Preliminary study of in vitro osteogenesis by co-culture of osteoblasts and bone marrow stromal cells

Aim:This study explored the feasibility of in vitro osteogenesis by co-culture of osteoblasts and BMSCs so as to confirm the hypothesis that osteoblasts can provide osteogenic microenvironment to induce osteogenic differentiation of BMSCs and thus promote in vitro osteogenesis of BMSCs.The lowest ratio when osteoblasts have fine capacity of inducing BMSCs osteogenic differentiation was also studied.In this way,a novel method in seeding cell culture for bone tissue engineering is expected. Methods: The newly born(1d) Sprague-Dawley(SD) rats were sacrificed and osteoblasts were obtained by modified enzymatic digestion and differential attachment technique.BMSCs were isolated and cultured from the 1-month-old SD rats through cell attachment to tissue culture.Osteoblasts and BMSCs were mixed at the ratio of 1∶9、2∶8、3∶7、1∶0(osteoblasts∶BMSCs) and co-cultured.The lowest effective ratio of osteoblasts on the proliferation of BMSCs and the differentiation of BMSCs into osteoblasts were judged by alkaline phosphatase(ALP) in the culture medium 3,6,9 days after co-culture.BMSCs and osteoblasts were mixed at the lowest osteogentic ratio.200 μL mixed cells(6.0×107/mL) were seeded onto a collage I and chitosan scaffold,9 mm in diameter and 3 mm in thickness,as co-culture group.Osteoblasts and BMSCs with the same cell number were seeded respectively onto the scaffolds as positive control(osteoblast group) and negative control(BMSCs group).200 μL osteoblasts(equal to the osteoblasts number of co-culture group) alone were seeded as low concentration osteoblast group.All specimens were harvested after in vitro culture for 8 weeks.Gross observation,histology and immunohistochemistry were used to evaluate the results.Results: The lowest effective ratio of osteoblasts on the proliferation and differentiation of BMSCs into osteoblasts is 3∶7(osteoblasts∶BMSCs).In both co-culture group and positive control group,the cell-scaffold constructs could maintain the original size and shape during in vitro culture and formed mature bone after 8 weeks of in vitro culture.Furthermore,the neo-bone in both groups were similar to each other in gross appearance and histological features(HE,ALP staining),and abundant collagen I type was also detected by immunohistochemistry in both groups.In negative control group,however,the constructs became smaller during in vitro culture and bone-like tissue could not be observed.In low concentration bone group,the constructs shrunk gradually during in vitro culture although histology showed a small amount bone formation.Conclusion:Osteoblasts can provide osteogenic microenvironment to promote in vitro osteogenesis of BMSCs.30% osteoblasts have fine capacity of inducing BMSCs osteogenic differentiation.