The Experimental Research Of Construction Of Tissue Engineered Osteochondral Composite

Objective:Observe the condition to construct the tissue engineered osteochondral composite through bone marrow mesenchymal stem cells (BMSCs) cultured on the poly-lactide-co-glycolic acid (PLGA), which is different interstice ratio and different diameter and modified with different collagen and different cellular growth factor. In order to explore a new method to construct the tissue engineered osteochondral composite.Methods:1. BMSCs were isolated and cultured from the 2-month-old Sprague-Dawley (SD) rats through density-gradient centrifugation and cell attachment to tissue culture.2. The cell exterior antigen,cell activities and cell cycle were observed with flow cytometry method.3. BMSCs were induced and differentiate into osteoblasts and cartilages by using specific induction culture medium. Thus the cells were stained with type I collagen, Alkaline phosphatase(ALP), Calcified nodules,typeâ…¡collagen and Toluidine Blue. Then analyse and determine the result of all staining.4. PLGA was made by phase separation, the condition of different part of the PLGA is different (The interstice ratio and diameter of the biomaterial's cartilages is bigger, and modified with typeâ…¡collagen,the basic fibroblast growth factor and ransforming growth factor-β; The interstice ratio and diameter of the biomaterial's osteoblasts is less, and modified with typeâ… collagen and the factor of bone morphogenetic protein-2), undergo observation of the biomaterial structure through scan electron microscope.5. The third era (P₃) BMSCs were cultured on PLGA modified with collagen, observe the adhesion of cell and biomaterial through scan electron microscope.6. PLGA-cellular composite biomaterial, which is different interstice ratio and diameter and modified with different collagen and different cellular growth factor, was implanted into the muscle pf SD mice, and observe the condition of cells' differentiation and up-growth regularly through it's general shape, HE,Toluidine Blue,typeâ…¡collagen,ALP staining, the cells shape through scan electron microscope and perspective electron microscope.Result:1. The BMSCs could be obtained through both density-gradient centrifugation and cell attachment methods.2. BMSCs express CD29,CD44,CD105,CD106, but not express CD34,CD45. The cell activities is 81.33 %, cells of G0-G1 phase are 86.41%.3. The cell morphology change from spindle to polygon after cells been induced by specific induction cultural medium, Positive and strong reaction in typeâ… collagen,ALP,calcified nodules,typeâ…¡collagen and Toluidine Blue staining.4. The PLGA made through phase separation method is a multi-interstice three-dimensional biomaterial, and the interstice ratio and diameter of the biomaterial's cartilages is 91% and 120μm; the interstice ratio and diameter of the biomaterial's osteoblasts is 85% and 100μm.5. The BMSCs has good adhesion to PLGA by scan electron microscope observation.6. Regular observation of the PLGA-cellular composite biomaterial implanted through all kinds staining, they show: there are many osteoblasts, cartilages and little costeoblasts.Conclusion:1. The BMSCs can be obtained through both density-gradient centrifugation and cell attachment methods.2. BMSCs can be induced into osteoblasts and cartilages by specific induction culture medium,3. PLGA modified with collagen has good adhesion with BMSCs.4. BMSCs cultured on PLGA, which is different interstice ratio and diameter modified with different collagen and different cellular growth factor, can construct the tissue engineered osteochondral composite on animals...