RESOURCES

Abstract
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.

Abstract
The development of synthetic bone substitutes that equal or exceed the efficacy of autologous graft remains challenging. In this study, a rat calvarial defect model was used as a reference to investigate the influence of composition and architecture of 3D-printed cement, with or without bioactives, on tissue regeneration. Printable cement pastes were formulated by combining hyaluronic acid and cement precursors. Cementitious scaffolds were printed with 3 different patterns. After 7 weeks of implantation with or without bone marrow, multiparametric qualitative and quantitative assessments were performed using µCT, SEM, and histology. None of the set-up strategies was as efficient as autologous cancellous bone graft to repair calvarial defects. Nonetheless, the presence of scaffold improved the skull vault closure, particularly when the scaffold was soaked in total bone marrow before implantation. No significant effect of scaffold macro-architecture was observed on tissue mineralization. Magnesium phosphate-based scaffolds (MgP) seemed to induce higher bone formation than their calcium-phosphate-based counterparts. They also displayed a quicker biodegradation and sparse remaining material was found after 7 weeks of implantation. Although further improvements are required to reach clinical settings, this study demonstrated the potential of organo-mineral cements for bone regeneration and highlighted the peculiar properties of MgP-based cements.

Abstract
Abstract Bioprinting is a booming technology, with numerous applications in tissue engineering and regenerative medicine. However, most biomaterials designed for bioprinting depend on the use of sacrificial baths and/or non-physiological stimuli. Printable biomaterials also often lack tunability in terms of their composition and mechanical properties. To address these challenges, the authors introduce a new biomaterial concept that they have termed “clickable dynamic bioinks”. These bioinks use dynamic hydrogels that can be printed, as well as chemically modified via click reactions to fine-tune the physical and biochemical properties of printed objects after printing. Specifically, using hyaluronic acid (HA) as a polymer of interest, the authors investigate the use of a boronate ester-based crosslinking reaction to produce dynamic hydrogels that are printable and cytocompatible, allowing for bioprinting. The resulting dynamic bioinks are chemically modified with bioorthogonal click moieties to allow for a variety of post-printing modifications with molecules carrying the complementary click function. As proofs of concept, the authors perform various post-printing modifications, including adjusting polymer composition (e.g., HA, chondroitin sulfate, and gelatin) and stiffness, and promoting cell adhesion via adhesive peptide immobilization (i.e., RGD peptide). The results also demonstrate that these modifications can be controlled over time and space, paving the way for 4D bioprinting applications.

Abstract
The survival and function of thick tissue engineered implanted constructs depends on pre-existing, embedded, functional, vascular-like structures that are able to integrate with the host vasculature. Bioprinting was employed to build perfusable vascular-like networks within thick constructs. However, the improvement of oxygen transportation facilitated by these vascular-like networks was directly quantified. Using an optical fiber oxygen sensor, we measured the oxygen content at different positions within 3D bioprinted constructs with and without perfusable microchannel networks. Perfusion was found to play an essential role in maintaining relatively high oxygen content in cell-laden constructs and, consequently, high cell viability. The concentration of oxygen changes following switching on and off the perfusion. Oxygen concentration depletes quickly after pausing perfusion but recovers rapidly after resuming the perfusion. The quantification of oxygen levels within cell-laden hydrogel constructs could provide insight into channel network design and cellular responses.