Mandibular Condylar Cartilage Cells Research Articles

Role of HIF-2α/NF-κB pathway in mechanical stress-induced temporomandibular joint osteoarthritis.

Many activities overload temporomandibular joint (TMJ) and cause mandibular condylar cartilage (MCC) degradation by inducing the expression of hypoxia-inducible factor-2α (HIF-2α). Although NF-κB signaling pathway has been reported to induce HIF-2α expression, the underlying mechanisms need to be verified. The aim was to investigate the effects of NF-κB/HIF-2α on MCC degradation induced by mechanical stress and the regulatory mechanism of NF-κB in the HIF-2α pathway. Chondrocytes were subjected to cyclic compressive forces in a hypoxic environment. Western blotting was used to test the effects of stress on the expression of NF-κB and HIF-2α. HIF-2α siRNA and shRNA were constructed and transfected into MCC cells in vitro and in vivo to inhibit HIF-2α expression. To test the regulatory effect of the NF-κB pathway on HIF-2α, siRNA p65 was transfected into MCC. The results showed that mechanical stress could cause cartilage degradation and significantly increased the expression of NF-κB, HIF-2α, and downstream degradation factors (MMP13 and ADAMTs-4). Blockade of HIF-2α decreased cartilage degradation and related degradation factors. Suppression of p65significantly decreased the expression of HIF-2α. Our results indicated that the upstream NF-κB pathway exerted a regulatory effect on HIF-2α in the degradation of MCC induced by stress.

Implications of zonal architecture on differential gene expression profiling and altered pathway expressions in mandibular condylar cartilage

Mandibular condylar cartilage (MCC) is a multi-zonal heterogeneous fibrocartilage containing different types of cells, but the factors/mechanisms governing the phenotypic transition across the zones have not been fully understood. The reliability of molecular studies heavily rely on the procurement of pure cell populations from the heterogeneous tissue. We used a combined laser-capture microdissection and microarray analysis approach which allowed identification of differential zone-specific gene expression profiling and altered pathways in the MCC of 5-week-old rats. The bioinformatics analysis demonstrated that the MCC cells clearly exhibited distinguishable phenotypes from the articular chondrocytes. Additionally, a set of genes has been determined as potential markers to identify each MCC zone individually; Crab1 gene showed the highest enrichment while Clec3a was the most downregulated gene at the superficial layer, which consists of fibrous (FZ) and proliferative zones (PZ). Ingenuity Pathway Analysis revealed numerous altered signaling pathways; Leukocyte extravasation signaling pathway was predicted to be activated at all MCC zones, in particular mature and hypertrophic chondrocytes zones (MZ&HZ), when compared with femoral condylar cartilage (FCC). Whereas Superpathway of Cholesterol Biosynthesis showed predicted activation in both FZ and PZ as compared with deep MCC zones and FCC. Determining novel zone-specific differences of large group of potential genes, upstream regulators and pathways in healthy MCC would improve our understanding of molecular mechanisms on regional (zonal) basis, and provide new insights for future therapeutic strategies.

The Antioxidant Resveratrol Protects against Chondrocyte Apoptosis by Regulating the COX-2/NF-κB Pathway in Created Temporomandibular Osteoarthritis.

Temporomandibular joint osteoarthritis (TMJOA) is characterized by chronic inflammatory degradation of mandibular condylar cartilage (MCC). Studies have found a positive correlation between inflammation and cyclooxygenase- (COX-) 2 in OA pathology. NF-κB is a crucial transcription factor of inflammatory and immune responses in the cause of TMJOA pathology. Resveratrol (RES) plays a critical role in antioxidation and anti-inflammation. But, studies on the effects of RES on TMJOA are very limited. So, the purpose of this study is to investigate the antioxidant and protective effects of RES against MCC degradation through downregulating COX-2/NF-κB expression. In vitro studies, the MCC cells were divided into three groups: the NC group, OA group, and RES group. The optimum dose of RES (10 μM) was determined. The TMJOA model of mice was created by injection of collagenase. And mice were injected with RES (100 μg/10 μl) 3 times one week for 4 weeks in the RES group. The expressions of COX-2, P65, MMP1, MMP13, COL2, and ACAN were measured by RT-PCR. Morphological changes of MCC were studied with HE staining. The results showed that inflammation could induce MCC degradation in vitro and vivo, while RES could reverse the degradation. Meanwhile, RES could downregulate COX-2/NF-κB/MMP expression and increase cartilage markers in vitro and vivo studies. The results indicated that RES treatment had antioxidant effects against chondrocyte apoptosis by downregulating the COX-2/NF-κB pathway in created TMJOA.

Estrogen receptor alpha mediates mandibular condylar cartilage growth in male mice.

In the appendicular skeleton, estrogen via ERα signalling has been shown to mediate endochondral growth plate fusion in both males and females. However, the role of ERα in mediating growth of the mandibular condylar cartilage is unknown. Thus, this study focuses on the characterization of the mandibular condylar cartilage phenotype in young and adult male ERαKO mice. Columbia University Medical Center. WT and ERαKO C57BL/6 male mice were sacrificed at 49days or 9months for phenotypic analysis. Changes to MCC thickness, cell number and cell density were measured using histomorphometric methods. Cartilage-specific gene expression and OARSI scores were investigated for 49-day and 9-month-old male ERαKO and WT mice. In young mice, a significant increase in the number of mandibular condylar cartilage cells and a significant decrease in the expression of Col10, Runx2 and DMP1 were observed in the male ERαKO mice compared to WT. In 9-month-old mice, we found a similar increase in the number of cells but no change in osteoarthritic histological scoring in ERαKO mice compared to WT mice. In summary, estrogen plays a role in mediating mandibular condylar maturation in young male mice. However, according to this study, it does not play a role in mediating long-term growth or age-related mandibular condylar cartilage degeneration in males.

MicroRNA-200a Regulates the Development of Mandibular Condylar Cartilage

Mandibular condylar cartilage (MCC) is classified as secondary cartilage, the histologic structure of which is unique from that of primary cartilage. MicroRNA (miRNA) is a small noncoding RNA that binds to the messenger RNA (mRNA) target to repress its translation and plays an important role in cell differentiation, proliferation, and death. Microarray analysis revealed that miR-200a was characteristically expressed during embryonic development. We hypothesized that miR-200a may be involved in regulating the formation of cartilage during MCC growth. We investigated the function of miR-200a by transfecting an inhibitor or mimic into MCC organ and cell cultures. A histologic examination revealed the localized inhibitory effects of the miR-200a mimic and widespread enhancing effects of the inhibitor on chondrocytic differentiation in the MCC organ culture system. An immunohistochemical examination and gene expression analysis demonstrated that the miR-200a inhibitor enhanced chondrogenesis, while the mimic had the opposite effect by enhancing cell proliferation. Quantitative reverse transcription polymerase chain reaction analysis revealed that miR-200a downregulated the gene expression of chondrocyte markers. Moreover, transfection of the miR-200a mimic into ATDC5 cells repressed the formation of the cartilaginous matrix. These results indicate that miR-200a contributed to chondrogenesis in developing MCC by controlling proliferation and differentiation in MCC cells.

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.

Hyaline cartilage cells outperform mandibular condylar cartilage cells in a TMJ fibrocartilage tissue engineering application

To compare temporomandibular joint (TMJ) condylar cartilage cells in vitro to hyaline cartilage cells cultured in a three-dimensional (3D) environment for tissue engineering of mandibular condylar cartilage. Mandibular condylar cartilage and hyaline cartilage cells were harvested from pigs and cultured for 6 weeks in polyglycolic acid (PGA) scaffolds. Both types of cells were treated with glucosamine sulfate (0.4 mM), insulin-like growth factor-I (IGF-I) (100 ng/ml) and their combination. At weeks 0 and 6, cell number, glycosaminoglycan (GAG) and collagen content were determined, types I and II collagen were visualized by immunohistochemistry and GAGs were visualized by histology. Hyaline cartilage cells produced from half an order to a full order of magnitude more GAGs and collagen than mandibular condylar cartilage cells in 3D culture. IGF-I was a highly effective signal for biosynthesis with hyaline cartilage cells, while glucosamine sulfate decreased cell proliferation and biosynthesis with both types of cells. In vitro culture of TMJ condylar cartilage cells produced a fibrous tissue with predominantly type I collagen, while hyaline cartilage cells formed a fibrocartilage-like tissue with types I and II collagen. The combination of IGF and glucosamine had a synergistic effect on maintaining the phenotype of TMJ condylar cells to generate both types I and II collagen. Given the superior biosynthetic activity by hyaline cartilage cells and the practical surgical limitations of harvesting cells from the TMJ of a patient requiring TMJ reconstruction, cartilage cells from elsewhere in the body may be a potentially better alternative to cells harvested from the TMJ for TMJ tissue engineering. This finding may also apply to other fibrocartilages such as the intervertebral disc and knee meniscus in applications where a mature cartilage cell source is desired.

Effects of growth factors and glucosamine on porcine mandibular condylar cartilage cells and hyaline cartilage cells for tissue engineering applications

Temporomandibular joint (TMJ) condylar cartilage is a distinct cartilage that has both fibrocartilaginous and hyaline-like character, with a thin proliferative zone that separates the fibrocartilaginous fibrous zone at the surface from the hyaline-like mature and hypertrophic zones below. In this study, we compared the effects of insulin-like growth factor-I (IGF-I), basic fibroblast growth factor (bFGF), transforming growth factor beta1 (TGF-β1), and glucosamine sulphate on porcine TMJ condylar cartilage and ankle cartilage cells in monolayer culture. In general, TMJ condylar cartilage cells proliferated faster than ankle cartilage cells, while ankle cells produced significantly greater amounts of glycosaminoglycans (GAGs) and collagen than TMJ condylar cartilage cells. IGF-I and bFGF were potent stimulators of TMJ cell proliferation, while no signals statistically outperformed controls for ankle cell proliferation. IGF-I was the most effective signal for GAG production with ankle cells, and the most potent upregulator of collagen synthesis for both cell types. Glucosamine sulphate promoted cell proliferation and biosynthesis at specific concentrations and outperformed growth factors in certain instances. In conclusion, hyaline cartilage cells had lower cell numbers and superior biosynthesis compared to TMJ condylar cartilage cells, and we have found IGF-I at 100 ng/mL and glucosamine sulphate at 100 μg/mL to be the most effective signals for these cells under the prescribed conditions.

Phenotypic switching of in vitro mandibular condylar cartilage during matrix mineralization.

In order to analyze the phenotypic conversion of chondrocytes, mandibular condyles of mice and rabbits were cultured under cell and organ culture systems, and then examined by a combination of morphological and biochemical procedures. In organ culture, mandibular condylar cartilage (MCC) obtained from newborn mice began to mineralize from the central zone and then progressively widened towards the peripheral zone. Electron microscopic observations showed that with the increasing duration of the organ culture, chondrocytes at the central zone converted into spindle-shaped osteoblastic cells accompanying the formation of the bone type of thick-banded collagen fibrils. To obtain a better understanding of the chondrocytic conversion, immunolocalizations for type I and type X collagens and osteocalcin (OC) were examined in mouse MCC cells in cell culture. Type X collagen and OC were expressed almost simultaneously at the late stage of culture, and type I collagen was detected along the calcified nodules after the production of these proteins. Northern blot analysis in cell cultures of rabbit MCC indicated that type II collagen and alkaline phosphatase (ALPase) messenger ribonucleic acids (mRNAs) were highly expressed at day 7, but subsequently decreased. In contrast, mRNA for type I collagen was expressed at a low level on day 7 and peaked on day 12. The present results suggest that, morphologically and biochemically, cellular modification in MCC cells under culture conditions occurs at a cellular morphological level and also at marker-gene-expression level.

Changes in phenotypic gene expression in rat mandibular condylar cartilage cells during long-term culture

Gene expression patterns have been investigated in prolonged cultures of rat mandibular condylar cartilage (MCC) cells to examine the possibility of culture-induced phenotypic changes. MCC cells were isolated from newborn rats and grown in the presence of 10% fetal bovine serum (FBS) and basic fibroblast growth factor (bFGF). MCC cells were passaged and cultured in the presence of 10% FBS and bFGF until confluent. After confluence, the medium was changed to that supplemented with 10% FBS, ascorbate, and beta-glycerophosphate (day 1). Mineralization and gene expression of MCC cells have been investigated. Mineralization, visualized by staining with Alizarin red, was observed to begin at day 13 in culture, and increased up to day 22 in culture, which was the length of this study. Type II collagen and aggrecan mRNAs were highly expressed at the start of culture (day 1-4) and decreased in a time-dependent manner. Type I collagen, alkaline phosphatase, and osteopontin mRNAs expressed biphasic patterns that peaked at the start of culture and the beginning of mineralization (day 13-16). Osteonectin mRNA was expressed throughout the culture period. Osteocalcin mRNA was expressed before the beginning of mineralization peaking at day 7. These observations suggest that the gene expression patterns of MCC cells can be categorized into two different periods in prolonged culture: maturation (day 1-10) and mineralization (day 13-22). The day culture system of MCC represents a new model system in which the differentiation of embryonic MCC cells can be examined.

Nodule formation and calcification of mandibular condylar cartilage cell cultures mimic in vivo ultrastructure.

Mandibular condylar cartilage (MCC) of growing mammals contains four layers of cells which display a series of increasingly differentiated phenotypes and which culminate in a terminally differentiated cell that produces a calcified matrix. In this study, MCC cells were placed into culture in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum and 50 micrograms/ml ascorbic acid. After 12 h in culture, transmission electron microscopy revealed the presence of multiple cell types that underwent differentiation with additional time in culture. By 7 days, fusiform-shaped cells were seen that contained numerous actin-like cytofilaments and micropinocytotic vesicles characteristic of myofibroblasts. Chondroblast-like cells were also observed. By 10 days, without addition of beta-glycerophosphate or dexamethasone, these cellular events culminated in the formation of mineralized nodules containing matrix vesicles. The nodular surface at day 13 consisted of two or more layers of myofibroblast-like cells, while the deeper zones of the nodule contained cells displaying a morphology typical of calcified hypertrophic chondroblasts. These ultrastructural observations are consistent with the hypothesis that cells from the MCC are capable of recapitulating in culture the maturational events seen in vivo. This cell culture model may be useful for investigating cell-mediated cartilage calcification without the addition of exogenous phosphate or other regulatory factors.

The behaviour of mandibular condylar cartilage in cell culture

This investigation aimed to identify the behaviour of primate mandibular condylar cartilage cells in an in vitro model. Cells were harvested from the mandibular condyles of 1-year-old marmosets and maintained in primary culture for up to 30 d. Cell proliferation, structure, and phenotype were assessed at regular intervals by phase-contrast microscopy, cytologic staining, and immunocytochemistry. Cell growth increased progressively, and cultures reached confluence after 22 d. Cells were initially small and round but later became enlarged and heterogeneous in shape. Polygonal and hypertrophic chondroblast-like cells dominated the mature cultures. Glycosaminoglycan synthesis increased as cultures aged. Type I collagen was produced by early cultures, whereas type II collagen predominated in mature confluent monolayers. It is suggested that this mixed population of cells underwent phenotypic changes in primary cell culture that closely resemble the normal in vivo maturation process.

Phenotypic Changes of Rabbit Mandibular Condylar Cartilage Cells in Culture

The present study describes the behavior of mandibular condylar cartilage (MCC) cells as a function of time in primary culture, since it is not yet clear whether these cells maintain their phenotype in culture. MCC cells from New Zealand white rabbits were seeded at high density and cultured in DMEM containing 50 micrograms/mL ascorbic acid and 10% fetal bovine serum. These cells appeared as a heterogeneous population and changed their shape, size, and refractivity as cultures aged. Cartilage-like cells, which always dominated the culture, were infiltrated with a minority of fibroblast-like cells. Cell number increased progressively, and cultures reached confluence at nine days. Antibody activity for cartilage-specific glycosaminoglycan was determined by ELISA assay. This reaction reached a maximum at six days and decreased thereafter. Cultures stained with Alcian blue (pH 1.0) supported these results. Cytoplasmic mRNA analysis indicated that the transcription of type II collagen gene was present at all time points. Type I collagen and alkaline phosphatase mRNA levels showed progressive increases from 12 h to nine days, with significantly higher values in cells cultured for six, nine, and 12 days than in cells collected from earlier time points. These results suggest that in our present culture system, MCC cells undergo phenotypic changes that resemble their maturation processes in vivo.