Cytotoxic and anti-inflammatory effects of chitosan and hemostatic gelatin in oral cell culture / Efectos citotóxicos y antiinflamatorios del quitosano y la gelatina hemostática en cultivo celular oral

ABSTRACT

ABSTRACT Chitosan is a biopolymer with bactericidal/bacteriostatic effect, biocompatible and biodegradable. It has been used in tissue engineering to replace tissues partially or completely by releasing bioactive materials or influencing cell growth, usually in regenerative medicine and dentistry. The aim of this study was to evaluate the cytotoxic and anti-inflammatory effect of chitosan alone or with hemostatic gelatin (Spongostand®) in cultures of human pulp cells (HPC), human gingival fibroblasts (HGF) and mouse pre-osteoblasts (MC3T3-E1, ATCC). HPC and HGF were isolated from patients. Cells were subcultured in DMEM. Chitosan was inoculated at different concentrations (0-0.5%) and hemostatic gelatins impregnated with chitosan (0.19%) were placed directly in the presence of cells and incubated for 24 hours. Cell viability was determined by MTT method and mean cytotoxic concentration (CC50) was calculated from the dose-response curve. Anti-inflammatory effect was calculated from the in vitro gingivitis model induced with interleukin 1beta (IL-1β) in HGF and protein detection. The data were subjected to Shapiro-Wilk, Kruskal-Wallis and Mann-Whitney tests. Experiments were performed in triplicate of three independent assays. Cell viability of HPC, HGF and MC3T3-E1 in contact with chitosan decreased significantly (p<0.05). The HPC were the most sensitive (CC50= 0.18%), followed by HGF (CC50= 0.18%) and MC3T3-E1 (CC50= 0.19%). The cytotoxicity of gelatins impregnated with chitosan decreased cell viability of HGF and HPC by 11% and 5%, respectively. The proinflammatory effect was reduced significantly in the gingivitis model. To conclude, chitosan induces moderate cytotoxic effects alone or with hemostatic gelatin at 0.19%, in dose-dependent manner, with anti-inflammatory effects on human gingival fibroblasts. The use of chitosan as a biomaterial can be an excellent choice for use in regenerative dentistry.

RESUMEN

RESUMEN El quitosano es un biopolímero con efecto bactericida/bacteriostático, biocompatible y biodegradable. Se ha utilizado en ingeniería de tejidos con el fin de reemplazar parcial o completamente los tejidos como material bioactivo o influyendo en el crecimiento celular, comúnmente, para medicina y odontología regenerativa. Evaluar el efecto citotóxico y antiinflamatorio del quitosano solo o con gelatina hemostática (Spongostand®) en cultivos con células pulpares humanas (HPC), fibroblastos gingivales humanos (HGF) y preosteoblastos de ratón (MC3T3-E1, ATCC). HPC, HGF se aislaron de pacientes. Las células se subcultivaron en DMEM. Se inoculó quitosano a diferentes concentraciones (0-0,5%) y se colocaron gelatinas hemostáticas impregnadas con quitosano (0,19%) directamente en presencia de células y se incubaron durante 24 horas. La viabilidad celular se determinó mediante el método MTT y se calculó la concentración citotóxica media (CC50) a partir de la curva dosis-respuesta. El efecto antiinflamatorio se calculó a partir del modelo de gingivitis in vitro inducido con interleucina 1β (IL-1β) en HGF. Los datos se sometieron a las pruebas de Shapiro-Wilk, Kruskal-Wallis y Mann-Whitney. Los experimentos se realizaron por triplicado de tres ensayos independientes. La viabilidad celular de HPC, HGF y MC3T3-E1 en contacto con el quitosano disminuyó significativamente la viabilidad celular (p <0.05). Las HPC fueron las más sensibles (CC50= 0,18%) seguido de HGF (CC50= 0,18%) y MC3T3-E1 (CC50= 0,19%). Las gelatinas impregnadas con quitosano mostraron una disminución en la viabilidad celular para HGF, HPC de 11% y 5% respectivamente y se redujo significativamente el efecto pro-inflamatorio en el modelo de gingivitis humano. El quitosano induce efectos citotóxicos moderados solo o con gelatina hemostática a 0,19% de forma dosis-dependiente con efectos antiinflamatorios en fibroblastos gingivales humanos. El uso de quitosano como biomaterial puede ser una excelente opción para su uso en odontología regenerativa.