Three-dimensional cell sheet model improves in vitro prediction accuracy of osteogenic potential for biodegradable magnesium-based metals

Biodegradable metals have been increasingly utilized clinically due to their biosafety and pro-osteogenic properties. However, conventional monolayer cell-based preclinical safety evaluation methods based on ISO10993-5 consistently indicate significant cytotoxicity that contradicts in vivo outcomes. In this study, we aimed to establish an in vitro evaluation model that better correlates with in vivo performance. Three-layer BMSC cell sheets were constructed using layer-by-layer assembly. Histological analyses revealed a stable three-dimensional structure with elevated cell-cell interaction proteins, including N-Cadherin, Fibronectin, and Vinculin, along with enhanced osteogenic potential. The cytotoxicity of 4N pure Mg was evaluated in both cell sheet and monolayer co-culture models. Flow cytometry showed higher Ki67 expression and lower ROS levels and apoptosis rate in cell sheets. ShRNA-mediated silencing of N-Cadherin in cell sheets significantly compromised their cytoprotective capacity against Mg metal-induced toxicity. Osteogenesis-related gene expression correlation analysis between in vitro co-culture models and in vivo femur implantation models was conducted using RNA-seq and qRT-PCR. Results showed that 4N pure Mg enhanced osteogenic genes (BMP2R, RUNX2, and SP7) in cell sheets, consistent with in vivo patterns but contrary to monolayer models. Various Mg-based metals (4N/5N Pure Mg, ZE21B, and WE43) were evaluated in cell sheet defect, monolayer defect, and cranial defect models. 5N Pure Mg, ZE21B, and WE43 promoted defect healing in both cranial defect and cell sheets, but showed no positive effect in monolayers. Collectively, cell sheet models correlated well with in vivo results, suggesting their potential as alternative in vitro evaluation models, thereby accelerating clinical translation of Mg-based biomaterials.