| Objectives:To establish the methods system of separation,culture and identification of hMSCs ; to investigate the biocompatibility and osteogenesis capability of bio-lu bioceramic compounds with hMSCs and to evaluate the feasibility of constructing tissue engineering bone with them and their practical value in clinic.Methods:1 To establish the methods system of separation,culture and identification of hMSCs in vitro.Bone marrow mononuclear cells were obtained by Percoll gradient centrifugation ,and then were purified,subcultured and expanded in incubator with 5%CO2 ,saturated humidity at 37oC. The cell's morphology was observed by inverted phase contrast microscope;the growth curve was drew by MTT;the cell surface antigens were examined by immunocytochemistry. Vigorous passage3 hMSCs were cultured in conditioned medium supplemented of Dex 10-8mol/L,β-GP10mmol/L and AA50μg/L.The cell growth status and morphology were observed under inverted microscope every day; cytochemical staining for the activity of alkaline phosphatase(AKP) and Von—kossa staining for calcified nodues. 2 The experimental study of bio-lu bioceramic combines hMSCs on biocompatibility and osteogenic capability in vitro.The study was divided into experimental groups and control groups,the experimental groups were bio-lu bioceramic combines hMSCs,while the control groups were the same numbers of cells seeded in 12 holes culture plate.Both of them were cultured and induced under the identical conditions. The cell growth status and morphology were observed under inverted microscope every day; scanning electron microscopy(SEM) was used to observe the growth and the compatibility of the hMSCs compounds with the scaffolds. the growth curve was drew by MTT . Samples of control groups and experimental groups were obtained randomly for the activity alkaline phosphatase(ALP) by cytochemical staining and quantitative analysis and for the calcified nodues by Von—kossa staining.Statistical analysis were performed,there is statistical significance if P<0.05.Reults:1 hMSCs can be effectively isolated and purified by density gradient centrifugation and adherence to the culture plastic.The primary hMSCs attached to the plates after 48 hours and grew into colonies with the appearance of fibroblastic cells.The morphology of hMSCs became analogic gradually with time went on;the growth and proliferation of hMCSs were active;immunocytochemistry showed hMSCs were positive for CD29 and CD71,while negative for CD34. Cytochemical staining for ALP) was positive after three days'induction and Von—kossa staining for calcified nodues was positive after a week's induction.2 hMSCs can attach to the surface and the inner of bioceramic; The shape of growth curve between experimental groups and control groups was similar.There was no statistical significance(P>0.05)between them during the early and middle period,while statistical significance(P<0.05) was detected during late period(9th and 10thday).The experimental groups and the control groups were positive for cytochemical staining after three days'induction, there was statistical significance (P<0.01) at every time point for quantitative analysis of alkaline phosphatase between them; there was notably difference in Von-kassa staining for calcified nodues at every time point .Conclusions:1 To make use of hMSCs was safe and convenient; hMSCs could be effectively isolated and purified by density gradient centrifugation and adherence to the culture plastic;hMSCs could be induced into osteoblast easily; hMSCs can provide ideal seeding cells for constructing tissue engineering bone.2 Bio-lu bioceramic has good biocompatibility with hMSCs;three dimensional space induction is superior to plane induction; bio-lu bioceramic has the ability of osteo-conduction and osteo-induction; bio-lu bioceramic can be used to construct tissue engineering bone as scaffolds in tissue engineering. |
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