Additive manufacturing of hydroxyapatite-chitosan-genipin composite scaffolds for bone tissue engineering applications

Additive manufacturing holds promise for the fabrication of three-dimensional scaffolds with precise geometry, to serve as substrates for the guided regeneration of natural tissue. In this work, a bioinspired approach is adopted for the synthesis of hybrid hydroxyapatite hydrogels, which were subsequently printed to form 3D scaffolds for bone tissue engineering applications. These hydrogels consist of hydroxyapatite nanocrystals, biomimetically synthesized in the presence of both chitosan and L-arginine. To improve their mechanical properties, chemical crosslinking was performed using a natural crosslinking agent (genipin), and their rheology was modified by employing an acetic acid/gelatin solution. Regarding the 3D printing process, several parameters (flow, infill and perimeter speed) were studied in order to accurately produce scaffolds with predesigned geometry and micro-architecture, while also applying low printing temperature (15 degrees C). Following the printing procedure, the 3D scaffolds were freeze dried in order to remove the entrapped solvents and therefore, obtain a porous interconnected network. Evaluation of porosity was performed using micro-computed tomography and nanomechanical properties were assessed through nanoindentation. Results of both characterization techniques, showed that the scaffolds' porosity as well as their modulus values, fall within the corresponding range of the respective values of cancellous bone. The biocompatibility of the 3D printed scaffolds was assessed using MG63 human osteosarcoma cells for 7 days of culturing. Cell viability was evaluated by MTT assay as well as double staining and visualized under fluorescence microscopy, while cell morphology was analyzed through scanning electron microscopy. Biocompatibility tests, revealed that the scaffolds constitute a cell-friendly environment, allowed them to adhere on the scaffolds' surface, increase their population and maintain high levels of viability.