The Studying Of The Adhesivness Of Osteoblasts And Vascular Endothelial Cells From Rat Bone Marrow Stromal Cells Co-cultivated On Allogeneic Freeze-dried Demineralizde Bone Matrix In Vitro

Objective To evaluate the feasibility and experimental evidence of rat bone marrow stromal cells(BMSCs) on formatting tissue engineering bone with allogeneic freeze-dried demineralized bone matrix in vitro, which through inducing rat BMSCs into vascular endothelial cell(VEC) and co-cultured with allogeneic freeze-dried demineralized bone matrix, and co-culture the induced rat BMSCs and scaffold coated by Collagen I to format tissue engineering active bone in vitro.Methods The BMSCs were isolated from bone marrow in SD rat about a mouth old and cultured in defined medium in vitro. The BMSCs in 3rd generation were cultured in conditional medium(test group)and in basic medium(control group). The morphological characteristics of BMSCs and induced cells were observed by inverted phase-contrast microscope. Immunohistochemistry was used to examine the expression of CD31, CD34. The growth and proliferation ability was analyzed by MTT examination and growth curve was drawn. The osteoblasts and VECs and the two kinds of cells of different densities were seeded onto scaffold coated by Collagen I. The induced BMSCs that compounded scaffold were detected by scanning electron microscope.Results 1. The morphological features of attached BMSCs indicated colonies with the appearance of spindle shape, fibroblast-like shape. There were different colonies in the shape, size, density and confluence ultimately. The contact inhibition was not observed.2. The vascular differentiation of BMSCs were observed and grew stronger with the time, while the induced cells proliferation became slow. There was positive expression in CD31 and CD34.W-P body was observed by TEM.3. The cell grow curve was drown by MTT : Within 1-2days that was latency period. It was no obvious cell proliferation; Three days later, the cells grew fast obviously and came into logarithmic phase. After 7 days, the cells grew slowly and this phase was called plain phase.4.The structure of allogeneic freeze-dried demineralized bone matrix was natural, polyporous. BMSCs grow well and spread extensively and proliferate rapidly on it.5. The induced and cultured osteoblast and VECs and the two kinds of cells were co-cultured in the allogeneic freeze-dried partially bone coated by Collagen I, the scaffold coated by Collagen I has higher cell adhesion.Conclusion 1. BMSCs from bone marrow with active proliferative abilities and high purity can be induced to differentiate into VEC with typical characteristic on morphology and function. They were optimal seed cells source of vascularized tissue engineering bone.2. Allogeneic freeze-dried demineralized bone matrix has good histocompatibility, and will be optimal biomaterial for tissue engineering bone construction.3.The adhesive rate of VEC combined with scaffold is obvious correlation to cell seeding density. The VEC don't increased when the densities of seeding cells reached 2×106/ml. It is suggested that scaffold loaded with cells is feasible to produce tissue engineering bone.4. There was higher cell adhesion in the scaffold. It is illustrated that Collagen I was an ideal surface modifying material.