Different bacterial models for in vitro induction of non-cavitated enamel caries-like lesions: microhardness and polarized light miscroscopy analyses

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Effect of mucoadhesive polymers on the in vitro performance of insulin-loaded silica nanoparticles: Interactions with mucin and biomembrane models

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Effect of trimetaphosphate and fluoride association on hydroxyapatite dissolution and precipitation in vitro

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

In vitro enamel remineralization capacity of composite resins containing sodium trimetaphosphate and fluoride

This study evaluated the in vitro enamel remineralization capacity of experimental composite resins containing sodium trimetaphosphate (TMP) combined or not with fluoride (F). Bovine enamel slabs were selected upon analysis of initial surface hardness (SH1) and after induction of artificial carious lesions (SH2). Experimental resins were as follows: resin C (control-no sodium fluoride (NaF) or TMP), resin F (with 1.6 % NaF), resin TMP (with 14.1 % TMP), and resin TMP/F (with NaF and TMP). Resin samples were made and attached to enamel slabs (n = 12 slabs per material). Those specimens (resin/enamel slab) were subjected to pH cycling to promote remineralization, and then final surface hardness (SH3) was measured to calculate the percentage of surface hardness recovery (%SH). The integrated recovery of subsurface hardness (ΔKHN) and F concentration in enamel were also determined. Data was analyzed by ANOVA and Student-Newman-Keuls test (p < 0.05). Resins F and TMP/F showed similar SH3 values (p = 0.478) and %SH (p = 0.336) and differed significantly from the other resins (p < 0.001). Considering ΔKHN values, resin TMP/F presented the lowest area of lesion (p < 0.001). The presence of F on enamel was different among the fluoridated resins (p = 0.042), but higher than in the other resins (p < 0.001). The addition of TMP to a fluoridated composite resin enhanced its capacity for remineralization of enamel in vitro. The combination of two agents with action on enamel favored remineralization, suggesting that composite resins containing sodium trimetaphosphate and fluoride could be indicated for clinical procedures in situations with higher cariogenic challenges.

Protective effects of the galectin-1 protein on in vivo and in vitro models of ocular inflammation

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Effect of Er:YAG laser on CaF2 formation and its anticariogenic action on human enamel: An in vitro study

Objective: The objective of this study was to evaluate the effect of Er: YAG laser on the formation of CaF2, after the application of acidulated phosphate fluoride (APF), and its influence on the anti-cariogenic action in human dental enamel. Background Data: Er:YAG laser was designed to promote ablation of the enamel. However, the possibility of using this energy to increase the enamel's resistance to caries has hardly been explored, and neither has its interaction with the use of fluorides. Materials and Methods: One hundred and twenty blocks of enamel were allocated to four groups of 30 blocks each: (1) C, control group; (2) Er:YAG, laser; (3) APF; and (4) Er:YAG+APF. Of these, 80 blocks were submitted to pH cycling for 14 days. In the other 40 blocks, fluoride (CaF2) was measured before cycling. After pH cycling, surface microhardness (SMH), microhardness in cross-section (converted to mineral contents % vol. min.), and fluoride after cycling (40 blocks) were also determined. Results: SMH decreased in all groups. The control group showed the highest decrease, and Er:YAG+APF showed the lowest decrease (p < 0.05). Groups APF and Er:YAG showed the same results (p > 0.05). Mineral content at depths 10, 20, and 40 μm was lower in the control and Er:YAG groups, and higher in groups APF and Er:YAG+APF. CaF2 (μgF/cm2) deposited before pH cycling was higher in the APF group when compared to the Er:YAG+APF group. Control and Er:YAG groups showed the lowest values (p > 0.05). Conclusion: It was concluded that Er:YAG laser influenced the deposition of CaF2 on the enamel and showed a superficial anti-cariogenic action, but not in depth.

Efeito da adição de nanopartículas de hexametafosfato de sódio em dentifrícios fluoretados sobre a desmineralização dentária: estudo in vitro

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Effect of NaF and TiF4 varnish and solution on bovine dentin erosion plus abrasion in vitro

Objectives. This in vitro study aimed to analyze the effect of TiF4 compared to NaF varnishes and solutions, to protect against dentin erosion associated with abrasion. Materials and methods. Bovine dentin specimens were pre-treated with NaF-Duraphat (2.26% F), NaF/CaF2-Duofluorid (5.63% F), experimental-NaF (2.45% F), experimental-TiF4 (2.45% F) and placebo varnishes; NaF (2.26% F) and TiF4 (2.45% F) solutions. Controls remained untreated. The erosive pH cycling was performed using a soft drink (pH 2.6) 4 x 90 s/day and the toothbrushing-abrasion 2 x 10 s/day, in vitro for 5 days. Between the challenges, the specimens were exposed to artificial saliva. Dentin tissue loss was measured profilometrically (mu m). Results. ANOVA/Tukey's test showed that all fluoridated varnishes (Duraphat, 7.5 +/- 1.1; Duofluorid, 6.8 +/- 1.1; NaF, 7.2 +/- 1.9; TiF4, 6.5 +/- 1.0) were able to significantly reduce dentin tissue loss (40.7% reduction compared to control) when compared to placebo varnish (11.2 +/- 1.3), control (11.8 +/- 1.7) and fluoridated (NaF, 9.9 +/- 1.8; TiF4, 10.3 +/- 2.1) solutions (p < 0.0001), which in turn did not significantly differ from each other. Conclusion. All fluoridated varnishes, but not the solutions, had a similar performance and a good potential to reduce dentin tissue loss under mild erosive and abrasive conditions in vitro. Risk patients for erosion and abrasion, especially those with exposed dentin, should benefit from this clinical preventive measure. Further research has to confirm this promising result in the clinical situation.

Impact of experimental Nano-HAP pastes on bovine enamel and dentin submitted to a pH cycling model

This in vitro study evaluated the preventive potential of experimental pastes containing 10% and 20% hydroxyapatite nanoparticles (Nano-HAP), with or without fluoride, on dental demineralization. Bovine enamel (n=15) and root dentin (n=15) specimens were divided into 9 groups according to their surface hardness: control (without treatment), 20 Nanop paste (20% HAP), 20 Nanop paste plus (20% HAP + 0.2% NaF), 10 Nanop paste (10% HAP), 10 Nanop paste plus (10% HAP + 0.2% NaF), placebo paste (without fluoride and HAP), fluoride paste (0.2% NaF), MI paste (CPP-ACP, casein phosphopeptide-amorphous calcium phosphate), and MI paste plus (CPP-ACP + 0.2% NaF). Both MI pastes were included as commercial control products containing calcium phosphate. The specimens were treated with the pastes twice a day (1 min), before and after demineralization. The specimens were subjected to a pH-cycling model (demineralization–6-8 h/ remineralization-16-18 h a day) for 7 days. The dental subsurface demineralization was analyzed using cross-sectional hardness (kgf/mm 2 , depth 10-220 µm). Data were tested using repeated-measures two-way ANOVA and Bonferroni's test (p<0.05). The only treatment able to reduce the loss of enamel and dentin subsurface hardness was fluoride paste (0.2% NaF), which differed significantly from the control at 30- and 50-µm depth (p<0.0001). The other treatments were not different from each other or compared with the control. The experimental Nanop pastes, regardless of the addition of fluoride, were unable to reduce dental demineralization in vitro.

Different protocols to produce artificial dentine carious lesions in vitro and in situ: hardness and mineral content correlation

This study compared dentine demineralization induced by in vitro and in situ models, and correlated dentine surface hardness (SH), cross-sectional hardness (CSH) and mineral content by transverse microradiography (TMR). Bovine dentine specimens (n = 15/group) were demineralized in vitro with the following: MC gel (6% carboxymethylcellulose gel and 0.1 m lactic acid, pH 5.0, 14 days); buffer I (0.05 m acetic acid solution with calcium, phosphate and fluoride, pH 4.5, 7 days); buffer II (0.05 m acetic acid solution with calcium and phosphate, pH 5.0, 7 days), and TEMDP (0.05 m lactic acid with calcium, phosphate and tetraethyl methyl diphosphonate, pH 5.0, 7 days). In an in situ study, 11 volunteers wore palatal appliances containing 2 bovine dentine specimens, protected with a plastic mesh to allow biofilm development. The volunteers dripped a 20% sucrose solution on each specimen 4 times a day for 14 days. In vitro and in situ lesions were analyzed using TMR and statistically compared by ANOVA. TMR and CSH/SH were submitted to regression and correlation analysis (p < 0.05). The in situ model produced a deep lesion with a high R value, but with a thin surface layer. Regarding the in vitro models, MC gel produced only a shallow lesion, while buffers I and II as well as TEMDP induced a pronounced subsurface lesion with deep demineralization. The relationship between CSH and TMR was weak and not linear. The artificial dentine carious lesions induced by the different models differed significantly, which in turn might influence further de- and remineralization processes. Hardness analysis should not be interpreted with respect to dentine mineral loss

Impact of different fluoride concentrations and pH of dentifrices on tooth erosion/abrasion in vitro

Background: This in vitro study analysed the effect of different fluoride concentrations in acidic or neutral liquid dentifrices in protecting enamel and dentine from erosive and abrasive wear. Methods: Bovine enamel and dentine specimens (n = 132) were randomly allocated to 11 groups (each n = 12): experimental liquid dentifrices with 550 ppm F, 1100 ppm F, 5000 ppm F or 0 ppm F/placebo (each at pH 4.5 and pH 7.0); and commercial dentifrices with 550 ppm F (Colgate Baby, pH 7.0), 1100 ppm F (Crest, pH 7.0) and 5000 ppm F (Duraphat, pH 7.0). The specimens were subjected to erosion for 90 seconds, 4 times/day, over 7 days. Immediately after the first and last erosion, the specimens were brushed for 15 seconds using one of the dentifrices. Tooth wear was measured profilometrically (lm) and analysed by ANOVA (p < 0.05). Results: All fluoridated liquid dentifrices significantly reduced enamel wear compared to the placebo and commercial dentifrices. Only liquid dentifrices with 1100 and 5000 ppm F significantly reduced dentine wear compared to placebo dentifrice. The pH had no effect, but the consistency had a significant impact on the effect of dentifrices. Conclusions: Liquid dentifrices with high F concentration appear to be a good option to prevent tooth wear

Micro-FEM models based on micro-CT reconstructions for the in vitro characterization of the elastic properties of trabecular bone tissue.

This master’s thesis describes the research done at the Medical Technology Laboratory (LTM) of the Rizzoli Orthopedic Institute (IOR, Bologna, Italy), which focused on the characterization of the elastic properties of the trabecular bone tissue, starting from october 2012 to present. The approach uses computed microtomography to characterize the architecture of trabecular bone specimens. With the information obtained from the scanner, specimen-specific models of trabecular bone are generated for the solution with the Finite Element Method (FEM). Along with the FEM modelling, mechanical tests are performed over the same reconstructed bone portions. From the linear-elastic stage of mechanical tests presented by experimental results, it is possible to estimate the mechanical properties of the trabecular bone tissue. After a brief introduction on the biomechanics of the trabecular bone (chapter 1) and on the characterization of the mechanics of its tissue using FEM models (chapter 2), the reliability analysis of an experimental procedure is explained (chapter 3), based on the high-scalable numerical solver ParFE. In chapter 4, the sensitivity analyses on two different parameters for micro-FEM model’s reconstruction are presented. Once the reliability of the modeling strategy has been shown, a recent layout for experimental test, developed in LTM, is presented (chapter 5). Moreover, the results of the application of the new layout are discussed, with a stress on the difficulties connected to it and observed during the tests. Finally, a prototype experimental layout for the measure of deformations in trabecular bone specimens is presented (chapter 6). This procedure is based on the Digital Image Correlation method and is currently under development in LTM.

Biomolecular studies in Alzheimer’s Disease models: investigations in vitro and in vivo

The Alzheimer’s disease (AD), the most prevalent form of age-related dementia, is a multifactorial and heterogeneous neurodegenerative disease. The molecular mechanisms underlying the pathogenesis of AD are yet largely unknown. However, the etiopathogenesis of AD likely resides in the interaction between genetic and environmental risk factors. Among the different factors that contribute to the pathogenesis of AD, amyloid-beta peptides and the genetic risk factor apoE4 are prominent on the basis of genetic evidence and experimental data. ApoE4 transgenic mice have deficits in spatial learning and memory associated with inflammation and brain atrophy. Evidences suggest that apoE4 is implicated in amyloid-beta accumulation, imbalance of cellular antioxidant system and in apoptotic phenomena. The mechanisms by which apoE4 interacts with other AD risk factors leading to an increased susceptibility to the dementia are still unknown. The aim of this research was to provide new insights into molecular mechanisms of AD neurodegeneration, investigating the effect of amyloid-beta peptides and apoE4 genotype on the modulation of genes and proteins differently involved in cellular processes related to aging and oxidative balance such as PIN1, SIRT1, PSEN1, BDNF, TRX1 and GRX1. In particular, we used human neuroblastoma cells exposed to amyloid-beta or apoE3 and apoE4 proteins at different time-points, and selected brain regions of human apoE3 and apoE4 targeted replacement mice, as in vitro and in vivo models, respectively. All genes and proteins studied in the present investigation are modulated by amyloid-beta and apoE4 in different ways, suggesting their involvement in the neurodegenerative mechanisms underlying the AD. Finally, these proteins might represent novel potential diagnostic and therapeutic targets in AD.

Comparison of immortalized bEnd5 and primary mouse brain microvascular endothelial cells as in vitro blood-brain barrier models for the study of T cell extravasation

Important insights into the molecular mechanism of T cell extravasation across the blood-brain barrier (BBB) have already been obtained using immortalized mouse brain endothelioma cell lines (bEnd). However, compared with bEnd, primary brain endothelial cells have been shown to establish better barrier characteristics, including complex tight junctions and low permeability. In this study, we asked whether bEnd5 and primary mouse brain microvascular endothelial cells (pMBMECs) were equally suited as in vitro models with which to study the cellular and molecular mechanisms of T cell extravasation across the BBB. We found that both in vitro BBB models equally supported both T cell adhesion under static and physiologic flow conditions, and T cell crawling on the endothelial surface against the direction of flow. In contrast, distances of T cell crawling on pMBMECs were strikingly longer than on bEnd5, whereas diapedesis of T cells across pMBMECs was dramatically reduced compared with bEnd5. Thus, both in vitro BBB models are suited to study T cell adhesion. However, because pMBMECs better reflect endothelial BBB specialization in vivo, we propose that more reliable information about the cellular and molecular mechanisms of T cell diapedesis across the BBB can be attained using pMBMECs.

CO2 laser-irradiation through topically applied fluoride increases acid resistance of demineralised human enamel in vitro

PURPOSE: To evaluate the effect of CO2 laser treatment through topically applied amine fluoride solution on demineralised enamel. MATERIALS AND METHODS: Sixty extracted human molar crowns were selected and cut longitudinally into half. One half was subjected to a 10-day pH-cycling procedure to create caries-like lesions, whereas the other was left non-demineralised. The following treatments were randomly assigned (one treatment per tooth, on respective non-demineralised and demineralised matched specimens): exposure to a 1% amine fluoride solution for 15 s without irradiation (group I), irradiation for 15 s with a continuous-wave CO2 laser (group II), or laser-treatment for 15 s through the amine fluoride solution applied immediately beforehand (group III). Fluoride uptake (n = 30) and acid resistance (n = 30) were determined after treatment. Enamel surface alterations after laser irradiation were monitored using scanning electron microscopy. RESULTS: In groups I and III, an increased fluoride uptake was detected (p < or = 0.05). Laser irradiation through topical fluoride resulted in an increased acid resistance of sound and demineralised enamel specimens in deeper layers (p < or = 0.05). In addition, less surface alterations were observed in SEM examination of specimens irradiated through the amine fluoride solution compared with counterparts treated with laser only. CONCLUSIONS: CO2 laser light application through an amine fluoride solution may be instrumental in enhancing acid resistance of sound and demineralised enamel.