Descontaminação de superfícies de titânio com Terpinen-4-ol e Carvacrol: estudo in vitro das propriedades físico-química, antimicrobiana e citotóxica
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Data
2018-04-04
Autores
Maquera Huacho, Patricia Milagros
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Editor
Universidade Estadual Paulista (Unesp)
Resumo
A doença peri-implantar é um processo inflamatório de etiologia bacteriana. Diferentes tratamentos antimicrobianos disponíveis buscam reduzir a carga microbiana e eliminar a inflamação. Terapias alternativas com extratos naturais abrem novas perspectivas para controlar e reduzir a carga microbiana. Terpinen-4-ol e Carvacrol, principais constituintes naturais de óleos essenciais vêm se destacando por sua propriedade antimicrobiana. O objetivo deste estudo foi avaliar: I) as modificações físico-químicas em superfícies de titânio, adesão e proliferação de fibroblastos e osteoblastos; II) as propriedades antimicrobianas sobre biofilme de P. gingivalis e F. nucleatum; e citotóxicas em fibroblastos. III) efeito inibitório de biofilmes simples e multi-espécies de S. sanguinis, S. gordonii, S. mitis, S. oralis , S. salivarius, S. mutans, S. sobrinus, A. viscosus, A. naeslundii, P. intermedia, P. gingivalis, F. nucleatum, A. actinomycetemcomitans e V. parvula a partir de concentrações não tóxicas em queratinócitos. Para isto: I)Discos de titânio foram descontaminados com T4ol e Carvacrol e a influência na modificação do ângulo de contato, energia livre de superfície, estrutura química por MEV-EDS, viabilidade e adesão celular de fibroblastos e osteoblastos pelo ensaio Alamar Blue foram avaliados. II) Concentração inibitória mínima (CIM), concentração bactericida mínima (CBM), concentração mínima de inibição do biofilme (CMIB) por ensaio XTT, e biofilme em superfícies de titânio foi quantificada em UFC e por Microscopia de laser de varredura confocal (CLSM) e a citotoxicidade em fibroblastos foi avaliada pelo ensaio MTT. III) A citoxicidade foi testada em queratinócitos orais por ensaio XTT, a inibição e erradicação do biofilmes simples foi analisada por cristal violeta e o efeito sobre biofilmes multi-espécie foi avaliado por qPCR. Resultados de MEV-EDS demonstraram presença de Na, O e Cl após o tratamento com T4ol e Carvacrol. Todas as superfícies apresentaram características hidrófilas e valores de Energia Livre de Superfície (ELS) entre 5,5 mN/m e 3,4 mN/m. Teste de viabilidade e microscopia de fluorescência não apresentaram diminuição da viabilidade celular e adesão. Os valores de CIM foram Carvacrol 0,007% e 0,002% respectivamente para P. gingivalis e F. nucleatum e T4ol 0,06% para ambos os microrganismos. Os valores de CBM foram semelhantes aos CIM. Os valores de CMIB foram Carvacrol 0,03%, 0,06% e T4ol 0,06%, 0,24%, respectivamente, para P. gingivalis e F. nucleatum. Observou-se por contagem de UFC e CLSM atividade antibiofilme usando Carvacrol (0,26%, 0,06%) e T4ol (0,95%, 0,24%) com atividade citotóxica semelhante à Clorexidina. T4ol 0.19% e Carvacrol 0.06% não afetaram a viabilidade das células epiteliais. Foi observada a inibição e erradicação de biofilmes simples e multi-espécie, especialmente em patógenos periodontais. Portanto, podemos concluir que T4ol e Carvacrol não alteram as propriedades da superfície do titânio e permitem interação adequada com células envolvidas no processo de re-osseointegração. Além disso, concentrações específicas destes compostos sem toxicidade sobre células humanas, reduziram o número de patógenos periodontais em biofilmes simples e multi-espécie.
Peri-implant disease is an inflammatory process of bacterial etiology. Different antimicrobial treatments available seek to reduce microbial load and eliminate inflammation. Alternative therapies with natural extracts open new perspectives to control and reduce microbial burden. Terpinen-4-ol and Carvacrol, the main natural constituents of essential oils; has been notable for its antimicrobial property. The objective of this study was to evaluate: I) physico-chemical modifications on titanium surfaces, adhesion and proliferation of fibroblasts and osteoblasts; II) the antimicrobial properties of P. gingivalis and F. nucleatum biofilm; and cytotoxic effects on fibroblasts. III) Inhibitory effect of single and multi-species biofilms of S. sanguinis, S. gordonii, S. mitis, S. oralis, S. salivarius, S. mutans, S. sobrinus, A. viscosus, A. naeslundii, P. intermedia, P. gingivalis, F. nucleatum, A. actinomycetemcomitans and V. parvula from non-toxic concentrations in keratinocytes. For this: I) Titanium discs were decontaminated with T4ol and Carvacrol and the influence on the modification of contact angle, surface free energy, chemical structure by MEV-EDS, viability and cellular adhesion of fibroblasts and osteoblasts by the Alamar Blue assay were evaluated. II) Minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), minimum concentration of biofilm inhibition (MBIC) by XTT assay, and biofilm on titanium surfaces was quantified in CFU and by Confocal Scanning Laser Microscopy (CLSM), and fibroblast cytotoxicity was evaluated by the MTT assay. III) Cytotoxicity was tested in oral keratinocytes by XTT assay, inhibition and eradication of single biofilms was analyzed by crystal violet and the effect on multi-species biofilms was evaluated by qPCR. SEM results showed the presence of Na, O and Cl after treatment with T4ol and Carvacrol. All surfaces showed hydrophilic characteristics and surface free energy (SFE) values between 5.5 mN/m and 3.4 mN/m. Viability test and fluorescence microscopy showed no decrease in cell viability and adhesion. MIC values were Carvacrol 0.007% and 0.002% respectively for P. gingivalis and F. nucleatum and T4ol 0.06% for both microorganisms. MBC values were similar to MICs. The MBIC values were Carvacrol 0.03%, 0.06% and T4ol 0.06%, 0.24%, respectively, for P. gingivalis and F. nucleatum. Anti-biofilm activity using Carvacrol (0.26%, 0.06%) and T4ol (0.95%, 0.24%) was observed through CFU and CLSM and showed cytotoxic activity like Chlorhexidine. T4ol 0.19% and Carvacrol 0.06% did not affect the viability of epithelial cells. Inhibition and eradication of simple and multispecies biofilms were observed, especially in periodontal pathogens. Therefore, we can conclude that T4ol and Carvacrol do not alter the surface properties of Ti surfaces and allow an adequate interaction with cells involved in the reosseointegration process; and specific concentrations of these compounds without toxicity to human cells, reduced the number of periodontal pathogens in single and multi-species biofilms.
Peri-implant disease is an inflammatory process of bacterial etiology. Different antimicrobial treatments available seek to reduce microbial load and eliminate inflammation. Alternative therapies with natural extracts open new perspectives to control and reduce microbial burden. Terpinen-4-ol and Carvacrol, the main natural constituents of essential oils; has been notable for its antimicrobial property. The objective of this study was to evaluate: I) physico-chemical modifications on titanium surfaces, adhesion and proliferation of fibroblasts and osteoblasts; II) the antimicrobial properties of P. gingivalis and F. nucleatum biofilm; and cytotoxic effects on fibroblasts. III) Inhibitory effect of single and multi-species biofilms of S. sanguinis, S. gordonii, S. mitis, S. oralis, S. salivarius, S. mutans, S. sobrinus, A. viscosus, A. naeslundii, P. intermedia, P. gingivalis, F. nucleatum, A. actinomycetemcomitans and V. parvula from non-toxic concentrations in keratinocytes. For this: I) Titanium discs were decontaminated with T4ol and Carvacrol and the influence on the modification of contact angle, surface free energy, chemical structure by MEV-EDS, viability and cellular adhesion of fibroblasts and osteoblasts by the Alamar Blue assay were evaluated. II) Minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), minimum concentration of biofilm inhibition (MBIC) by XTT assay, and biofilm on titanium surfaces was quantified in CFU and by Confocal Scanning Laser Microscopy (CLSM), and fibroblast cytotoxicity was evaluated by the MTT assay. III) Cytotoxicity was tested in oral keratinocytes by XTT assay, inhibition and eradication of single biofilms was analyzed by crystal violet and the effect on multi-species biofilms was evaluated by qPCR. SEM results showed the presence of Na, O and Cl after treatment with T4ol and Carvacrol. All surfaces showed hydrophilic characteristics and surface free energy (SFE) values between 5.5 mN/m and 3.4 mN/m. Viability test and fluorescence microscopy showed no decrease in cell viability and adhesion. MIC values were Carvacrol 0.007% and 0.002% respectively for P. gingivalis and F. nucleatum and T4ol 0.06% for both microorganisms. MBC values were similar to MICs. The MBIC values were Carvacrol 0.03%, 0.06% and T4ol 0.06%, 0.24%, respectively, for P. gingivalis and F. nucleatum. Anti-biofilm activity using Carvacrol (0.26%, 0.06%) and T4ol (0.95%, 0.24%) was observed through CFU and CLSM and showed cytotoxic activity like Chlorhexidine. T4ol 0.19% and Carvacrol 0.06% did not affect the viability of epithelial cells. Inhibition and eradication of simple and multispecies biofilms were observed, especially in periodontal pathogens. Therefore, we can conclude that T4ol and Carvacrol do not alter the surface properties of Ti surfaces and allow an adequate interaction with cells involved in the reosseointegration process; and specific concentrations of these compounds without toxicity to human cells, reduced the number of periodontal pathogens in single and multi-species biofilms.
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Palavras-chave
Biofilmes, Doenças periodontais, Titânio, Medicamentos fitoterápicos, Técnicas in vitro, Biofilms, Periodontal diseases, Titanium, Phytotherapeutic medicines, In vitro techniques