Effect of the application of arginine on the composition and metabolism of salivary microcosm biofilms to prevent the enamel demineralization

Author(s)

Daniela Alejandra Cusicanqui Mendez

• Document type: Doctoral Thesis
• Press: Bauru.
• Institution: Universidade de São Paulo (USP). Faculdade de Odontologia de Bauru (FOB/SDB)
• Defense date: 2022-03-30

Examining board members

Thiago Cruvinel da Silva; Daniela Rios Honório; Eliana Mitsue Takeshita Nakagawa; Gláuber Campos Vale

• Advisor: Thiago Cruvinel da Silva

Abstract

The aim of the present study was to determine the effect of two different concentrations of arginine (2.5% and 8%), combined or not with 1,450 ppm sodium fluoride, on the composition and metabolism of salivary microcosm biofilms, and prevention and control of the enamel demineralization. Saliva samples collected from 3 free-caries adults were used as inocula to obtain a microbial pool to grow microcosm biofilms on bovine enamel blocks. This study was divided in 3 phases: i) to determine the adequate growth conditions of salivary microcosm biofilms, ii) to evaluate the effect of arginine combined or not with sodium fluoride on biofilm growth, and iii) to assess the effect of treatments with arginine combined or not with sodium fluoride on 3-day biofilms. The composition and viability of biofilms were determined by the colony forming unit counts of total microorganisms, total streptococci, mutans streptococci, and total lactobacilli grown on Tryptic Soy Blood Agar, Mitis Salivarius Agar, Mitis Salivarius Bacitracin Agar, and Rogosa Agar with 0.13% glacial acetic acid, respectively. The vitality of intact biofilms was determined in a confocal scanning laser microscope (CSLM). The metabolism of biofilms was determined by the quantification of insoluble extracellular polysaccharides (AlexaFluor and CalcoFluor in CSLM), resazurin assay (colorimetric method), and determination of the concentration of L-lactate in culture media (spectrophotometric enzymatic method). The enamel demineralization was assessed by the concentration of calcium released in culture media by Arsenazo III method, and integrated mineral loss and lesion depth by transversal microradiography. Statistical analyses were performed by Kruskal Wallis and Dunns post-hoc tests (P<0,05). The biofilms grown in modified McBain with 0.2% sucrose and 25 mmol/L PIPES in anaerobic conditions presented adequate outcomes to laboratorial essays with arginine, regarding composition, viability, and vitality of biofilms, as well as the reproductibility of results. It was observed a synergic antibiofilm effect of arginine and sodium fluoride by the reduction of the viability, vitality, extracellular polysaccharides, and the metabolism of resazurin in biofilms that grew or were treated by the reagents. However, the production of lactic acid was significantly reduced only in treatments with 8% arginine and both combinations of arginine and sodium fluoride. The concentration of calcium released in culture media was significantly lower in all groups of biofilm growth, except for 2.5% arginine, and in the treatment groups with 8% arginine and both combinations of arginine and sodium fluoride. Distinctly, the other parameters of enamel demineralization were similar among all groups. Therefore, these findings showed a synergic effect of arginine plus sodium fluoride during the biofilm growth, and also to treat salivary microcosm biofilms, reducing the viability, vitality, and metabolism, slowing the mineral loss even in conditions with high availability of sucrose; however, no avoiding the development of enamel lesions. (AU)

• FAPESP's process: 18/01654-8 - Effect on the application of arginine on the composition and metabolism of saliva microcosm biofilms and prevention of enamel demineralization
• Grantee: Daniela Alejandra Cusicanqui Méndez
• Support Opportunities: Scholarships in Brazil - Doctorate