Experimental Study Of The Differentiation Effect Of TGF-β1 Inducing BMSCs On Acetabulum Chondrocytes In DDH

[Background and Objective]Developmental dysplasia/dislocation of the hip (DDH) is one of the most common lower limb malformations in pediatric orthopedics. It cause acetabular cartilage development arrested in the early stage, which probably lead to Osteoarthritis (OA) in long term. Compared with the normal population, about 50 percents with DDH OA have to receive total hip arthroplasty (THA), which bring great economic burden and physical sufferings to the patients.Cartilage engineering has emerged as a potential effectively way to repair osteochondral defect caused by articular degeneration, trauma, OA. In recent years, a lot of studies showed that TGF-β1 can regulate the concentration of mesenchymal stem cells (MSCs) to induce chondrogenesis. Sox9, the downstream factor of TGF-β1, is the master regulator of cartilage development. Overexpression of Sox9 in MSCs directly activate transcription of genes required for cartilage development including extracellular matrix genes Col2al, GAG and Aggrecan. But the application of cartilage engineering on DDH has not been reported.Based on Our previous studies, we design the co-culture between BMSCs induced by TGF-β1 and normal or DDH acetabular chondrocytes and detect the changes of the expression of BMP-2 in the co-cuculture models to explore whether BMSCs induced by TGF-β1can improve the maturation of DDH or normal acetabular chondrocytes and to present theory evidence for further experiment in vivo.[Methods]Part 1. To establish animal model of DDH, and acquire acetabular chondrocyte.1. According to the precious methods to establish animal model of DDH. we modified swadding position on 20 newborn SD rats though extension fixation of bilateral hips and knees with medical tape for 7 days as the DDH group. Another 20 newborn SD rats without any treatment are the normal group.2. That rats were sacrificed at ages of 8 days repectively with over-dose anesthesia. The hips were taken out in sterility and the hips which failed to form DDH models were discharged in DDH group.4 normal hips and 4 DDH hips were sent for HE stain, and acetabular were taken out from the rest hips, which would be digested for acetabular chondrocytes.Part 2. The isolation, culturing and identifieation of rat bone marrow derived MSCs1. Another 10 ratswere sacrificed at ages of 4 weeks, Whole bone marrow adherent cultivation was used to isolate bone marrow MSCs from rats.2. The third passage BMSCs identified the positivity of CD34, CD90, and CD29 by flow cytometry.Part 3. The co-culture system was formed, and BMP-2 were detected.1. Direct co-culture system:The acetabular chondrocytes in DDH group or normal group were added in relevant wells, co-cultured with The P3 BMSCs. Meanwhile, DACs and NACs and BMSCs were cultured respectively as the control groups. According to the different concentration of TGF-β1 (0,10 ng/ml), the cultured groups were distributed into 10 groups, and each had 3 wells repeated.2. Transwell indirect co-culture system:groups were setted as the direct coculture system. DACs or NACs were added into the transwell inserts, while the BMSCs were added into the plate wells.3. The BMP-2 were detected by enzyme linked immunosorbent assay (ELISA) after 3 days culturing.Part 4. The results were analyzed using SPSS 16.0 software.[Results]1. The achievement ratio of establishing animal model of DDH achieved 90%(18/20). The hips of rats in DDH model showed the lateral acetabulum surface irregular flat, shallow and hypertrophy, embedding of softtissue. HE stain showed irregular actabular chondrocytes and cracks in extracellular matrix.2. The results of flow cytometry on BMSCs passage 3 showed that the ratio of positive of CD9O, CD29 and negative of CD34 was 98.5%.3. When cultured alone, DACs was fusiform and unevenly distributed. No significant chondrocyte hypertrophy was observed until the seventh day of coculture. Suggesting DACs were dedifferentiation when cultured alone. In co-cultured with BMSCs and in the presence of TGF-β1, DACs from turned round, and in the third day of culture appeared hypertrophic chondrocytes. This morphological changes were not observed in co-culture system without TGF-β1.4. Increased BMP-2 level was abserved in normal groups cocultured with BMSCs compared to DDH groups in the direct coculture system in 3 days, P<0.001. When added in TGF-01 (10ng/ml), the BMP-2 level of DDH groups was higher than in normal groups. Similar results were detected in the transwell coculture systems in 7 days.[Conclusions]1. The chondrocyte morphologic change indicated that the chondrocytes shifted from differentiation to dedifferentiation.2. BMSCs may promote differentiation of NACs but DACs, suggest extra factors needed to promote differentiation of DACs.3. BMSCs induced by TGF-β1 may promote differentiation of DACs which turned round, and hypertrophic chondrocytes were observed, with BMP-2 level increased.4. Cell-cell interaction may promote the effect of BMSCs or BMSCs induced by TGF-β1 on normal or DDH acetabular chondrocytes. Thus suggest that further in vivo experiments, TGF-β1-induced BMSCs with DACs in direct contact, can promote DACs differentiation and maturation more efficient.