Despite bone's innate self-renewal capability, some periodontal pathologic and traumatic defects' size inhibits full spontaneous regeneration. This current research characterized a 3D porous biodegradable nano-hydroxyapatite/chitosan (nHAp/CS, 70/30) scaffold for periodontal bone regeneration, which preparation method includes the final solvent extraction and sterilization through supercritical CO2 (scCO2). Micro-CT analysis revealed the fully interconnected porous microstructure of the nHAp/CS scaffold (total porosity 78 %, medium pore size 200 μm) which is critical for bone regeneration. Scanning electron microscopy (SEM) showed HAp crystals forming on the surface of the nHAp/CS scaffold after 21 days in simulated body fluid, demonstrating its bioactivity in vitro. The presence of nHAp in the scaffolds promoted a significantly lower biodegradation rate compared to a plain CS scaffold in PBS. Dynamic mechanical analysis confirmed their viscoelasticity, but the presence of nHAp significantly enhanced the storage modulus (42.34 ± 6.09 kPa at 10 Hz after 28 days in PBS), showing that it may support bone ingrowth at low-load bearing bone defects. Both scaffold types significantly inhibited the growth, attachment and colony formation abilities of S. aureus and E. coli, enhancing the relevance of chitosan in the grafts' composition for the naturally contaminated oral environment. At SEM and laser scanning confocal microscopy, MG63 cells showed normal morphology and could adhere and proliferate inside the biomaterials' porous structure, especially for the nHAp/CS scaffold, reaching higher proliferative rate at day 14. MG63 cells seeded within nHAp/CS scaffolds presented a higher expression of RUNX2, collagen A1 and Sp7 osteogenic genes compared to the CS samples. The in vivo subcutaneous implantation in mice of both scaffold types showed lower biodegradability with the preservation of the scaffolds porous structure that allowed the ingrowth of connective tissue until 5 weeks. Histology shows an intensive and progressive ingrowth of new vessels and collagen between the 3rd and the 5th week, especially for the nHAp/CS scaffold. So far, the scCO2 method enabled the production of a cost-effective and environment-friendly ready-to-use nHAp/CS scaffold with microstructural, chemical, mechanical and biocompatibility features that make it a suitable bone graft alternative for defect sites in an adverse environment as in periodontitis and peri-implantitis.
Keywords: Antimicrobial activity; Biomaterials; Biomimetics; Highly-porous 3D microstructure; Nano-hydroxyapatite/chitosan scaffold; Osteogenic differentiation.
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