Sustainable Fabrication of Tailored Bone Substitutes: From High‐Throughput Scaffold Manufacturing, Scaled‐Up HMSC Expansion to Dynamic Cultivation in a Perfusion Bioreactor

ABSTRACT The demand for off‐the‐shelf biocompatible bone substitutes has driven the development of numerous independent in vitro technologies to generate products resembling physiological tissues. Due to technical challenges and overly simplified cultivation approaches/niches, the end‐products are often uniformly shaped and inferior to native bone tissue. In this report, three major technologies are implemented cohesively to address these shortfalls: (1) Spinner flasks for scaled‐up stem cell expansion, (2) Manufacturing via 3D‐printing and cast‐molding processes, large modular collagen‐based (COL) scaffolds +/− chondroitin sulfate A (CSA) of tailored dimensions, (3) Perfusion bioreactor with controlled oxygen tension (pO 2 ) to support high cell density and osteochondral differentiation. We report an oxygenated in vitro niche within the bioreactor that supports high cell density and self‐induced osteogenic differentiation for 60 days. Enhanced mineralization and osteogenic gene expression in COL scaffolds were observed, while COL + CSA scaffolds exhibited elevated Col10a gene expression for hypertrophic chondrocytes, a representative indicator of active endochondral ossification. In summary, this report describes an integral approach to consistently and rapidly achieve physiologically relevant bone substitutes of tailored dimensions. Furthermore, the pivotal effect of recapitulating endochondral ossification in vitro through dynamic bioreactor culture is demonstrated, paving the way to generate complex tissue structures in the future.