Formation of three dimensional marrow stromal cell-polymer constructs in bioreactors for bone tissue engineering

摘要

The aim of this study is to investigate the ability of rat bone marrow stromal cells (MSCs) seeded in three-dimensional PLGA biodegradable scaffolds to be cultured in a spinner-flask or a rotating vessel. The studied bioreactors generate good mixing and thus better nutrient transport to the seeded cells. Poly (DL-lactic-co-glycolic acid) (PLGA) copolymer scaffolds have been selected for this study because they are osteoconductive and biocompatible, degrading into products that can be either metabolized or excreted. Rat bone MSCs were harvested from femurs and tibias of six-week old male Sprague-Dawley rats and expanded in the presence of primary culture media. Seven days after the MSC harvest the cells were lifted from the primary culture and they were seeded 01) porous 75:25 PLGA cylindrical scaffolds. The cell-polymer constructs were cultured in a spinner-flask, a rotary vessel and under static conditions in the presence osteogenic supplements. On the 7th, 14th and 21st day of culture, foams were removed and the cell number and the alkaline phosphatase (AP) activity were measured. During every media change samples were assayed for osteocalcin (OC) secretion. The spinner flask culture had the highest cell number and AP activity (with a maximum occurring on day 14) and the highest OC secretion with a peak on day 18. The spinner flask culture had also the highest mineralization. Histological sections of the foams on the 21st day showed higher cell densities at the exterior of the foam providing strong evidence for the existence of nutrient concentration gradients at the interior of the scaffolds. Although the good mixing provided in the spinner-flask accelerated the proliferation and differentiation of marrow stromal osteoblasts, high proliferation and mineralization was limited to the external surface of the scaffold.