To date, chitosan-based hemostatic agents have gained increasing interest from their biocompatibility, inexpensiveness and hemostatic capability, especially in coagulopathic conditions. We have developed a functionally improved Calcium Alginate/N,O-carboxymethylchitosan (CA/NOCC) hemostatic sponge, shown to be biocompatible and biodegradable.
Objectives: To ensure its safe use with the gingival tissue, this study aimed to evaluate the cytotoxicity of CA/NOCC sponge on primary human gingival fibroblasts (GFs).
Methods: Human GFs were cultured with or without the CA/NOCC sponge. Cell morphology was assessed by scanning electron microscopy. Cell viability and proliferation were determined by MTT assays. The levels of Ca2+ released into the culture medium were also measured.
Results: Gingival fibroblasts cultured with the CA/NOCC sponge demonstrated lowered cell density, and significant ultrastructural changes of the cell membrane, by forming numerous blebs and fibrils. From MTT assays, approximately 30% decrease in the proliferation rate was observed. Moreover, the levels of Ca2+, up to 4.6 mM, were detected in the medium of GFs cultured with the CA/NOCC sponge.
Conclusions: It could be implicated that the cytopathic effects on the morphology and proliferative ability of GFs may result from the high level of Ca2+ released from the CA/NOCC hemostatic sponge.
1. Kamoh A, Swantek J. Hemostasis in oral surgery. Dent Clin North Am. 2012;56(1):17-23.
2. Kumar S. Local hemostatic agents in the management of bleeding in oral surgery. Asian J Pharm Clin Res. 2016;9(3):35-41.
3. Brodbelt AR, Miles JB, Foy PM, Broome JC. Intraspinal oxidised cellulose (Surgicel) causing delayed paraplegia after thoracotomy--a report of three cases. Ann R Coll Surg Engl. 2002;84(2):97-9.
4. Cicciù M, Fiorillo L, Cervino G. Chitosan use in Dentistry: A systematic review of recent clinical studies. Mar Drugs. 2019;17(7):417.
5. Khan MA, Mujahid M. A review on recent advances in chitosan based composite for hemostatic dressings. Int J Biol Macromol. 2019;124:138-47.
6. Chen KY, Chen YC, Lin TH, Yang CY, Kuo YW, Lei U. Hemostatic enhancement via chitosan is independent of classical clotting pathways—A quantitative study. Polymers. 2020;12(10):2391.
7. Koksal O, Ozdemir F, Cam Etoz B, Buyukcoskun NI, Sigirli D. Hemostatic effect of a chitosan linear polymer (Celox®) in a severe femoral artery bleeding rat model under hypothermia or warfarin therapy. Ulus Travma Acil Cerrahi Derg. 2011;17(3):199-204.
8. Millner RW, Lockhart AS, Marr R, Jones K. Omni-Stat®(Chitosan) arrests bleeding in heparinised subjects in vivo: an experimental study in a model of major peripheral vascular injury. Eur J Cardiothorac Surg. 2011;39(6):952-4.
9. Kumar KR, Kumar J, Sarvagna J, Gadde P, Chikkaboriah S. Hemostasis and post-operative care of oral surgical wounds by hemcon dental dressing in patients on oral anticoagulant therapy: A split mouth randomized controlled clinical trial. J Clin Diagn Res. 2016;10(9):Zc37-40.
10. Sharma S, Kale TP, Balihallimath LJ, Motimath A. Evaluating effectiveness of axiostat hemostatic material in achieving hemostasis and healing of extraction wounds in patients on oral antiplatelet drugs. J Contemp Dent Pract. 2017;18(9):802-6.
11. Pippi R, Santoro M, Cafolla A. The use of a chitosan-derived hemostatic agent for postextraction bleeding control in patients on antiplatelet treatment. J Oral Maxillofac Surg. 2017;75(6):1118-23.
12. Shariatinia Z. Carboxymethyl chitosan: Properties and biomedical applications. Int J of Biol Macromol. 2018;120:1406-19.
13. Janvikul W, Thavornyutikarn B. New route to the preparation of carboxymethylchitosan hydrogels. J Appl Polym Sci. 2003;90:4016-20.
14. Janvikul W, Uppanan P, Thavornyutikarn B, Krewraing J, Prateepasen R. In vitro comparative hemostatic studies of chitin, chitosan, and their derivatives. J Appl Polym Sci. 2006;102(1):445-51.
15. Liu L, Lv Q, Zhang Q, Zhu H, Liu W, Deng G, et al. Preparation of carboxymethyl chitosan microspheres and their application in hemostasis. Disaster Med Public Health Prep. 2017;11(6):660-7.
16. Zhong QK, Wu ZY, Qin YQ, Hu Z, Li SD, Yang ZM, et al. Preparation and properties of carboxymethyl chitosan/alginate/tranexamic acid composite films. Membranes. 2019;9(1):11.
17. Janvikul W, Uppanan P, Thavornyutikarn B. Calcium alginate, N,O-carboxymethylchitosan and their blends: In vitro biocompatibility and biodegradability. Proceedings of The World Congress on Bioengineering 2007 (WACBE); 2007, July 9-11; Bangkok, Thailand.
18. Janvikul W, Uppanan P, Kosorn W, Phusuksombati D, Prateepasen R. Evaluation of efficacy of chitosan derivative based hemostat: In vitro and in vivo studies. Proceedings of The 2nd International Symposium on Biomedical Engineering (ISBME 2006); 2006, November 8-10; Bangkok, Thailand.
19. Thavornyutikarn B, Kosorn W, Surattanawanich P, Wongparami U, Janvikul W. Chitosan derivative-based hemostat: Irritation, hypersensitivity and absorbability study. J Met Mater Miner. 2013;23:49-52.
20. Janvikul W, Thavornyutikarn B, Kosorn W, Surattanawanich P. Clinical study of chitosan-derivative-based hemostat in the treatment of split-thickness donor sites. Maejo Int J Sci Technol. 2013;7:385-95.
21. Phaewphala K, Kosorn W, Janvikul W, Kitrueangphatchara K, Sricholpech M. Biocompatibility of a chitosan-derived hemostatic agent with human alveolar osteoblasts. M Dent J. 2020;40(3):277-88.
22. Taskin AK, Yasar M, Ozaydin I, Kaya B, Bat O, Ankarali S, et al. The hemostatic effect of calcium alginate in experimental splenic injury model. Ulus Travma Acil Cerrahi Derg. 2013;19(3):195-9.
23. Szekalska M, Puciłowska A, Szymańska E, Ciosek P, Winnicka K. Alginate: current use and future perspectives in pharmaceutical and biomedical applications. Int J Polym Sci. 2016; Article ID 7697031. doi:10.1155/2016/7697031
24. Li Z, Ramay HR, Hauch KD, Xiao D, Zhang M. Chitosan-alginate hybrid scaffolds for bone tissue engineering. Biomaterials. 2005;26(18):3919-28.
25. Barros LF, Kanaseki T, Sabirov R, Morishima S, Castro J, Bittner CX, et al. Apoptotic and necrotic blebs in epithelial cells display similar neck diameters but different kinase dependency. Cell Death Differ. 2003;10(6):687-97.
26. Mills JC, Stone NL, Erhardt J, Pittman RN. Apoptotic membrane blebbing is regulated by myosin light chain phosphorylation. J Cell Biol. 1998;140(3):627-36.
27. Messer RL, Bishop S, Lucas LC. Effects of metallic ion toxicity on human gingival fibroblasts morphology. Biomaterials. 1999;20(18):1647-57.
28. Li X, Kong X, Zhang Z, Nan K, Li LL, Wang XH, et al. Cytotoxicity and biocompatibility evaluation of N,O-carboxymethyl chitosan/oxidized alginate hydrogel for drug delivery application. Int J Biol Macromol. 2012;50(5):1299-305.
29. Rasad MSBA, Halim AS, Hashim K, Rashid AHA, Yusof N, Shamsuddin S. In vitro evaluation of novel chitosan derivatives sheet and paste cytocompatibility on human dermal fibroblasts. Carbohydr Polym. 2010;79(4):1094-100.
30. Anitha A, Rani VD, Krishna R, Sreeja V, Selvamurugan N, Nair SV, et al. Synthesis, characterization, cytotoxicity and antibacterial studies of chitosan, O-carboxymethyl and N,O-carboxymethyl chitosan nanoparticles. Carbohydr Polym. 2009;78(4):672-7.
31. Rosdy M, Clauss LC. Cytotoxicity testing of wound dressings using normal human keratinocytes in culture. J Biomed Mater Res. 1990;24(3):363-77.
32. Suzuki Y, Nishimura Y, Tanihara M, Suzuki K, Kitahara AK, Yamawaki Y, et al. Development of alginate gel dressing. J Artif Organs. 1998;1(1):28-32.
33. Suzuki Y, Nishimura Y, Tanihara M, Suzuki K, Nakamura T, Shimizu Y, et al. Evaluation of a novel alginate gel dressing: cytotoxicity to fibroblasts in vitro and foreign-body reaction in pig skin in vivo. J Biomed Mater Res. 1998;39(2):317-22.
34. Paddle-Ledinek JE, Nasa Z, Cleland HJ. Effect of different wound dressings on cell viability and proliferation. Plast Reconstr Surg. 2006;117(7 Suppl):110S-8S; discussion 9S-20S.
35. Grasdalen H, Larsen B, Smisrod O. 13C-NMR studies of monomeric composition and sequence in alginate. Carbohydr Res. 1981;89(2):179-91.
36. Whitfield JF, Bird RP, Chakravarthy BR, Isaacs RJ, Morley P. Calcium-cell cycle regulator, differentiator, killer, chemopreventor, and maybe, tumor promoter. J Cell Biochem Suppl. 1995;22:74-91.
37. Kulesz-Martin MF, Fabian D, Bertram JS. Differential calcium requirements for growth of mouse skin epithelial and fibroblast cells. Cell Tissue Kinet. 1984;17(5):525-33.
38. Sank A, Chi M, Shima T, Reich R, Martin GR. Increased calcium levels alter cellular and molecular events in wound healing. Surgery. 1989;106(6):1141-7; discussion 7-8.
39. Orrenius S, McCabe MJ, Jr., Nicotera P. Ca(2+)-dependent mechanisms of cytotoxicity and programmed cell death. Toxicol Lett. 1992;64-65 Spec No:357-64.
40. Maeno S, Niki Y, Matsumoto H, Morioka H, Yatabe T, Funayama A, et al. The effect of calcium ion concentration on osteoblast viability, proliferation and differentiation in monolayer and 3D culture. Biomaterials. 2005;26(23):4847-55.
Mahatchariyapong T, Phaewpala K, Kosorn W, Kitrueangphatchara K, Sricholpech M. Cytotoxicity of the Calcium Alginate/N,O-carboxymethylchitosan Hemostatic Sponge on Primary Human Gingival Fibroblasts: Original articles. CM Dent J [Internet]. 2022 Jan 27 [cited 2024 Nov 18];43(1):19-28. Available from: https://www.dent.cmu.ac.th/cmdj/frontend/web/?r=site/viewarticle&id=158
Mahatchariyapong, T., Phaewpala, K., Kosorn, W., Kitrueangphatchara, K. & Sricholpech, M. (2022). Cytotoxicity of the Calcium Alginate/N,O-carboxymethylchitosan Hemostatic Sponge on Primary Human Gingival Fibroblasts. CM Dent J, 43(1), 19-28. Retrieved from: https://www.dent.cmu.ac.th/cmdj/frontend/web/?r=site/viewarticle&id=158
Mahatchariyapong, T., Phaewpala Kaltimas,Kosorn Wasana,Kitrueangphatchara Kitwat and Sricholpech Marnisa. 2022. "Cytotoxicity of the Calcium Alginate/N,O-carboxymethylchitosan Hemostatic Sponge on Primary Human Gingival Fibroblasts." CM Dent J, 43(1), 19-28. https://www.dent.cmu.ac.th/cmdj/frontend/web/?r=site/viewarticle&id=158
Mahatchariyapong, T. et al. 2022. 'Cytotoxicity of the Calcium Alginate/N,O-carboxymethylchitosan Hemostatic Sponge on Primary Human Gingival Fibroblasts', CM Dent J, 43(1), 19-28. Retrieved from https://www.dent.cmu.ac.th/cmdj/frontend/web/?r=site/viewarticle&id=158
Mahatchariyapong, T., Phaewpala, K., Kosorn, W., Kitrueangphatchara, K. and Sricholpech, M. "Cytotoxicity of the Calcium Alginate/N,O-carboxymethylchitosan Hemostatic Sponge on Primary Human Gingival Fibroblasts", CM Dent J, vol.43, no. 1, pp. 19-28, Jan. 2022.
Mahatchariyapong, T., Phaewpala, K., Kosorn, W., et al. "Cytotoxicity of the Calcium Alginate/N,O-carboxymethylchitosan Hemostatic Sponge on Primary Human Gingival Fibroblasts." CM Dent J, vol.43, no. 1, Jan. 2022, pp. 19-28, https://www.dent.cmu.ac.th/cmdj/frontend/web/?r=site/viewarticle&id=158
