Engineering Multi-Spheroid Bone Constructs Through the Fusion of Large MSC Spheroids

Bone tissue engineering offers the promise of creating alternatives to bone grafts to facilitate defect repair. Although many different approaches have been investigated, recent work has focused on harnessing the potential of scaffold-free approaches, such as spheroids, to create viable engineered bone tissue. In this study, we explored the possibility of developing bone constructs from large MSC spheroids. Spheroids were developed from human MSCs and were sequentially cultured for 42 days in vary degrees of chondrogenic and osteogenic media to induced endochondral and intramembranous bone formation. After differentiation, five single MSC spheroids (from each group) were then further cultured for 14 days in osteogenic media to investigate the potential for fusion into a larger bone construct. Single and multi-spheroid constructs were harvested and analyzed biochemically and histologically as well as assessed by μCT imaging to determine the degree of mineralization. With increasing exposure to osteogenic media, single spheroids were smaller, expressed collagens indicative of bone formation (types I and X) and had lower DNA contents. Similarly, greater degree of fusion also occurred with spheroids that were initiated cultivated under longer exposures to osteogenic media. Evidence of mineralization was observed in all multi-spheroid constructs, again with the greatest mineralization occurring under longer exposure times to osteogenic media. The findings of this study demonstrate that bone constructs can be created by large differentiated MSC spheroids, with greater fusion and mineralization occurring with longer osteogenic exposure prior to multi-spheroid aggregation.