Bone Graft Paste Nanohydroxyapatite Chitosan-Gelatin (nHA/KG) for Periodontal Regeneration: Study on Three-Dimensional Cell Culture.

Objective:  Regenerative periodontal surgical approaches require scaffolds in a form that can fill narrow and irregular defects. Each scaffold must be specially designed to conform to the shape of the specific defect. The aim of this study was to fabricate nanohydroxyapatite chitosan-gelatin (nHA/KG) pastes with different composition percentages and to analyze the differences in physical, chemical, and biological characteristics in response to periodontal tissue regeneration in vitro.

Materials and methods:  The nHA/KG paste was prepared at three different concentrations of inorganic and organic contents (70/30; 75/25; and 80/20) by mixing nHA powder, chitosan flakes, and gelatin powder. The ratio of chitosan and gelatin on all nHA/KG pastes is 1:1. The three nHA/KG pastes were tested for the following rheology and bioactivity properties in simulated body fluid (SBF): pH value, swelling, degradability, surface morphology, and cell attachment by scanning electron microscopy and chemical structure by Fourier transform infrared (FTIR). Osteoblasts and fibroblasts were analyzed for proliferation using the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) assay and for cell proliferation by reverse transcription quantitative real-time polymerase chain reaction of COL1, alkaline phosphatase (ALP), osteocalcin (OCN), and RUNX2.

Statistical analysis:  Analysis of variance followed by Tukey's post hoc, Kruskal-Wallis, Wilcoxon, and paired sample t-tests were performed according to each data type.

Results:  The nHA/KG paste showed gel-like physical characteristics. The pH value after SBF immersion was stable at pH ± 7.0, although the pH of the nHA/KG 80/20 paste decreased to pH 6.3 on day 14. The three paste preparations showed significant differences in swelling (p < 0.05) on days 1 and 14 and in the degradability ratio on days 1, 2, and 7 (p < 0.05). The three-dimensional scaffold surface morphology differed depending on the immersion time. The FTIR test showed the presence of PO₄ ^3-, CO₃ ^2-, -OH, amide I, and amide II functional groups in all paste variants. The nHA/KG 75/25 paste had the most stable structure during the immersion period. Biological tests showed a viability ratio of osteoblasts and fibroblasts ≥ 70%. The paste could stimulate the messenger ribonucleic acid expression of the COL1, ALP, OCN, and RUNX2.

Conclusion:  The nHA/KG bone graft paste showed good potential as an injectable scaffold, with the nHA/KG 75/25 paste being the best of the three pastes tested here.