Eggshell-derived amorphous calcium phosphate : Synthesis, characterization and bio-functions as bone graft materials in novel 3D osteoblastic spheroids model

Funding Information: We acknowledge financial support from the Baltic Research Programme Project No. EEA-RESEARCH-85 “Waste-to-resource: eggshells as a source for next generation biomaterials for bone regeneration (EGGSHELL)” under the EEA Grant of Iceland, Liechtenstein and Norway No. EEZ/BPP/VIAA/2021/1 and National Natural Science Foundation of China (No. 8197175 2). The authors acknowledge the access to the infrastructure and expertise of the BBCE – Baltic Biomaterials Centre of Excellence (European Union's Horizon 2020 research and innovation programme under grant agreement No. 857287). Publisher Copyright: © 2023 The AuthorsA multitude of autogenous/allogeneic and semi-synthetic bone graft materials have been developed to reconstruct the defective bone tissue but with high bio-cost and potential environmental pollution. With high calcium content and several trace elements, chicken eggshells are no longer considered as wastes but attractive sources of high-value-added biomaterials. This study used chicken eggshells and synthetic hydroxyapatite (HAp) to synthesize amorphous calcium phosphate (ACP) bone graft materials, namely Control and Eggshell. The physiochemical characteristics, biosafety, and immunocompatibility of synthetic ACP particles were inspected. Their osteogenic activity was further investigated in a novel osteoblastic spheroids model. Eggshell ACP particles exhibited ideal cytocompatibility compared to the control ACP and were more resistant to re-crystallization. In osteoblastic spheroids, Eggshell ACP mediated typical osteogenic mRNA profiles of MC-3T3-E1 cells, accompanied by the increased formation of mineralized nodules and boosted synthesis of ECM proteins represented by OPN and collagen I. This study establishes a promising technique to synthesize stable, safe, and osteoinductive ACP graft particles from eggshell waste. Furthermore, the osteoblastic spheroids constructed in the present study provide a more practical model for biomaterial research, which reflect the three-dimensional interaction between host bone tissue and graft materials more realistically.Peer reviewe