| Part I. The isolation, cultivation, identification and differentiation of rabbit bone mesenchy mal stem cells invitroObjective:To explore the method of isolating, cultivating and identification of rabbit bone mesenchymal stem cells (BMSCs), and study their capacity of proliferation and potential of differentiation in vitro.Method:Rabbit BMSCs in rinse solution of bone marrow was isolated by density gradient centrifugation and bone marrow culture, cultured in vitro. Flow cytometry was used to examine the expression of CD29, CD34 and CD44 cell surface antigens. The proliferation ability of the 4th and 8th passage cell was determined by MTT assay and the growth curve was drawed based on it. The 3rd passage cell was induced osteogenesis and chondrogenesis respectively, then alkaline phosphatase and VonKossa staining, immunocytochemical stain and RT-PCR of collegen I were used to examined the osteogenesis cells and toluidin blue and safranin O staining, immunocytochemical stain and RT-PCR of collegen II were used to examined the chondrogenesis cells.Results:The morphous of BMSCs obtained by density gradient centrifugation and bone marrow culture are spindle-shaped or polygon-shape uniformly, when the cells reached almost confluence, they presented colony whirlpool. Flow cytometry showed that more than 90% cells expressed CD44 and CD29, while only 5% of the cells expressed CD34. The growth curve showed that cells of the 4rd and 8th generation have high proliferation. For osteogenesis cells, the alkaline phosphatase staining and collegen I immunocytochemical stain was positive, VonKossa staining showed the calcium nodule, and mRNA of collegen I was observed in RT-PCR. For chondrogenesis cells, the toluidin blue staining showed the metachromasia of the cells, safranin O staining and collegen II immunocytochemical stain was positive, and the mRNA of collegen II was observed in RT-PCR.Conclusion:Density gradient centrifugation combined with bone marrow culture can isolate and purify the BMSCs ideally. Under the effect of inducer, BMSCs can represent specific cyte phenotype of chondrocytes or osteocytes, and can be used in tissue engineering to restore the osteochondral defect. BMSCs have satisfactory proliferation, self-renewal and cell differentiation potentiality in vitro, which are ideal seed cells of tissue engineering. Part II. The study of preparation and biological characteristics of PLGA-PCL/PLGA-HA-βTCP biphasicscaffoldObjective:To research and prepare a novel biphasic PLGA scaffold, and examine the structure and biological characteristics, explore the feasibility to be a tissue engineering scaffold.Method:Scaffold preparation:particle leaching and casting method were used to prepare the biphasic PLGA scaffold, hyaluronic acid was vac-sorb on the surface of the scaffold. Structure observe:observe the scaffold appearance in general and scanning electron microscopy examine the incisal surface. Biocompatibility:implant the scaffold into the subcutaneous tissue, observe the reaction of the local tissue and the speed of scaffold degradation. Examine the sells implanted scaffold with scanning electron microscopy and HE staining of paraffin section.Results:The appearance of the scaffold is white spongioid material, include two layers. Scanning electron microscopy showed the aperture consistent with the design. The implanted scaffold has not induce the inflammation, and degradation gradually. Until 12 weeks, the scaffold has degraded premodinantly. Both scanning electron microscopy and HE staining of paraffin section indicate the biphasic scaffold has satisfactory biocompatibility.Conclusion:In our study, the method to prepare the scaffold is simple, and the scoffold parameters are easy to control. The two layers are integrated tightly, which improve the flaw of the common biphasic scaffold. The biocompatibility of the biphasic scaffold is satisfactory, which facilitate the adsorption and proliferation of BMSCs. With the modification of the both layer, the BMSCs can differentiate to the purpose cells. The structure of the biphasic PLGA scaffold is more similar to the normal joint, the degradation time is satisfactory, so the biphasic PLGA scaffold can restore the osteochondral defect ideally. PartⅢ. Restore the osteochondral defect of rabbit knee joint with the PLGA-PCL/PLGA-HA-βTCP biphasic scaffold combined with BMSCsObjective:To investigate the reparation effect of osteochondral defect with biphasic PLGA scaffold combined with BMSCs. Explore the feasibility that the biphasic PLGA scaffold, loaded with BMSCs, work as tissue engineering scaffold to restore the osteochondral defect.Method:Thirty healthy rabbit, sixty knees are randomly devided into three groups (A, B, C group). On femoral trochlea of each knee joint, a diameter 5mm, depth 4-5mm cylindrical hole was made to cause osteochondral defect. A group is blank, which spontaneous restoration; and implant only biphasic scaffold in the osteochondral defect in B group, and the compomers of scaffold and BMSCs in C group. After 6w and 12w, sacrifice the rabbit respectively, compare the score of appearance and histological staining of specimen. Biomechanics test was proceed to examine the sample of B group, C group and normal joint. All the properties are compred with the normal joint tissue respectively.Result:6 weeks postoperative, A group the score of appearance in general is 4.3±0.9, score of histology is 0.8±0.2; B group the score of appearance in general is 6.8±0.7, score of histology is 5.1±0.8; C group the score of appearance in general is 8.1±0.4, score of histology is 12.4±0.9.12 weeks postoperative, A group the score of appearance in general is 5.2±1.2, score of histology is 3.7±0.5; B group the score of appearance in general is 7.7±0.8, score of histology is 10.3±0.6; C group the score of appearance in general is, score 9.2±0.3 of histology is 17.2±0.3. In the whole, the restore effect of C group is the best, and A group is the worst. Biomechanics test show:the creep time of normal joint is 2763±649s, stress relaxation time 2142±402s, Young's modulus 0.36±0.22MPa; the creep time of B group is 2189±533s, stress relaxation time 1647±381s, Young's modulus 0.29±0.17MPa; the creep time of C group is 2314±592s, stress relaxation time 1936±334s, Young's modulus 0.32±0.13MPa. The creep time and stress relaxation time of B group and C group is shorter than the normal joint, and Young's modulus is smaller the normal joint but there is no significance difference between them.Conclusion:The xenogenous BMSCs are satisfactory seed cells of tissue engineering, which can be used in restoration of osteochondral defect. The biphasic scaffold is a satisfactory scaffold, which can carry BMSCs to the defect area, provide structure support, induce the regeneration of new tissue, and restore the tissue defect. The restore effect of biphasic scaffold combined with BMSCs is ideal, and the biological properties of the new tissues are close to the normal.
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