Cytotoxic and genotoxic effects of contemporary esthetic restorative materials on pulpal cells and mucosal cells of oral cavity

Kompozitni dentalni materijali nakon svoje primjene dolaze u izravan dodir s oralnim tkivima. Zbog ovog bliskog i dugotrajnog kontakta, kompozitne materijale bi trebao karakterizirati visok stupanj biokompatibilnosti. Svrha ovog doktorskog rada bila je utvrditi koliko su kompoziti biokompatibilni i sigurni za kliničko korištenje kroz procjenu njihovog citotoksičnog i/ili genotoksičnog djelovanja. Citotoksično i genotoksično djelovanje istraživalo se na stanicama humane kulture gingive i pulpe, ex vivo, ovisno o koncentraciji korištenog materijala primjenom diferencijalnog bojanja akridin-narančastom bojom i etidijevim bromidom te primjenom komet testa. Isti materijali ispitivani su i u in vivo uvjetima. Pacijentima su izrađene kompozitne restauracije klase V te se pratio genotoksični učinak materijala na epitelnim stanicama gingive uz rub postavljenog ispuna. Uzorci stanica za ispitivanje uzimani su 0, 7, 30 i 180 dana nakon postavljanja restauracije. Oštećenje DNK analizirano je dvjema metodama, mikronukleus i komet testom. Kompozitni materijali u testiranim koncentracijama pokazali su genotoksični učinak u uvjetima ex vivo za oba ispitivana parametra komet testa, dužina i intenzitet repa. Kompozitni materijal Kalore pokazao je veću genotoksičnost u odnosu na Vertise flow. Rezultati in vivo dijela istraživanja pokazali su značajno više parametre komet testa unutar razdoblja od 30 i 180 dana. Mikronukleus test za isto vrijeme ekspozicije pokazao je znatno veći broj stanica s mikronukleusom, kariolizom i nuklearnim pupovima. Među testiranim materijalima nije uočena razlika u djelovanju. Na temelju rezultata, možemo zaključiti da upotreba kompozita uzrokuje DNK oštećenje u stanicama pulpe i gingive, međutim uzimajući u obzir visoku učinkovitost mehanizama popravka DNK, rezultate ne bi trebalo smatrati apsolutnim pokazateljima genotoksičnosti.Objectives: The most important requirement for the materials to be used in medical applications is its biocompatibility. Dental composite materials come into close contact with oral tissues, especially with gingival cells. Due to this close and long-term contact, the materials should exhibit a high degree of biocompatibility. The aim of this doctoral thesis was to certify that the modern composite materials are biocompatible and safe for clinical use and also, was to evaluate their cytotoxic and/or genotoxic effect on human fibroblasts ex vivo and gingival cells in vivo. Materials and Methods: Ex vivo cytotoxicity and genotoxicity of two contemporary commercial composite materials (Kalore, Vertise flow) on human gingival and pulpal fibroblasts depending on the concentration of the used material (20 mg/ml, 40 mg/ml), were tested using the ethidium bromide/acridin orange viability staining and an alkaline single-cell gel electrophoresis technique (comet assay). Genotoxicity was also assessed in gingival epithelial cells in vivo. Class V composite restorations were placed in 30 adult patients using two different composite resins. Cell samples were collected by gentle brushing gingival area along the composite restoration prior to, 7 days, 30 days, and 180 days following the restoration of the tooth. DNA damage was analysed for each cell sample by comet and micronucleus assay. Results: Ex vivo tested dental composites revealed statistically significant increase in the tail length and tail intensity in treated human gingival and pulpal fibroblasts, independent of the applied concentration. Cultures treated with nano-hybrid composite material Kalore (20 mg/ml) have been showed more genotoxic than those treated with self-adhesive flowable composite Vertise flow. In vivo results showed significantly higher comet assay parameters (tail length and % DNA in the tail) within 30, and 180 days of treatment. While the micronucleus tests for the same exposure time showed a higher number of cells with micronuclei, karyolysis and nuclearbuds. Results did not reveal the difference between the two composite materials for the same exposure time. Conclusion: We can conclude based on the results that the use of composite resins causes cellular damage. However, taking into consideration the high efficiency of DNA repair mechanisms, the results should not be considered as an absolute indicator of genotoxicity potential. As dental composite resins remain in close contact with oral tissue over a long period of time, further research on their possible genotoxicity are desirable