Effects of connective tissue growth factor (CTGF/CCN2) on condylar chondrocyte proliferation, migration, maturation, differentiation and signalling pathway.

CCN2, also known as connective tissue growth factor (CTGF), is a 38 kDa cysteine-rich extracellular matrix protein that regulates a sequence of cellular functions and participates in multiple complex biological processes, such as chondrogenesis and osteogenesis. In the present study, we provided the first evidence describing the physiological role of CCN2 in condylar chondrocyte proliferation, migration, maturation and differentiation. CCN2 was widely expressed throughout the whole layers of condylar cartilage and predominantly distributed in the proliferative zone. Recombinant CCN2 promoted the proliferation, migration, proteoglycan synthesis and differentiation capacity of isolated condylar chondrocytes. The stimulatory effect of CCN2 on chondrocyte proliferation was associated with the activation of phosphatidylinositol 3-kinase/Akt signalling pathway. The blocking of this pathway by its inhibitor LY294002 impaired the proliferative effect of CCN2 on chondrocytes. These results suggested a novel physiological role of CCN2 in the development of condylar cartilage.

Role of endoplasmic reticulum stress pathway in hydrostatic pressure-induced apoptosis in rat mandibular condylar chondrocytes.

Excessive mechanical loads induce chondrocyte apoptosis and irreversible cartilage degeneration, but the underlying molecular mechanism is poorly understood. The aim of this study was to investigate the possible role of endoplasmic reticulum (ER) stress pathway in hydrostatic pressure (HP)-induced apoptosis in rat mandibular condylar chondrocytes. Chondrocytes were isolated from rat mandibular condylar cartilage and subjected to HP. Cell viability and apoptosis were assessed by Cell Counting Kit-8 and flow cytometry assay. Expression of ER stress-associated molecules was detected by quantitative real-time PCR and western blot analysis. In addition, expression of apoptosis-related proteins (bax, bcl-2, and cleaved-caspase-3) was assessed by western blot. To explore ER stress function, chondrocytes were pretreated with salubrinal before exposure to HP. Expression of type II collagen, aggrecan, MMP-13, and ADAMTS-5 was evaluated by real-time PCR. The results indicated that HP reduced cell viability in a magnitude- and time-dependent manner. HP-induced activation of ER stress pathway by increasing expression of GRP78, CHOP, caspase-12, PERK, and peIF2α in chondrocytes. Moreover, the expression of bax and cleaved-caspase-3 was increased, while the expression of bcl-2 was decreased in response to HP as the stress time prolonged. In addition, salubrinal suppressed HP-induced apoptosis, upregulated type II collagen and aggrecan mRNA expression, and downregulated MMP-13 and ADAMTS-5 mRNA expression in response to HP. These results demonstrate that HP induces apoptosis in mandibular condylar chondrocytes through ER stress-mediated apoptotic pathway. Suppression of ER stress by salubrinal prevents chondrocytes from undergoing apoptosis and matrix degradation induced by HP.

Hydrostatic Compress Force Enhances the Viability and Decreases the Apoptosis of Condylar Chondrocytes through Integrin-FAK-ERK/PI3K Pathway.

Reduced mechanical stimuli in many pathological cases, such as hemimastication and limited masticatory movements, can significantly affect the metabolic activity of mandibular condylar chondrocytes and the growth of mandibles. However, the molecular mechanisms for these phenomena remain unclear. In this study, we hypothesized that integrin-focal adhesion kinase (FAK)-ERK (extracellular signal-regulated kinase)/PI3K (phosphatidylinositol-3-kinase) signaling pathway mediated the cellular response of condylar chondrocytes to mechanical loading. Primary condylar chondrocytes were exposed to hydrostatic compressive forces (HCFs) of different magnitudes (0, 50, 100, 150, 200, and 250 kPa) for 2 h. We measured the viability, morphology, and apoptosis of the chondrocytes with different treatments as well as the gene, protein expression, and phosphorylation of mechanosensitivity-related molecules, such as integrin α2, integrin α5, integrin ß1, FAK, ERK, and PI3K. HCFs could significantly increase the viability and surface area of condylar chondrocytes and decrease their apoptosis in a dose-dependent manner. HCF of 250 kPa resulted in a 1.51 ± 0.02-fold increase of cell viability and reduced the ratio of apoptotic cells from 18.10% ± 0.56% to 7.30% ± 1.43%. HCFs could significantly enhance the mRNA and protein expression of integrin α2, integrin α5, and integrin ß1 in a dose-dependent manner, but not ERK1, ERK2, or PI3K. Instead, HCF could significantly increase phosphorylation levels of FAK, ERK1/2, and PI3K in a dose-dependent manner. Cilengitide, the potent integrin inhibitor, could dose-dependently block such effects of HCFs. HCFs enhances the viability and decreases the apoptosis of condylar chondrocytes through the integrin-FAK-ERK/PI3K pathway.

Celecoxib exerts protective effects on extracellular matrix metabolism of mandibular condylar chondrocytes under excessive mechanical stress.

OBJECTIVE: Excessive mechanical stress is considered a major cause of temporomandibular joint osteoarthritis (TMJ-OA). High magnitude cyclic tensile strain (CTS) up-regulates pro-inflammatory cytokines and matrix metalloproteinases (MMPs) in chondrocytes, while selective cyclooxygenase (COX)-2 inhibition has been shown to be beneficial to cytokine-induced cartilage damage. However, the effect of selective COX-2 inhibitors on mechanically stimulated chondrocytes remains unclear. This study evaluated the effect of celecoxib, a selective COX-2 inhibitor, on extracellular matrix (ECM) metabolism of mandibular condylar chondrocytes under CTS. METHODS: Porcine mandibular chondrocytes were subjected to CTS of 0.5 Hz, 10% elongation with celecoxib for 24 h. The gene expressions of COX-2, MMPs, aggrecanase (ADAMTS), type II collagen and aggrecan were examined by real-time PCR. Also, prostaglandin E2 (PGE2) concentrations were determined using enzyme immunoassay kit. The levels of MMP and transcription factor NF-κB were measured by western blot while MMP activity was determined by casein zymography. RESULTS: The presence of celecoxib normalized the release of PGE2 and diminished the CTS-induced COX-2, MMP-1, MMP-3, MMP-9 and ADAMTS-5 gene expressions while recovered the downregulated type II collagen and aggrecan gene expressions. Concurrently, celecoxib showed inhibition of NF-κB and suppression of MMP production and activity. CONCLUSIONS: Celecoxib exerts protective effects on mandibular condylar chondrocytes under CTS stimulation by diminishing degradation and restoring synthesis of ECM.

Critical role of Bmpr1a in mandibular condyle growth.

The importance of Bone Morphogenetic Proteins (BMPs) in the regulation of cell fate, differentiation and proliferation in the growth plate is well-known. However, in secondary cartilages (such as that in the temporomandibular joint) that grow by proliferation of prechondrocytes and differ in their pattern of growth, the role of BMPs is largely unexplored. To examine this question, we ablated Bmpr1a in the condylar cartilage of neonatal mice and assessed the consequences for mandibular condyle growth and organization at intervals over the ensuing 4 weeks. Bmpr1a deficiency caused significant chondrodysplasia and almost eliminated the chondrocytic phenotype in the TMJ. Expression of Sox9, collagen II, proteoglycan were all greatly reduced, and cell proliferation as detected by BrdU was almost non-existent in the knockout mice. Primary bone spongiosa formation was also disturbed and was accompanied by reduced Osterix expression. These findings strongly suggest that Bmpr1a is critical for the development and growth of the mandibular condyle via its effect on proliferation of prechondroblasts and chondrocyte differentiation.

Chondrogenic potential of superficial versus cartilage layer cells of the temporomandibular joint condyle in photopolymerizable gelatin-based hydrogels.

The objectives of this study were to compare the chondrogenic potential of cells derived from different layers of Mandibular condyle cartilage and to gain further understanding of the impact of chondrogenic cues when embedded into a novel hydrogel scaffold (PGH, a polymer blend of poly (ethylene glycol), gelatin, and heparin) compared to a gelatin hydrogel scaffold (GEL). Cartilage layer cells (CLCs) and fibroblastic superficial layer cells (SLCs) were harvested from the mandibular condyle of boer goats obtained from a local abattoir. After expansion, cells were seeded into PGH and GEL hydrogels and cultured in chondrogenic media for 3 weeks. Scaffolds were harvested at 0, 1, and 3 week(s) and processed for gross appearance, histochemical, biochemical, and mechanical assays. In terms of chondrogenesis, major differences were observed between scaffold materials, but not cell types. Glycosaminoglycan (GAG) staining showed GEL scaffolds deposited GAG during the 3 week period, which was also confirmed with the biochemical testing. Moreover, GEL scaffolds had significantly higher compressive modulus and peak stress than PGH scaffolds at all time points with the largest difference seen in week 3. It can be concluded that GEL outperformed PGH in chondrogenesis. It can also be concluded that materials play a more important role in the process of chondrogenesis than the tested cell populations. Fibroblastic SLCs were shown to have similar chondrogenic potential as CLCs cells, suggesting a rich pool of progenitor cells in the superficial fibroblastic layer capable of undergoing chondrogenesis given appropriate physical and chemical cues.

Double-stranded RNA-dependent protein kinase regulates insulin-stimulated chondrogenesis in mouse clonal chondrogenic cells, ATDC-5.

Double-stranded RNA-dependent protein kinase (PKR) is an interferon-induced protein that has been identified and characterized as a translational inhibitor in an interferon-regulated antiviral pathway. PKR is also reported to play important roles in the regulation of cell growth and differentiation. We have previously demonstrated that PKR inactivation suppresses osteoblast calcification and osteoclast formation. However, reports concerning the roles of PKR in chondrogenesis are limited. In this study, we have demonstrated that PKR is required for the in vitro differentiation of the mouse clonal chondrogenic cell line ATDC-5. ATDC-5 cells treated with insulin differentiated into chondrocytes and produced an alcian-blue-positive cartilage matrix. The protein expression of signal transducers and activators of transcription (STAT) peaked at day 7 of differentiation, whereas the expression of SRY-box-containing gene 9 (Sox-9), which is a transcription factor for chondrocyte differentiation, increased gradually. When the cells were treated with a PKR inhibitor (2-aminopurine), the cartilage matrix formation decreased. The protein expression of STAT1 continued to increase up to day 21, whereas the expression of Sox-9 was low and did not increase. We also demonstrated that PKR was localized to a marginal region of the mandibular condyle cartilage in mouse embryos. Our findings suggest that PKR has important functions in the differentiation of chondrocytes through the modulation of STAT1 and Sox-9 expression.

Effects of intermittent versus continuous parathyroid hormone administration on condylar chondrocyte proliferation and differentiation.

Endochondral ossification is a complex process involving chondrogenesis and osteogenesis regulated by many hormones and growth factors. Parathyroid hormone (PTH), one of the key hormones regulating bone metabolism, promotes osteoblast differentiation and osteogenesis by intermittent administration, whereas continuous PTH administration inhibits bone formation. However, the effects of PTH on chondrocyte proliferation and differentiation are still unclear. In this study, intermittent PTH administration presented enhanced effects on condylar chondrocyte differentiation and bone formation, as demonstrated by increased mineral nodule formation and alkaline phosphatase (ALP) activity, up-regulated runt-related transcription factor 2 (RUNX2), ALP, collagen type X (COL10a1), collagen type I (COL1a1), osteocalcin (OCN), bone sialoprotein (BSP), bone morphogenetic protein 2 (BMP2) and osterix (OSX) mRNA and/or protein expression. On the contrary, continuous PTH administration promoted condylar chondrocyte proliferation and suppressed its differentiation, as demonstrated by up-regulated collagen type II (COL2a1) mRNA expression, reduced mineral nodule formation and down-regulated expression of the mRNAs and/or proteins mentioned above. Our data suggest that PTH can regulate condylar chondrocyte proliferation and differentiation, depending on the type of PTH administration. These results provide new insight into the effects of PTH on condylar chondrocytes and new evidence for using local PTH administration to cure mandibular asymmetry.

Age-dependent physiological changes in the histoarchitecture of the articular cartilage of the rabbit mandibular condyle: a morphological and morphometric study.

Mandibular condyle articular cartilage participates in condylar postnatal growth and is responsible for adaptations to anatomical and/or biomechanical alterations throughout life. In a preliminary study in rabbits, differences were observed in the thickness of the layers of articular cartilage in control animals at 5 and 6 months (generally considered adults for this purpose). This study aimed to describe sagittally sectioned condylar cartilages stained with Picrosirius-hematoxylin in rabbits at 40 days and 5, 6, 8, 13, and 18 months to determine when histological maturity is reached. At 40 days, 5 layers were seen: fibrous, proliferative, transition, maturation, and hypertrophic. Older animals (5-18 months) lacked the transition layer. Fibrous, proliferative, and hypertrophic regions were considered for morphometric analysis. The thickness of the fibrous region did not change during the analyzed period (p = 0.1899). When proliferative and hypertrophic regions and the total thickness of the cartilage were compared, a difference was detected (p < 0.001). The thickness of the proliferative region was greatest at 40 days and decreased at 5 months; however, it increased at 6 months, when it was significantly thicker than at 5, 8, 13, and 18 months. Both the hypertrophic region and the total thickness were thickest at 40 days, intermediate at 5, 6, and 8 months, and thinnest at 13 and 18 months. In summary, our data suggest a physiological period of increased cartilage growth at 6 months. Additionally, rabbits at this age should be avoided in experiments involving condylar cartilage. Finally, 13-month-old rabbits have reached histological maturity of the condylar cartilage.

Isolation and characterization of murine mandibular condylar cartilage cell populations.

OBJECTIVES: The mandibular condylar cartilage is a heterogeneous tissue containing cells at various stages of chondrocyte maturation organized into 4 zones: superficial, polymorphic, flattened, and hypertrophic. The goal of this study was to use transgenic mice containing chondrocyte maturation markers fused to fluorescent protein transgenes to isolate and characterize homogenous cell populations of the mandibular condylar cartilage. METHODS: Fluorescent reporter expression in the mandibular condylar cartilage of transgenic mice containing the 3.6-kb fragment of the rat collagen type 1 promoter fused to a topaz-fluorescent protein (Col3.6-tpz), collagen type 2 promoter fused to a cyan-fluorescent protein (Col2-cyan), and/or collagen type 10 promoter fused to cherry-fluorescent protein (Col10-cherry) was examined. Mandibular condylar cartilage cells were analyzed by fluorescence-activated cell sorting (FACS) and either used for gene expression analysis or plated in cell cultures and exposed to adipogenic, osteogenic, or chondrogenic conditions. To determine cell fate, transgenic mice containing the Col3.6-cre recombinase were bred with cre reporter mice. RESULTS: Localization and analysis of gene expression revealed that Col3.6-tpz-positive cells corresponded to the polymorphic/flattened zones and Col2-cyan-positive cells corresponded to the flattened/hypertrophic zones of the mandibular condylar cartilage. Mandibular condylar cartilage FACS-sorted Col3.6-tpz-positive cells have the potential to differentiate into bone, cartilage, and fat. Cell fate mapping revealed that Col3.6 cells are precursors of some of the hypertrophic chondrocytes in the mandibular condylar cartilage. CONCLUSION: Col3.6-tpz cells represent an earlier stage of the mandibular condylar cartilage maturation pathway.

GLUT4 in murine bone growth: from uptake and translocation to proliferation and differentiation.

Skeletal growth, taking place in the cartilaginous growth plates of long bones, consumes high levels of glucose for both metabolic and anabolic purposes. We previously showed that Glut4 is present in growing bone and is decreased in diabetes. In the present study, we examined the hypothesis that in bone, GLUT4 gene expression and function are regulated via the IGF-I receptor (IGF-IR) and that Glut4 plays an important role in bone growth. Insulin and IGF-I actions on skeletal growth and glucose uptake were determined using mandibular condyle (MC) organ cultures and MC-derived primary cell cultures (MCDC). Chondrogenesis was determined by following proliferation and differentiation activities using immunohistochemical (IHC) analysis of proliferating cell nuclear antigen and type II collagen expression, respectively. Overall condylar growth was assessed morphometrically. GLUT4 mRNA and protein levels were determined using in situ hybridization and IHC, respectively. Glut4 translocation to the cell membrane was assessed using confocal microscopy analysis of GFP-Glut4 fusion-transfected cells and immunogold and electron microscopy on MC sections; glucose uptake was assayed by 2-deoxyglucose (2-DOG) uptake. Both IGF-I and insulin-stimulated glucose uptake in MCDC, with IGF-I being tenfold more potent than insulin. Blockage of IGF-IR abrogated both IGF-I- and insulin-induced chondrogenesis and glucose metabolism. IGF-I, but not insulin, induced Glut4 translocation to the plasma membrane. Additionally, insulin induced both GLUT4 and IGF-IR gene expression and improved condylar growth in insulin receptor knockout mice-derived MC. Moreover, silencing of GLUT4 gene in MCDC culture abolished both IGF-I-induced glucose uptake and chondrocytic proliferation and differentiation. In growing bone, the IGF-IR pathway stimulates Glut4 translocation and enhances glucose uptake. Moreover, intact Glut4 cellular levels and translocation machinery are essential for early skeletal growth.

Sex differences in chondrocyte maturation in the mandibular condyle from a decreased occlusal loading model.

Temporomandibular joint disorders (TMDs) predominantly afflict women of childbearing age. Defects in mechanical loading-induced temporomandibular joint (TMJ) remodeling are believed to be a major etiological factor in the development of TMD. The goal of this study was to determine if there are sex differences in CD-1 and C57BL/6 mice exposed to a decreased occlusal loading TMJ remodeling model. Male and female CD-1 and C57BL/6 mice, 21 days old, were each divided into two groups. They were fed either a normal pellet diet (normal loading) or a soft diet and had their incisors trimmed out of occlusion (decreased occlusal loading) for 4 weeks. The mandibular condylar cartilage was evaluated by histology, and the subchondral bone was evaluated by micro-CT analysis. Gene expression from both was evaluated by real-time PCR analysis. In both strains and sexes of mice, decreased occlusal loading caused similar effects in the subchondral bone, decreases in bone volume and total volume compared with their normal loading controls. However, in both strains, decreased occlusal loading caused a significant decrease in the expression of collagen type II (Col2) and Sox9 only in female mice, but not in male mice, compared with their normal loading controls. Decreased occlusal loading causes decreased bone volume in both sexes and a decrease in early chondrocyte maturation exclusively in female mice.

An immunohistochemical study on the localization of type II collagen in the developing mouse mandibular condyle.

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.

Morphological observation of process of mouse temporomandibular joint formation.

The aim of this study was to clarify the developmental mechanism of the temporomandibular joint (TMJ) cavity, using the relationship between Meckel's cartilage and the mandible to morphologically observe the process of TMJ formation in mouse fetuses. We investigated the involvement of apoptosis in the development of the mouse TMJ cavity. We attempted to 3-dimensionally clarify the developmental process of the mandible and Meckel's cartilage by observing the developmental process optically and reconstructing 3-dimensional images to observe 3-dimensional locations of the mandible and Meckel's cartilage. Formation of the upper joint cavity began on embryonal day 16, and a complete joint cavity was formed on embryonal day 18. Formation of the lower joint cavity began on embryonal day 18, and formation was almost completed on embryonal day 19. Meckel's cartilage adjacent to the mandible decreased with development of the mandible but was vestigial on embryonal day 19. The posterior region of Meckel's cartilage developed toward the posterior direction, and it was 3-dimensionally confirmed that the mandible and Meckel's cartilage were separated. Histological observation by the TUNEL method revealed the presence of solitary and diffuse apoptotic cells not only in the joint cavity, but also around the condyle.

Regenerative Potential of Mandibular Condyle Cartilage and Bone Cells Compared to Costal Cartilage Cells When Seeded in Novel Gelatin Based Hydrogels.

The field of temporomandibular joint (TMJ) condyle regeneration is hampered by a limited understanding of the phenotype and regeneration potential of cells in mandibular condyle cartilage. It has been shown that chondrocytes derived from hyaline and costal cartilage exhibit a greater chondro-regenerative potential in vitro than those from mandibular condylar cartilage. However, our recent in vivo studies suggest that mandibular condyle cartilage cells do have the potential for cartilage regeneration in osteochondral defects, but that bone regeneration is inadequate. The objective of this study was to determine the regeneration potential of cartilage and bone cells from goat mandibular condyles in two different photocrosslinkable hydrogel systems, PGH and methacrylated gelatin, compared to the well-studied costal chondrocytes. PGH is composed of methacrylated poly(ethylene glycol), gelatin, and heparin. Histology, biochemistry and unconfined compression testing was performed after 4 weeks of culture. For bone derived cells, histology showed that PGH inhibited mineralization, while gelatin supported it. For chondrocytes, costal chondrocytes had robust glycosaminoglycan (GAG) deposition in both PGH and gelatin, and compression properties on par with native condylar cartilage in gelatin. However, they showed signs of hypertrophy in gelatin but not PGH. Conversely, mandibular condyle cartilage chondrocytes only had high GAG deposition in gelatin but not in PGH. These appeared to remain dormant in PGH. These results show that mandibular condyle cartilage cells do have innate regeneration potential but that they are more sensitive to hydrogel material than costal cartilage cells.

Proteomic analysis of early-response to mechanical stress in neonatal rat mandibular condylar chondrocytes.

The objectives of this study were to investigate the early response to mechanical stress in neonatal rat mandibular chondrocytes by proteomic analysis. To evaluate its molecular mechanism, chondrocytes were isolated and cultured in vitro, then loaded mechanical stress by four-point bending system on different patterns. Morphological observation, flow cytometric analysis, and MTT assays indicated that 4,000 microstrain loading for 60 min was an appropriate mechanical stimulus for the following proteome analysis, which produced a transient but obvious inhibitory effect on the cell cycle. Therefore, we took a proteomic approach to identify significantly differential expression proteins in chondrocytes under this mechanical stress. Using 2-DE and MALDI-TOF, we identified seven differentially expressed proteins including the MAPK pathway inhibitor RKIP, cytoskeleton proteins, actin and vimentin, and other selected proteins. Some differentially expressed proteins were validated by both Western blot analysis and fluorescent staining of cytoskeleton at different loading times. The vimentin and RKIP responsive expression were also proven in vivo in oral orthopedic treatment rats, which was in line with the result in vitro. The histological changes in cartilage also showed the inhibition effect. Furthermore, the expressional level of phosphorylated ERK was increased, which demonstrates the changes in MAPK activity. Taken together, these data indicate that mechanical stress resulted in vimentin expression changes first and then led to the subsequent changes in actin expression, MAPK pathway regulated by RKIP and heat shock protein GRP75. All those changes contributed to the cytoskeleton remolding and cell cycle inhibition, finally led to condylar remodeling.

Expression of Ten-m/Odz3 in the fibrous layer of mandibular condylar cartilage during postnatal growth in mice.

It has been speculated that the mandibular condyle develops via the differentiation of the fibroblast-like cells covering the condyle into chondrocytes; however, the developmental mechanisms behind this process have not been revealed. We used laser-capture microdissection and cDNA microarray analysis to elucidate the genes that are highly expressed in these fibroblast-like cells. Among these genes, the transcription of Ten-m/Odz3 was significantly increased in the fibroblast-like cells compared with other cartilage tissues. For the first time, we describe the temporal and spatial expression of Ten-m/Odz3 mRNA in relation to the expression of type I, II, and X collagen mRNA, as determined by in-situ hybridization in mouse mandibular condylar cartilage and mouse femoral cartilage during the early stages of development. Ten-m/Odz3 was expressed in the fibrous layer and the proliferating and mature chondrocyte layers, which expressed type I and II collagen, respectively, but was not detected in the hypertrophic chondrocyte layer. Furthermore, we evaluated the in-vitro expression of Ten-m/Odz3 using ATDC5 cells, a mouse chondrogenic cell line. Ten-m/Odz3 was expressed during the early stage of the differentiation of mesenchymal cells into chondrocytes. These findings suggest that Ten-m/Odz3 is involved in the differentiation of chondrocytes and that it acts as a regulatory factor in the early stages of the development of mandibular condylar cartilage.

Applying an excessive mechanical stress alters the effect of subchondral osteoblasts on chondrocytes in a co-culture system.

Osteoarthritis (OA) sometimes occurs as a consequence of repeated microtrauma involved in parafunction, which may lead to microfracture in the subchondral bone. The aim of this in vitro study was to evaluate the effects of subchondral osteoblasts in loading with repeated excessive mechanical stress on the metabolism of overlying chondrocytes. A high-magnitude cyclic tensile stress of 15 kPa (30 cycles min(-1)) was applied to the cultured osteoblasts obtained from porcine mandibular condyles. The chondrocytes in alginate beads were then co-cultured with mechanically stressed or unstressed osteoblasts. Chondrocytes co-cultured with unstressed osteoblasts showed a phenotypic shift to hypertrophic chondrocytes, characterized by decreased expression of type II collagen, aggrecan, Sry-related HMG box (SOX-9), and cartilage oligomeric matrix protein (COMP) genes and increased expression of type X collagen and bone sialoprotein (BSP) genes, suggesting that the co-culture may change the chondrocyte differentiation to some extent. These changes were more distinct in chondrocytes co-cultured with excessively mechanically stressed osteoblasts. After co-culture with stressed osteoblasts, the expressions of matrix metalloproteinase (MMP)1, MMP3 and MMP13 genes were also enhanced and the synthesis of DNA, proteoglycan and collagen were significantly decreased in chondrocytes. These results demonstrate that alterations in cartilage metabolism can be induced by stressed osteoblasts, indicating a possible explanation for the onset and progression of OA.

Differential regulation of proteoglycan-4 expression by IL-1α and TGF-ß1 in rat condylar chondrocytes.

Proteoglycan 4 (PRG4) is a multifaceted glycoprotein that mediates boundary lubrication of articular cartilage and its dysregulation is associated with impaired lubrication and cartilage destruction in multiple synovial joints. However, the spatiotemporal expression of PRG4 and the associated regulatory networks remain largely unknown in the mandibular condylar cartilage that is responsible for homeostasis and functions of the temporomandibular joint. We here investigated the possible regulatory effects of the interleukin-1α (IL-1α) or/and transforming growth factor-ß1 (TGF-ß1) on the expression of PRG4 in primary chondrocytes that were isolated from the superficial layer of the condylar cartilage of the 20-day-old male Sprague-Dawley rats. Both IL-1α and TGF-ß1 have been implicated in joint destruction and repair. Treatment of primary chondrocytes for 24 h with recombinant human (rh) IL-1α (10 ng/ml) resulted in pronounced reduction in the expression levels of PRG4 mRNA and protein, whereas stimulation with rhTGF-ß1 (10 ng/ml) significantly increased the expression levels, as measured by RT-PCR and ELISA, respectively. Moreover, rhTGF-ß1 was capable to antagonize the inhibitory effects on the PRG4 expression caused by rhIL-1α and robustly restored its abundance in the cultured condylar chondrocytes. Taken together, our data indicate that PRG4 is synthesized and secreted by condylar cartilage chondrocytes and its expression is differentially regulated by IL-1α and TGF-ß1. The rhIL-1α-mediated PRG4 repression is reversible and potently antagonized by rhTGF-ß1 in condylar chondrocytes. The observed up-regulation of PRG4 upon rhTGF-ß1 treatment further supports the therapeutic application of rhTGF-ß1 in the treatment of temporomandibular joint osteoarthritis.

The effect of 904 nm low level laser on condylar growth in rats.

A growth center of the mandible that contributes to its length and height is the mandibular condyle. Proliferation of prechondroblasts, followed by synthesis of the extracellular matrix and hypertrophy of the cartilage cells, governs the major part of condylar growth. The sample consisted of 54 male rats, weighing between 60 g and 80 g, divided randomly into three groups. Group I was the control group, group II was irradiated bilaterally, and group III was irradiated on the right side. Laser irradiation (lambda = 904 nm, 2000 Hz, pulse length 200 ns and output power 4 mW) was performed, and the procedure was repeated after a 50-day interval. Two months later, the rats were killed. In a single blind manner the lengths of denuded mandibles and the lengths of mandibles on soft tissue were measured. The growth of the mandibles in the unilaterally irradiated group (P < 0.001) and the bilaterally irradiated group (P < 0.05) was significantly more than that in the control group. There was no significant difference between right and left condylar growth in the bilaterally irradiated group (P = 0.3). Soft tissue analysis also verified these results (P < 0.001). Histomorphometric results also revealed a significant difference between laser-irradiated groups and the control group (P < 0.01). We concluded that particular laser irradiation with the chosen parameters can stimulate condylar growth and subsequently cause mandibular advancement. These findings might be clinically relevant, indicating that low level laser irradiation can be used for further improvement of mandibular retrognathism.