Physical, Mechanical, and Anti-Biofilm Formation Properties of CAD-CAM Milled or 3D Printed Denture Base Resins: In Vitro Analysis.

PURPOSE: To investigate surface characteristics (roughness and contact angle), anti-biofilm formation, and mechanical properties (mini-flexural strength) of computer-aided design and computer-aided manufacturing (CAD-CAM) polymethylmethacrylate (PMMA) polymer, and three-dimensional (3D) printed resin for denture base fabrication compared with conventional heat polymerized denture base resins. MATERIALS AND METHODS: A total of 60 discs and 40 rectangular specimens were fabricated from one CAD-CAM (AvaDent), one 3D printed (Cosmos Denture), and two conventional heat polymerized (Lucitone 199 and VipiWave) materials for denture base fabrication. Roughness was determined by Ra value; the contact angle was measured by the sessile drop method. The biofilm formation inhibition behavior was analyzed through Candida albicans adhesion, while mini-flexural strength test was done using a three-point bending test. The data were analyzed using descriptive and analytical statistics (α = 0.05). RESULTS: The CAD-CAM PMMA group showed the lowest C. albicans adhesion (log CFU/mL: 3.74 ± 0.57) and highest mini-flexural strength mean (114.96 ± 16.23 MPa). 3D printed specimens presented the highest surface roughness (Ra: 0.317 ± 0.151 µm) and lowest mini-flexural strength values (57.23 ± 9.07 MPa). However, there was no statistical difference between CAD-CAM PMMA and conventional groups for roughness, contact angle, and mini-flexural strength. CONCLUSIONS: CAD-CAM milled materials present surface and mechanical properties similar to conventional resins and show improved behavior in preventing C. albicans adhesion. Nevertheless, 3D printed resins present decreased mini-flexural strength.

Twice-daily red and blue light treatment for Candida albicans biofilm matrix development control.

Phototherapy has been proposed as a direct means of affecting local bacterial infections. However, the use of phototherapy to prevent fungal biofilm development has received comparatively less attention. This study aimed to determine the effects of red light treatment and blue light treatment, without a photosensitizer, on the development of Candida albicans biofilm. During the development of 48-h biofilms of C. albicans SN 425 (n = 10), the biofilms were exposed twice-daily to noncoherent blue and red light (LumaCare; 420 nm and 635 nm). The energy density applied was 72 J cm-2 for blue light and 43.8 J cm2, 87.6 J cm2, and 175.5 J cm2 for red light. Positive control (PC) and negative control (NC) groups were treated twice-daily for 1 min with 0.12% chlorhexidine (CHX) and 0.89% NaCl respectively. Biofilms were analyzed for colony forming units (CFU), dry-weight, and exopolysaccharides (EPS-soluble and EPS-insoluble). Data was analyzed by one-way ANOVA and Tukey post hoc test (α = 0.05). Dry-weight was lower than NC (p < 0.001) and approached PC levels with both red and blue light treatments. CFU were also lower in groups exposed to blue light and higher durations of red light (p < 0.05). EPS-soluble and EPS-insoluble measures were variably reduced by these light exposures. In conclusion, twice-daily exposure to both blue and red lights affect the biofilm development and physiology of polysaccharide production and are potential mechanisms for the control of C. albicans biofilm matrix development.

Effect of mechanical toothbrushing combined with different denture cleansers in reducing the viability of a multispecies biofilm on acrylic resins.

PURPOSE: To investigate the efficacy of immersion and brushing with different cleansing agents in reducing the viability of multispecies biofilm on acrylic resins. METHODS: Lucitone 550 (L) and Tokuyama Rebase Fast II (T) specimens (10 x 2 mm) were prepared, sterilized, and inoculated with a suspension of Candida albicans, Candida glabrata, and Streptococcus mutans. Specimens were incubated for 48 hours at 37 degrees C for biofilm formation. Then, they were divided into groups (n = 12) and subjected to brushing or immersion for 10 seconds in distilled water (W), 0.2% peracetic acid-Sterilife (Ac), 1% chlorhexidine digluconate (CHX), 1:1 water/dentifrice solution (D), 1% sodiumhypochlorite (NaOCl), and sodium perborate/Corega Tabs (Pb). Viable microorganisms were evaluated by the XTT assay and colony counts (cfu/mL). Data were performed by ANOVA and Tukey test with 5% significance level. RESULTS: The multispecies biofilm on L and T were killed by brushing or immersion in Ac, CHX, and NaOCl for only 10 seconds.

Effects of short-term immersion and brushing with different denture cleansers on the roughness, hardness, and color of two types of acrylic resin.

PURPOSE: To investigate the cumulative effects of brushing (B) or immersion (I), using different cleansing agents, on the surface roughness, hardness and color stability of a heat-polymerized denture resin, Lucitone 550 (L), and a hard chairside reline resin, Tokuyama Rebase Fast II (T). METHODS: A total of 316 specimens (10 x 2 mm) were fabricated. The specimens (n = 9) were divided into brushing or immersion groups according to the following agents: dentifrice/distilled water (D), 1% sodium hypochlorite (NaOCl), Corega Tabs (Pb), 1% chlorhexidine gluconate (Chx), and 0.2% peracetic acid (Ac). Brushing and immersion were tested independently. Assays were performed after 1, 3, 21, 45 and 90 brushing cycles or immersion of 10 seconds each. Data were evaluated statistically by repeated measures ANOVA. Tukey's honestly significant difference (HSD) post-hoc test was used to determine differences between means (α = 0.05). RESULTS: For L there was no statistically significant difference in roughness, except a significant decrease in roughness by brushing with D. T showed a significant effect on the roughness after 90 immersions with Ac. Hardness values decreased for L when specimens were immersed or brushed in NaOCl and Pb. The hardness of T decreased with increases in the repetitions (immersion or brushing), regardless of the cleaning method. Values of color stability for L resin showed significant color change after brushing with and immersion in Ac and Pb. Brushing with D exhibited a higher incidence of color change. For T there were no significant differences between cleaning agents and repetitions in immersion. A color change was noted after three brushings with the Ac, Chx, and D. Brushing with dentifrice decreased roughness of L. Immersion in or brushing with NaOCl and Pb decreased the hardness of L. For T, hardness decreased with increases in immersions or brushing. Color changes after the immersion in or brushing with cleaning agents were clinically acceptable according to National Bureau of Standards parameters for both resins.

Candida biofilm matrix as a resistance mechanism against photodynamic therapy.

BACKGROUND: Antimicrobial photodynamic therapy (aPDT) efficiency on Candida albicans is recognized in free-floating cultures. Though, the lack of aPDT effectiveness against C. albicans organized in biofilms is still unclear. This study aimed to explore the role of the extracellular matrix (ECM) in the protection against aPDT in C. albicans biofilms. METHODS: C. albicans SN 425 wild-type and two mutant strains CNJ 2302; Δ/Δefg1 and CJN 2330; Δ/Δtec1 (ECM deficient) were used. Biofilms were grown on 24-well plates and exposed twice-daily to aPDT with 44 µM toluidine blue-O (TBO) for 5 min followed by red light (635 nm) for 1 min (87.6 J/cm²) or 2 min (175.2 J/cm2). Application of just TBO, light, 0.12% chlorhexidine, and ultrapure water were used as controls. After 48 h, biofilms were assessed for dry-weight (DW), colony forming units (CFU), extracellular DNA (eDNA), soluble and insoluble protein (SP/IP), water-insoluble (alkali-soluble) polysaccharide (ASP), water-soluble polysaccharides (WSP), and confocal scanning laser microscopy. RESULTS: The strains with ECM deficient were affected by aPDT. For the mutant strain Δ/Δefg1, aPDT significantly reduced CFU, ASP, DW, eDNA, WSP and IP when compared to NC (p<0.001) and for the Δ/Δtec1, aPDT significantly reduced CFU, eDNA, IP and SP. Whereas CFU, DW, ASP of the wild-type strain biofilms were not reduced (p>0.05). CONCLUSIONS: C. albicans strains with reduced ECM compounds were more sensitive to aPDT suggesting that the ECM may have a significant protection role from aPDT in C. albicans biofilms.

Regimen and different surfaces interfere with photodynamic therapy on Candida albicans biofilms.

The aim of this study is to compare antimicrobial photodynamic therapy (aPDT) against Candida albicans biofilms formed on two different substrates - acrylic resin or bottom of polystyrene plate; and two aPDT application regimens - twice-daily over the course of 48 h or single treatment after 48 h biofilm formation. C. albicans SN425 biofilms cultivated on Roswell Park Memorial Institute medium were incubated for 5 min with toluidine blue O (44 µM) used as a photosensitizer before red light (635 nm; 175.2 J/cm2) exposure for 2 min. As negative control, ultrapure water, and as positive control 0.12% chlorhexidine (CHX) were used. Biofilms were analyzed for colony forming units (CFU) and cells morphology by confocal scanning laser microscopy. Single treatment and twice-daily aPDT on polystyrene plate and single treatment on acrylic resin did not significantly reduce the CFU (p > 0.05); in contrast, twice-daily aPDT on acrylic resin has reduced C. albicans below the detection limit, similarly to CHX treatment. Single aPDT treatment on polystyrene plate and on the resin presented a bulky and homogeneous biofilm predominantly formed by pseudohyphae. In contrast, in the resin group, the biofilm treated twice-daily with aPDT was predominantly formed by yeast cells, whilst pseudohyphae were occasionally visible. In conclusion, biofilms formed on polystyrene plates are more resistant to aPDT than biofilms formed on acrylic resin. Moreover, applying aPDT twice-daily reduces C. albicans biofilm development on acrylic resin and is a better approach against C. albicans biofilms than one single application on the mature biofilm.

Fluconazole impacts the extracellular matrix of fluconazole-susceptible and -resistant Candida albicans and Candida glabrata biofilms.

Background: Fluconazole (FLZ) is a drug commonly used for the treatment of Candida infections. However, ß-glucans in the extracellular matrices (ECMs) hinder FLZ penetration into Candida biofilms, while extracellular DNA (eDNA) contributes to the biofilm architecture and resistance. Methods: This study characterized biofilms of FLZ-sensitive (S) and -resistant (R) Candida albicans and Candida glabrata in the presence or absence of FLZ focusing on the ECM traits. Biofilms of C. albicans American Type Culture Collection (ATCC) 90028 (CaS), C. albicans ATCC 96901 (CaR), C. glabrata ATCC 2001 (CgS), and C. glabrata ATCC 200918 (CgR) were grown in RPMI medium with or without FLZ at 5× the minimum inhibitory concentration (37°C/48 h). Biofilms were assessed by colony-forming unit (CFU)/mL, biomass, and ECM components (alkali-soluble polysaccharides [ASP], water-soluble polysaccharides [WSP], eDNA, and proteins). Scanning electron microscopy (SEM) was also performed. Data were analyzed by parametric and nonparametric tests (α  =  0.05). Results: In biofilms, FLZ reduced the CFU/mL of all strains (p < 0.001), except for CaS (p = 0.937). However, the ASP quantity in CaS was significantly reduced by FLZ (p = 0.034), while the drug had no effect on the ASP levels in other strains (p > 0.05). Total biomasses and WSP were significantly reduced by FLZ in the ECM of all yeasts (p < 0.001), but levels of eDNA and proteins were unaffected (p > 0.05). FLZ affected the cell morphology and biofilm structure by hindering hyphae formation in CaS and CaR biofilms, by decreasing the number of cells in CgS and CgR biofilms, and by yielding sparsely spaced cell agglomerates on the substrate. Conclusion: FLZ impacts biofilms of C. albicans and C. glabrata as evident by reduced biomass. This reduced biomass coincided with lowered cell numbers and quantity of WSPs. Hyphal production by C. albicans was also reduced.

An in vitro model of Fusobacterium nucleatum and Porphyromonas gingivalis in single- and dual-species biofilms.

PURPOSE: The goal of this study was to develop and validate a standardized in vitro pathogenic biofilm attached onto saliva-coated surfaces. METHODS: Fusobacterium nucleatum (F. nucleatum) and Porphyromonas gingivalis (P. gingivalis) strains were grown under anaerobic conditions as single species and in dual-species cultures. Initially, the bacterial biomass was evaluated at 24 and 48 hours to determine the optimal timing for the adhesion phase onto saliva-coated polystyrene surfaces. Thereafter, biofilm development was assessed over time by crystal violet staining and scanning electron microscopy. RESULTS: The data showed no significant difference in the overall biomass after 48 hours for P. gingivalis in single- and dual-species conditions. After adhesion, P. gingivalis in single- and dual-species biofilms accumulated a substantially higher biomass after 7 days of incubation than after 3 days, but no significant difference was found between 5 and 7 days. Although the biomass of the F. nucleatum biofilm was higher at 3 days, no difference was found at 3, 5, or 7 days of incubation. CONCLUSIONS: Polystyrene substrates from well plates work as a standard surface and provide reproducible results for in vitro biofilm models. Our biofilm model could serve as a reference point for studies investigating biofilms on different surfaces.

Inactivation of genes TEC1 and EFG1 in Candida albicans influences extracellular matrix composition and biofilm morphology.

Background: Infections caused by Candida spp. have been associated with formation of a biofilm, i.e. a complex microstructure of cells adhering to a surface and embedded within an extracellular matrix (ECM). Methods: The ECMs of a wild-type (WT, SN425) and two Candida albicans mutant strains, Δ/Δ tec1 (CJN2330) and Δ/Δ efg1 (CJN2302), were evaluated. Colony-forming units (cfu), total biomass (mg), water-soluble polysaccharides (WSPs), alkali-soluble polysaccharides (ASPs), proteins (insoluble part of biofilms and matrix proteins), and extracellular DNA (eDNA) were quantified. Variable-pressure scanning electron microscopy and confocal scanning laser microscopy were performed. The biovolume (µm3/µm2) and maximum thickness (µm) of the biofilms were quantified using COMSTAT2. Results: ASP content was highest in WT (mean ± SD: 74.5 ± 22.0 µg), followed by Δ/Δ tec1 (44.0 ± 24.1 µg) and Δ/Δ efg1 (14.7 ± 5.0 µg). The protein correlated with ASPs (r = 0.666) and with matrix proteins (r = 0.670) in the WT strain. The population in Δ/Δ efg1 correlated with the protein (r = 0.734) and its biofilms exhibited the lowest biomass and biovolume, and maximum thickness. In Δ/Δ tec1, ASP correlated with eDNA (r = 0.678). Conclusion: ASP production may be linked to C. albicans cell filamentous morphology.

Eficácia da imersão e da escovação mecânica combinada com diferentes agentes de limpeza de próteses na redução da viabilidade de biofilme multiespécies / Effectiveness of immersion and mechanical brushing combined with different denture cleansers in reducing viability of multispecies biofilm

O objetivo deste estudo foi investigar a eficácia da escovação com diferentes agentes de limpeza para reduzir a viabilidade de um biofilme multiespécies em resinas acrílicas. Amostras de Lucitone 550 (L) e Tokuyama Rebase Fast II (T) (10 mm x 2 mm) foram preparadas, esterilizadas e inoculadas com uma suspensão de 107 células/mL de Candida albicans e Candida glabrata e de 108 células/mL de Streptococcus mutans. As amostras foram incubadas por 48 h a 37 °C para a formação do biofilme. Em seguida, foram divididas (n = 12) e submetidas à escovação ou imersão durante 10 s nas soluções: água destilada (A); ácido peracético 0,2% (Ac), digluconato de clorexidina 1% (Chx), solução água/dentifrício 1:1 (D), hipoclorito de sódio a 1% (NaOCl) e perborato de sódio (Pb). Os microrganismos viáveis foram avaliados por ensaio de XTT e contagem de colônias (ufc /mL). Os dados foram verificados por ANOVA e teste de Tukey com nível de significância de 5%. Neste estudo, a eficácia da remoção do biofilme foi aumentada pela escovação combinada com agentes químicos de limpeza, por outro lado, a imersão por 10 s em AC e NaOCl mostrou-se tão eficaz quanto a escovação em L e T. Este resultado é bastante interessante, pois a imersão nestes agentes de limpeza por um curto período poderia ser uma alternativa viável para a desinfecção de próteses de pacientes com dificuldades motoras para executar a escovação.

The aim of this study was to investigate the efficacy of brushing with different cleaning agents in reducing the viability of multispecies biofilm in acrylic resins. Lucitone 550 (L) and Tokuyama Rebase Fast II (T) specimens (10 mm x 2 mm) were prepared, sterilized and inoculated with a Candida albicans and Candida glabrata suspension of 107 cells/mL and a suspension of 108 cells/mL of Streptococcus mutans. Specimens were incubated for 48 h at 37°C for the biofilm formation. Then, they were divided (n=12) and subjected to brushing or immersion for 10 s in the solutions: distilled water (W), 0.2% peracetic acid (Ac), 1% chlorhexidine digluconate (Chx), 1:1 water/dentifrice solution (D), 1% sodium hypochlorite (NaOCl) and sodium perborate (Pb). Viable microorganisms was evaluated by XTT assay and colony counts (cfu/mL). Data were performed by ANOVA and Tukey test with 5% significance level. In this study, the efficacy of biofilm removal was increased by brushing combined with chemical cleaning agents, on the other hand, immersion for 10 sec in NaOCl and AC has shown to be as effective as brushing for L and T. This result is quite interesting, inasmuch as immersion in these cleaning agents for a short period could be a viable alternative for prostheses disinfection of patients with motor disabilities to perform the brushing

Influência das partículas de carga inorgânica nas propriedades físicas, químicas e mecânicas de resinas compostas - revisão sistemática / Influence of inorganic particles in the physical, chemical and mechanical properties of composite resins - systematic review

O objetivo desta revisão sistemática foi fazer um levantamento sobre trabalhos de pesquisa que investigaram a influência das partículas de carga inorgânica nas propriedades físicas, químicas e mecânicas de resinas compostas. Utilizou-se de fontes eletrônicas de catalogação bibliográfica e de outras fontes de informação (comunicação pessoal, busca manual), a partir de 2000 até Outubro de 2007. Como estratégias de busca foram empregados os termos: resina composta e partícula inorgânica (descritores de assunto) e (1) opalescência ou translucidez; (2) análise química; (3) desgaste ou resistência à tração diametral ou resistência à compressão ou resistência à flexão ou resistência à fadiga ou dureza (limites de assunto). A maior parte dos trabalhos encontrados foi de investigação das propriedades mecânicas; seguida das propriedades físicas e químicas. Como amplamente descrito na literatura, partículas de carga inorgânca exercem função de melhorar propriedades mecânicas, as quais dependem do aumento da fração volumétrica, da distribuição, da morfologia e da qualidade da união química na interface partícula/matriz resinosa. Trabalhos que avaliaram o desempenho de resinas compostas comerciais ou experimentais com nanopartículas dão sustentação ao fato de que elas por sí só ou combinadas com outros tamanhos, resultaram em melhores propriedades físicas e mecânicas. De acordo com esta revisão, estudos adicionais precisam ser realizados para comparar os efeitos das frações de nanopartículas na radiopacidade e translucidez das resinas compostas.