| Objective:(1) To investigate the biological effects of sustained release bFGF microspheres on the mesenchymal stem cells from the bone marrow(BMSCs) of rabbits in vitro in order to offer experimental reference for their using in vivo . (2) We investigated the effect of bFGF microspheres on the healing of different sizes of articular cartilage defects in rabbits. (3) Investigating the effect of bFGF microspheres combine with type-â…¡ collagen sponge on the healing of the sizes of articular cartilage defects which can not be repired by using bFGF microspheres only in rabbits. Method:(1)The secondary cultured BMSCs of rabbits were divided into three groups according to the different ingredients being added to the DMEM culture medium , ie , control group , bFGF group , bFGF- PLGA microspheres group. The proliferation of the cultured BMSCs of rabbits was measured with cell counting method, MTT method and flow cytometry at 1 ,2, 4, 7 and 10 days. (2) BMSCs of rabbits were divided into three groups according to the different ingredients being added to the same condition of inducing BMSCs to differentiate into chondrocytes , ie , control group , bFGF group , bFGF- PLGA microspheres group. Matrix of cartilage cells were detected by alcian blue staining, and cartilage specific collagen â…¡ wasdetested by immunohistochemistry and in situ hybridization . (3) Full-thickness defects were made in 48 male New Zealand white rabbits.According different sizes and methods,the rabbits were divided into four groups(A,free-bFGF group :5x5x3mm; microspheres group : B group, 5x5x3mm , C group, 5x7x3mnu D group, 5x9x3mm). The speciments were collected in 4,8,12 weeks post-operation. General observation, histological grading and immunohistochemistry and in situ hybridization for type â…¡ collagen were applied and the glycosaminoglycan content was quantified to the repire of defects in different groups. (4) Investigating the effect of bFGF microspheres combine with type-â…¡ collagen sponge on the healing of the sizes of articular cartilage defects which can not be repired by using bFGF microspheres only in rabbits.Result:(1) The in vitro cellular study showed no significent difference in the cell number and cell viability of three groups one day after plate culture. The cell number and cell liability in the free-bFCF group were more than those in other two groups at two days. The bFCF/PLGA group were more than those in other two groups four and seven and ten days after plate culture with significant difference. The flow cytometrical examination showed that the PI in the free-bFCF group reached the highest two days after plate culture and the bFGF/PLCA group went the highest after four days. (2) It was confirmed that these was expression of type II collagen mRNA in bFGF/PLCA group and free-bFCF group at seven days after inducing with insignificant difference ,but with significant difference at 2 weeks after inducing (bFGF/PLCA group> free-bFCF group>control group ).(3) The average total scores on the histological grading scale were significantly better (p < 0.05) for the defects treated with bFGF microspheres than for the untreated defects at all time-points. At 12 weeks, the thickness of the repair cartilage wassimilar wirh the normal adjacent articular cartilage and a new tidemark usually had formed between the repair cartilage and the underlying subchondral bone in group B . Immunostaining with an antibody against type-â…¡ collagen showed the diffuse presence of this cartilage-specific collagen throughout the repair cartilage in the treated defects and positive rate in group B was highest then the others with significant difference. The histological scores and GAG content in group B was more than those in other two groups with significant difference. Conclution:(1)bFGF/PLGA microspheres can promote the proliferation and differentiation to chondrocyte of BMSCs through a long periond of controlled release of bFGF and superior to free- bFGF with significant difference. (2)This study indicated that the intraarticular injection of bFGF/PLGA microspheres could repair of articular cartilage defects in the knees of rabbits with like-cartilage tissue.But this repairment related to the size of the defect,it can only repire the size of the defect under 5x7x3mm ,can not repir the size more than this. (3) Using bFGF microspheres combine with type-â…¡ collagen sponge together can repire the sizes of osteochondral defects(5x7x3mm) which can not be repired by using bFGF microspheres only in rabbits with like-cartilage tissue. |
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