Refined in vivo model for bone regeneration: insights into scaffold architecture and porosity.

BACKGROUND: The architecture of bone substitute scaffolds-particularly pore size and organization-plays a crucial role in orchestrating immune responses, osteogenesis and angiogenesis. Yet, the mechanisms linking scaffold design to the temporal dynamics of bone regeneration remain partially understood. To address this, we established a refined in vivo model that integrates histological, molecular, and immunological analyses from a single explant, enabling spatially resolved insight into the bone healing process and dynamics. METHODS: Using a dynamic rabbit calvarial model, we investigated 3D-printed calcium phosphate cement scaffolds designed with concomitant macroarchitectures of 250 μm and 500 µm pores within a single construct, allowing direct intra-animal comparison. The model recapitulated three vertically migrating zones of regeneration-regenerative, osteogenic, and granulation-captured at 2 and 4 weeks. Histomorphometric analyses quantified bone ingrowth, while laser microdissection enabled zone-specific transcriptomic profiling from paraffin-embedded sections previously used for (immuno-)histology. Gene expression was further validated by qPCR and complemented with immunohistochemical characterization of macrophage and neutrophil populations. RESULTS: Histological analysis revealed a consistent spatial organization of bone regeneration across conditions. After 4 weeks, scaffolds with 250 µm pores exhibited more homogeneous and advanced bone formation than those with 500 µm pores or particulate substitutes. Transcriptomic analysis identified 280-381 differentially expressed genes between microporous architectures, with over half being non-coding RNAs, suggesting an important role for post-transcriptional regulation. Enrichment analyses indicated modulation of pathways involved in immune activity, ossification, calcium signaling and autophagy. Immunohistochemistry confirmed similar inflammatory mechanisms across both macroarchitectures but revealed earlier M1-to-M2 macrophage transition and faster inflammatory resolution with the finest porous network. CONCLUSION: This integrative in vivo model provides a robust workflow for correlating structural, cellular, and molecular dimensions of bone regeneration within the same specimen. The findings show that scaffold macroarchitecture influences both the extent and timing of immune and osteogenic processes. While scaffolds with 250 μm and 500 µm pores supported regeneration, the finer design consistently promoted more advanced tissue formation and maturation. These results underscore the key role of scaffold design in modulating bone healing and highlight this model as a platform for studying structure-function relationships in bone tissue engineering.