Cytotoxicity of β-tricalcium phosphate chitosan gelatin composite scaffold as a bone substitute: in vitro study

Tansza Setiana Putri, Eddy Eddy, Deviyanti Pratiwi, Florencia Livia Kurniawan, Thet Thet Swe

Abstract


Introduction: Beta-tricalcium phosphate (βTCP) has higher solubility than hydroxyapatite (HA), allowing it to be more easily resorbed and replaced by newly formed bone. This higher solubility enables the release of calcium and phosphate ions that play important roles in bone remodeling and osteoblast activity; however, excessive ion release may lead to cytotoxic effects. Limestone, mainly composed of calcium carbonate (CaCO₃), can serve as a calcium source for the fabrication of βTCP. βTCP scaffolds can be combined with organic components such as chitosan and gelatin to form composite scaffolds for bone tissue engineering. Therefore, this study aimed to analyze the cytotoxicity of a β-tricalcium phosphate–chitosan–gelatin composite scaffold as a bone substitute. Methods: Type of research was experimental laboratory. Freeze-drying method was used to produce a composite scaffold which was divided into two groups: chitosan-gelatin scaffold as control group and βTCP-chitosan-gelatin scaffold (each group consisted of three samples) To evaluate cytotoxicity, composite scaffolds were tested on osteoblast cells and the MTT assay was measured and assessed based on time evaluation at 24 hours and 72 hours. Cytotoxicity was determined based on the percentage of viable cells obtained from the MTT assay. Results: Viable cells percentage on the chitosan-gelatin scaffold was 70.32% at 24 h and increased to 99.52% at 72 h. While on the chitosan-gelatin-βTCP scaffold there were 85.11% viable cells at 24 h and increased to 89.54% at 72 h. Statistical analysis using one-way ANOVA showed no significant difference among all groups (p>0.05). However, Fisher’s LSD test indicated a significant difference in cell viability between 24 hours and 72 hours within the chitosan gelatin group. Conclusion: The βTCP-chitosan-gelatin composite scaffold demonstrated no cytotoxic effect on osteoblast cells, indicating its biocompatibility and potential suitability as a bone substitute material.


Keywords


Cytotoxicity, β-tricalcium phosphate, chitosan, gelatin, scaffold

Full Text:

PDF

References


Islam MM, Shahruzzaman M, Biswas S, Nurus Sakib M, Rashid TU. Chitosan based bioactive materials in tissue engineering applications-A review. Bioact Mater. 2020;5(1):164–83. https://doi.org/10.1016/j.bioactmat.2020.01.012

Lou CW, Wen SP, Lin JH. Chitosan/gelatin porous bone scaffolds made by crosslinking treatment and freeze-drying technology: Effects of crosslinking durations on the porous structure, compressive strength, and in vitro cytotoxicity. J Appl Polym Sci. 2015;132(17):1–11. https://doi.org/10.1002/app.41851

Wang S, Sun C, Guan S, Li W, Xu J, Ge D, Zhuang M, Liu T, Ma X. Chitosan/gelatin porous scaffolds assembled with conductive poly(3,4-ethylenedioxythiophene) nanoparticles for neural tissue engineering. J Mater Chem B. 2017;5(24):4774–88. https://doi.org/10.1039/C7TB00608J

Nieto-Suárez M, López-Quintela MA, Lazzari M. Preparation and characterization of crosslinked chitosan/gelatin scaffolds by ice segregation induced self-assembly. Carbohydr Polym. 2016;141:175–83. http://dx.doi.org/10.1016/j.carbpol.2015.12.064

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):1190–209. https://doi.org/10.1080/09205063.2015.1082809

Maji K, Dasgupta S, Pramanik K, Bissoyi A. Preparation and characterization of gelatin-chitosan-nanoβ-TCP based scaffold for orthopaedic application. Mater Sci Eng C. 2018;86:83–94. https://doi.org/10.1016/j.msec.2018.02.001

Serra IR, Fradique R, Vallejo MCS, Correia TR, Miguel SP, Correia IJ. Production and characterization of chitosan / gelatin / β -TCP scaffolds for improved bone tissue regeneration. Mater Sci Eng C. 2015;55:592–604. https://doi.org/10.1016/j.msec.2015.05.072

Fukuda N, Tsuru K, Mori Y, Ishikawa K. Fabrication of self-setting β-tricalcium phosphate granular cement. J Biomed Mater Res - Part B Appl Biomater 2018;106:800–7. https://doi.org/10.1002/jbm.b.33891.

Putri TS, Hayashi K, Ishikawa K. Bone regeneration using β -tricalcium phosphate ( β -TCP ) block with interconnected pores made by setting reaction of β -TCP granules. J Biomed Mater Res - Part A 2020;108:625–32. https://doi.org/10.1002/jbm.a.36842.

Jerban S, Lapczyna H, Müller R, Galea L, Hofmann S, Doebelin N, et al. Effect of grain size and microporosity on the in vivo behaviour of β-tricalcium phosphate scaffolds. Eur Cells Mater 2016;28:299–319. https://doi.org/10.22203/ecm.v028a21.

Wei LJ, Shariff KA, Momin SA, Bakar MHA, Cahyanto A. Self-setting β-tricalcium phosphate granular cement at physiological body condition: effect of citric acid concentration as an inhibitor. J Aust Ceram Soc 2021;57:687–96. https://doi.org/10.1007/s41779-021-00575-4.

Jeong J, Kim JH, Shim JH, Hwang NS, Heo CY. Bioactive calcium phosphate materials and applications in bone regeneration. Biomater Res. 2019;23:4. https://doi.org/10.1186/s40824-018-0149-3

Bohner M, Santoni BLG, Döbelin N. β-tricalcium phosphate for bone substitution: Synthesis and properties. Acta Biomater. 2020;113:23–41. https://doi.org/10.1016/j.actbio.2020.06.022

Bellucci D, Sola A, Cannillo V. Hydroxyapatite and tricalcium phosphate composites with bioactive glass as second phase: State of the art and current applications. J Biomed Mater Res - Part A. 2016;104:1030–56. https://doi.org/10.1002/jbm.a.35619

Ishikawa K, Putri TS, Tsuchiya A, Tanaka K, Tsuru K. Fabrication of interconnected porous β-tricalcium phosphate (β-TCP) based on a setting reaction of β-TCP granules with HNO3 followed by heat treatment. J Biomed Mater Res - Part A. 2018;106(3):797–804. https://doi.org/10.1002/jbm.a.36285

Putri TS, Sunarso, Hayashi K, Tsuru K, Ishikawa K. Feasibility study on surface morphology regulation of β-tricalcium phosphate bone graft for enhancing cellular response. Ceram Int. 2022;48(9):13395–9. https://doi.org/10.1016/j.ceramint.2022.02.200

Shariff KA, Tsuru K, Ishikawa K. Fabrication of dicalcium phosphate dihydrate-coated β-TCP granules and evaluation of their osteoconductivity using experimental rats. Mater Sci Eng C 2017;75:1411–9. https://doi.org/10.1016/j.msec.2017.03.004.

Mohd Pu’ad NAS, Koshy P, Abdullah HZ, Idris MI, Lee TC. Syntheses of hydroxyapatite from natural sources. Heliyon. 2019;5(5):e01588. https://doi.org/10.1016/j.heliyon.2019.e01588

Putri TS, Ratnasari A, Sofiyaningsih N, Nizar MS, Yuliati A, Shariff KA. Mechanical improvement of chitosan–gelatin scaffolds reinforced by β-tricalcium phosphate bioceramic. Ceram Int. 2022;48(8):11428–34. https://doi.org/10.1016/j.ceramint.2021.12.367

Habibie S, Santosa Wargadipura AH, Gustiono D, Herdianto N, Riswoko A, Nikmatin S, et al. Production and Characterization of Hydroxyapatite Bone Substitute Material Performed from Indonesian Limestone. Int J Biomed Eng Sci 2017;4:11–23. https://doi.org/10.5121/ijbes.2017.4102.

Herdianto N, Rianti W, Ulfah IM, Kurniawati F, Gustiono D, Lukmana, et al. Effect of different calcination temperature on limestone-derived betha tricalcium phosphate prepared by wet chemical precipitation method. AIP Conf Proc 2024;3003:20078. https://doi.org/10.1063/5.0186093.

Putri TS, Rianti D, Rachmadi P, Yuliati A. Effect of glutaraldehyde on the characteristics of chitosan–gelatin–β-tricalcium phosphate composite scaffolds. Mater Lett. 2021;304:130672. https://doi.org/10.1016/j.matlet.2021.130672

Putri TS, Elsheikh M. Flexural Strength Evaluation of Chitosan-Gelatin-Β-Tricalcium Phosphate-Based Composite Scaffold. J Int Dent Med Res. 2022;15(1):31–4.

Sunarso, Toita R, Tsuru K, Ishikawa K. Immobilization of calcium and phosphate ions improves the osteoconductivity of titanium implants. Mater Sci Eng C 2016;68:291–8. https://doi.org/https://doi.org/10.1016/j.msec.2016.05.090.

Sirait M, Sinulingga K, Siregar N, Doloksaribu ME, Amelia. Characterization of hydroxyapatite by cytotoxicity test and bending test. J Phys Conf Ser. 2022;2193(1):12039. https://doi.org/10.1088/1742-6596/2193/1/012039

Tang Z, Li X, Tan Y, Fan H, Zhang X. The material and biological characteristics of osteoinductive calcium phosphate ceramics. Regen Biomater. 2018;5(1):43–59. https://doi.org/10.1093/rb/rbx024

Tang Z, Tan Y, Ni Y, Wang J, Zhu X, Fan Y, Chen X, Yang X, Zhang X. Comparison of ectopic bone formation process induced by four calcium phosphate ceramics in mice. Mater Sci Eng C. 2017;70:1000–10. https://doi.org/10.1016/j.msec.2016.06.097

Barba A, Maazouz Y, Diez-Escudero A, Rappe K, Espanol M, Montufar EB, Öhman-Mägi C, Persson C, Fontecha P, Manzanares MC, Franch J, Ginebra MP. Osteogenesis by foamed and 3D-printed nanostructured calcium phosphate scaffolds: Effect of pore architecture. Acta Biomater. 2018;79:135–47. https://doi.org/10.1016/j.actbio.2018.09.003

Hao Y, Yang N, Sun M, Yang S, Chen X. The role of calcium channels in osteoporosis and their therapeutic potential. Front Endocrinol (Lausanne) 2024;15:1–11. https://doi.org/10.3389/fendo.2024.1450328.

Pezeshki-Modaress M, Zandi M, Rajabi S. Tailoring the gelatin/chitosan electrospun scaffold for application in skin tissue engineering: an in vitro study. Prog Biomater. 2018;7(3):207–18. https://doi.org/10.1007/s40204-018-0094-1




DOI: https://doi.org/10.24198/pjd.vol38no1.63795

Refbacks

  • There are currently no refbacks.


 

Creative Commons License All publications by the Universitas Padjadjaran [e-ISSN: 2549-6212, p-ISSN: 1979-0201] are licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.This license requires that reusers give credit to the creator. It allows reusers to distribute, remix, adapt, and build upon the material in any medium or format, for noncommercial purposes only.

Visitor Stat