Experimental Study To Fabricate Tissue Engineered Adipose With Rat Mesenchymal Stem Cells And Poly Lactic-co-glycolic Acid.

Objective:1. To explore the suitable conditions to isolate,purificate and culture rat mesenchymal stem cells( MSCs) in vitro,and to provide an experimental basis for further study.2. To study the relationship between the ability of differentiation into adipocytes and passages of rat bone marrow mesenchymal stem cells (MSCs).3. To explore an ideal method to isolaten and purificate of bone marrow MSCs in vitro by comparison of proliferation ability and adipose differentiation potential of the cells obtained by two different methods.4. To fabricate adipose grafts by rat bone marrow-derived mesenchymal stem cells combined with poly lactic-co-glycolic acid (PLGA) scaffold using tissue engineering method.Methods:1. MSCs were acquired by adhesive screening methods and density gradient and centrifugation methods. The growth of MSCs was observed at different plate density, different serum concentration and different culture media changing time respectively. The growth curves were charted and the cell cycle was detected by flow cylmeter.2. MSCs were isolated from SD rats by full marrow culture. The cells were induced in culture solution added with dexamethazone insulin indomethine 3-isobutyl- methylzanthine. Observation of morphological changes and Oil Red O stain were carried out at ten days after induced. At the same time, cell counting and optical density were measured. 3. Density gradient centrifugation and plastic adherence methods were used for isolation of bone marrow MSCs from adult SD rats and the proliferation ability of the cells was analyzed by MTT examination. In order to observe the adipose differentiation ability of MSCs abtaind by two methods,cells of passes 3 were indulsed.4. Male rat bone marrow-derived mesenchymal stem cells cultured adiposis were seeded on to the scaffold for one week in vitro. The adhesion and growth of cells were observed with scanning electronmicro scope. Meanwhile,the cell - scaffold complexes were allografted to female rat. In situ hybridization technique was employed to identify tissue engineering adipose and its growth condition.Results:1. The purity of cell obtaind by Density gradient and centrifugation method was higher than that of adhesive screening. The cells,which were plated at 1×10⁶cells/ml and cultured in MEM medium supplemented with 12% fetal bovine scrum,grew well.At 37℃,5% CO₂ incubator,cells grew slowly in first several days after plated.2. The value of cell count by Oil red O stain coloration and optical density declined as the passages increased.3. High purity of MSCs were obtained by gradient centrifugation. And the cells proliferated fast and got confluence in7 to 10 days.Other cells like red blood cells and osteoctast cells could be seen in primary MSCs obtained by plastic adherent method. These MSCs proliferated slowly and even after several passages red blood cells and other cells could also be observed. But similar adipose differentiation ability of MSCs obtaind by two methods was observed after they were induced. No significant difference was found in Oil red O coloration and optical density.4. The adhesion and growth of adiposis cluster were observed on the surface of scaffolds. New adipose formation was observed at one month post operatively and mature adipose tissue was found on the surface of the newly formed adipose at three month in vivo.Conclusion:1. Isolation,purification and culture of rat mesenchymal stem cell(MSCs) in vitro should adopt density gradient and centrifugation methods. In suitable conditions in vitro,MSCs show stable growth and rapid proliferation.2. The differentiation ability of bone marrow mesenchymal stem cell(MSCs) to into adipocytes decrease along with passages increasing.3.Gradient centrifugation might be an ideal method to isolate and purificate MSCs from bone marrow. The cells show better proliferation ability than those isolated by plastic adherent methods.Meanwhile,the cells obtained from differend methods show the similar adipose differentation potential.4.The complex of PLGA is an ideal biological and biodegradable scaffolds. Mesenchymal stem cells seeded in PLGA scaffold can be used in the fatty tissue engineering research.