGF-beta and BMP-2 in chick upper sternal chondrocytes, and mediates chondrocyte hypertrophy at least partly through activation of Runx2 which in turn may induce col10a1 expression., (Copyright 2006 Wiley-Liss, Inc.)

Canonical Wnt signaling (beta-catenin/TCF) has emerged as a key regulator of skeletogenesis. In this study, chondrogenesis is examined in a mouse model in which the Wnt antagonist secreted frizzled related protein 1 (sFRP1) is non-functional and results in a high bone mass phenotype and activation through the canonical pathway of the Runx2 transcription factor that is essential for bone formation. We find during the period of rapid post-natal growth, shortened height of the growth plate and increased calcification of the hypertrophic zone (HZ) in the sFRP1-/- mouse, indicating accelerated endochondral ossification. Using mouse embryo fibroblasts (MEFs) induced into the chondrogenic lineage, increased chondrogenesis and accelerating differentiation of hypertrophic chondrocytes in the sFRP1-/- MEFs was observed compared to WT cells. The induced maturation of hypertrophic chondrocytes in sFRP1(-/-) MEFs was inversely correlated to phospho-beta-catenin levels, indicating involvement of activated canonical Wnt signaling characterized by an increased expression of collagen type 2a1 and Sox 9. However, an absence of Indian hedgehog expression which occurs in WT cells was found. SFRP1-/- cells also exhibited an early induction of collagen type 10a1. Thus, these modifications in gene expression are contributing mechanism(s) for increased chondrocyte differentiation in SFRP1-/- cells. These studies have identified sFRP1 as a critical negative regulator of Wnt signaling for the normal progression of chondrocyte differentiation. Microarray gene profiling provided additional novel insights into the regulatory factors for appropriate Wnt signaling necessary for the control of chondrocyte maturation., (Copyright 2006 Wiley-Liss, Inc.)

Interaction between chondrocytes and extracellular matrix is considered a key factor in the generation of grafts for matrix-associated chondrocyte transplantation. Therefore, our objective was to study the influence of differentiation status on cellular attachment. Adhesion of chondrocytes to collagen type II increased after removal from native cartilage up to the third day in monolayer in a dose-dependent manner. Following dedifferentiation after the second passage, adhesion to collagen types I (-84%) and II (-46%) decreased, whereas adhesion to fibrinogen (+59%) and fibronectin (+43%) increased. A cartilage construct was developed based on a clinically established collagen type I scaffold. In this matrix, more than 80% of the cells could be immobilized by mechanisms of adhesion, filtration, and cell entrapment. Confocal laser microscopy revealed focal adhesion sites as points of cell-matrix interaction, as well as collagen type II expression in the cartilage graft after two weeks of in vitro cultivation. Basic fibroblast growth factor (bFGF) treated chondrocytes showed increased adhesion to collagen types I and II, fibronectin, and fibrinogen. Attachment to these investigated proteins significantly enhanced cell proliferation. Matrix design in cartilage engineering must meet the biological demands of amplified cells, because adhesion of chondrocytes depends on their differentiation status and is regulated by bFGF.

Recent studies have demonstrated that intermittent administration of parathyroid hormone (PTH) enhances osteogenesis (hard callus formation) and increases mechanical strength in experimental fracture healing. Thus far, however, effects of PTH on chondrogenesis (soft callus formation) during fracture healing have not been fully elucidated. In the present study, we analyzed the underlying molecular mechanism by which exogenous PTH would affect chondrogenesis in a model of experimental fracture healing. Unilateral femoral fractures were produced in 2-month-old Sprague-Dawley rats. Daily subcutaneous injections of 10 microg/kg of recombinant human PTH(1-34) [rhPTH(1-34)] were administered over a 28-day period of fracture healing. Control animals were injected with vehicle solution (normal saline) alone. The results showed that, on day 14 after fracture, cartilage area in the PTH-treated group was significantly increased (1.4-fold) compared with the controls, but this increase was not observed at days 21 and 28. In the early stage of chondrogenesis (days 4-7), cell proliferation, expressed as the rate of proliferating cell nuclear antigen-positive cells, was increased in mesenchymal (chondroprogenitor) cells but not chondrocytes in the PTH-treated group compared with controls. In addition, gene expression of SOX-9 was up-regulated in the PTH-treated group on day 4 (1.4-fold), and this was accompanied by enhanced expression of pro-alpha1 (II) collagen (1.8-fold). After 14 days, there were no significant differences between groups in either cell proliferation or the expression levels of cartilage differentiation-related genes (SOX-9, pro-alpha1 (II) collagen, pro-alpha1 (X) collagen and osteopontin). These results suggest that intermittent treatment with low-dose rhPTH(1-34) induces a larger cartilaginous callus but does not delay chondrocyte differentiation during fracture healing.

The purpose of this study was to establish diagnostic criteria for Stickler syndrome. Ninety patients from 38 families had complete evaluations for possible Stickler syndrome. Molecular confirmation of COL2A1 mutation status (type I Stickler syndrome) was available on 25 patients from six families. In the remaining 65 patients, 47 from 25 families were affected with Stickler syndrome and 18 from seven families were unaffected with Stickler syndrome. A diagnostic nosology based on type I Stickler patients with known COL2A1 mutations was applied to clinically affected and unaffected patients. A diagnostic scale of 9 points evaluated molecular data or family history data and characteristic ocular, orofacial, auditory, and musculoskeletal findings. A score of > or =5 was diagnostic of Stickler syndrome. These criteria demonstrate 100% sensitivity when applied to type I Stickler syndrome patients with known COL2A1 mutations, 98% sensitivity when applied to clinically affected Stickler patients, and 86% specificity when applied to patients unaffected based on clinical and/or molecular analysis. We conclude that diagnostic criteria based on type I Stickler patients with molecularly confirmed COL2A1 mutations appear to be sensitive and specific for the diagnosis of this syndrome and should be helpful to clinicians when making the diagnosis.

Immunoreactivity to collagen type II (CII) has been implicated in the pathogenesis of rheumatoid arthritis. Patients have been described to have an acidic pH in their inflamed synovial tissue. It is known that protein structures are modified by environmental pH, thus it is plausible that changes in synovial pH could affect the conformation of proteins like CII. Posttranslational modifications could alter the biophysical properties of cartilage proteins leading to autoimmunity. In this study we investigated if arthritogenicity of CII was affected by changes in pH, and if so, this could be correlated to altered protein conformation. Immunisation with CII at neutral pH induced a milder disease than did CII at acidic pH. All animals elicited a humoral response to CII, although with a significantly higher IgG1/IgG2b-ratio in the pH 7.4 group. Analysis by circular dichroism and electron microscopy indicated less fibrillation of CII at low pH as compared to neutral pH. Our results suggest that CII is more immunogenic and arthritogenic in an acidic environment than in a neutral environment. We can correlate these findings to pH-induced conformational changes of CII. Hence, self-tolerance to CII might be affected by changes in pH leading to altered and increased arthritogenicity.

Aritcular cartilage damage results in degradation of macromolecular in cartilage tissue such as aggrecan and type II collagen that are mediated with aggrecanases and matrix metalloproteinases. As the activity of MMP-13 against Type II collagen is very strong, so it plays significant role in cartilage collagen degradation. However, it is noteworthy that the inhibition of both aggrecanse-1. 2 can prevent cartilage degradation. Aggrecan hydrates the collagen network and provides the cartilage tissue with its characteristic pressibility and elasticity. It become now clear that aggrecan plays protective role in preventing degradation of collagen fibrils. Aggrecan and type II collagen molecules cooperatively maintain the cartilage integrity and function.

Vascular calcification is a pathological calcification process. Its pathogenesis involves active mineralization by chondrogenic and osteogenic cells. Since cartilaginous metaplasia has been found in several vascular diseases, this process may represent one of vascular remodeling in response to vascular injury. Endochondral ossification following cartilaginous metaplasia play an important role in the progression of vascular calcification.

Type II collagen and hyaluronan are the two major components of extracellular molecules in cartilage and play an important role in mechanical functions of extracellular matrix. Currently, their mechanical properties have been investigated only at the gross-level. In this study, the mechanical properties of single type II collagen and hyaluronan molecules were directly measured using optical tweezers technique. The persistence length was found to be 11.2+/-8.4 nm in type II collagen and 4.5+/-1.2 nm in hyaluronan. This result suggested that type II collagen is stiffer than hyaluronan at the individual molecule level, which supports the general concept that collagen is responsible for resisting tensile force. The experimental system developed here also provides a powerful tool for quantifying mechanical properties of extracellular matrix at the single molecule level.

Unlabelled: A missense mutation in the mouse Col2a1 gene has been discovered, resulting in a mouse phenotype with similarities to human spondyloepiphyseal dysplasia (SED) congenita. In addition, SED patients have been identified with a similar molecular mutation in human COL2A1. This mouse model offers a useful tool for molecular and biological studies of bone development and pathology., Introduction: A new mouse autosomal recessive mutation has been discovered and named spondyloepiphyseal dysplasia congenita (gene symbol sedc)., Materials and Methods: Homozygous sedc mice can be identified at birth by their small size and shortened trunk. Adults have shortened noses, dysplastic vertebrae, femora, and tibias, plus retinoschisis and hearing loss. The mutation was mapped to Chr15, and Col2a1 was identified as a candidate gene., Results: Sequence analyses revealed that the affected gene is Col2a1, which has a missense mutation at exon 48 causing an amino acid change of arginine to cysteine at position 1417. Two human patients with spondyloepiphyseal dysplasia (SED) congenita have been reported with the same amino acid substitution at position 789 in the human COL2A1 gene., Conclusions: Thus, sedc/sedc mice provide a valuable model of human SED congenita with molecular and phenotypic homology. Further biochemical analyses, molecular modeling, and cell culture studies using sedc/sedc mice could provide insight into mechanisms of skeletal development dependent on Col2a1 and its role in fibril formation and cartilage template organization.

The local mechanical environment is a crucial factor in determining cell and tissue differentiation during vertebrate skeletal development and repair. Unlike the basic response of bone to mechanical load, as described in Wolff's law, the mechanobiological relationship between the local mechanical environment and tissue differentiation influences everything from tissue type and molecular architecture to the formation of complex joints. This study tests the hypothesis that precisely controlled mechanical loading can regulate gene expression, tissue differentiation and tissue architecture in the adult skeleton and that precise manipulation of the defect's local mechanical environment can initiate a limited recapitulation of joint tissue development. We generated tissue type predictions using finite element models (FEMs) interpreted by published mechanobiological fate maps of tissue differentiation. The experiment included a custom-designed external fixator capable of introducing daily bending, shear or a combination of bending and shear load regimens to induce precisely controlled mechanical conditions within healing femoral defects. Tissue types and ratios were characterized using histomorphometrics and molecular markers. Tissue molecular architecture was quantified using polarized light and Fourier transforms, while immunological staining and in situ hybridization were used to characterize gene expression. The finite element models predicted the differentiation of cartilage within the defects and that substantial fibrous tissues would develop along the extreme excursion peripheries in the bending group. The three experimentally induced loading regimens produced contiguous cartilage bands across all experimental defects, inhibiting bony healing. Histomorphometric analysis of the ratios of cartilage to bone in the experimental groups were not significantly different from those for the knee joint, and Fourier transform analysis determined significantly different collagen fibril angle specializations within superficial, intermediate and deep layers of all experimental cartilages (P<0.0001), approximating those for articular cartilage. All stimulations resulted in the expression of collagen type II, while the bending stimulation also resulted in the expression of the joint-determining gene GDF-5. These findings indicate that the local mechanical environment is an important regulator of gene expression, tissue differentiation and tissue architecture.

The cartilage extracellular matrix is composed of a dense collagen network that entraps a range of other specialized proteins important for the proper formation and function of the tissue. Loss of two abundant cartilage components, type II collagen and perlecan, has drastic effects on skeletal development. Both collagen II and perlecan mutants have severe and lethal chondrodysplasia characterized by disorganized growth plate, lack of collagen network, defective endochondral bone formation, and abnormal intervertebral disk development. To test whether the reduced collagen density in the perlecan-null cartilage is due to enhanced activity of collagen-degrading proteinases, we have analyzed gelatinase expression and activity in the mutant tissue. Immunohistochemical analysis revealed a weak, but clear, expansion of MMP-9 deposition into the hypertrophic zone of the perlecan-null growth plate. However, in situ and SDS-PAGE zymography showed that the activity of gelatinases (MMP-2 and MMP-9) is not altered in perlecan-null cartilage, suggesting that they are not primarily linked to the reduced fibrillar network observed in the mutant. Likewise, intercrossing of perlecan mutants onto an MMP-9-null background could not rescue the ultrastructural abnormalities of the perlecan-deficient cartilage.

Adult human bone marrow-derived stem cells, having the ability to differentiate into cells of multiple lineages, have been isolated and propagated by varied protocols, including positive (CD105(+))/negative (CD45(-)GlyA(-)) selection with immunomagnetic beads, or direct plating into selective culture media. Each substratum-adherent cell population was subjected to a systematic analysis of their cell surface markers and differentiation potential. In the initial stages of culture, each cell population proliferated slowly, reaching confluence in 10-14 days. Adherent cells proliferated at similar rates whether cultured in serum-free medium supplemented with basic fibroblast growth factor, medium containing 2% fetal bovine serum (FBS) supplemented with epidermal growth factor and platelet-derived growth factor, or medium containing 10% FBS alone. Cell surface marker analysis revealed that more than 95% of the cells were positive for CD105/endoglin, a putative mesenchymal stem cell marker, and negative for CD34, CD31, and CD133, markers of hematopoietic, endothelial, and neural stem cells, respectively, regardless of cell isolation and propagation method. CD44 expression was variable, apparently dependent on serum concentration. Functional similarity of the stem cell populations was also observed, with each different cell population expressing the cell type-specific markers beta-tubulin, type II collagen, and desmin, and demonstrating endothelial tube formation when cultured under conditions favoring neural, cartilage, muscle, and endothelial cell differentiation, respectively. On the basis of these data, adult human bone marrow-derived stem cells cultured in adherent monolayer are virtually indistinguishable, both physically and functionally, regardless of the method of isolation or proliferative expansion.