Root canal dressings for revascularization influence in vitro mineralization of apical papilla cells

Abstract Endodontic revascularization is based on cell recruitment into the necrotic root canal of immature teeth after chemical disinfection. The clinical outcome depends on the ability of surviving cells from the apical tissue to differentiate and promote hard tissue deposition inside the dentinal walls. Objective To investigate the effect of calcium hydroxide (CH) and modified triple antibiotic paste (mTAP - ciprofloxacin, metronidazole and cefaclor) on the viability and mineralization potential of apical papilla cells (APC) in vitro . Material and Methods APC cultures were kept in contact with CH or mTAP (250-1000 µg/mL) for 5 days, after which cell viability was assessed using 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Next, APCs were subjected to CH or mTAP at 250 µg/mL for 5 days before inducing the differentiation assay. After 14 and 21 days, calcium deposition was assessed by the Alizarin Red S staining method, followed by elution and quantification using spectrophotometry. Data were analyzed using ANOVA followed by Tukey post hoc test. Results CH induced cell proliferation, whereas mTAP showed significant cytotoxicity at all concentrations tested. APC treated with CH demonstrated improved mineralization capacity at 14 days, while, for mTAP, significant reduction on the mineralization rate was observed for both experimental periods (14 and 21 days). Conclusion Our findings showed that CH induces cell proliferation and improves early mineralization, whereas mTAP was found cytotoxic and reduced the mineralization potential in vitro of APCs.

Comparison of primary human gingival fibroblasts from an older and a young donor on the evaluation of cytotoxicity of denture adhesives

Abstract Denture adhesives (DA) improve the retention and stability of ill-fitting dentures, especially for older adults. These materials should be biocompatible, i.e., they cannot cause undesired biological responses and be non-cytotoxic to oral tissues. However, in vitro testing of DA biocompatibility employing primary cell culture may possibly be affected by other factors, such as the donor age. Objective To compare the cytotoxicity of three different denture adhesives when assessed in primary gingival fibroblasts from a young donor or from an older donor, as well as the release of the basic fibroblast growth factor (bFGF), and the inflammatory response marker interleukin-6 (IL-6). Material and Methods Gingival fibroblasts isolated from a 30- and a 62-year-old donor were assayed for proliferation (1-7 days) and sensitivity to latex (positive control). Fibroblasts were indirectly exposed to Corega Ultra (cream), Corega powder and Fixodent Original for a 24 h period and assayed by XTT and Crystal Violet tests. The release of IL-6 and bFGF by exposed cells was determined by ELISA. Results While cells from the young donor presented higher cell growth after 7 days, the sensitivity to increasing concentrations of latex extracts was very similar between young and older cells. Both XTT and CVDE detected no difference between the DA and the control group. All materials induced higher levels of IL-6 and bFGF compared to control. Cells from the older donor exposed to Corega Ultra released lower levels of cytokine and growth factor. Conclusions All materials were considered non-cytotoxic, but affected cytokine and growth factor release. The biological differences found between fibroblasts from both donors could be due to individual or age-related factors. The authors suggest the use of cells from older donors on studies of dental products aimed at older patients, to better simulate their physiological response.

Efficacy of relacin combined with sodium hypochlorite against Enterococcus faecalis biofilms

Abstract Objective Relacin is a synthetic molecule that targets RelA, an essential protein in a conserved bacterial stress response system. It was shown to inhibit bacterial growth. The aims of this study were to evaluate the antimicrobial effect of relacin combined with sodium hypochlorite (NaOCl) on Enterococcus faecalis biofilms and to evaluate the cytotoxicity of relacin. Material and Methods 48-h E. faecalis OG1RF biofilms were treated by various concentrations of relacin in order to determine its inhibitory concentration. Then, the 48-h biofilms were treated either with 1-min NaOCl (0.01%, 0.05%) alone, or in combination of relacin. As a means of comparison, the biofilms of ΔrelA were also treated by 1-min NaOCl (0.01%, 0.05%, 0.25%). The treatment efficacy was determined by agar plate count assays. The cytotoxicity of relacin was examined on human gingival epithelial cells Ca9-22 and murine fibroblasts NIH-3T3 by a methyl thiazolyltetrazolium (MTT) assay and a lactate dehydrogenase assay. Statistical analysis was performed by one-way or two-way analysis of variance (ANOVA) with Bonferroni's post-hoc test and an independent Student's t-test. A significance level of p<0.05 was used. Results Relacin inhibited the growth of OG1RF biofilms partially at 8 mM and fully at 14 mM. The relacin (14 mM) and NaOCl combined treatment resulted in significantly higher treatment efficacy than NaOCl treatment alone. At 0.05% NaOCl, the combined treatment resulted in 5.65 (±0.19) log reduction in biofilm viability. The ΔrelA biofilms were more susceptible to NaOCl treatment than the wild type biofilms at 0.25% NaOCl. Relacin at 14 mM was not toxic to host epithelial cells and fibroblasts. Conclusions The combination of relacin with a low concentration of NaOCl was effective and not cytotoxic.

Repair of bone defects using adipose-derived stem cells combined with alpha-tricalcium phosphate and gelatin sponge scaffolds in a rat model

Abstract Objectives This study aimed to evaluate the potential of adipose-derived stem cells (ASCs) combined with a modified α-tricalcium phosphate (α-TCP) or gelatin sponge (GS) scaffolds for bone healing in a rat model. Material and Methods Bone defects were surgically created in the femur of adult SHR rats and filled with the scaffolds, empty or combined with ASCs. The results were analyzed by histology and histomorphometry on days seven, 14, 30, and 60. Results Significantly increased bone repair was observed on days seven and 60 in animals treated with α-TCP/ASCs, and on day 14 in the group treated with GS/ASCs, when compared with the groups treated with the biomaterials alone. Intense fibroplasia was observed in the group treated with GS alone, on days 14 and 30. Conclusions Our results showed that the use of ASCs combined with α-TCP or GS scaffolds resulted in increased bone repair. The higher efficacy of the α-TCP scaffold suggests osteoconductive property that results in a biological support to the cells, whereas the GS scaffold functions just as a carrier. These results confirm the potential of ASCs in accelerating bone repair in in vivo experimental rat models. These results suggest a new alternative for treating bone defects.

Influência da adição de nanotubos de TiO2 na biocompatibilidade de cerâmicas Y-TZP e de materiais a base de resina / Influence of the addition of TiO2 nanotubes on the biocompatibility of Y-TZP ceramics and resin-based materials

O objetivo deste estudo in vitro foi avaliar a biocompatibilidade de uma zircônia tetragonal policristalina estabilizada por ítrio (Y-TZP), um cimento à base de resina (RelyX ™ Ultimate) e um adesivo à base de 10-MDP (Single Bond Universal) modificados ou não por nanotubos de titânio (TiO2), por meio dos testes de viabilidade celular MTT e Cristal Violeta. Para este fim, foram obtidos discos de 13 mm x 2 mm de zircônia Y-TZP (IPS e.max ZirCAD). Os discos de cimento resinoso e adesivo com 10-MDP foram obtidos através de um molde metálico com as mesmas dimensões. Para Y-TZP, a incorporação de nanotubos de TiO2 ocorreu antes da sinterização, enquanto para os materiais à base de resina, 0,3% em peso de nanotubos foram adicionados antes da fotopolimerização. Os espécimes foram divididos em 8 grupos (n = 8). A avaliação in vitro foi feita através de testes nos quais as células da linhagem de fibroblastos NIH 3T3 foram colocadas em contato indireto com estes materiais. Para a viabilidade celular foram realizados MTT e Cristal Violeta em duplicata e após 24, 48 e 72 horas a absorbância foi analisada por espectrofotometria em leitora Elisa. Os dados obtidos foram submetidos a ANOVA a dois critérios, seguido do teste de Tukey (α = 0,05). Os resultados mostraram que em 24 e 48 hs todos os materiais mostraram-se biocompatíveis. No período de 72 hs os maiores aumentos de absorbância aconteceram para os grupos Y-TZP sem nanotubos de TiO2 e adesivo com nanotubos de TiO2 comparados aos demais grupos. De modo geral, a incorporação de nanotubos a estes materiais não interferiu na viabilidade celular em ambos os testes.(AU)

The aim of this in vitro study was to evaluate the biocompatibility of yttrium-stabilized tetragonal zirconia polycrystal (Y-TZP), a resin-based cement (RelyX™ Ultimate) and a 10- MDP-based adhesive (Single Bond Universal) modified or not by titanium nanotubes (TiO2), by means of the MTT cell viability test and Crystal Violet. For this purpose, disks of 13 mm in diameter per 2 mm is thickness of pre-sintered Y-TZP zirconia (IPS e.max ZirCAD) were obtained. The resin-cement and a 10-MDP adhesive disks were obtained through a metal mold with the same dimensions. For Y-TZP, the incorporation of TiO2 nanotubes occured before sinterization, while for the resin-based materials 0.3wt% of nanotubes were added to the uncured materials. The specimens were divided into 8 groups (n = 8). The in vitro evaluation was carried out by means of tests in which fibroblast line NIH 3T3 cells were placed into indirect contact with these materials. For cell viability were made MTT assay tests and Crystal Violet in duplicate and after 24, 48 and 72 hours the absorbance levels were analyzed by spectrophotometry Elisa reader. The data obtained were submitted to two-way ANOVA, followed by Tukey test (α = 0.05). In the period of 72 the highest increases of absorbance happened for the groups Y-TZP without TiO2 nanotubes and adhesive with TiO2 nanotubes when compared to the other groups. In general, the incorporation of nanotubes into these materials did not interfere with cell viability in both tests.(AU)

Effects of low-level laser therapy on stem cells from human exfoliated deciduous teeth

ABSTRACT Low-Level Laser Therapy stimulates the proliferation of a variety of types of cells. However, very little is known about its effect on stem cells from human exfoliated deciduous teeth (SHED). Objective This study aimed to evaluate the influence of different laser therapy energy densities on SHED viability and proliferation. Material and Methods SHED were irradiated according to the groups: I (1.2 J/cm2 - 0.5 mW – 10 s), II (2.5 J/cm2 – 10 mW – 10 s), III (3.7 J/cm2 – 15 mW – 10 s), IV (5.0 J/cm2 – 20 mW – 10 s), V (6.2 J/cm2 – 25 mW – 10 s), and VI (not irradiated – control group). Cell viability was assessed 6 and 24 h after irradiation measuring the mitochondrial activity and using the Crystal Violet assay. Cell proliferation was assessed after 24, 48, and 72 h of irradiation by SRB assay. Results MTT assay demonstrated differences from 6 to 24 hours after irradiation. After 24 h, groups I and IV showed higher absorbance values than those of control group. Crystal Violet assay showed statistically differences in the absorbance rate from 6 to 24 h after irradiation for groups III and VI. At 24 h after irradiation, Group III absorbance rate was greater than that of groups I, II, and IV. Group VI absorbance rate was greater than that of groups I and IV. SRB assay showed that the group I had higher rates than those of groups II, III, V, and VI, at 24 h after irradiation. After 48 h, group I exhibited the greatest cell proliferation rate followed by groups III, V, and VI. After 72 h, group III exhibited the lowest cell proliferation rate than those of groups II, IV, and V. Conclusions The Low-Level Laser Therapy energy densities used in this study did not cause loss of cell viability and stimulated SHED proliferation within the parameters described in this study.

Efeitos da fotobiomodulação com laser e LED na proliferação e migração de fibroblastos gengivais de pacientes com e sem Síndrome de Down / Effects of photobiomodulation with laser and LED on the proliferation and migration of gingival fibroblasts from patients with and without Down syndrome

A terapia de fotobiomodulação tem sido vastamente utilizada em cultura de fibroblastos com o objetivo de se verificar seu real efeito na cicatrização de feridas e de se estabelecer os melhores parâmetros de luz. Pacientes com síndrome de Down (SD) possuem alta prevalência da doença periodontal (DP) e importantes alterações imunológicas, as quais podem interferir no processo de cicatrização. O objetivo do presente estudo foi de avaliar os efeitos da utilização de Laser e LED em fibroblastos gengivais de pacientes com e sem SD (FSD e FGH, respectivamente), verificando a viabilidade celular e o processo In vitro de cicatrização de feridas. As células foram cultivadas em placas de 96 poços (1x103 célula/poço) e colocadas em estado de aquiescência (meio DMEM com 1% de soro fetal bovino) 24h antes da irradiação e retomando sua condição inicial de 10% de soro fetal bovino (SFB) instantes antes da aplicação de Laser (AlGaAs - 660nm e AlGaInP - 810nm) e LED (637 ±15nm) com exceção do controle negativo (C-) que continuou com 1% de SFB. Os grupos foram divididos em: C+ (sem irradiação, 10% SFB), C- (sem irradiação, 1% SFB), LIV5 (5 J/cm2 por 3s), LIV8 (8 J/cm2 por 5s), LV5 (5 J/cm2, por 3s), LV8 (8 J/cm2 por 5s), LED3 (0,03 J/cm2 por 3s) e LED5 (0,05J/cm2 por 5s). A potência utilizada foi a mesma tanto para o Laser como para o LED (40mW). A viabilidade celular foi avaliada através dos testes colorimétricos MTT e Cristal Violeta, nos períodos de 24,48,72 e 96h. O teste de cicatrização de feridas In vitro (placas de 24 poços) para avaliação da migração dos fibroblastos, foi nos períodos de 12, 24, 36 e 48h. A análise estatística foi realizada através do teste ANOVA de medidas repetidas complementado pelo teste de Tukey (p<0,05). Os grupos experimentais, em grande parte dos períodos, obtiveram melhor viabilidade celular em relação ao C+, com exceção do grupo LIV8 que apresentou crescimento celular próximo de zero, em todos os períodos. Para FSD os melhores resultados foram com LIV5, LED3 e LED5 (p<0,05), enquanto que para FGH, foi o LV5 (p>0,05). No ensaio de cicatrização de feridas os melhores resultados foram LIV5 para FGH (p<0,05) e todos os tratamentos com exceção do LIV8 para FSD (p<0,05). Os FSD apresentaram maior fechamento da ferida em relação ao FGH nos períodos de 24 e 36h (p<0,05). Como conclusão, a fotobiomodulação por Laser e LED mostrou ser efetiva para viabilidade celular, tanto para o FGH como para o FSD, com exceção do Laser infravermelho de maior densidade de energia e maior tempo de exposição (LIV8). Na migração celular, a fotobiomodulação foi efetiva no maior fechamento da ferida para os FSD. Logo, a terapia de fotobiomodulação por Laser e LED, com os parâmetros adequados, parecer ser um tratamento adjuvante promissor para pacientes com SD.(AU)

The photobiomodulation therapy has been widely used in fibroblast culture in order to verify its effects on wound healing and to establish the best parameters of light. Down's syndrome patients (DS) present high prevalence of periodontal disease (PD) and important immunological changes, which could interfere on the wound healing process. The aim of this study was to evaluate the Laser and LED effects on gingival fibroblasts cultures from patients with or without DS (FSD and FGH, respectively), through cell viability tests and in vitro wound healing test. Cells were grown in 96-well plates (1x103 cells / well) and then, put in a quiescence environment, (DMEM medium with 1% fetal bovine serum) for 24 hours before irradiation. After that an initial condition of 10% fetal bovine serum (FBS) was set before Laser (Red -AlGaAs - 660nm and Infrared - AlGaInP - 810nm) and LED (637 ± 15nm) application, with the exception of the negative control (C-) which still remained with 1% FBS. The groups were divided in: C+ (no irradiation, 10% FBS), C (no irradiation 1% FBS), LIV5 (5J/cm2 for 3s), LIV8 (8 J / cm2 for 5s), LV5 (5J/cm2 for 3s), LV8 (8J/cm2 for 5s), LED3 (0.03J/cm2 for 3 seconds) and LED5 (0,05J / cm2 for 5s). The power output was the same for both Laser and LED (40mW). Cell viability was evaluated through MTT and Crystal Violet colorimetric tests, in periods of 24,48,72 and 96h. The in vitro wound healing assay (24 well plates), measured the fibroblasts migration, during 12, 24, 36 and 48 hours. Statistical analysis was performed using repeated measures ANOVA complemented by Tukeys test (p <0.05). The experimental groups, in most periods, presented higher cell viability compared to C+, except for the LIV8 group that exhibited cell growth near to zero, in all periods. In relation to FSD, the best results were with LIV5, LED 3 and LED 5 (p<0.05), while to FGH, the LV5 presented higher viability (p<0.05). The best results for the wound healing test were LIV5 for FGH (p<0,05) and all groups but LIV8 for FSD (p<0,05). FSD cells presented higher wound closure in relation to FGH at 24 and 36h (p<0,05). In conclusion, the Laser and LED photobiomodulation was effective for cell growth, for both FGH and FSD cells, except for the infrared laser with higher energy density and longer exposure time (LIV8). Photobiomodulation was more effective for wound closure by FSD cells. Therefore, laser and LED photobiomodulation therapy, with appropriate parameters, seems to be a promising adjunctive treatment for patients with DS.(AU)

Biocompatibility and bioactivity of calcium silicate-based endodontic sealers in human dental pulp cells

Mineral Trioxide Aggregate (MTA) is a calcium silicate-based material. New sealers have been developed based on calcium silicate as MTA Fillapex and MTA Plus.Objective The aim of this study was to evaluate biocompatibility and bioactivity of these two calcium silicate-based sealers in culture of human dental pulp cells (hDPCs).Material and Methods The cells were isolated from third molars extracted from a 16-year-old patient. Pulp tissue was sectioned into fragments with approximately 1 mm3 and kept in supplemented medium to obtain hDPCs adherent cultures. Cell characterization assays were performed to prove the osteogenic potential. The evaluated materials were: MTA Plus (MTAP); MTA Fillapex (MTAF) and FillCanal (FC). Biocompatibility was evaluated with MTT and Neutral Red (NR) assays, after hDPCs exposure for 24 h to different dilutions of each sealer extract (1:2, 1:3 and 1:4). Unexposed cells were the positive control (CT). Bioactivity was assessed by alkaline phosphatase (ALP) enzymatic assay in cells exposed for one and three days to sealer extracts (1:4 dilution). All data were analyzed by ANOVA and Tukey post-test (p≤0.05%).Results MTT and NR results showed suitable cell viability rates for MTAP at all dilutions (90-135%). Cells exposed to MTAF and FC (1:2 and 1:4 dilutions) showed significant low viability rate when compared to CT in MTT. The NR results demonstrated cell viability for all materials tested. In MTAP group, the cells ALP activity was similar to CT in one and three days of exposure to the material. MTAF and FC groups demonstrated a decrease in ALP activity when compared to CT at both periods of cell exposure.Conclusions The hDPCs were suitable for the evaluation of new endodontic materialsin vitro. MTAP may be considered a promising material for endodontic treatments.

Evaluation of caries-associated virulence of biofilms from Candida albicans isolated from saliva of pediatric patients with sickle-cell anemia

A previous study demonstrated that the amount of Candida spp. in saliva is higher in children with sickle-cell disease. The results from a recent study demonstrate its participation in the etiology of dental caries. Objective This study assessed caries-associated virulence (production of acid, extracellular polysaccharides, proteins and metabolic activity) of biofilms from Candida albicans isolated from saliva of patients with sickle-cell anemia in comparison to isolates obtained from matched healthy children. Material and Methods The isolates were previously obtained from 25 children (4-6 years) and their matched controls (healthy children). One isolate of C. albicans per children was used, totaling 25 isolates per group. The C. albicans biofilms were grown for five days and analyzed regarding the production of lactic acid, extracellular polysaccharides, proteins and metabolic activity. The production of lactic acid was determined by the enzymatic method. The concentration of extracellular polysaccharides was determined by the phenol-sulphuric acid method, and the concentration of the protein was analyzed using the QuantiPro BCA kit. The XTT reduction was used to verify the metabolic activity. The data were analyzed with GraphPad Prism at 5%. Results The Mean±standard deviation for acid production, extracellular polysaccharides, proteins and metabolic activity of isolates from sickle-cell group was, respectively: 7.1±5.0 mmol/L; 15.6±2.5 μg glucose/mg biofilm; 7,503±3,097 μg/mL; A490 3.5±0.7. For isolates from control group the values obtained were: 3.5±3.3 mmol/L; 12.8±3.4 μg glucose/mg biofilm; 4,995±682 μg/mL; A490 3.4±0.5. The C. albicans isolates from patients with sickle-cell anemia produced a significantly greater quantity of acids (p=0.025), polysaccharides (p=0.025) and proteins (p=0.047) compared with the isolates from control group. However, there was ...

Radiopacity and cytotoxicity of Portland cement associated with niobium oxide micro and nanoparticles

Objective Mineral Trioxide Aggregate (MTA) is composed of Portland Cement (PC) and bismuth oxide (BO). Replacing BO for niobium oxide (NbO) microparticles (Nbµ) or nanoparticles (Nbη) may improve radiopacity and bioactivity. The aim of this study was to evaluate the radiopacity and cytotoxicity of the materials: 1) PC; 2) White MTA; 3) PC+30% Nbµ; 4) PC+30% Nbη. Material and Methods For the radiopacity test, specimens of the different materials were radiographed along an aluminum step-wedge. For cell culture assays, Saos-2 osteoblastic-cells (ATCC HTB-85) were used. Cell viability was evaluated through MTT assay, and bioactivity was assessed by alkaline phosphatase activity assay. Results The results demonstrated higher radiopacity for MTA, followed by Nbµ and Nbη, which had similar values. Cell culture analysis showed that PC and PC+NbO associations promoted greater cell viability than MTA. Conclusions It was concluded that the combination of PC+NbO is a potential alternative for composition of MTA. .