Eggshell Microparticle Reinforced Scaffolds for Regeneration of Critical Sized Cranial Defects

Scaffold-based approaches for bone regeneration have been studied using a wide range of biomaterials as reinforcing agents to improve the mechanical strength and bioactivity of the 3D constructs. Eggshells are sustainable and inexpensive materials with unique biological and chemical properties to support bone differentiation. The incorporation of eggshell particles within hydrogels yields highly osteoinductive and osteoconductive scaffolds. This study reveals the effects of microparticles of whole eggshells, eggshells without a membrane, and a pristine eggshell membrane on osteogenic differentiation in protein-derived hydrogels. The in vitro studies showed that gels reinforced with eggshells with and without a membrane demonstrated comparable cellular proliferation, osteogenic gene expression, and osteogenic differentiation. Subsequently, in vivo studies were performed to implant eggshell microparticle-reinforced composite hydrogel scaffolds into critical-sized cranial defects in Sprague Dawley (SD) rats for up to 12 weeks to study bone regeneration. The in vivo results showed that the eggshell microparticle-based scaffolds supported an average bone volume of 60 mm³ and a bone density of 2000 HU 12 weeks post implantation. Furthermore, histological analyses of the explanted scaffolds showed that the eggshell microparticle-reinforced scaffolds permitted tissue infiltration and induced bone tissue formation over 12 weeks. The histology staining also indicated that these scaffolds induced significantly higher bone regeneration at 6 and 12 weeks as compared to the blank (no scaffold) and pristine gel scaffolds. The eggshell microparticle-reinforced scaffolds also supported significantly higher bone formation, remodeling, and vascularization over 6 and 12 weeks as confirmed by immunohistochemistry analysis. Collectively, our results indicated that eggshell microparticle-reinforced scaffolds facilitated significant bone regeneration in critical-sized cranial defects.