Mesenchymal stem cell spheroids incorporated with collagen and black phosphorus promote osteogenesis of biodegradable hydrogels

Mesenchymal stem cell (MSC)-spheroids have sparked significant interest in bone tissue engineering due to their resemblance to natural bone tissue, especially in terms of cell-cell and cell-extracellular matrix interactions. Many biomaterials or biomolecules have been incorporated into MSC-spheroids to enhance their osteogenic abilities. In this respect, we assessed the osteogenic responses of MSC spheroids leveraged through the unique combination of collagen and black phosphorus (BP). The MSC spheroids were successfully constructed with 6 μg/mL collagen and/or a concentration gradient (0 μg/mL, 4 μg/mL, 8 μg/mL, and 16 μg/mL) of BP and were evaluated for MSC viability and their osteogenic differentiation over a time period of 14 days. Improved MSC viability and osteogenic ability were observed for the spheroids with collagen and BP at the concentration of 4 μg/mL and 8 μg/mL. Next, blank spheroids (Control) or the optimized MSC spheroids with 6 μg/mL collagen and 4 μg/mL BP (Col+BP4) were further encapsulated into two types of hydrogel scaffolds: porous oligo[poly(ethylene glycol) fumarate] (OPF) hydrogel and hydroxyapatite-collagen I scaffold (HE-COL). The osteogenic abilities of these four groups were evaluated after 14 and 21 days of osteogenic induction. The MSC spheroids incorporated with collagen and BP implanted into OPF porous hydrogel (Col+BP/OPF) elicited a higher expression of Runx2, osteopontin, and alkaline phosphatase than blank spheroids implanted into OPF porous hydrogel (Control/OPF). Enhanced osteogenesis was also observed in the Col+BP/HE-COL group as compared to Control/HE-COL. Taken together, the results from this study showed the perspectives of collagen and BP incorporated MSC spheroids for the development of injectable cellular therapies for bone regeneration.