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

Abstract

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.

Keywords

periodontal regenerative therapy - nanohydroxyapatite - chitosan - gelatin - bone graft paste

Publication History

Article published online:
12 March 2025

• References

• 1 Shalini M, Gajendran P. The role of scaffolds in periodontal regeneration. Int J Pharm Sci Rev Res 2017; 45 (01) 135-140
• 2 Yamada S, Shanbhag S, Mustafa K. Scaffolds in periodontal regenerative treatment. Dent Clin North Am 2022; 66 (01) 111-130
• 3 Pan Y, Zhao Y, Kuang R. et al. Injectable hydrogel-loaded nano-hydroxyapatite that improves bone regeneration and alveolar ridge promotion. Mater Sci Eng C 2020; 116: 111158
• 4 Przekora A. Current trends in fabrication of biomaterials for bone and cartilage regeneration: materials modifications and biophysical stimulations. Int J Mol Sci 2019; 20 (02) 435
• 5 Kamboj M, Arora R, Gupta H. Comparative evaluation of the efficacy of synthetic nanocrystalline hydroxyapatite bone graft (Ostim^®) and synthetic microcrystalline hydroxyapatite bone graft (Osteogen^®) in the treatment of human periodontal intrabony defects: a clinical and denta scan study. J Indian Soc Periodontol 2016; 20 (04) 423-428
• 6 Husain S, Al-Samadani KH, Najeeb S. et al. Chitosan biomaterials for current and potential dental applications. Materials (Basel) 2017; 10 (06) 1-20
• 7 Astuty SW, Sunarto H, Amir L, Idrus E. Evaluation of regenerative therapy using cell sheet through cementum protein-1 expression on macaca nemestrina. Int J Appl Pharmaceut 2017; 9 (02) 107-109
• 8 Rianti D, Fanny G, Nathania RV. et al. The characteristics, swelling ratio and water content percentage of chitosan-gelatin/limestone-based carbonate hydroxyapatite composite scaffold. Int J Integ Eng 2022; 14 (02) 13-23
• 9 Sharma C, Dinda AK, Potdar PD, Chou CF, Mishra NC. Fabrication and characterization of novel nano-biocomposite scaffold of chitosan-gelatin-alginate-hydroxyapatite for bone tissue engineering. Mater Sci Eng C 2016; 64: 416-427
• 10 Mohamed KR, Beherei HH, El-Rashidy ZM. In vitro study of nano-hydroxyapatite/chitosan-gelatin composites for bio-applications. J Adv Res 2014; 5 (02) 201-208
• 11 Thorpe AA, Creasey S, Sammon C, Le Maitre CL. Hydroxyapatite nanoparticle injectable hydrogel scaffold to support osteogenic differentiation of human mesenchymal stem cells. Eur Cell Mater 2016; 32: 1-23
• 12 Cortellini P. Minimally invasive surgical techniques in periodontal regeneration. J Evid Based Dent Pract 2012; 12 (03) 89-100
• 13 Hu NM, Chen Z, Liu X. et al. Mechanical properties and in vitro bioactivity of injectable and self-setting calcium sulfate/nano-HA/collagen bone graft substitute. J Mech Behav Biomed Mater 2012; 12: 119-128
• 14 Rezwan K, Chen QZ, Blaker JJ, Boccaccini AR. Biodegradable and bioactive porous polymer/inorganic composite scaffolds for bone tissue engineering. Biomaterials 2006; 27 (18) 3413-3431
• 15 Chavan PN, Bahir MM, Mene RU, Mahabole MP, Khairnar RS. Study of nanobiomaterial hydroxyapatite in simulated body fluid: formation and growth of apatite. Mater Sci Eng B 2010; 168 (01) 224-230
• 16 Baino F, Yamaguchi S. The use of simulated body fluid (SBF) for assessing materials bioactivity in the context of tissue engineering: Review and challenges. Biomimetics (Basel) 2020; 5 (04) 1-19
• 17 Weinreb M, Nemcovsky CE. In vitro models for evaluation of periodontal wound healing/regeneration. Periodontol 2000 2015; 68 (01) 41-54
• 18 Koch F, Meyer N, Valdec S, Jung RE, Mathes SH. Development and application of a 3D periodontal in vitro model for the evaluation of fibrillar biomaterials. BMC Oral Health 2020; 20 (01) 148
• 19 Bachtiar EW, Bachtiar BM, Abbas B, Harsas NA, Sadaqah NF, Aprilia R. Biocompatibility and osteoconductivity of injectable bone xenograft, hydroxyapatite and hydroxyapatite-chitosan on osteoblast culture. Dent J (Majalah Kedokteran Gigi) 2010; 43 (04) 176-180
• 20 Bachtiar EW, Suniarti DF, Fadhilah ND, Ulfiana R, Abbas B. Potency of injectable hydroxyapatite chitosan scaffold for bone regeneration. J Clin Diagn Res 2017; 11 (12) ZF01-ZF03
• 21 Harsas NA, Yosefa V, Savvyana M, Abbas B, Winiati Bachtiar E, Soeroso Y. Physicochemical characterization of nanohydroxyapatite powder in simulated body fluid immersion: a pilot study. Int J Nanoelectron Mater 2023; 16: 905-922
• 22 Kokubo T, Yamaguchi S. Simulated body fluid and the novel bioactive materials derived from it. J Biomed Mater Res A 2019; 107 (05) 968-977
• 23 Hatakeyama W, Taira M, Chosa N, Kihara H, Ishisaki A, Kondo H. Effects of apatite particle size in two apatite/collagen composites on the osteogenic differentiation profile of osteoblastic cells. Int J Mol Med 2013; 32 (06) 1255-1261
• 24 Chen J, Zhang C, Feng Y. et al. Studies on culture and osteogenic induction of human mesenchymal stem cells under CO2-independent conditions. Astrobiology 2013; 13 (04) 370-379
• 25 Sohrabi M, Eftekhari Yekta B, Rezaie H. et al. Enhancing mechanical properties and biological performances of injectable bioactive glass by gelatin and chitosan for bone small defect repair. Biomedicines 2020; 8 (12) 1-19
• 26 Maas M, Hess U, Rezwan K. The contribution of rheology for designing hydroxyapatite biomaterials. Curr Opin Colloid Interface Sci 2014; 19 (06) 585-593
• 27 Ramli H, Zainal NFA, Hess M, Chan CH. Basic principle and good practices of rheology for polymers for teachers and beginners. Chem Teach Int 2022; 4 (04) 307-326
• 28 Kumar BYS, Isloor AM, Kumar GCM, Inamuddin, Asiri AM. Nanohydroxyapatite reinforced chitosan composite hydrogel with tunable mechanical and biological properties for cartilage regeneration. Sci Rep 2019; 9 (01) 15957
• 29 Maulida HN, Hikmawati D, Budiatin AS. Injectable bone substitute paste based on hydroxyapatite, gelatin and streptomycin for spinal tuberculosis. J Spine 2015; 04 (06) 266
• 30 Araújo M, Miola M, Baldi G, Perez J, Verné E. Bioactive glasses with low Ca/P ratio and enhanced bioactivity. Materials (Basel) 2016; 9 (04) 266
• 31 Danoux CB, Barbieri D, Yuan H, de Bruijn JD, van Blitterswijk CA, Habibovic P. In vitro and in vivo bioactivity assessment of a polylactic acid/hydroxyapatite composite for bone regeneration. Biomatter 2014; 4: e27664
• 32 Galow AM, Rebl A, Koczan D, Bonk SM, Baumann W, Gimsa J. Increased osteoblast viability at alkaline pH in vitro provides a new perspective on bone regeneration. Biochem Biophys Rep 2017; 10: 17-25
• 33 Kumar P, Saini M, Dehiya BS. et al. Fabrication and in-vitro biocompatibility of freeze-dried CTS-nHA and CTS-nBG scaffolds for bone regeneration applications. Int J Biol Macromol 2020; 149: 1-10
• 34 Li TT, Zhang Y, Ren HT, Peng HK, Lou CW, Lin JH. Two-step strategy for constructing hierarchical pore structured chitosan-hydroxyapatite composite scaffolds for bone tissue engineering. Carbohydr Polym 2021; 260: 117765
• 35 Wattanutchariya W, Changkowchai W. Characterization of porous scaffold from chitosan-gelatin/hydroxyapatite for bone grafting. In: Ao SI, Castillo O, Douglas C, Feng DD, Lee JA. eds. Proceedings of the International MultiConference of Engineers and Computer Scientists. 2014
• 36 Kong L, Gao Y, Lu G, Gong Y, Zhao N, Zhang X. A study on the bioactivity of chitosan/nano-hydroxyapatite composite scaffolds for bone tissue engineering. Eur Polym J 2006; 42 (12) 3171-3179
• 37 Kasaj A, Willershausen B, Junker R, Stratul SI, Schmidt M. Human periodontal ligament fibroblasts stimulated by nanocrystalline hydroxyapatite paste or enamel matrix derivative. An in vitro assessment of PDL attachment, migration, and proliferation. Clin Oral Investig 2012; 16 (03) 745-754
• 38 Maji K, Dasgupta S, Kundu B, Bissoyi A. Development of gelatin-chitosan-hydroxyapatite based bioactive bone scaffold with controlled pore size and mechanical strength. J Biomater Sci Polym Ed 2015; 26 (16) 1190-1209
• 39 Souto-Lopes M, Grenho L, Manrique YA. et al. Full physicochemical and biocompatibility characterization of a supercritical CO₂ sterilized nano-hydroxyapatite/chitosan biodegradable scaffold for periodontal bone regeneration. Biomater Adv 2023; 146: 213280
• 40 Liu J, Zhao Z, Ruan J. et al. Stem cells in the periodontal ligament differentiated into osteogenic, fibrogenic and cementogenic lineages for the regeneration of the periodontal complex. J Dent 2020; 92: 103259
• 41 Gupta SK, Dinda AK, Potdar PD, Mishra NC. Modification of decellularized goat-lung scaffold with chitosan/nanohydroxyapatite composite for bone tissue engineering applications. BioMed Res Int 2013; 2013: 651945