Experimental Study On Osteoinduction Of Recombinant Human Bone Morphogenetic Protein-2 And Recombinant Human Bone Morpho Genetic Protein -7 On Rabbit Bone Marrow Mesenchymal Stem Cell

Objective To induce rabbit bone marrow mesenchymal stem cell(BM-MSC) osteogenic in vitro by recombinant human bone morphogenetic protein-2(rhBMP-2) and recombinant human bone morphogenetic protein-7(rhBMP-7)having osteoinduction activity and observe the growth, morphology characteristic and proliferation of BM-MSC, and examine related osteogenesis index in order to understand the growth and morphology characteristic of BM-MSC under the stimulation of extraneous growth factor and osteoinduction ability of different kind and density's rhBMP in vitro and establish the experiment foundation for applying BM-MSC to sclerotin repair.Methods Rabbit thighbone marrow was extracted, BM-MSC were isolated with modified whole bone marrow culture, BM-MSC were cultured in vitro and the growth and morphology characteristic were observed. And BM-MSC were induced separately with culture media containing 10ng/ml, 50ng/ml, 100ng/ml rhBMP-2 and 10ng/ml, 50ng/ml, 100ng/ml rhBMP-7 in vitro, we observed the morphological change of BM-MSC every day, counted cells on second, 4th, 6th, 8th day and compared proliferation of groups. Simultaneously cell culture supernatant were collected on the 4th, 7th, 10th, 14th, 18th day after joining rhBMP. Alkaline phosphatase and osteocalcin were measured quantificationally.At the same time control group was cultured by DMEM.Results1.Rabbit BM-MSC were isolated and cultured successfully with modified whole bone marrow culture. The second generation of BM-MSC were dyed by the fluorescence immunity, CD44 positive, CD45 negative. And primary cell and fourth generation cell were analysed by flow cytometry, CD29 and CD44 positive, CD45 negative, which was corresponding to phenotype of mesenchymal stem cell.2.RhBMP-2 and rhBMP-7 induced BM-MSC to the osteoid cell directionally. In the course of culture, adherent cells changed from elongateon to polygon gradually, cells accumulated, cytoplasm was dark and included the thick pellets.3.Compared with control group, cell counts of 10ng/ml rhBMP-2 group, 10ng/ml rhBMP-7 group and 100ng/ml rhBMP-7 group had no obvious difference, these growth factors had no enhancement to proliferation of BM-MSC, but cell counts of 50ng/ml rhBMP-2 group, 100ng/ml rhBMP-2 group and 50ng/ml rhBMP-7 group increased obviously, these growth factors had enhancement to proliferation of BM-MSC. The effect of 100ng/ml rhBMP-2 was strongest.4. Alkaline phosphatase and osteocalcin in cell culture supernatant were measured on the 4th, 7th, 10th, 14th, 18th day. The result showed that 100ng/ml rhBMP-2 had highest osteoinduction activity.Conclusions Higher-purity rabbit BM-MSC were isolated with modified whole bone marrow culture, which had phenotype of mesenchymal stem cell.The isolated BM-MSC could proliferate largely in vitro. RhBMP-2 and rhBMP-7 had activity of enhancing proliferation and osteoinduction, and rhBMP could make BM-MSC differentiate the osteoid cell directionally, which established the fine experiment foundation for applying BM-MSC to repairing dentale damage.