Attachment and differentiation of mouse embryonic stem cells in a 3D scaffold.

Abstract

Current strategies to correct bone defects present various drawbacks, leading to the emergence of bone tissue engineering to create de novo tissue out of embryonic stem cells (ESCs). In this study, we demonstrated the in vitro attachment and differentiation of mESCs on 3D polycaprolactone-tricalcium phosphate (PCL-TCP) scaffolds over 32 days. These scaffolds were cultured in either basal (mESCs basal 3D) or osteogenic medium (mESCs OST 3D), which consisted of dexamethasone, ascorbic-acid and glycerophosphate supplements. 2D cultures in the respective media were adopted as controls. Osteoblast precursors were detected in all experimental groups on day 4. Hereafter, osteoblasts with elongated morphology were detected. Confocal and scanning electron microscopy confirmed cellular viability and adhesion on the PCL-TCP scaffolds throughout the experiment. Osteoblast differentiation markers alkaline phosphatase and osteocalcin were also significantly upregulated at day 26, in accordance to mineralization profile monitored with von Kossa and alizarin red staining. Staining for mineral nodules was most intense at day 32. mESCs OST 3D demonstrated higher osteocalcin expression and enhanced mineralization, suggesting that osteogenic supplements further enhanced osteogenic differentiation. In conclusion, 3D PCL-TCP scaffolds help facilitate the in vitro attachment, expansion and osteogenic differentiation of mESCs, and show promise as suitable 3D carriers for mESC-based therapies.