Abstract:

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