Morphological, Thermal, Mechanical and Cytotoxic Investigation of Hydroxyapatite Reinforced Chitosan/Collagen 3D Bioprinted Dental Grafts

Abstract

Dental tissue regeneration, particularly alveolar bone and gingival repair, remains a major challenge in regenerative medicine. 3D bioprinting offers patient-specific and anatomically precise constructs, representing an advanced alternative to conventional grafting. In this study, nanohydroxyapatite (nHA), chitosan (CS), and collagen (CoL) were combined to fabricate and characterize 3D bioprinted dental grafts. SEM revealed a highly porous, interconnected architecture favorable for cell infiltration and nutrient exchange. EDS confirmed Ca/P ratios of 2.06 for nHA/CoL and 1.83 for nHA/CS/CoL, both of which are above the stoichiometric 1.67, indicating the presence of additional mineral phases and ion substitutions. FTIR and XRD verified characteristic functional groups and crystalline phases, including B-type HA with carbonate substitution. Mechanical testing showed that pure nHA exhibited the lowest compressive strength, whereas CoL incorporation improved stiffness. The nHA/CS/CoL composite achieved the highest compressive strength, elastic modulus, and toughness, demonstrating superior mechanical resilience. DSC analysis indicated endothermic peaks at 106.49 ◦C and 351.91 ◦C, with enthalpy values (264.91 J/g and 15.09 J/g) surpassing those of nHA alone. TGA revealed ~28.8% weight loss across three degradation stages, confirming enhanced thermal stability. In vitro cytocompatibility testing using L929 fibroblasts validated the biocompatibility of the composites. Collectively, the synergy between bioceramics and biopolymers markedly improved both mechanical and thermal performance. These findings position the nHA/CS/CoL scaffold as a promising candidate for clinical applications in dental tissue regeneration. Unlike conventional grafting materials, this study introduces a synergistically optimized nHA/CS/CoL bio-ink formulation specifically designed for extrusion-based 3D bioprinting of patient-specific dental constructs. The core innovation lies in the precise integration of nHA within a dual-polymer matrix (CS/CoL), which bridges the gap between mechanical resilience and biological signaling, achieving a compressive strength that mimics native alveolar bone while maintaining high cytocompatibility.