Investigation Of Rat Bone Marrow Mesenchymal Stem Cells In Tissue Engineering

To improve a method for animal operation, isolation and cultivation of mesenchymal stem cells (MSCs) from rat bone marrow. Induce MSCs osteogenicly to offer functional cell for bone tissue engineering. The aim of this investigation was to repair a SD rat model of critical size calvarial defects by using bone tissue engineering technique with autogenous BMSCs and coral hydroxyapatite(CHA); hydroxyapatite/tricalcium phosphate (HA/TCP); polylacticcoglycollic acid(PLGA); and alginate gelatin. To evaluate potential of rat bone marrow cells for cartilage tissue engineering by examining their chondrogenic properties within a three-dimensional scaffold of alginate gel. To compare the osteogenenic potential of the compounds of autogenous BMSCs with PLGA or alginate gelatin in nude mice.Separated and purified the BMSCs by density gradient centrifugation and adhering to the culture plastic. After the in vitro subculture, expansion, induction observed the living characteristic of the cells, drew the growth curve according to the result of MTT chromatometry, observed the effect of the osteogenic induction on the growth of MSCs. Osteogenic characteristics: akaline phosphatase in quality and in quantity; Von Kossa's mineralized nodules staining; immunohistochemistrical detection for Collegen I and III were detected.Bone marrow was obtained from the right tibias from all the rats under general anesthesia and sterilized condition. Autogenous BMSCs were differentiated for 14 days. The cells were then seeded and subcultured for another 2 days in 4 kinds of scaffolds (CHA, HA/TCP, PLGA and alginate gel). Two full thickness cranial plate defects were created without damage of dura mater on each rat. Then the cell-scaffold compounds were set in the cranial critical size defects of SD rat autgenously. Animals being made cranial defect but received no implant served as same operated controls. With postsurgical radiographic and histological analysis completed at 4 and 8 weeks after implant experiments.Bone marrow was obtained from the right tibias of all the SD rats. After expanding and culturing 3 passages,BMSCs was induced by chondrogenic culture medium for 10d. Suspended the induced cells in alginate. added a certain amount of CaCl₂ to make into gel, and injected the gel into subcutaneous tissues of rats' backs. The grafts were taken out for examinations 4 and 8 weeks after the operations. No cartilage was produced 4 weeks after operations. Considerable cartilage appeared 8 weeks after operations. Histological and immunohistochemical analysis of the complex grafts showed development of a cartilaginous phenotype as demonstrated by colocalization of Alcian blue staining with collagen type II and cartilage proteoglycan link protein. 4. Bone marrow was obtained from the right tibias from all the rats under general anesthesia and sterile condition. Autogenous BMSCs were differentiated for 14 days. The cells were then seeded and subcultured for another 2 days in above 2 kinds of scaffolds. Then the cell-scaffold compounds were implanted subcultaneously into the back of nude mice. The implanted grafts were taken out for osteogenesis examinations including histological and immunochemistrical methods four weeks and eight weeks after the operations.Using the refined animal operation method and regular density gradient centrifugation and by adhering the MSCs . can be isolated, purified expanded safely, simply and effectively. The osteogenic iduction did have inhibiting effect on the growth of MSCs. The detection confirmed that the osteogenic induction realy endued MSCs with osteogenic characteristics.Radiographs showed obviously better increased calcification in defects repaired by alginate compounds than those repaired by PLGA compounds. Histology and immunohisto- chemistry of collagen I and III showed that a great amount of new bone in growth took place in CHA or alginate compounds. The newly formed bone appeared not only at the edges of the defect margins but also the center part of the compounds. And the newly formed bone had the typical structure of marrow cavity somewhere. Also there were new bone tissue formed in HA/TCP or PLGA compounds too but the quality and quantity of new bone were much less than those in CHA and alginate comound.Regular HE and alcian staining showed a great deal of cartilage holding chondrocyte masses surrounded by abundant matrix. Alginate gelatin decompounded obviously, and the rest distributed among cartilage. Eight weeks after operation, a great amount of mature bone tissue was found in the implanted compounds of alginate gelatin. A relatively less amount bone tissue occurred in the PLGA compounds.The marrow MSCs could be simply, safely and effectively isolated and purified by the improved method combining with density gradient centrifugation and adhering. The osteogenicly induced MSCs had the characteristics of osteoblasts.Transplantation of syngeneic BMSCs with CHA or alginate gel can serve as an example of a cell based treatment for skeletal reformation and wouldbe especially useful for augmenting or regenerating bone in skeletal defects. Syngeneic BMSCs with CHA or alginate gel exhibited potential techniques of repairing variety of skeletal defects occurring in different clinical situation.3. These findings suggest that alginate gelatin is a potential candidate bioactive scaffold for cartilage tissue engineering applications.4. Alginate gelatin is a favorable bone tissue engineering scaffold, and compound of autogenous BMSCs and alginate gelatin is a promising approach in bone tissue engineering.