Influência do substrato dentinário humano e bovino sobre a citotoxicidade de materiais resinosos utilizados para cimentação de peças protéticas
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Data
2021-07-08
Autores
Souza, Isabela dos Reis
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Universidade Estadual Paulista (Unesp)
Resumo
Devido às questões éticas e à dificuldade de se obter dentes humanos íntegros, pesquisadores têm usado dentes bovinos para avaliar as propriedades físicas e mecânicas de diferentes materiais dentários. Todavia, ainda não está comprovado se este modelo de dente animal também pode ser usado, com segurança, para determinar in vitro, as propriedades biológicas de materiais e produtos recomendados para aplicação sobre o substrato dentinário. Portanto, o objetivo deste estudo foi avaliar e comparar a citotoxicidade transdentinária de diferentes materiais resinosos usados para cimentação de peças protéticas, bem como determinar, por meio de um modelo laboratorial que mimetiza situações clínicas, se a dentina bovina (DB) tem potencial para substituir a dentina humana (DH) em testes biológicos de toxicidade indireta. Para tanto, discos padronizados de DB e DH (0,4 mm de espessura) foram adaptados em dispositivos que simulam câmaras pulpares artificiais (CPAs), as quais foram colocadas, individualmente, em placas de 24 compartimentos. Em seguida, células odontoblastóides MDPC-23 foram semeadas na superfície pulpar dos discos. Após 48 horas de incubação, os cimentos resinosos foram aplicados na superfície oclusal dos discos, o que possibilitou estabelecer os seguintes grupos: G1- DH sem tratamento (controle negativo); G2- DB sem tratamento (controle negativo); G3- Single Bond Universal sobre DH (controle positivo); G4- Single Bond Universal sobre DB (controle positivo); G5- RelyX Luting 2 sobre DH; G6- RelyX Luting 2 sobre DB; G7- RelyX U200 sobre DH; G8- RelyX U200 sobre DB; G9- RelyX Ultimate sobre DH; e G10- RelyX Ultimate sobre DB. Células MDPC-23, bem como células da polpa dentária humana (HDPCs), também foram semeadas em compartimentos de placas esterilizadas de 96 compartimentos. Após 24 horas de incubação, os efeitos tóxicos foram determinados nas células MDPC-23 semeadas sobre os discos por meio da análise de viabilidade (AlamarBlue) e morfologia celular (MEV). Os extratos (meio de cultura + componentes dos materiais que se difundiram pelos discos) foram coletados e aplicados sobre as MDPC-23 e HDPCs semeadas nos compartimentos. Essas células foram avaliadas quanto a viabilidade (Live/Dead), adesão e espalhamento celular (F-actina), atividade de fosfatase alcalina (ALP, Timolftaleína Monofosfato) e deposição de nódulos de mineralização (Alizarin Red). Os dados numéricos obtidos foram submetidos aos testes estatísticos ANOVA a um critério, complementados por Tukey (nível de significância de 5%). A maior redução na viabilidade das células MDPC-23 cultivadas sobre os discos de dentina ocorreu quando o sistema adesivo Single Bond Universal (G3 e G4) e o cimento RelyX Luting 2 (G5 e G6) foram usados em comparação aos respectivos controles negativos (G1 e G2, p<0,05). Não se observou diferença entre os materiais quando eles foram aplicados sobre DH ou DB (p>0,05). Redução significativa da viabilidade, adesão e atividade de ALP pelas células MDPC-23 cultivadas nos compartimentos, ocorreu nos grupos G3, G4, G5, G6, G9, G10, quando comparados a G1 e G2 (p<0,05). Porém, G7 e G8 foram estatisticamente semelhantes aos controles G1 e G2 (p>0,05). Redução significativa da viabilidade, adesão e atividade de ALP pelas HDPCs ocorreu nos grupos G5 e G6 quando comparados a G1 e G2 (p<0,05). De maneira geral, foi observado que as células MDPC-23 são mais sensíveis aos extratos dos cimentos resinosos do que as HDPCs. Não houve diferença significativa de citotoxicidade quando os mesmos materiais resinosos foram aplicados na superfície oclusal dos discos de DH e DB (p>0,05). De acordo com a metodologia usada e baseado nos resultados obtidos nesse estudo laboratorial, foi possível concluir que o cimento RelyX U200 foi o único material resinoso não citotóxico testado, sendo que para este modelo de teste in vitro de citotoxicidade indireta, dentina bovina pode ser usada para substituir dentina humana.
Based upon ethical questions and because of the difficulty of getting intact human teeth, researchers have employed bovine teeth to assess some physic and mechanic properties of diverse dental materials. However, lack of scientific data about using such animal teeth model for biological tests of products recommended to be applied on different teeth substrates still remains. Therefore, the aim of this study was to evaluate and compare the transdentinal cytotoxicity of different resin luting cements as well as to use an in vitro model that mimic clinical situations to determine whether bovine dentin (BD) has potential to replace human dentin (HD) for biological tests of indirect toxicity. For this purpose, standardized HD and BD discs (0.4 mm thick) were adapted in artificial pulp chamber devices (APCs), which were individually placed in wells of 24- well plates. Then, odontoblast-like MDPC-23 cells were seeded on the pulpal surface of the dentin discs. After 48- hour incubation, the luting cements were applied on the occlusal surface of the discs, giving rise to the following groups: G1- No treatment on HD (negative control); G2- No treatment on BD (negative control); G3- Single Bond Universal on HD (positive control); G4- Single Bond Universal on BD (positive control); G5- RelyX Luting 2 on HD; G6- RelyX Luting 2 on BD; G7- RelyX U200 on HD; G8- RelyX U200 on BD; G9- RelyX Ultimate on HD; and G10- RelyX Ultimate on BD. MDPC-23 cells as well as human dental pulp cells (HDPCs) were also seeded in wells of 96-well plates. After 24 hours incubation, the transdentinal toxicity on the MDPC-23 cells seeded at the pulp side of the discs was immediately determined by the analysis of cell viability (AlamarBlue assay) and morphology (SEM). The extracts (culture medium + components released from the dental materials that diffused through the dentin discs) were collected and applied on the MDPC-23 cells and HDPCs seeded in wells. Then, these cells were assessed concerning their viability (Live/Dead assay), adhesion and spreading (F-actin), as well as the phenotypic expression of alkaline phosphatase (ALP, thymolphthalein monophosphate) and mineralized nodules formation (Alizarin Red). MN). The numerical data were submitted to ANOVA one-way statistical tests, complemented by Tukey (significance level of 5%). Lower viability of the MDPC-23 cells seeded on dentin was observed after using Single Bond Universal (G3 and G4) and RelyX Luting 2 (G5 e G6) in comparison with their respective negative controls (G1 and G2, p<0.05). There was no significant difference between each dental material when they were applied on HD or BD (p>0.05). The viability of those MDPC23 cells seeded in wells, as well as their adhesion, spreading and ALP activity was significantly lower in G3, G4, G5, G6, G9 and G10 than in G1 and G2 (p<0.05). However, G7 and G8 were not different from the controls G1 and G2 (p>0.05). The viability, adhesion, spreading and ALP activity of the HPDCs was significantly lower in G5 and G6 than in G1 and G2 (p<0.05). Overall, the MDPC-23 cells were more sensitive to the extracts obtained from the dental materials assessed in this study than the HDPCs. No statistical difference occurred when the same dental material was applied on HD or BD (p>0.05). According to the methodology used and the results obtained in this laboratory investigation, one may conclude that only RelyX U200 luting cement does not cause transdentinal toxic effects to pulp cells and that bovine dentin can be employed to replace human dentin for assessing the indirect cytotoxicity of resin-based luting cements
Based upon ethical questions and because of the difficulty of getting intact human teeth, researchers have employed bovine teeth to assess some physic and mechanic properties of diverse dental materials. However, lack of scientific data about using such animal teeth model for biological tests of products recommended to be applied on different teeth substrates still remains. Therefore, the aim of this study was to evaluate and compare the transdentinal cytotoxicity of different resin luting cements as well as to use an in vitro model that mimic clinical situations to determine whether bovine dentin (BD) has potential to replace human dentin (HD) for biological tests of indirect toxicity. For this purpose, standardized HD and BD discs (0.4 mm thick) were adapted in artificial pulp chamber devices (APCs), which were individually placed in wells of 24- well plates. Then, odontoblast-like MDPC-23 cells were seeded on the pulpal surface of the dentin discs. After 48- hour incubation, the luting cements were applied on the occlusal surface of the discs, giving rise to the following groups: G1- No treatment on HD (negative control); G2- No treatment on BD (negative control); G3- Single Bond Universal on HD (positive control); G4- Single Bond Universal on BD (positive control); G5- RelyX Luting 2 on HD; G6- RelyX Luting 2 on BD; G7- RelyX U200 on HD; G8- RelyX U200 on BD; G9- RelyX Ultimate on HD; and G10- RelyX Ultimate on BD. MDPC-23 cells as well as human dental pulp cells (HDPCs) were also seeded in wells of 96-well plates. After 24 hours incubation, the transdentinal toxicity on the MDPC-23 cells seeded at the pulp side of the discs was immediately determined by the analysis of cell viability (AlamarBlue assay) and morphology (SEM). The extracts (culture medium + components released from the dental materials that diffused through the dentin discs) were collected and applied on the MDPC-23 cells and HDPCs seeded in wells. Then, these cells were assessed concerning their viability (Live/Dead assay), adhesion and spreading (F-actin), as well as the phenotypic expression of alkaline phosphatase (ALP, thymolphthalein monophosphate) and mineralized nodules formation (Alizarin Red). MN). The numerical data were submitted to ANOVA one-way statistical tests, complemented by Tukey (significance level of 5%). Lower viability of the MDPC-23 cells seeded on dentin was observed after using Single Bond Universal (G3 and G4) and RelyX Luting 2 (G5 e G6) in comparison with their respective negative controls (G1 and G2, p<0.05). There was no significant difference between each dental material when they were applied on HD or BD (p>0.05). The viability of those MDPC23 cells seeded in wells, as well as their adhesion, spreading and ALP activity was significantly lower in G3, G4, G5, G6, G9 and G10 than in G1 and G2 (p<0.05). However, G7 and G8 were not different from the controls G1 and G2 (p>0.05). The viability, adhesion, spreading and ALP activity of the HPDCs was significantly lower in G5 and G6 than in G1 and G2 (p<0.05). Overall, the MDPC-23 cells were more sensitive to the extracts obtained from the dental materials assessed in this study than the HDPCs. No statistical difference occurred when the same dental material was applied on HD or BD (p>0.05). According to the methodology used and the results obtained in this laboratory investigation, one may conclude that only RelyX U200 luting cement does not cause transdentinal toxic effects to pulp cells and that bovine dentin can be employed to replace human dentin for assessing the indirect cytotoxicity of resin-based luting cements
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Palavras-chave
Polpa dentária, Cimentos dentários, Toxicidade, Dentina, Dental pulp, Dental cement, Toxicity, Dentin