Humanized in Vivo Bone Tissue Engineering: In Vitro Preculture Conditions Control the Structural, Cellular, and Matrix Composition of Humanized Bone Organs

Subcutaneous animal models for ectopic bone tissue engineering (BTE) are invaluable tools for studying bone biology and disease processes. However, clear guidelines for optimizing BTE design are lacking, which hinders reproducibility in regenerative medicine and disease modeling. We proposed that the duration and composition of in vitro preculture play a pivotal role in shaping the structural, cellular, and extracellular matrix (ECM) properties of humanized bone constructs by modulating osteogenic differentiation and mineralization of biomaterial-embedded osteoblasts prior to implantation. To investigate the hypothesis, we used two biomaterials seeded with human osteoprogenitors (2.2×10 6 cells/mL), each mimicking distinct ECM strategies: protein mimicry (GelMA hydrogels) and mineral mimicry (CaP-mPCL scaffolds). Constructs underwent preculture for one or four weeks in osteogenic media (OM) with or without a three-day mineralization boost (OM+). Afterward, the constructs were implanted into NSG mice with BMP2 supplementation and cultured in vivo for 11 weeks. Post-implantation analyses assessed structural, cellular, and ECM properties. The in vitro preculture conditions significantly influenced the in vivo recapitulation of human bone structure, cellular composition, ECM organization, and bone marrow content. In both GelMA and CaP-mPCL constructs, collagen organization in vivo was shaped by the preculture conditions. Constructs precultured under high osteogenic differentiation and mineralization (OM+) developed a more mature, lamellar-like collagen architecture, while those cultured under OM-only conditions showed a greater prevalence of immature, woven-like collagen, regardless of biomaterial type. The alignment of the osteocyte network with collagen fibers was also enhanced under OM+ conditions, leading to a more organized ECM with improved elastic modulus, resembling native bone properties. This study underscores the critical role of in vitro preculture composition in directing ECM organization and improving the structural fidelity of engineered bone constructs in vivo . Specifically, the inclusion of a mineralization boost in the preculture medium provides a cost-effective and efficient method to enhance the quality and native-like properties of BTE models. This approach offers significant potential for accelerating the development of high-fidelity bone models for research and clinical applications.