In vitro micro-mineralized tissue formation by the combinatory condition of adipose-derived stem cells, macroporous PLGA microspheres and a bioreactor

Abstract

For the final purpose of accelerating the restoration of defected bone tissue, researches for developing in vitro three dimensional (3D) mineralized tissue using various stem cells, scaffolds and culture systems have been extensively done. In this research, an integrated bioprocess to generate stem cell-based 3D mineralized construct was developed using adipose-derived stem cell (ADSC), highly porous biodegradable poly(D,L-lactide-co-glycolide) (PLGA) microspheres and a high-aspect ratio vessel (HARV) bioreactor system. First, ADSCs adhered uniformly on poly-L-ornithine-coated macroporous PLGA microspheres, and the ADSC/microsphere composites were cultured in the presence of osteogenic supplements in a HARV bioreactor. Alkaline phosphatase (ALPase) activity assay, immunocytochemical staining and quantitative real-time polymerase chain reaction (qPCR) analysis showed temporal increase of ALPase activity, molecular expressions of type I collagen, osteocalcin and runx2 and upregulation of runx2, Sp7, type I collagen and bone sialoprotein mRNA during 3D osteogenic culture of ADSC/microsphere composites. Finally, 3D dynamic osteogenic culture generated highly mineralized micro tissues as validated by alizarin red-S staining and SEM-EDS. The results demonstrated that cell-based 3D micro-mineralized tissue could be generated by integrated bioprocess, and potentially utilized for bone tissue regeneration. The integrated bioprocess in this study may provide an efficient and scalable culture system for application in bone tissue engineering. Open image in new window