SURFACE MARKERS AND GENE EXPRESSION TO CHARACTERIZE THE DIFFERENTIATION OF MONOLAYER EXPANDED HUMAN ARTICULAR CHONDROCYTES

Autologous chondrocyte implantation (ACI) is a method of cartilage repair. To improve the quality of regenerated tissue by ACI, it is essential to identify surface marker expression correlated with the differentiation status of monolayer expanded human articular chondrocytes and to define the index for discriminating dedifferentiated cells from monolayer expanded human articular chondrocytes. Normal human articular chondrocytes were cultured in monolayer until passage 4. At each passage, mRNA expression of collagen type I, II, and X and aggrecan was analyzed by real-time quantitative PCR, and the surface marker expression of CD14, CD26, CD44, CD49a, CD49c, CD54, and CD151 was analyzed by fluorescence-activated cell sorting (FACS). The ratios of mRNA levels of collagen type II to I (Col II/Col I) represented the differentiation status of chondrocytes more appropriately during monolayer culture. The surface marker expression of CD44, CD49c, and CD151 was upregulated according to the dedifferentiation status, whereas that of CD14, CD49a, and CD54 was downregulated. The most appropriate combination of the ratio of Col II/Col I was CD54 and CD44. Cell sorting was performed using a magnetic cell sorting system (MACS) according to CD54 and CD44, and real-time quantitative PCR was performed for the cell subpopulations before and after cell sorting. The expression of collagen type II and aggrecan of the chondrocytes after MACS was higher than that before sorting, but not significantly. The mean fluorescence intensity (MFI) ratio of CD54 to CD44 could be an adequate candidate as the index of the differentiation status

MicroRNA-125b regulates the expression of aggrecanase-1 (ADAMTS-4) in human osteoarthritic chondrocytes

INTRODUCTION: Increased expression of aggrecanase-1 (ADAMTS-4) has emerged as an important factor in osteoarthritis (OA) and other joint diseases. This study aimed to determine whether the expression of ADAMTS-4 in human chondrocytes is regulated by miRNA. METHODS: MiRNA targets were identified using bioinformatics. Chondrocytes were isolated from knee cartilage and treated with interleukin-1 beta (IL-1β). Gene expression was quantified using TaqMan assays and protein production was determined by immunoblotting. Luciferase reporter assay was used to verify interaction between miRNA and target messenger RNA (mRNA). RESULTS: In silico analysis predicted putative target sequence of miR-125b on ADAMTS-4. MiR-125b was expressed in both normal and OA chondrocytes, with significantly lower expression in OA chondrocytes than in normal chondrocytes. Furthermore, IL-1β-induced upregulation of ADAMTS-4 was suppressed by overexpression of miR-125b in human OA chondrocytes. In the luciferase reporter assay, mutation of the putative miR-125b binding site in the ADAMTS-4 3'UTR abrogated the suppressive effect of miR125. CONCLUSIONS: Our results indicate that miR-125b plays an important role in regulating the expression of ADAMTS-4 in human chondrocytes and this identifies miR-125b as a novel therapeutic target in OA