Cuttlefish Bone as a Sustainable Osteoinductive Biomaterial in 3d-Printed Biocomposite Scaffolds Promoted Osteogenesis

Sustainability focuses on reducing waste products by reintegrating them in other useful production cycles, thereby providing financial advantages and minimizing environmental pollution levels. Marine derived food waste from shrimps, crabs, squids, lobsters, and cuttlefish are usually discarded away. In particular, cuttlefish bones consist of aragonite, a calcium carbonate polymorph with osteoinductive properties. Aragonite is a common skeletal mineral in marine organisms, while calcium carbonates have been used as bio-fillers in polymers 3D printing. Considering these points, the aim of this work was to develop 3D biocomposite scaffolds consisting of the thermoplastic polylactic acid and the cuttlefish bone with tailored properties for bone tissue engineering. Aragonite powder was obtained from the bones, purified from organic compounds and impurities by alkaline treatment. PLA and composite PLA/aragonite filaments were prepared, containing 2.5, 5 and 10% w/w of aragonite. 3D printed scaffolds were manufactured using the fused filament fabrication (FFF) technology. All raw and composite materials were characterized by Differential Scanning Calorimetry, Thermogravimetric analysis, X-ray Diffraction, Scanning Electron Microscopy and Fourier Transform Infrared spectroscopy. The hydrophilicity and compression modulus were also evaluated. The cell adhesion and proliferation were investigated by culturing pre-osteoblasts on the scaffolds. The osteogenic potential was examined by quantification of the alkaline phosphatase activity, collagen and calcium production in vitro. Alkaline treatment led to the removal of organic compounds and did not alter the aragonite polymorph. The incorporation of aragonite increased the hydrophilicity of PLA and the Young modulus. Pre-osteoblastic cells cultured on 3D printed PLA/aragonite scaffolds exhibited high adhesion. Alkaline phosphatase activity, calcium and collagen production were significantly elevated with increasing aragonite content, indicating enhanced osteogenic differentiation. Calcium production, detected by alizarin red staining, showed an almost two-fold increase from day 7 to day 14, indicating that PLA/aragonite scaffolds stimulated matrix mineralization. Moreover, biogenic aragonite significantly increased collagen production, particularly at the highest aragonite concentrations of 5 and 10% w/w, supporting extracellular matrix formation. In this study, different composite scaffold formulations were produced containing a thermoplastic PLA matrix and three different concentrations of aragonite (2.5, 5 and 10% w/w) from cuttlebones as a sustainable filler with osteoinductive properties. The results demonstrate that 3D biocomposite PLA/aragonite scaffolds promote cell adhesion, proliferation and osteogenesis in vitro, with the highest aragonite content of 5 and 10% w/w indicating significantly higher levels of all osteogenic markers.