| Background:OA is a joint disease of relatively frequent occurrence that is characterized by progressive damage to the articular cartilage and subchondral bone, causing pain and disability in older adults. Several tissues of the joint, including the cartilage, synovial membrane, and subchondral bone, play significant roles in the development of OA pathology. MicroRNAs (miRNAs) are a large family of small (21-25-nucleotide) noncoding RNAs. Recent evidence indicates that miRNAs have important roles in regulating osteogenic and chondrogenic differentiation and proliferation, eventually influencing the catabolism and anabolism of bone and cartilage. In this study, we focused on detecting the expression level of miR-214 in cartilage and subchondral bone of different graded osteoarthritis (OA), which is classified by Outerbridge criteria, and demonstrating the function of miR-214 in the development of OA.Methods:(1) Human tibial plateaus were collected from patients undergoing total knee arthroplasty (TKA), from October 2013 to December 2014. Then, classified these samples by Outerbridge criteria, and detected the different expression of miR-214 in cartilage and subchondral bone of different degree. (2) Extracted chondrocytes from 3 days year old C57 mouse, which were then calculated by IL-1β or IL-1β plus antagomir-214 in different time, and analysis the viability of chondrocytes. (3) 40 C57 mouses were divided into two groups, control group and experiment group, which were collected knee joint respectively after 2 weeks and 4weeks to analysis the difference in histological staining, immunohistological staining, and RT-PCR. (4) Knee joint chondrocytes were isolated from New Zealand white rabbits,2 months old, and expanded in vitro. The chondrocytes at passage 2 were seeded onto a scaffold of articular cartilage extracellular matrix in the concentration of 1×106/L to prepare cell-scaffold composites. Cell-scaffold composites were cultivated in an Instron bioreactor with mechanical compression (1 Hz,3 hours per day,10% compression) as experimental group for 7.14.24.28 days or cultured statically for 1 day as control group.Results:(1) After detected the different outerbridge degree samples, it showed that miR-214 was examined in all cartilage and subchondral bone. Compared the expression level of miR-214, it had significant difference between 4 groups, and showed a rising trend (P<0.05). And it had a positive correlation between the severity of OA and the expression level of miR-214. In grade Ⅲ group, the level of miR-214 in subchondral bone was higher than other groups (P<0.05). (2) After different cultured by IL-1βand IL-1β+antagomir-214, it showed that in histological staining, normal chondrocytes have a clearly morphology and obviously positive staining in extracellular. In IL-1β group, with the time extend of cultured, the safranin "O" staining and toluidine blue (TB) staining were decreased. And in IL-1β+antagomir-214 group, it was increased with the culture time extending. In the immunohistological staining, it showed that the COL-I, COL-X, MMP-13 positive staining were increased with the time prolonged, but the COL-II staining was decreased. But in IL-1β+antagomir-214 group, with the culture time prolonged, the staining of COL-I, COL-X, MMP-13 were decreased, and higher than normal group; COL-II staining was increased. In the result of RT-PCR, it showed in IL-1β group, the expression of COL-II was decreased progressively, and the expression of COL-I, COL-X, MMP-13 were increased gradually (P<0.05). At the same time, it showed the expression of miR-214 was increased gradually. In IL-1β+antagomir-214 group, the expression of COL-II was increased progressively; the expression of COL-I, COL-X, MMP-13 were reduced gradually (P<0.05). (3) After constructed the OA model, the knee joints were analyzed by X-Ray, and it showed the local bone hyperplasia, change of force line, narrow of joint space, and it was obvious in the progression of OA. After injected antagomir-214 in the articular cavity, the lesion and degradation of joint in OA model was decreased, comparing with the without injection group. The expression of miR-214 in OA model was increased gradually with the time extend (P<0.05). In the histological staining, the cartilage cells were proliferated, hypertrophic, arranged in disorder, and the extracellular matrix positive staining was reduced in the late. And in the 4weeks ACLT group, it had more serious degradation in cartilage than that in ACLT+antagomir-214 group. In the immunohistological staining, it showed that the collegen type Ⅱ staining was decreased progressively in ACLT group than that in ACLT+antagomir-214 group. (4) Morphological observations demonstrated that the thickness, elastic modulus and maximum load of the composite in the experimental group were significantly higher than those in the control group, which were positively related to time (P< 0.05). Histological staining showed the proliferation of chondrocytes, formation of cartilage lacuna and synthesis of proteoglycan in the experimental group through hematoxylin-eosin staining and safranin-O staining, which were increased gradually with mechanical stimulation time. Real-time quantitative PCR revealed that mRNA expressions of collagen type Ⅰ and collagen type Ⅱ were significantly higher in the experimental group than the control group (P< 0.05). The experimental group showed the highest mRNA expression of collagen type Ⅰ and collagen type Ⅱ at 21 and 28 days of mechanical stimulation, respectively (P< 0.05). And the expression of miR-214 was increased after 21d with the mechanical stimulation.Conclusion:(1) The expression level of miR-214 in 4 groups is keeping rising, which is relative with the severity of OA progression. And the expression level of miR-214 also has obvious change in subchondral bone. (2) The antagomir-214 has significant function in slow down the progression of OA. Our findings indicate that miR-214 has intertwined relationship with the development and progression of OA, and provide theoretical foundation for OA development and treatment. |
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