Hybrid Biofabrication of a Hierarchically Organized Periosteum-Inspired In Vitro Platform

The periosteum is a hierarchically organized, vascularized membrane essential for skeletal maintenance and regeneration, yet in vitro models that reproduce its compartmentalized and multicellular architecture remain limited. In this study, we present a hierarchically organized, periosteum-inspired in vitro platform fabricated by integrating melt electrowriting (MEW) with extrusion-based 3D bioprinting. The platform integrates a human osteoblast-laden plasma-containing alginate/methylcellulose hydrogel compartment, a configurable MEW-PCL microfibrous interface, and an endothelial cell-fibroblast-laden fibrin/gelatin hydrogel compartment. MEW scaffold architectures and compartment-specific bioink-cell configurations were screened based on their ability to support osteogenic maturation-associated readouts and CD31⁺ endothelial cell organization. The resulting tri-layer construct maintained high cytocompatibility and structural integrity during culture for up to 49 days. The osteogenic compartment supported osteogenic differentiation-associated readouts and mineralized matrix deposition, while the endothelial cell-containing compartment supported interconnected CD31⁺ endothelial cell organization accompanied by endothelial cell-associated gene expression. Overall, this hybrid MEW-3D bioprinted platform provides an engineerable in vitro model for periosteum-inspired tissue modeling, biomaterial screening, and investigation of osteogenic and endothelial cell-associated processes under controlled conditions.