Preparation, Physicochemical Assessment, and Cytotoxicity of Bone Powder Derived from Cuttlefish Bone via Hydrothermal and Chemical-Enzymatic Transformation

Background The rising demand for effective bone graft materials, particularly in dentistry, stems from the limitations of autografts, such as limited availability and donor site morbidity. Cuttlebone, a natural and abundant calcium carbonate source, exhibits potential as an alternative due to its structural and mechanical properties. This study investigates the physicochemical and biological properties of bone powder derived from cuttlebone using two processing methods: hydrothermal and chemical-enzymatic transformation, aiming to explore its potential for oral and maxillofacial bone graft applications. Methods The thermal processing method removed all organic components, while the enzymatic method retained collagen proteins. The resulting bone powders were characterized using scanning electron microscopy (SEM), X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), energy-dispersive X-ray spectroscopy (EDX), and Brunauer–Emmett–Teller (BET) analysis. Cytotoxicity and biocompatibility were evaluated using the MTT assay and cell adhesion tests on L929 fibroblasts and C2C12 osteoblast precursor cells. Results SEM and XRD analyses revealed that the thermal samples had a crystalline, polyhedral structure, while the enzymatic samples were amorphous and sheet-like. FTIR confirmed the presence of calcium phosphate in both samples, but the enzymatic group retained collagen-associated amide bonds. BET analysis indicated that the enzymatic samples had a larger surface area and pore volume, which supported better cell adhesion. MTT assays demonstrated the enzymatic group showed over 100% cell viability, indicating excellent biocompatibility, while thermal samples exhibited below 70%. Conclusion The enzymatically processed Cuttlebone-derived bone powder showed superior biocompatibility and structural properties suitable for bone tissue engineering applications. In contrast, the thermal samples, although crystalline, were cytotoxic. These findings suggest that enzymatic processing of Cuttlebone may offer a promising, biocompatible alternative for bone grafting applications.