[Experimental study on adhesiveness of osteoblasts and vascular endothelial cells from rat BMSCs co-cultured on allogeneic freeze-dried partially bone in vitro].

Objective: To investigate the adhesiveness of osteoblasts and vascular endothelial cells from rat BMSCs.

Methods: The BMSCs were isolated from 4-week-old SD co-cultured on allogeneic freeze-dried partially bone in vitro. rats (weighing 100-110 g) and cultured in vitro. The third generation of BMSCs were induced into osteoblasts and vascular endothelial cells. The osteoblasts and vascular endothelial cells after being induced for 7 days in a ratio of 1 to 1 were directly co-cultured (experimental group), while the second generation of uninduced BMSCs was used as a control (control group). The growth and proliferation ability were analyzed by MTT examination and the growth curve was drawn at 1-8 days. The osteoblasts and vascular endothelial cells after being induced for 14 days were implanted in the allogeneic freeze-dried partially bone coated by 20% Col I or not at different densities (0.25 x 10(6)/mL, 0.50 x 10(6)/mL, 1.00 x 10(6)/mL, 2.00 x 10(6)/mL, 4.00 x 10(6)/mL), as modified group and unmodified group, the cell adherence rate was calculated after 24 hours. These two kinds of cells were implanted in the pre-disposal treated allogeneic freeze-dried partially bone and observed by scanning electron microscope.

Results: ALP staining of osteoblasts showed that there were blue grains in cytoplasm at 7 days. CD31 and CD34 immunocytochemical staining of vascular endothelial cell showed that there were positive signals in the cytoplasm at 14 days. The MTT test showed that the proliferation level of the experimental group was lower than those of the control group. There were significant differences in absorbance value between two group from 3 days to 8 days (P < 0.05). The cell adherence rate increased with increasing seeding density when the seeding density was (0.25-1.00) x 10(6)/mL. The cell adherence rate reached the peak when the seeding density was 1.00 x 10(6)/mL. The cell adherence rate decreased when the seeding density was more than 2.00 x 106/mL. There were significant differences in cell adherence rate between modified group and unmodified group at different seeding densities (P < 0.05). The proliferation of the osteoblasts and endothelial cells presented better growth and histocompatibility under scanning electron microscope.

Conclusion: The growing behavior of two kinds of cells is good in the allogeneic freeze-dried partially bone coated by 20% Col I, which can be used in reconstruction of vascularized tissue engineered bone.