Gelatin-Based Microribbon Hydrogels Support Robust MSC Osteogenesis across a Broad Range of Stiffness

Scaffold macroporosity has been shown to be critical for promoting bone regeneration. Although injectable materials are preferred for minimally invasive delivery, conventional macroporous scaffolds were not injectable and do not support homogeneous cell encapsulation. We recently reported a gelatin-based microribbon (μRB) scaffold that offers macroporosity while also supporting homogeneous cell encapsulation. Compared to conventional gelatin hydrogels, macroporous gelatin μRB scaffolds demonstrated great advantage in enhancing mesenchymal stem cell (MSC)-based cartilage formation. However, whether gelatin-based μRBs support MSC osteogenesis and bone formation remains unknown. The goal of this study is to assess the potential of gelatin-based μRBs for supporting MSC-based osteogenesis and bone formation in vitro. Given recent evidence from the literature that osteogenesis is sensitive to substrate stiffness, we further investigate how varying μRB stiffness modulates MSC osteogenesis. We first determine the maximal stiffness range of gelatin μRBs that can be fabricated (13-57 kPa), which supports both retention of μRB shape and macroporosity within scaffolds after inter-cross-linking. Interestingly, varying μRB stiffness across a broad range of stiffness did not significantly impact osteogenesis, with all groups supporting upregulation of bone markers and extensive collagen deposition. All gelatin μRBs also supported a comparable level of cell spreading and upregulation of mechanosensing markers. However, soft μRB (13 kPa) scaffolds did not maintain structural integrity and condensed into a pellet over time. Both intermediate and stiff gelatin μRB-based scaffolds maintained their integrity and supported robust bone formation, leading to a more than 10-fold increase in the compressive moduli of engineered bone after 5 weeks of culture in osteogenic media. Incorporating hydroxyapatite (HA) nanoparticle coating onto the gelatin μRB surface further accelerated the maturation of MSCs into osteoblasts and mineralization. Together, these results validate that gelatin μRBs can support MSC osteogenesis across a broad range of stiffness and offers an injectable macroporous scaffold for enhancing stem-cell-based bone regeneration.