Sustainable Eggshell-Based Amorphous Calcium Phosphate Scaffolds and Membrane Protein Hydrogel for Regeneration in Rabbit Femoral and Calvarial Defects

Critical-sized bone defects require grafts with ideal spatial supporting. We upcycled eggshell waste sustainably into porous amorphous calcium phosphate (ACP) scaffolds and evaluated their early bone-regeneration performance alone and in combination with flexible and bioactive adjuncts <i>in vivo</i>. ACP scaffolds with ∼60% porosity were fabricated by pressure-densification, using ice microspheres as space holders. Before implantation, ACP scaffolds were vacuum-infiltrated with a hyaluronic acid (HA) hydrogel to minimize internal voids. Bilateral critical-size femoral and calvarial defects were created in New Zealand White rabbits and reconstructed with one of four treatments: (1) Sham, (2) ACP scaffold (ACP), (3) ACP infused with HA hydrogel containing a proline-rich peptide P2 (ACP + HA/P2), or (4) ACP infused with HA/P2 supplemented with soluble eggshell membrane proteins (prepared by our lab, ACP + HA/P2 + SEPs). After 6 weeks, micro-CT and histology were performed. All ACP-containing groups showed greater defect filling and mineralization than the Sham group. Micro-CT analysis demonstrated that ACP + HA/P2 + SEPs achieved the highest bone volume and trabecular number and the lowest trabecular separation. Histology confirmed more extensive mineralized collagenous deposition on ACP scaffolds and a more integrated bone-material fusion zone in this group. In femoral defects with graft displacement, ACP scaffolds induced cortical-scaffold bone bridging within the marrow, most evident with SEPs. Eggshell-derived ACP scaffolds, particularly when paired with SEP-enriched HA hydrogel, enhance early regeneration of critical-sized bone defects. Upcycling the inorganic and organic constituents of eggshell waste yields a complementary, single-origin biomaterials package for sustainable bone repair.