Influence of salivary conditioning and sucrose concentration on biofilm-mediated enamel demineralization

Abstract The acquired pellicle formation is the first step in dental biofilm formation. It distinguishes dental biofilms from other biofilm types. Objective To explore the influence of salivary pellicle formation before biofilm formation on enamel demineralization. Methodology Saliva collection was approved by Indiana University IRB. Three donors provided wax-stimulated saliva as the microcosm bacterial inoculum source. Acquired pellicle was formed on bovine enamel samples. Two groups (0.5% and 1% sucrose-supplemented growth media) with three subgroups (surface conditioning using filtered/pasteurized saliva; filtered saliva; and deionized water (DIW)) were included (n=9/subgroup). Biofilm was then allowed to grow for 48 h using Brain Heart Infusion media supplemented with 5 g/l yeast extract, 1 mM CaCl2.2H2O, 5% vitamin K and hemin (v/v), and sucrose. Enamel samples were analyzed for Vickers surface microhardness change (VHNchange), and transverse microradiography measuring lesion depth (L) and mineral loss (∆Z). Data were analyzed using two-way ANOVA. Results The two-way interaction of sucrose concentration × surface conditioning was not significant for VHNchange (p=0.872), ∆Z (p=0.662) or L (p=0.436). Surface conditioning affected VHNchange (p=0.0079), while sucrose concentration impacted ∆Z (p<0.0001) and L (p<0.0001). Surface conditioning with filtered/pasteurized saliva resulted in the lowest VHNchange values for both sucrose concentrations. The differences between filtered/pasteurized subgroups and the two other surface conditionings were significant (filtered saliva p=0.006; DIW p=0.0075). Growing the biofilm in 1% sucrose resulted in lesions with higher ∆Z and L values when compared with 0.5% sucrose. The differences in ∆Z and L between sucrose concentration subgroups was significant, regardless of surface conditioning (both p<0.0001). Conclusion Within the study limitations, surface conditioning using human saliva does not influence biofilm-mediated enamel caries lesion formation as measured by transverse microradiography, while differences were observed using surface microhardness, indicating a complex interaction between pellicle proteins and biofilm-mediated demineralization of the enamel surface.

Effect of hydroalcoholic extract of Myracrodruon urundeuva All. and Qualea grandiflora Mart. leaves on the viability and activity of microcosm biofilm and on enamel demineralization

Abstract Objectives: The aim of this study was to assess the effect of Myracrodruon urundeuva All. and Qualea grandiflora Mart. leaves hydroalcoholic extracts on viability and metabolism of a microcosm biofilm and on enamel demineralization prevention. Methodology: Microcosm biofilm was produced on bovine enamel using inoculum from pooled human saliva mixed with McBain saliva, under 0.2% sucrose exposure, for 14 days. The biofilm was daily-treated with the extracts for 1 min. At the end, it was analyzed with respect to viability by fluorescence, CFU counting and extracellular polysaccharides (phenol-sulphuric acid colorimetric assay) and lactic acid (enzymatic assay) production. The demineralization was measured by TMR. The data were compared using ANOVA or Kruskal-Wallis (p<0.05). Results: M. urundeuva All. at 100, 10 and 0.1 μg/mL and Q. grandiflora Mart. at 100 and 0.1 μg/mL reduced biofilm viability similarly to positive control (chlorhexidine) and significantly more than the negative-vehicle control (35% ethanol). M. urundeuva at 1000, 100 and 0.1 μg/mL were able to reduce both lactobacilli and mutans streptococci CFU counting, while Q. grandiflora (1000 and 1.0 μg/mL) significantly reduced mutans streptococci CFU counting. On the other hand, the natural extracts were unable to significantly reduce extracellular polysaccharides and lactic acid productions neither the development of enamel carious lesions. Conclusions: The extracts showed antimicrobial properties on microcosm biofilm, however, they had no effect on biofilm metabolism and caries protection.

Role of Candida albicans on enamel demineralization and on acidogenic potential of Streptococcus mutans in vitro biofilms

Abstract There is growing evidence that C. albicans is associated with dental caries, but its role on caries development needs to be better clarified. Objective: To evaluate at the hard tissue level the effect of C. albicans on the cariogenic potential of S. mutans biofilms focusing on the mineral profile of induced carious lesions. This study also aimed to evaluate the effect of C. albicans on the acidogenic potential of S. mutans biofilms. Methodology: Dual-species (CA+SM) and single-species biofilms (CA or SM) were grown on the surface of enamel slabs in the presence of glucose/sucrose supplemented culture medium for 24, 48 and 72 hours. Demineralization was evaluated through percentage of surface microhardness change (%SMC) and transversal microradiography analysis (ILM and LD) and pH of the spent medium was recorded daily. Data were analyzed by two-way ANOVA followed by Bonferroni correction. Results: %SMC was statistically different among the biofilms at each time point being the highest for SM biofilms and the lowest for CA biofilms which also differed from CA+SM biofilms [SM (24 h: 47.0±7.3; 48 h: 66.3±8.3; 72 h: 75.4±3.9); CA (24 h: 7.3±3.3; 48 h: 7.1±6.4; 72 h: 6.6±3.6); CA+SM (24 h: 35.9±7.39.1; 48 h: 47.2±9.5; 72 h: 47.6±9.5)]. pH of spent medium was statistically lower for SM biofilms compared to the other biofilms at each time point and remained constant over time while pH values increased from 24 to 72 h for both CA and CA+SM biofilms [SM (24 h: 4.4±0.1; 48 h: 4.4±0.1; 72 h: 4.5±0.1); CA (24 h: 6.9±0.3; 48 h: 7.2±0.2; 72 h: 7.5±0.2); CA+MS (24 h: 4.7±0.2; 48 h: 5.1±0.1; 72 h: 6.1±0.6)]. IML and LD for SM biofilms increased over time while no difference was observed from 24 to 72 h for the other biofilms. Conclusions: The present data suggest that C. albicans has low enamel demineralization potential and the presence of C. albicans can reduce both the cariogenic and acidogenic potentials of S. mutans biofilms.

Comparação de diferentes pré-tratamentos de superfície na remineralização de esmalte com flúor em tratamento de lesões com mancha branca e sua resistência após desafio ácido / Comparison of different pretreatments on remineralization of enamel white spot lesions with fluoride and its resistance after new acidic challenge

A aplicação de flúor tópico é a principal estratégia de natureza química para a remineralização de lesões incipientes (LI) clinicamente visíveis como manchas brancas (MB) por cárie. Abordagens para aumentar a retenção de F no substrato pode favorecer sua ação e para isso, tratamentos prévios da superfície do esmalte podem ser usados. O objetivo deste trabalho foi comparar a capacidade de remineralização do flúor sem e com pré-tratamento do esmalte com ácido fosfórico e nitrato de alumínio e a sua resistência após novo desafio. Sessenta espécimes de esmalte bovino foram preparados (6mm x 4mm) e selecionados por meio de análise de microdureza de superfície (MS). LI foram produzidas através de ciclagem Desmineralização-Remineralização (DES-RE) e os espécimes divididos aleatoriamente em cinco grupos, de acordo com o tratamento (n=12): V- controle (verniz de fluoreto de sódio 5% por 4 horas), F (sem pré-tratamento); P-F(condicionamento ácido com ácido fosfórico por 30s); Al-F (nitrato de alumínio a 0,05M por 1min); P-Al-F (condicionamento com ácido fosfórico a 37% + nitrato de alumínio a 0,05M). Os tratamentos foram repetidos semanalmente durante quatro semanas. Após o tratamento, os espécimes foram submetidos à nova ciclagem ácida. Após cada etapa, nova MS foi realizada e ao final das análises, um corte transversal dos espécimes foi realizado. Uma das metades foi submetida à análise da microdureza longitudinal (ML) e a outra preparada para realização da microrradiografia transversal (TMR) para a análise de conteúdo mineral perdido. O percentual de perda de dureza de superfície (%PDS) foi analisado por ANOVA a 2 critérios de medidas repetidas e Tukey e o percentual de perda de dureza longitudinal (%PDL) por ANOVA a 3 critérios de medidas repetidas e Tukey (p<0,05). Os dados de TMR no parâmetro LD (profundidade da lesão) foram analisados por ANOVA a dois critérios (p<0,05). Os resultados mostraram que a ciclagem DES-RE resultou em significante %PDS e %PDL em todos os grupos. Grupo F revelou menor perda de MS após tratamento e, F, Al-F e P-Al-F, mostraram menor perda de dureza final, após o novo desafio ácido. Na análise de %PDL, o grupo V apresentou menor perda de dureza final quando comparado com os demais grupos, nas diferentes profundidades. A análise do conteúdo mineral não revelou nenhuma diferença entre os tratamentos e fases. Nenhum dos pré tratamentos propostos foram capazes de otimizar a atuação do gel APF na remineralização de MB.(AU)

The topical fluoride is the main chemical strategy to remineralize incipient caries lesions (ICL) visible as carious white spot lesions (WSL). Approaches to increase the fluoride retention may favor its action and therefore, enamel pretreatments can be used for this purpose. The aim of this study was to compare the potential of fluoride remineralization with and without previous enamel pretreatment with aluminum nitrate and phosphoric acid as well as their resistance after a new acid challenge. Sixty bovine enamel specimens were prepared (6mm x 4mm) and selected by the surface hardness (SH) analysis. The ICL were produced using DE-RE cycling and the specimens were randomized in five groups, according to the treatment (n=12): Vcontrol (5% sodium fluoride varnish during 4h), and four groups previously preatreated with topical application of acidulated phosphate fluoride (APF) during 4min: F (without pretreatment), P-F (phosphoric acid etching during 30s), Al-F (0.05M aluminum nitrate during 1min); P-Al-F (phosphoric acid etching + aluminum nitrate). The treatments were performed weekly during four weeks. After the treatment, the specimens were submitted to the new acid challenge. After each step, a new SH analysis was performed followed by the transversal cut of the specimens. A half was submitted to the longitudinal cross-sectional hardness analysis (LH) and the other half was prepared to transverse microradiography assessments (TMR) to determine the loss of mineral content. The percentage of surface mineral loss (%SH) was analyzed using two-way repeated-measures ANOVA and the percentage of crosssectional mineral loss (%LH) by the three-way repeated-measures ANOVA and Tukey (p<0.05). Data of TMR analysis by LD (lesion depth) parameter was analyzed with two-way ANOVA (p<0.05). The results showed significant %SH and %LH after DE-RE cycling for all groups. F group showed the lowest %SH after treatment, with no significant difference to Al-F and P-Al-F after the new acid challenge. V group showed the lowest %LH compared to the other groups, in the different depths, suggesting more resistance. Mineral content assessment did not reveal any difference among treatments and phases. None pretreatments were able to increase the potential of fluoride remineralization.(AU)