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By producing instability in mouse knee joints, we attempted to determine the involvement of runt‐related transcription factor 2 (RUNX‐2), which is required for chondrocyte hypertrophy, in the development of osteoarthritis (OA).An experimental mouse OA model was created by surgical transection of the medial collateral ligament and resection of the medial meniscus of the knee joints of heterozygous RUNX‐2–deficient (Runx2+/−) mice and wild‐type littermates. Cartilage destruction and osteophyte formation in the medial tibial cartilage were compared by histologic and radiographic analyses. Localization of type X collagen and matrix metalloproteinase 13 (MMP‐13) was examined by immunohistochemistry. Localization of RUNX‐2 was determined by X‐Gal staining in heterozygous RUNX‐2–deficient mice with the lacZ gene insertion at the Runx2‐deletion site (Runx2+/lacZ). Messenger RNA levels of type X collagen, MMP‐13, and RUNX‐2 were examined by real‐time reverse transcriptase–polymerase chain reaction analysis.RUNX‐2 was induced in the articular cartilage of wild‐type mice at the early stage of OA, almost simultaneously with type X collagen but earlier than MMP‐13. Runx2+/− and Runx2+/lacZ mice showed normal skeletal development and articular cartilage; however, after induction of knee joint instability, they exhibited decreased cartilage destruction and osteophyte formation, along with reduced type X collagen and MMP‐13 expression, as compared with wild‐type mice.RUNX‐2 contributes to the pathogenesis of OA through chondrocyte hypertrophy and matrix breakdown after the induction of joint instability. [ABSTRACT FROM AUTHOR]

Background: Early detection of oral squamous cell carcinomas (OSCCs) is urgently needed to improve the prognosis and quality of life (QOL) of patients. Oral leukoplakias (OLs), known as the most common premalignant lesions in the oral cavity, often precede OSCCs. Especially, OLs with dysplasia are known to have a high risk of malignant transformation. Here, we searched for the promoter methylation characteristic of high-risk OLs.Methods: To identify methylation-silenced genes, a combined analysis of methylated DNA immunoprecipitation (MeDIP) - CpG island (CGI) microarray analysis and expression microarray analysis after treatment with a demethylating agent was performed in two OSCC cell lines (Ca9-22 and HSC-2). The methylation statuses of each gene were examined by methylation-specific PCR.Results: A total of 52 genes were identified as candidates for methylation-silenced genes in Ca9-22 or HSC-2. The promoter regions of 13 genes among the 15 genes randomly selected for further analysis were confirmed to be methylated in one or more of five cell lines. In OSCC tissues (n = 26), 8 of the 13 genes, TSPYL5, EGFLAM, CLDN11, NKX2-3, RBP4, CMTM3, TRPC4, and MAP6, were methylated. In OL tissues (n = 24), seven of the eight genes, except for EGFLAM, were found to be methylated in their promoter regions. There were significantly greater numbers of methylated genes in OLs with dysplasia than in those without dysplasia (p < 0.0001).Conclusions: OLs at high risk for malignant transformation were associated with aberrant promoter methylation of multiple genes. [ABSTRACT FROM AUTHOR]

BACKGROUND: Transmission and tomographic X-ray measurements are useful in assessing bone structures, but only a few studies have examined cartilage growth because of the poor contrast in conventional X-ray imaging. OBJECTIVE: In this study, we attempted to use the linear attenuation coefficient (LAC) as a metric of tissue-engineered cartilage development, which would be useful in high-throughput screening of cartilage products. METHODS: Assuming that the LAC is related to the amount of extracellular matrix (ECM) in terms of the density and its atomic components, we measured X-ray absorption through tissue-engineered cartilage constructs. Characteristic X-ray beams from a molybdenum microfocus X-ray tube were employed to avoid beam hardening. The correlation of the LAC with mechanical properties was analyzed for verification. RESULTS: The LAC was higher for chondrocyte constructs and lower for fibroblast-dominant constructs and was consistent with the quantification of toluidine blue staining, which is a proof of ECM production. The LAC was positively correlated with the bending modulus but negatively correlated with the dynamic elastic modulus and stiffness, possibly because of the remaining scaffold. CONCLUSIONS: The LAC has the potential to be used as a metric of development of tissue-engineered cartilage. However, the calcified regions should be excluded from analysis to avoid decreasing the correlation between the LAC and the amount of ECM. [ABSTRACT FROM AUTHOR]

Although autologous chondrocyte implantation has already been in clinical use, chondrocyte dedifferentiation is problematic during proliferation culture. We attempted a three-dimensional (3D) collagen gel culture under chondrocyte proliferation with repeated passaging to prevent the chondrocytes dedifferentiation. Human auricular chondrocytes were cultured in 3D or conventional monolayer conditions, which reached a 1000-fold increase in cell numbers at passages 3 and 4, respectively. During multiplication, the chondrocytes in 3D culture showed greater suppression of collagen type I (COL1) and preservation of collagen type II (COL2) than those in monolayer. Tissue-engineered cartilage made of 3D cells also abundantly accumulated COL2 or proteoglycan and possessed favorable mechanical properties. The advantage of 3D cells may result from the similarity of microenvironments in cell-to-matrix adhesion or cell-to-cell contacts with that of native cartilage. The up-regulation of integrins and down-regulation of cadherins in the 3D cells mimicked the expression pattern of native cartilage, rather than that of monolayer cells. The silencing of integrin β1 and Ob-cadherin expression by small interfering ribonucleic acid in the cultured chondrocytes led to the promotion of dedifferentiation and redifferentiation, respectively, indicating that the 3D collagen gel culture provided sufficient cell preparation and reduced chondrocyte dedifferentiation, which is regarded as a feasible strategy in autologous chondrocyte implantation. [ABSTRACT FROM AUTHOR]

Because a temporal arrest in the G1-phase of the cell cycle is a prerequisite for cell differentiation, this study investigated the involvement of cell cycle factors in the differentiation of cultured mouse prechondrocyte cell line ATDC5. Among the G1 cell cycle factors examined, both protein and mRNA levels of cyclin-dependent kinase (Cdk6) were downregulated during the culture in a differentiation medium. The protein degradation of Cdk6 was not involved in this downregulation because proteasome inhibitors did not reverse the protein level. When inhibitors of p38 MAPK, ERK-1/2, and PI3K/Akt were added to the culture, only a p38 MAPK inhibitor SB203580 blocked the decrease in the Cdk6 protein level by the differentiation medium, indicating that the Cdk6 inhibition was mediated by p38 MAPK pathway. In fact, p38 MAPK was confirmed to be phosphorylated during differentiation of ATDC5 cells. Enforced expression of Cdk6 in ATDC5 cells blocked the chondrocyte differentiation and inhibited Sox5 and Sox6 expressions. However, the Cdk6 overexpression did not affect the proliferation or the cell cycle progression, suggesting that the inhibitory effect of Cdk6 on the differentiation was exerted by a mechanism largely independent of its cell cycle regulation. These results indicate that Cdk6 may be a regulator of chondrocyte differentiation and that its p38-mediated downregulation is involved in the efficient differentiation. 2005 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]