Preparation of canine bone marrow stromal stem cell sheets and investigation on their osteoblastic differentiation / 中国组织工程研究

ABSTRACT

BACKGROUND:Conventional methods,including trypsin digestion and cells transfer using single call suspension,have many drawbacks,which limit the development of bone tissue engineering.OBJECTIVE:To culture bone marrow stromal stem calls,induce osteoblastic differentiation,and prepare cell sheets.METHODS:Canine bone marrow stromal calls were isolated by density gradient centrifugation technique,inoculated into DMEM medium,and induced to differentiate into osteoblasts.Complete call sheets were harvested by call sheet engineering based on the temperature change of temperature-responsive medium.RESULTS AND CONCLUSION:Immediately after inoculation,primary calls were scattered on the bottom of culture flask,presenting a transparent spherical body with a good refractive capacity.At 12 hours,calls exhibited a long shuttle shape,reached complete confluency,and grew in a whirlpool-like fashion.After osteoblastic induction,the majority of bone marrow stromal stem calls appeared tetragonal,polygonal,and squamose.At 21-28 days,round or oval-shaped calcified nodules formed.When the bone marrow stromal stem calls in the temperature-responsive culture dishes were cooled below the critical temperature 32℃,cells were gradually detached from the bottom of culture flask and formed complete bone marrow stromal stem call sheets.These findings indicate that density gradient cantrifugation technique can be used to successfully isolate and culture canine bone marrow stromal stem cells to differentiate into osteoblasts and call sheet engineering enables to harvest complete bone marrow stromal stem call sheets.