The impact of chitosan in experimental resin with different photoinitiator systems.

OBJECTIVE: to investigate the effect of different concentrations of chitosan added to experimental resins containing either BAPO or camphorquinone (CQ) as photoinitiators, regarding degree of conversion (DC), flexural strength (FS), flexural elastic modulus (E), Knoop microhardness (KHN), cytotoxicity, genotoxicity and antimicrobial activity. METHODS: Experimental resins with polymeric matrix of BisGMA and TEGDMA was added either 0.5 wt% BAPO or 0.5 wt% camphorquinone/0.2% amine along with and chitosan concentrations of 0.5%; 1.0% or 2.0%. Degree of conversion was measured using Fourier transformed infrared spectroscopy. Flexural strength and elastic modulus were obtained through three-point bending test and Knoop microhardness was measured in a microidenter. Direct cytotoxicity was performed in human keratinocytes and genotoxicity test was done in murine macrophages cells. Antimicrobial activity was acessed against Staphylococcus aureus and Streptococcus mutans through the inhibition halo. Data were analyzed using two-way ANOVA and Tukey teste (α = 0.05). RESULTS: The materials containing photoinitiator BAPO showed higher values of DC, FS, E, and KHN compared to resins with CQ. The addition of chitosan did not affect the properties of these materials. However, in resins containing CQ, the addition of chitosan improve these properties compared to control group. For the groups containing BAPO the chitosan reduced cytotoxicity and genotoxicity compared to materials with camphorquinone. The materials with 1.0% and 2.0% chitosan showed increased antibacterial activity in the materials containing BAPO as photoinitiator for both bacteria. SIGNIFICANCE: The alternative photoinitiator BAPO and chitosan can improve physical and biological properties of photoactivated resins when compared with the materials with photoinitiator camphorquinone.

Effect of accelerated stability on the physical, chemical, and mechanical properties of experimental bleaching gels containing different bioadhesive polymers.

OBJECTIVE: To evaluate the physical-chemical (weight, pH, quantification of hydrogen peroxide) and mechanical (texture profile and rheology tests) properties of the experimental bleaching gel based on the bioadhesive polymer Aristoflex® AVC, after accelerated stability testing. MATERIALS AND METHODS: A total of 300 syringes of bleaching gels were divided into 5 groups (n = 60): Whiteness Perfect® 10%-FGM (WP); carbamide peroxide 10% with aristoflex (CPa); carbamide peroxide 10% with Carbopol (CPc); aristoflex thickener (A); and Carbopol thickener (C). According to the following requirements and time, the accelerated stability test was performed: in an incubator at 40 °C and 75% humidity per 1, 3, and 6 months, and baseline (refrigerator at 5 °C and 25% humidity). The variables were analyzed following the statistical tests: Two-way ANOVA and Tukey's test were applied to pH; weight data were analyzed using a mixed model for repeated measurements over time and the Tukey-Kramer test; one-way ANOVA and Tukey's test analyzed the rheology test; generalized linear models were used to quantify the peroxide amount and texture profile data. A significance level of 5% was considered. RESULTS: The experimental bleaches CPa and CPc had the highest pH values when compared to the others in 6 months. Thickeners A and C did not change the pH, weight, and active content over the accelerated stability times (p > 0.05). Furthermore, there was weight loss after 3 months of storage for CPa and CPc (p < 0.05). In the quantification of hydrogen peroxide, the WP group showed the highest values over time (p < 0.0001), only showing a significant loss after the 3rd month. Meanwhile, CPa and CPc showed a reduction in quantification from the 1st month. CONCLUSIONS: Temperature and humidity directly influenced the active content and properties of bleaching gels. In addition, the presence of components regardless of thickeners, such as stabilizers, in the commercial gel allowed for greater stability over time. CLINICAL RELEVANCE: The development of experimental bleaching gels for clinical use requires careful testing. Therefore, accelerated stability testing represents a valuable tool in the development and evaluation of cosmetic formulations.

Mucoadhesive, Thermoreversible Hydrogel, Containing Tetracaine-Loaded Nanostructured Lipid Carriers for Topical, Intranasal Needle-Free Anesthesia.

Recent advances have been reported for needle-free local anesthesia in maxillary teeth by administering a nasal spray of tetracaine (TTC) and oxymetazoline, without causing pain, fear, and stress. This work aimed to assess whether a TTC-loaded hybrid system could reduce cytotoxicity, promote sustained permeation, and increase the anesthetic efficacy of TTC for safe, effective, painless, and prolonged analgesia of the maxillary teeth in dental procedures. The hybrid system based on TTC (4%) encapsulated in nanostructured lipid carriers (NLC) and incorporated into a thermoreversible hydrogel of poloxamer 407 (TTCNLC-HG4%) displayed desirable rheological, mechanical, and mucoadhesive properties for topical application in the nasal cavity. Compared to control formulations, the use of TTCNLC-HG4% slowed in vitro permeation of the anesthetic across the nasal mucosa, maintained cytotoxicity against neuroblastoma cells, and provided a three-fold increase in analgesia duration, as observed using the tail-flick test in mice. The results obtained here open up perspectives for future clinical evaluation of the thermoreversible hybrid hydrogel, which contains TTC-loaded NLC, with the aim of creating an effective, topical, intranasal, needle-free anesthesia for use in dentistry.

Resistivity Technique for the Evaluation of the Integrity of Buccal and Esophageal Epithelium Mucosa for In Vitro Permeation Studies: Swine Buccal and Esophageal Mucosa Barrier Models.

Permeation assays are important for the development of topical formulations applied on buccal mucosa. Swine buccal and esophageal epithelia are usually used as barriers for these assays, while frozen epithelia have been used to optimize the experimental setup. However, there is no consensus on these methods. In transdermal studies, barrier integrity has been evaluated by measuring electrical resistance (ER) across the skin, which has been demonstrated to be a simple, fast, safe, and cost-effective method. Therefore, the aims here were to investigate whether ER might also be an effective method to evaluate buccal and esophageal epithelium mucosa integrity for in vitro permeation studies, and to establish a cut-off ER value for each epithelium mucosa model. We further investigated whether buccal epithelium could be substituted by esophageal epithelium in transbuccal permeation studies, and whether their permeability and integrity were affected by freezing at -20 °C for 3 weeks. Fresh and frozen swine buccal and esophageal epithelia were mounted in Franz diffusion cells and were then submitted to ER measurement. Permeation assays were performed using lidocaine hydrochloride as a hydrophilic drug model. ER was shown to be a reliable method for evaluating esophageal and buccal epithelia. The esophageal epithelium presented higher permeability compared to the buccal epithelium. For both epithelia, freezing and storage led to decreased electrical resistivity and increased permeability. We conclude that ER may be safely used to confirm tissue integrity when it is equal to or above 3 kΩ for fresh esophageal mucosa, but not for buccal epithelium mucosa. However, the use of esophageal epithelium in in vitro transmucosal studies could overestimate the absorption of hydrophilic drugs. In addition, fresh samples are recommended for these experiments, especially when hydrophilic drugs are involved.

Promising potential of articaine-loaded poly(epsilon-caprolactone) nanocapules for intraoral topical anesthesia.

To determine whether the permeation capacity and analgesic efficacy of articaine (ATC) could be increased and cytotoxicity decreased by encapsulation in poly(ɛ-caprolactone) nanocapsules (ATCnano), aiming at local or topical anesthesia in dentistry. Cellular viability was evaluated (using the MTT test and fluorescence microscopy) after 1 h and 24 h exposure of HaCaT cells to ATC, ATCnano, ATC with epinephrine (ATCepi), and ATC in nanocapsules with epinephrine (ATCnanoepi). The profiles of permeation of 2% ATC and 2% ATCnano across swine esophageal epithelium were determined using Franz-type vertical diffusion cells. Analgesic efficacy was evaluated with a von Frey anesthesiometer in a postoperative pain model in rats, comparing the 2% ATC, 2% ATCnano, 2% ATCepi, and 2% ATCnanoepi formulations to 4% ATCepi (a commercially available formulation). We show that use of the nanocapsules decreased the toxicity of articaine (P<0.0001) and increased its flux (P = 0.0007). The 2% ATCepi and 4% ATCepi formulations provided higher analgesia success and duration (P<0.05), compared to 2% ATC, 2% ATCnano, and 2% ATCnanoepi. Articaine-loaded poly(ɛ-caprolactone) nanocapsules constitute a promising formulation for intraoral topical anesthesia (prior to local anesthetic injection), although it is not effective when injected in inflamed tissues for pain control, such as irreversible pulpitis.

Palatal needle-free anesthesia for upper molars extraction. A randomized clinical trial.

BACKGROUND: The aim of this study was to compare the ability of liposomal and non-liposomal lidocaine and prilocaine in hydrogel formulations to promote topical anesthesia in palatal mucosa during upper molar extractions. METHODS: In this randomized, cross over, triple-blinded clinical trial, a liposomal and a non-liposomal formulation of the eutectic mixture of local anesthetics, 2.5% lidocaine and 2.5% prilocaine, were used to promote palatal anesthesia without the local anesthetic infiltration during bilateral upper molars extractions. RESULTS: From the total of 40 patients included in this study, the non-liposomal eutectic lidocaine-prilocaine formulation failed in 40% of cases, unlike the liposomal formulation, which was effective for all patients (Fisher's exact test, p < 0.0001). Furthermore, the liposomal formulation (26.75 ± 7,47 min) induced longer anesthesia duration (t-test, p < 0.0001) than the non-liposomal formulation (16.78 ± 4.75 min). No mucosal ulceration or discomfort was reported for both formulations. CONCLUSION: The liposomal formulation was able to induce adequate anesthesia in palatal mucosa during dental extraction, avoiding the local anesthetic infiltration. For the first time, a topical formulation allowed upper molars surgical removal without injection of any local anesthetic agent into palatal mucosa in adults.

Full-Thickness Intraoral Mucosa Barrier Models for In Vitro Drug-Permeation Studies Using Microneedles.

The use of permeation enhancers such as microneedles (MNs) to increase drug penetration across intraoral mucosa has increased in recent years. Permeation studies, commonly performed using vertical diffusion cells, are a well-established way to preview formulations and enhance their performance during the development stage. However, to our knowledge, the existing intraoral mucosa barrier models do not permit permeation using MN-pretreated mucosa due to their insufficient thickness. Therefore, the objective of this study was to develop a barrier model using thick palate tissues to perform in vitro permeation studies, with physical enhancement of the permeability of intraoral mucosa by pretreatment with MNs. The adapted Franz-type cells used in the permeation experiments were validated (cell dimensions and volume, sealing effectiveness, stirring and dissolution efficiency, temperature control, and establishment of uniaxial flux). Commercially available MNs were used in the palatal mucosa. Optical images of the mucosa were acquired to analyze the microperforations created. In vitro permeation studies were conducted with the MN-pretreated mucosa. This work presents a new in vitro method for the evaluation of MNs as permeation enhancers, with the aim of improving the absorption of drug formulations topically applied within the oral cavity.

Hybrid Hydrogel Composed of Polymeric Nanocapsules Co-Loading Lidocaine and Prilocaine for Topical Intraoral Anesthesia.

This study reports the development of nanostructured hydrogels for the sustained release of the eutectic mixture of lidocaine and prilocaine (both at 2.5%) for intraoral topical use. The local anesthetics, free or encapsulated in poly(ε-caprolactone) nanocapsules, were incorporated into CARBOPOL hydrogel. The nanoparticle suspensions were characterized in vitro in terms of particle size, polydispersity, and surface charge, using dynamic light scattering measurements. The nanoparticle concentrations were determined by nanoparticle tracking analysis. Evaluation was made of physicochemical stability, structural features, encapsulation efficiency, and in vitro release kinetics. The CARBOPOL hydrogels were submitted to rheological, accelerated stability, and in vitro release tests, as well as determination of mechanical and mucoadhesive properties, in vitro cytotoxicity towards FGH and HaCaT cells, and in vitro permeation across buccal and palatal mucosa. Anesthetic efficacy was evaluated using Wistar rats. Nanocapsules were successfully developed that presented desirable physicochemical properties and a sustained release profile. The hydrogel formulations were stable for up to 6 months under critical conditions and exhibited non-Newtonian pseudoplastic flows, satisfactory mucoadhesive strength, non-cytotoxicity, and slow permeation across oral mucosa. In vivo assays revealed higher anesthetic efficacy in tail-flick tests, compared to a commercially available product. In conclusion, the proposed hydrogel has potential for provision of effective and longer-lasting superficial anesthesia at oral mucosa during medical and dental procedures. These results open perspectives for future clinical trials.

The effect of two drug delivery systems in ropivacaine cytotoxicity and cytokine release by human keratinocytes and fibroblasts.

OBJECTIVES: Modified drug delivery systems have been developed to improve pharmacological properties of local anaesthetics. However, the inflammatory potential of these formulations was not investigated. This study compared the in-vitro effects of ropivacaine (ropi) in plain, liposomal (MLV) or 2-hydroxypropyl-ß-cyclodextrin (HP-ß-CD) formulations on cell viability, apoptosis and cytokine (IL-1α, TNF-α, IL-6 and IL-10) release. METHODS: Human immortalized keratinocytes (HaCaT) and human immortalized gingival fibroblasts (HGF) were exposed to 1-100 µm ropi concentrations. The cell viability was measured by XTT and LIVE/DEAD assay. Apoptosis was performed by flow cytometry, and cytokine release was measured by ELISA assay. KEY FINDINGS: Human immortalized keratinocyte viability was reduced by ropi and both drug delivery systems. However, none of the formulations induced apoptosis. Results showed a differential regulation of IL-1α TNF-α, IL-6 and IL-10 by HaCaT and HGF. Ropi-HP-ß-CD increased twofold the IL-6 release by HGF in comparison with the control, while 100 µm ropi-MLV led to an increased release of all pro-inflammatory cytokines by HGF. CONCLUSION: The loss in cell viability was not related to cellular apoptosis. Ropi complexed with HP-ß-CD showed a similar cytokine release pattern when compared to the plain formulation. Thus, the HP-ß-CD form was a better drug carrier than the MLV form for ropivacaine drug delivery.

Comparison of liposomal and 2-hydroxypropyl-ß-cyclodextrin-lidocaine on cell viability and inflammatory response in human keratinocytes and gingival fibroblasts.

OBJECTIVES: The aim of this study was to observe the effect multilamellar liposomes (MLV) and 2-hydroxypropyl-ß-cyclodextrin (HP-ß-CD) in the in-vitro effects of lidocaine in cell viability, pro-inflammatory cytokines and prostaglandin E2 release of both human keratinocytes (HaCaT) and gingival fibroblasts (HGF) cells. METHODS: HaCaT and HGF cells were exposed to lidocaine 100-1 µm in plain, MLV and HP-ß-CD formulations for 6 h or 24 h. The formulation effects in cell viability were measured by XTT assay and by fluorescent labelling. Cytokines (IL-8, IL-6 and TNF-α) and PGE2 release were quantified by ELISA. KEY FINDINGS: MLV and HP-ß-CD formulations did not affect the HaCaT viability, which was significantly decreased by plain lidocaine after 24 h of exposure. Both drug carriers increased all cytokines released by HGF after 24-h exposure, and none of the carriers was able to reduce the PGE2 release induced by lidocaine. CONCLUSION: The effect of drug carrier in the lidocaine effects was dependent on the cell type, concentration and time of exposure. MLV and HP-ß-CD showed benefits in improving cell viability; however, both of them showed a tendency to increase cytokine release when compared to the plain solution.

Effect of 4-(N,N-dimethylamino)phenethyl Alcohol on Degree of Conversion and Cytotoxicity of Photo-Polymerized CQ-Based Resin Composites

The aim of this study was to evaluate the degree of conversion (DC) and the cytotoxicity of photo-cured experimental resin composites containing 4-(N,N-dimethylamino)phenethyl alcohol (DMPOH) combined to the camphorquinone (CQ) compared with ethylamine benzoate (EDAB). The resin composites were mechanically blended using 35 wt% of an organic matrix and 65 wt% of filler loading. To this matrix was added 0.2 wt% of CQ and 0.2 wt% of one of the reducing agents tested. 5x1 mm samples (n=5) were previously submitted to DC measurement and then pre-immersed in complete culture medium without 10% (v/v) bovine serum for 1 h or 24 h at 37 °C in a humidifier incubator with 5% CO2 and 95% humidity to evaluate the cytotoxic effects of experimental resin composites using the MTT assay on immortalized human keratinocytes cells. As a result of absence of normal distribution, the statistical analysis was performed using the nonparametric Kruskal-Wallis to evaluate the cytotoxicity and one-way analysis of variance to evaluate the DC. For multiple comparisons, cytotoxicity statistical analyses were submitted to Student-Newman-Keuls and DC analysis to Tukey's HSD post-hoc test (=0.05). No significant differences were found between the DC of DMPOH (49.9%) and EDAB (50.7%). 1 h outcomes showed no significant difference of the cell viability between EDAB (99.26%), DMPOH (94.85%) and the control group (100%). After 24 h no significant difference were found between EDAB (48.44%) and DMPOH (38.06%), but significant difference was found compared with the control group (p>0.05). DMPOH presented similar DC and cytotoxicity compared with EDAB when associated with CQ.

O objetivo deste estudo foi avaliar o grau de conversão (GC) e a citotoxicidade de resinas compostas experimentais utilizando o álcool 4-(N,N-dimetilamino) fenil etílico (DMPOH) associado à canforoquinona (CQ) como sistema fotoiniciador (SF) comparado à versão comercial utilizando o benzoato de etilamina (EDAB). Para tanto, as resinas compostas experimentais foram mecanicamente misturadas utilizando (em peso): 35% de matriz orgânica e 65% em peso de partículas de carga. Posteriormente, foram adicionados 0,2% de CQ e 0,2% de um dos agentes redutores testados. Amostras de 5 x 1 mm (n=5) foram previamentes submetidas à análise de GC e posteriormente, esterilizadas e colocadas no meio de cultura completo sem soro fetal bovino estéril por 1 h ou 24 h a 37 °C em encubadora com 5% de CO2 and 95% de umidade para avaliar os efeitos citotóxicos das resinas compostas experimentais utilizando o método MTT emcélulas células humanas imortalizadas de queratinócitos. Os dados de citotoxicidade foram submetidos à análise estatística de Kruskal-Wallis e de GC à análise de variância com um fator. Em virtude da ausência de normalidade, a análise estatística da citotoxicidade foi realizada utilizando-se o teste não-paramétrico de Kruskal-Wallis. Para o GC, os dados foram submetidos à análise de variaância de 1 fator. Posteriormente para múltiplas comparações, os dados de citotoxicidade foram submetidos ao teste Student-Newman-Keuls e o GC ao teste de Tukey's HSD post-hoc (=0.05). Não foi observada diferença estatística entre o GC de DMPOH (49,9%) e EDAB (50,7%). Para os resultados de 1 h não houve diferença na viabilidade celular entre EDAB (99,26%), DMPOH (94,85%) e o grupo controle (100%). Após 24 h, nenhuma diferença estatística foi encontrada entre EDAB (48,44%) e DMPOH (38,06%), entretanto, diferença significativa foi encontrada em relação ao grupo controle (p>0,05). O DMPOH apresentou GC e citotoxicidade semelhante à EDAB quando associado à CQ.

Effect of 4-(N,N-dimethylamino)phenethyl alcohol on degree of conversion and cytotoxicity of photo-polymerized CQ-based resin composites.

The aim of this study was to evaluate the degree of conversion (DC) and the cytotoxicity of photo-cured experimental resin composites containing 4-(N,N-dimethylamino)phenethyl alcohol (DMPOH) combined to the camphorquinone (CQ) compared with ethylamine benzoate (EDAB). The resin composites were mechanically blended using 35 wt% of an organic matrix and 65 wt% of filler loading. To this matrix was added 0.2 wt% of CQ and 0.2 wt% of one of the reducing agents tested. 5x1 mm samples (n=5) were previously submitted to DC measurement and then pre-immersed in complete culture medium without 10% (v/v) bovine serum for 1 h or 24 h at 37 °C in a humidifier incubator with 5% CO2 and 95% humidity to evaluate the cytotoxic effects of experimental resin composites using the MTT assay on immortalized human keratinocytes cells. As a result of absence of normal distribution, the statistical analysis was performed using the nonparametric Kruskal-Wallis to evaluate the cytotoxicity and one-way analysis of variance to evaluate the DC. For multiple comparisons, cytotoxicity statistical analyses were submitted to Student-Newman-Keuls and DC analysis to Tukey's HSD post-hoc test (=0.05). No significant differences were found between the DC of DMPOH (49.9%) and EDAB (50.7%). 1 h outcomes showed no significant difference of the cell viability between EDAB (99.26%), DMPOH (94.85%) and the control group (100%). After 24 h no significant difference were found between EDAB (48.44%) and DMPOH (38.06%), but significant difference was found compared with the control group (p>0.05). DMPOH presented similar DC and cytotoxicity compared with EDAB when associated with CQ.