Repair Of Cartilage Defects Of Osteoarthritis Animal Models In Rabbit Knee Joints And Releated Research

1. Comparison of Two Osteoarthritis Models in Rabbit[Objective] To establish osteoarthritis model in rabbit knees by two kinds of surgical methods, and to explore their applicable conditions. [Methods] 17 rabbits were divided into three groups (Hulth group, ligament-excised group and control group), they were anesthetized and operated on differently according to their group. The rabbits were sacrificed at 1, 3 and 6 weeks after surgery. The femoral condylars were harvested and studied in both gross morphological and pathohistological aspect. [Result] The degeneration of articular cartilage of the two surgery groups got worse by time, and their Mankin's scores were significantly higher than those in the control group (P<0.05). When compared with the ligament-excised group, degeneration of the articular cartilage of the Hulth group were more severe, and they resembled the advanced stage of osteoarthritis, while the ligament-excised osteoarthritis model resembled the early or intermediate stage of osteoarthritis. [Conclusion] Both surgical methods can successfully induce osteoarthritis in rabbit knees. The Hulth model could be fit for surgical treatment research, while the ligament-excised osteoarthritis model could be suited to study of the pathogenesis and medication of osteoarthritis. 2. Establishment of a Novel Osteoarthritis Model in Rabbit[Objective] To establish a novel osteoarthritis model in rabbit knees for the research of cartilage defect repair in osteoarthritis by tissue engineering. [Methods] 22 rabbits were divided into two groups (Upgrade group and control group). The rabbits were operated on differently according to their group and sacrificed at 3 weeks or 6 weeks after surgery. The specimens from femoral condylars and femoropatellar grooves were harvested and studied in both gross morphological and pathohistological aspect. [Result] The degeneration of articular cartilage of the two groups got worse with time, and Mankin's scores of the femoropatellar grooves in Upgrade group were significantly higher than those in the control group (P<0.01). But there were no significant difference between the femoral condylars scores of the two groups(P>0.05). [Conclusion] Both methods can develop a satisfactory experimental animal model of osteoarthritis. The novel osteoarthritis model is a good option for the research of cartilage tissue engineering.3. Autologous BMSC culture and induct into chondrocyte:[Objective] To study the isolation of bone marrow mesenchymal stem cells (BMSCs) and in vitro differentiation into chondrocytes using HA and cell aggregate, in order to achieve separation of induction scaffold from therapy scaffold. [Methods] Bone marrow was aspirated from bilateral femoral trochanters of rabbit. BMSCs were isolated and cultured in flasks and sub-cultured until the second passage. Then those cells were analyzed and identified if they were BMSCs by flow cytometry (FCM). After being centrifuged at 1000 rpm for 10 min, the cells were incorporated into the Sodium Hyaluronate. They were induced as a pelleted micromass with the inductive medium. Specific cell surface markers were identified by flow cytometry after the cells were treated with inductive medium for 7-14 days, microscopic, histoloical and immunohistochemical studies were performed for chondrogenic phenotype identification. [Result] Primary cultures of human MSCs express CD29 and CD44 positively and meanly, but CD34, CD45 and HLA DR negatively After 14 days of induction; the cells were positively stained by HE, Toluidine blue staining and Immunohistochemical analysis proved there were chondrocytes emerging and cartilage matrix secreted. There was little effect on cell cycle for 3D cell aggregate. [Conclusions] BMSCs from bone marrow aspirates can be expanded and purified. 3D cell aggregate can be induced in vitro to differentiate into chondrocytes by inductive medium and achieve separation of the induction scaffold from the therapy scaffold.4. Three-dimensional induction and differentiation of BMSCs using an injectable scaffold in vitro[Objective] To study the construction method of injectable scaffold from HA and FG, and to evaluate the 3D induction and differentiation of BMSCs into chondrocytes using an injectable scaffold in vitro. [Methods] 3D cell-scaffold Complex, composed of BMSCs, HA, FG and AM, was put into the chondrogenic medium. High-density BMSCs were induced into chondrocytes in the composite. Then we surveyed the growth of BMSCs, the degradation of scaffold, the quality change of the composite. Meanwhile,we studied the growth of chondrocyte and synthesis of cartilage matrix by histological section staining. By flow cytometry, we clarified how the scaffold affected the cell cycle and DNA contents, and caculated DNA index (DI). Using scanning electron microscope (SEM), we observated the size and shape of the scaffold cells and the number of BMSCs in the scaffold. [Result] The rate of degradation was slowwed significantly in test group. There were obvious differences in weight between two groups, after cultured at 14d, 28d or 42d in vitro(P<0.001). SEM showed the porosity of the scaffold was high; the density of BMSCs in scaffold was high. Flow cytometry showed there was no heteroploid cell; there was no harmful effect on the reproduction and matrix secretion of BMSCs-differentiated chondrocytes for those materials. [Conclution] The scaffold has no harmful effect on the reproduction of chondrocyte, maintenance of phenotype and secretion of matrix. The scaffold could keep its morphous at least 4 weeks at 37℃, which could meet the growth requirement of chondrocyte.5 Repair of cartilage defects in the rabbit osteoarthritis models[Objection] To study the possibility of using a new type scaffold after combination with autologous bone marrow stromal cells (ABMSCs) to repair articluar osteochondral defects of osteoarthritis (OA). [Method] 42 rabbits (84 knees) osteoarthritis models were established and divided into three groups (CFH group, drilling group and FG Group). Bone marrow was harevested from 14 rabbits of Group CFH, ABMSCs were cultured and in vitro expanded and induced. After induction, cells were seeded in new type scaffold and implanted into the osteochondral defects of Group CFH. In Group FG, only fibrin glue (FG) scaffolds without BMSC were implanted. The osteochondral defect bottom of Group D was drilled to reach medullary cavity. Rabbits were sacrificed at 6(n = 24), 12 (n = 30) and 24(n = 30) weeks after implantation. The specimens from femoropatellar grooves of the knee joints were harvested and studied for the histological scores. [Result] At 12 week, gross observation and histology showed that the defects in most of group CFH were completely repaired by engineered cartilage and cancellous bone, and a few defects were repaired by fibrocartilage and cancellous bone. However, in most of group D, the defects were repaired by fibrocartilage and cancellous bone with an irregular surface. No repair or only fibrous tissue was observed in groups FG. The mean scores showed: The total histological scores in the Group CFH were significant difference from those in group D or group FG at 6w, 12w and 24w after operation (p < 0.001 respectively). There was no significant difference between the Group CFH and the Group D at 6w after operation (p = 0.375). There was significant difference between group D and Group FH at 24w (p=0.015). In group CFH, The histological scores at 6w were significant difference from those at 12w or 24w after operation (p=0.23 and p=0.043 respectively). At 24w, neogenetic cartilage and femoropatellar grooves cartilage in 19 out of 30 knees degenerated obviously compared with those of 12 weeks, there were 3 out of 10 knees in the Group CFH, 6 out of 10 knees in the Group D, and all of 10 knees degenerated obviously in the Group FG. But the neocartilage became more mature and ill-demarcated to the surrounding cartilage in the Group CFH. In the Group D, The neogenetic cartilaginous tissues degenerated obviously compared with those of 12 weeks. In the Group FG, the defect existed in a long term. [Conclusion] Our study has considerable relevance to the treatment of large defects of articular cartilage and provides the basis for the development of repair technology for the regeneration of such large defects in the environment of severe OA. In Group CFH, the articular cartilage healing capacity appeared to augment and the articular cartilage degeneration rate slowed down obviously after ABMSCs were transplanted into the defect. Compared with D group, the Group CFH had better reconstitution of the surface of articular cartilage.