OJST  Vol.3 No.1 , March 2013
Insights into chitosan hydrogels on dentine bond strength and cytotoxicity
Abstract: Contemporary dental adhesives show favorable immediate results in terms of bonding effectiveness. However, the durability of resin-dentin bonds is their major problem. Materials and Methods: Preparation of 3 chitosan-antioxidant hydrogels was achieved using modified hydrogel preparation method. Their effect on the bond strength to dentine both short term (after 24 hours) and long term (after 6 months) were evaluated using shear bond strength measurements using Instron Universal Testing Mascine). The SEM was used to study the surface of the hydrogels. The cell survival rate (cytotoxicity) of the antioxidants resveratrol, β-carotene and propolis towards Balb/c 3T3 mouse fibroblast cells was also assessed using the standard MTT assay. Results: It was found that chitosan-H treated dentine gives significantly (p < 0.05; Non-parametric ANOVA test) higher shear bond values than dentine treated or not treated with phosphoric acid. The anti-oxidants chitosan hydrogels improved the shear bond strength. Overall, there was a relapse in the shear bond strength after 6 months. The SEM study showed that the hydrogel formulations have a uniform distribution of drug content, homogenous texture and yellow color. The pH of the growth medium adjusted to relevant values had a highly significant influence (Tukey-Kramer Multiple-Comparison Test; p < 0.01) on the cell survival rate of Balb/c mouse 3T3 fibroblast cells and therefore most probably also to tooth pulp fibroblast cells. The lower the pH value the higher the negative influence. Furthermore, the sequence of survival rate was found to be: β-carotene (92%) > propolis (68%) > resveratrol (33%). Conclusion: the antioxidant-chitosan hydrogels significantly improved bonding to dentine with or without phosphoric acid treatment. The pH of the growth medium had a high influence on the cell survival rate of Balb/c mouse 3T3 fibroblast cells. The release of the antioxidant β-carotene would not have an influence on the pulp cells. These materials might address the current perspectives for improving bond durability.
Cite this paper: Perchyonok, V. , Grobler, S. , Zhang, S. , Olivier, A. and Oberholzer, T. (2013) Insights into chitosan hydrogels on dentine bond strength and cytotoxicity. Open Journal of Stomatology, 3, 75-82. doi: 10.4236/ojst.2013.31014.

[1]   Schmalz, G. (1997) Concepts in biocompatibility testing of dental restorative materials. Clinical Oral Investigations, 1, 154-162. doi:10.1007/s007840050027

[2]   Peumans, M., Kanumilli, P., De-Munck, J., Van Landuyt, K., Lambrechts, P. and Van Meerbeek, B. (2005) Clinical effectiveness of contemporary adhesives: A systematic review of current clinical trials. Dental Materials, 21, 864-881. doi:10.1016/

[3]   Murray, P.E., Windsor, L.J., Hafez, A.A., Stevenson, R.G. and Cox, C.F. (2003) Comparison of pulp responses to resin composites. Operative Dentistry, 28, 242-250.

[4]   Schweikl, H., Spagnuolo, G. and Schmalz, G. (2006) Genetic and cellular toxicology of dental resin monomers. Journal of Dental Research, 85, 870-877. doi:10.1177/154405910608501001

[5]   Chen, R.S., Liu, C.C., Tseng, W.Y., Jeng, J.H. and Lin, C.P. (2003) Cyto toxicity of three dentin bonding agents on human dental pulp cells. Journal of Dentistry, 31, 223-229. doi:10.1016/S0300-5712(02)00088-X

[6]   Chen, R.S., Liuiw, C.C., Tseng, W.Y., Hong, C.Y., Hsieh, C.C. and Jeng, J.H. (2001) The effect of curing light intensity on the cytotoxicity of a dentin-bonding agent. Operative Dentistry, 26, 505-510.

[7]   De Souza Costa, C.A., Vaerten, M.A., Edwards, C.A. and Hanks, C.T. (1999) Cytotoxic effects of current dental adhesive systems on immortalized odontoblast cell line MDPC-23. Dental Materials, 15, 434-441. doi:10.1016/S0109-5641(99)00071-8

[8]   Vajrabhaya, L.O., Pasasuk, A. and Harnirattisai, C. (2003) Cytotoxicity evaluation of single component dentin bonding agents. Operative Dentistry, 28, 440-444.

[9]   Perchyonok, V.T., Zhang, S. and Oberholzer, T. (2012) Alternative chitosan based drug delivery system to fight oral mucositis: Synergy of conventional and bioactives towards the optimal solution. Current Nanoscience, 8, 541-547. doi:10.2174/157341312801784320

[10]   Mosmann, T. (1983) Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays. Journal of Immunological Methods, 65, 55-63. doi:10.1016/0022-1759(83)90303-4

[11]   Titley, K.C., Smith, D.C., Chernecky, R., Maric, B. and Chan, A. (1995) An SEM examination of etched dentin and the structure of the hybrid layer. Journal of Canadian Dental Association, 61, 887-894.

[12]   Dabas, D., Patil, A.C. and Uppin, V.M. (2011) Evaluation of the effect of concentration and duration of application of sodium ascorbate hydrogel on the bond strength of composite resin to bleached enamel. Journal of Conservative Dentistry, 14, 356-360. doi:10.4103/0972-0707.87197

[13]   Ravi Kumar, M.N.V. (2001) A review of chitin and chitosan applications. Reactive and Functional Polymers, 46, 1-27. doi:10.1016/S1381-5148(00)00038-9

[14]   Perchyonok, V.T., Zhang, S. and Oberholzer, T. (2012) Towards development of novel chitosan based drug delivery prototypes devices for targeted delivery drug therapy at the molecular level in aqueous media. Current Organic Chemistry, 16, 2437-2439.

[15]   Petri, D.F., Donegá, J., Benassi, A.M. and Bocangel, J.A. Preliminary study on chitosan modified glass ionomer restoratives. Dental Materials, 23, 1004-1010. doi:10.1016/

[16]   Grobler, S.R., Olivier, A., Moodley, D. and van Wyk Kotze, T.J. (2008) Cytotoxicity of recent bonding agents on mouse fibroblast cells. Quintessence International, 39, 511-516.

[17]   Grobler, S.R., Olivier, A., Moodley, D. and van Wyk Kotze, T.J. (2004) Cytotoxicity of two concentrations of a dentine bonding agent on mouse 3T3 and human pulp fibroblast cell-lines. Journal of the South African Dental Association, 59, 368-372.

[18]   Grobler, S.R., Oberholzer, T.G., Rossouw, R.J. and van Wyk Kotze, T.J. (2007) Microleakage and confocal laser studies of two single step self-etching bonding agents/ systems. Quintessence International, 38, 525.

[19]   Coe, F.G., Parikh, D.M., Johnson, C.A. and Anderson, G.J. (2012) The good and the bad: Alkaloid screening and brine-shrimp bioassays of aqueous extracts of 31 medicinal plants of eastern Nicaragua. Pharmaceutical Biology, 50, 384-392. doi:10.3109/13880209.2011.608077

[20]   Ruffa, M.J., Ferraro, G., Wagner, M.L, Calcagno, R.H. and Campo, L. (2002) Cavallaros cytotoxic effect of Argentine medicinal plant extracts on human hepatocellular carcinoma cell line. Journal of Ethnopharmacology, 79, 335-339. doi:10.1016/S0378-8741(01)00400-7

[21]   Chen, C., Weng, M., Wu, C. and Lin, J. (2004) Comparison of radical scavenging activity, cytotoxic effects and apoptosis induction in human melanoma cells by Taiwanese propolis from different sources. Evidence-Based Complementary and Alternative Medicine, 1, 175-185. doi:10.1093/ecam/neh034

[22]   Alija, A.J., Bresgen, N., Sommerburg, O., Siems, W. and Eckl, P.M. (2004) Cytotoxic and genotoxic effects of β-carotene breakdown products on primary rat hepatocytes. Oxford Journals, 25, 827-831. doi:10.1093/carcin/bgh056

[23]   Schwartz, J. and Shklar, G. (1992) The selective cytotoxic effect of carotenoids and α-tocopherol on human cancer cell lines in vitro. Journal of Oral and Maxillofacial Surgery, 50, 367-373. doi:10.1016/0278-2391(92)90400-T

[24]   Freshney, I. (2007) Culture of animal cells. John Wiley and Sons, New Jersey.

[25]   Gao, X., Xu, Y.X., Janakiraman, N., Chapman, R.A. and Gautam, S.C. (2001) Immunomodulatory activity of resveratrol: Suppression of lymphocyte proliferation, development of cell-mediated cytotoxicity, and cytokine production. Biochemical Pharmacology, 62, 1299-1308. doi:10.1016/S0006-2952(01)00775-4

[26]   Clément, M., Hirpara, J.L., Chawdhury, S. and Pervaiz, S. (1998) Chemopreventive agent resveratrol, a natural product derived from grapes, triggers CD95 signalling-dependent apoptosis in human tumor cells. Blood, 92, 996-1002.

[27]   Scneider, Y., Vincent, F., Duranton, B., Badolo, L., Gossé, F., Bergmann, C., Seiler, N. and Raaul, F. (2002) Anti-proliferative effect of resveratrol, a natural component of grapes and wine, on human colonic cancer cells. Cancer Letters, 158, 85-91. doi:10.1016/S0304-3835(00)00511-5

[28]   Nakagawa, H., Uemura, Y., Shikata, H., Hioki, K. and Tsubura, A. (2001) Resveratrol inhibits human breast cancer cell growth and may mitigate the effect of linoleic acid, a potent breast cancer cell stimulator. Journal of Cancer Research and Clinical Oncology, 126, 258-264. doi:10.1007/s004320000190

[29]   Iqbal, M.A. and Bamezai, R.N.K. (2012) Resveratrol inhibits cancer cell metabolism by down regulating pyruvate kinase M2 via inhibition of mammalian target of rapamycin. PLoS ONE, 7, e36764. doi:10.1371/journal.pone.0036764

[30]   Yoshino, J., Conte, C., Fontana, L., Mittendorfer, B., Imai, S., Schechtman, K.B., Charles, G., Kunz, I., Fanelli, F.R., Patterson, B.W. and Samuel, K.S. (2012) Resveratrol supplementation does not improve metabolic function in nonobese women with normal glucose tolerance. Cell Metabolism, 16, 658-664.

[31]   Banskota, A.H., Tezuka, Y., Prasain, J.K., Matsushige, K., Saiki, I. and Kadota, S. (1998) Chemical constituents of Brazilian propolis and their cytotoxic activities. Journal of Natural Products, 61, 896-900.

[32]   Giamalia, I., Steinberg, D., Grobler, S. and Gedalia, I. (1999) The effect of propolis exposure on microhardness of human enamel in vitro. Journal of Oral Rehabilitation, 26, 472-475. doi:10.1046/j.1365-2842.1999.00472.x

[33]   Medline Plus, “Propolis,” (2012). http//