Lyophilized Bone Graft Material Generated From IPSCs

Objectives: By leveraging the self-organization capabilities of induced pluripotent stem cells (iPSCs), we have developed a fabrication method for bone-like 3D cell constructs from iPSCs. These constructs would contain abundant bone matrix components even after lyophilization that could be utilized as a source of bone graft materials (BGMs). We hypothesized that the drying condition during lyophilization may affect the bone-regenerative potency of iPSC-derived bone graft materials (iPS-BGMs), and aimed to develop stable iPSC-derived biomaterials for bone regeneration using lyophilization.
Methods: Embryoid bodies (EBs) were generated from mouse iPSCs and cultured in osteogenic differentiation medium. Osteogenically induced EBs were inactivated by lyophilization (iPS-BGMs). To evaluate the osteogenic-induction potency of iPS-BGMs, the osteogenic differentiation of MSCs cultured for 1 week in osteogenic differentiation medium containing ground iPS-BGMs was assessed. The iPS-BGMs were subjected to analysis of morphology and protein content to compare the characteristics of iPS-BGMs lyophilized under different conditions. The in vivo bone regeneration potential of iPS-BGMs was evaluated using a rat femur defect model. Bone formation was evaluated through μCT and histological analysis.
Results: iPS-BGMs lyophilized at subzero temperatures for primary drying significantly retained proteins compared to that lyophilized at room temperature, and induced significant osteogenic differentiation and migration of MSCs. The temperature during primary drying affected iPS-BGM ECM structure, conferring a relatively uniform structure with high mechanical strength at subzero temperatures. In in vivo experiments, iPS-BGMs prepared at subzero temperatures during primary drying were specifically surrounded by macrophages, an optimal condition for modulating innate immune responses that promoted bone regeneration.
Conclusions: Primary drying conditions have been optimized to improve the in vivo bone-replacement properties of iPS-BGMs. Optimization of the primary drying conditions will further improve the quality and cost-effectiveness of bio-derived products, ultimately contributing to advancements in tissue regeneration in the future.