Preliminary Study On Formation Of CHA-Fang Complex In Vitro And Reimplanting

ObjectiverThe main objective of this study was as follows:(1) To induce Bone marrow stromall cells(BMSCs) isolated from Beagles differentiated into osteoblasts in vitro and identify the osteogenic potential and bioactivity of BMSCs. (2) To discuss the physical characters of BMSCs'growth combined with fang and Coralline Hydroxyapatite(CHA) .(3) To study the possibility of CHA absorbing BMSCs acting as carrier of periodontal tissue engineering.(4) To evoluate the periodontal tissue's regeneration effects of implanting BMSCs-fang-CHA in order to give theoretical evidence and basic experimental reference for further improving the study of periodontal tissue engineering.Methods: (1) Bone marrow stromal cells isolated from Beagle dogs were cultured in vitro for the primary culture,in basic medium consisted ofDMEM^iew horning cattle'erum,etc. Parts of subculture were cultured in mineralization medium which additionally contained 50 y g/ml ascorbic acid, 10mmol/LNa- beta -Glycerophosphate and 10-8mol/L dexamethasone in order to induce cells differentiating into osteoblasts. The morphological characters of cells and conditions of cells proliferation were observed by phase contrast microscope.The osteogenic activity of cells has been detected by means of colouration of the mineralized nodules and the Alkaline phosphatase activities. (2) Some bone marrow stromal cells cultured in basic medium were seeded into fangs,and the others were seeded into CHA. The osteogenic activity of cells has been detected by means of the Alkaline phosphatase activities and the shapes of cell-complexes were observed with phase contrast microscope and scanning electron microscope. (3) Experiment were divided into two groups: test group : BMSCs +fang+CHA;the control groups: fang+CHA.The complexes were implanted into alveolus after 7days of co-culture of cells ,fang,and CHA in medium only for test groups.(4)All Peagles were killed after implanted for 16 weeks,and the condition of periodontal regeneration in every group were histologically assessed and stained.Result:(1) Bone marrow stromal cells cultured in vitro clearly showed the ability of osteogenic in DMEM essential medium.Both VonKossa coloration of mineralized nodules in vitro and Alkaline phosphatase of the second-cultured cells were positive.(2) BMSCs grew well combined with fang and CHA,associating with matrix secreting. There was a good biocompatibiliry between the CHA,fang and BMSCs.(3) The histological staining showed that new bone formation in test group was significantlyhigher than that of control group. The calcification of new bone in test group was more mature than control group. There was a clearly wealthy likely-cementum formation only in test group. There was a clearly likely-periodontalligament tissue formation between fang and new bone in test group,and reversely in the control group.Conclusion:(1) Bone marrow stromal cells cultured in vitro contained osteogenic precusor cells.The second-cultured cells had the ability of osteogenic potential. (2) There was a good biocompatibility between CHA,fang and Bone marrow stromal cells.The CHA can be acted as one of the carrier materials in periodontal tissue engineering. (3) The complex of BMSCs,fang,and CHA could be able to improve the periodontal tissue' regeneration containing alveolar bone .and the formation of likely periodontalligament. (4) It will be possible that the way of tissue engineering promotes periodontal regeneration. (5)The BMSCs will be expected to become source of periodontalligament cell in vitro.