Effects Of Osteoblasts And Inductor On Proliferation And Osteodifferentiation Of Marrow Stromal Cells

Objective To investigate the effects of osteoblasts and inductor on proliferation and osteodifferentiation of marrow stromal cells,so try to find a new culture system to induce seed cells in vitro for bone tissue engineering.Methods Because of it's character of powerful proliferation and pluripotent differentiation, marrow stromal cells(MSCs) became seed cells for bone tissue engineering.There were four groups in this experiment which to study the effects of osteoblasts from rat cranium and osteogenic inductor(dexamethasone,beta-glycerophosphate,retinoic acid) on prolifecation and osteodifferentiation of MSCs isolated from rat femur and tibia.MSCs were cultured in DMEM medium(control group)and in osteoinductive culture medium(Inductor group);Furthermore MSCs were cocultured with osteoblasts at ratio of 2:1 in DMEM medium(Osteoblast group) and in osteoinductive culture medium(Combine group).The cell coculture system prevented cell-cell contact but permited diffusion of cytokines through the pores of millicell membrane.Cells number of four groups were counted at every other day for 8 days to draw growth curve.Alkaline phosphatase (ALP)activity and total cellular protein of MSCs at 10th day were measured to calculate relative activity of ALP.MRNA expression of osteocalcin(OC) at 2rd week were assayed with reverse transcript polymase chain reaction(RT-PCR). Results The growth curve showed that cells number of all four groups increased from 1st day to 8th day.Comparing three treatment groups with control group:MSCs of Osteoblast group was more than that of control group(P<0.05) but MSCs of Iductor group was fewer than (P<0.05) during the late 4 days;Cells number of Combine group was no different from control group(P>0.05).At the 10th day of cultivation,all four groups express alkaline phosphatase(ALP).Combine group showed the highest ALP activity. in four groups. ALP relative activity of Iductor group and Combine group were higher than that of Osteoblast group (P<0.05).Comparing Osteoblast group with control group in ALP activity and ALP relative activity showed no statistical significance(P>0.05).After being induced for 2 weeks,Three treatment goups expressed osteocalcin mRNA but control group didn't..Iductor group expressed higher osteocalcin mRNA than Osteoblast group did(P<0.05),There was no statistical significance between Combine group and Iductor group in mRNA expressionn of osteocalcin.Conclusion Osteoblasts powerfully promote proliferation of MSCs but weakly induce osteodifferentiation of MSCs.Inductor inhabits proliferation of MSCs but strongly induces osteodifferentiation of MSCs.The joint action of two induce agent doesn't affect the normal proliferation but obviously promotes osteodifferentiation of MSCs.Comebine group has the best inductive results,which meets the requirement on quantity and quality of seed cells for bone tissue engineering.Useing both osteoblasts and inductor to enhance proliferation and osteodifferentiation of MSCs may become a new culture system in vitro for bone tissue engineering.