Ion release, biocompatibility, and bioactivity of resin-modified calcium hydroxide cavity liners.

BACKGROUND: The placement of liners near the pulp area is essential for therapeutic effects and maintaining pulp health while stimulating the formation of tertiary dentin. This in vitro study aimed to evaluate the calcium release, pH, biocompatibility, solubility, and bioactivity of three resin-modified calcium hydroxide cavity liners. METHODS: The disc specimens of each cavity liner were prepared using polyethylene molds of 7 mm in diameter and 2 mm in height (n = 10). Three light-cure liners evaluated include Ultra-Blend Plus (UB), Base-it (BI), and Master Dent (MD). The samples were then immersed in flasks containing 10 mL of distilled water. Calcium ion release, pH, and solubility were evaluated in two weeks of incubation. The cytotoxicity of extracts adjacent to the specimens was evaluated by MTT assay using NIH/3T3 cells after 1, 3, and 7 days of incubation. The ability to induce the nucleation of calcium phosphates (CaPs) after 28-day immersion in a simulated body fluid was investigated by SEM-EDX analysis. Statistical analysis was performed using ANOVA, Kruskal-Wallis, and repeated measures tests at the significant level of 0.05. RESULTS: There was a significant difference in the release of calcium ions among the three liners investigated on days 1, 7, and 14 (p < 0.05). UB liners exhibited a significantly higher amount of calcium release than the other two liners, followed by BI, and MD. On day 1, there was no significant difference in the average pH among the three liners. However, after day 7, the MD liner showed a significant decrease in pH compared to the other two liners. BI liner demonstrated the highest level of biocompatibility, followed by the MD and UB liners. UB showed a high calcium release, solubility with no alkalizing activity, and the formation of more mature Ca-rich apatite deposits than the other two liners. CONCLUSION: Based on the results of this study, the cavity liner material's performance is material dependent. It can impact ion release, biocompatibility, and bioactivity which are important factors to consider in clinical practice. Further studies are needed to investigate the long-term effects of different liner materials on oral tissues.

Disinfection and Biocompatibility of Titanium Surfaces Treated with Glycine Powder Airflow and Triple Antibiotic Mixture: An In Vitro Study.

The aim of this in vitro study was to compare three disinfection protocols of biofilm-coated machined (MAC) and acid etched (SLA) commercial pure Grade 4 Titanium disks. Samples were infected with a vial of polymicrobial biofilm to simulate peri-implantitis in vitro. Seventeen MAC and twenty SLA titanium disks were randomly assigned to: (1) glycine powder air-flow (GYPAP) for 1 min; (2) a local delivered triple paste antibiotic composed by a gel mixture with ciprofloxacin, metronidazole, and clarithromycin (3MIX) for 1 h; and (3) a combination of both (GYPAP + 3MIX). Biocompatibility of the titanium disks after each treatment protocol was assessed by measurement of adhesion and growth of adipose-derived mesenchymal stem cells (ASCs) after 24 and 72 h. A confocal laser scanning microscope (CLSM) assessed the antibacterial effect of each treatment. Data of the antibacterial efficacy and cell viability were presented as mean with standard deviation and calculated by one-way ANOVA with multiple comparisons via Bonferroni tests. Results were considered significant with p < 0.05. The higher cell viability was achieved by the 3MIX and GYPAP combination on the SLA surfaces after 72 h. CLSM analysis showed a mean ratio of dead bacteria statistically higher in the 3MIX + GYPAP group compared with the GYPAP and 3MIX subgroups (p < 0.05). In conclusion, data showed that the combination of GYPAP and 3MIX could be preferred to the other protocols, especially in presence of SLA titanium surface.

Evaluation of Different Polymerisation Methods of Ocular Prosthesis Acrylic Resins on Subcutaneous Tissue Inflammatory Response in Rats.

Objectives: This study aimed to evaluate the influence of different polymerisation methods of acrylic resin for ocular prostheses on the subcutaneous tissue inflammatory response of rats. Methods: The study was conducted at the Basic Sciences Department, Araçatuba Dental School, São Paulo State University (UNESP), Brazil, from 2016 to 2018. The samples were prepared by water bath (WB), microwave energy (MW) or autopolymerisation (AP) (n = 20 samples per group). The inflammatory response (cell count and immunohistochemical analysis of interleukin [IL]-1ß, IL-6, tumour necrosis factor [TNF]-α, IL-17 and macrophage inflammatory protein-3α) was analysed by the implantation of a sample from each group in the subcutaneous tissue of 20 Wistar rats and evaluated after seven, 15, 30 and 60 days. The quantitative and qualitative data were analysed using analysis of variance and Tukey tests (P <0.05) and visual comparison, respectively. Results: There was a moderate inflammatory infiltrate for the MW and AP groups and a light infiltrate for the WB group after seven days. The inflammatory infiltrate and the immunolabeling of tested targets decreased gradually during the 60-day period. The AP group had the highest immunolabeling of TNF-α (seven days), IL-1ß and IL-17 (at 15 and 30 days) and IL-6 (at 30 and 60 days). The WB and MW groups showed greater immunolabeling at 15 and 30 days, while the MW group also had high results at 60 days. Conclusion: Polymerisation by microwave energy and by chemical activation resulted in a higher inflammatory response.

A comparison of the inflammatory host response to particulate debris adjacent to unlocked and locked screws of a growth guidance system for early onset scoliosis.

PURPOSE: The SHILLA™ Growth Guidance system is a stainless-steel rod and screw system used for Early Onset Scoliosis which incorporates a unique flanged set screw designed to capture the rod, while allowing it to slide as the patient grows. Concomitant with this design is the potential for generation of wear debris and for an inflammatory host response. We hypothesized that the magnitude of the host response adjacent to the unlocked screws and rods would be greater than the host response to the locked rod/screws. METHODS: Seven tissue samples adjacent to locked (3) and unlocked screws (4) from three SHILLA patients (mean implantation time of 19 post-operative months) with infantile idiopathic scoliosis were obtained as part of an explant analysis protocol during a PMDA-approved clinical trial in Japan. Gross appearance, high-resolution radiographs, and histology were assessed. ISO Standard 10993 Part 6 was used to assess the host response. RESULTS: All three locked screw had no metallosis. In contrast, metallosis for unlocked screw tissue samples were rated as "ubiquitous" (2/4), "focal" (1/4), or "absent" (1/4). Microscopic metallic debris was found intracellularly and within interstices of fibrous connective tissues more frequently adjacent to unlocked screws compared to locked screws. Cell type and population scoring consistently showed a modestly larger inflammatory response (macrophages) in the unlocked tissue samples. CONCLUSIONS: The peri-prosthetic tissue response to the unlocked rods/screws had a higher reactivity grade (slight reaction, Δ = 4.0) per ISO 10993 Part 6 compared to the locked screws in three patients with the SHILLA™ Growth Guidance scoliosis system.

On the biocompatibility of endodontic sealers.

Periapical tissue may be exposed to root canal filling materials in consequence of root canal therapy. There is scant scientific data about the biocompatibility of root canal filling materials of various chemistry on the periapical area. This study aimed to investigate the effects of different root canal sealers and their eluates on human alveolar osteoblasts in terms of cell proliferation, adhesion, morphology and gene expression in vitro. Five endodontic sealers (AH Plus®, Apexit®, Tubli-Seal®, Real Seal SE®, EndoRez®) and one gutta-percha obturation material (BeeFill®) were tested. Human alveolar osteoblasts derived from 3 different donors following incubation with sealer eluates after 24 h and 72 h were investigated by means of qPCR (gene expression). Morphological reactions of the alveolar osteoblasts were measured by culturing the cells for 3 d, and 7 d and 14 d, respectively, followed by scanning electron microscopy (morphology, adhesion) and fluorescence imaging of the actin cytoskeleton (morphology, proliferation). A repeated measures analysis was performed and p-values were adjusted by Tukey. While all sealers influenced the cell morphology and the expression of genes associated with apoptosis (Casp3), proliferation (histone H3), and inflammation (interleukin-6 and matrix metalloproteinases 1 and 3), mainly AH Plus® and Apexit® yielded a regular actin cytoskeleton and beneficial gene expression patterns. Regarding cell adhesion, only AH Plus® supported proper anchorage for alveolar osteoblasts. Our results provide evidence for the biocompatibility of epoxy resin-based endodontic sealers, i.e. AH Plus®, while other sealers proved cytotoxic for alveolar osteoblasts. Further studies are needed for understanding the bone cell reactions after endodontic treatment and the clinical decision-making regarding the sealer of choice for root canal fillings.

A comparative in vitro study on monomer release from bisphenol A-free and conventional temporary crown and bridge materials.

This study aimed to investigate the release of common monomers from two conventional and two bisphenol A (BPA)-free temporary crown and bridge materials. Cylindrical samples of all materials were prepared (N = 90; five samples for each material and cycle of analysis). All samples were immersed in high-performance liquid chromatography (HPLC)-grade water and incubated for 1 h, 12 h, 24 h, and 7 days in an incubation shaker at 37°C and 112 rpm. Extraction was performed in accordance with ISO 10993-12. Eluted monomers were detected and quantified by HPLC coupled with ultraviolet-visible spectroscopy and mass spectrometry (HPLC-UV/Vis-MS). Analysis of BPA was performed by HPLC coupled with ultraviolet-visible spectroscopy (HPLC-UV/Vis) and positive results were verified by HPLC-tandem mass spectrometry (HPLC-MS/MS). Neither bisphenol A-glycidyl methacrylate (Bis-GMA) nor BPA was quantifiable in any of the crown and bridge samples investigated in the present study. However, all samples contained triethylene glycol dimethacrylate (TEGDMA) and/or urethane dimethacrylate (UDMA) after 24 h of incubation. Statistical analysis showed that significantly more UDMA was released from the BPA-free materials than from the conventional materials. All concentrations of UDMA measured were below the effective cytotoxic concentrations previously reported. However, for a few materials, especially BPA-free temporary crown and bridge materials, the levels of UDMA were above previously reported potentially harmful concentrations for local cells. As BPA-free materials were introduced as being more biocompatible than materials containing BPA, substitution of Bis-GMA with UDMA should be further investigated.

EMbedding and Backscattered Scanning Electron Microscopy: A Detailed Protocol for the Whole-Specimen, High-Resolution Analysis of Cardiovascular Tissues.

Currently, an ultrastructural analysis of cardiovascular tissues is significantly complicated. Routine histopathological examinations and immunohistochemical staining suffer from a relatively low resolution of light microscopy, whereas the fluorescence imaging of plaques and bioprosthetic heart valves yields considerable background noise from the convoluted extracellular matrix that often results in a low signal-to-noise ratio. Besides, the sectioning of calcified or stent-expanded blood vessels or mineralised heart valves leads to a critical loss of their integrity, demanding other methods to be developed. Here, we designed a conceptually novel approach that combines conventional formalin fixation, sequential incubation in heavy metal solutions (osmium tetroxide, uranyl acetate or lanthanides, and lead citrate), and the embedding of the whole specimen into epoxy resin to retain its integrity while accessing the region of interest by grinding and polishing. Upon carbon sputtering, the sample is visualised by means of backscattered scanning electron microscopy. The technique fully preserves calcified and stent-expanded tissues, permits a detailed analysis of vascular and valvular composition and architecture, enables discrimination between multiple cell types (including endothelial cells, vascular smooth muscle cells, fibroblasts, adipocytes, mast cells, foam cells, foreign-body giant cells, canonical macrophages, neutrophils, and lymphocytes) and microvascular identities (arterioles, venules, and capillaries), and gives a technical possibility for quantitating the number, area, and density of the blood vessels. Hence, we suggest that our approach is capable of providing a pathophysiological insight into cardiovascular disease development. The protocol does not require specific expertise and can be employed in virtually any laboratory that has a scanning electron microscope.

Development of a decellularized porcine bone graft by supercritical carbon dioxide extraction technology for bone regeneration.

A series of novel decellularized porcine collagen bone graft (DPB) materials in a variety of shapes and sizes were developed by the supercritical carbon dioxide (SCCO2 ) extraction technique. The complete decellularization of DPB was confirmed by hematoxylin and eosin staining, 4,6-diamidino-2-phenylindole (DAPI) staining, and residual DNA analysis. The native intact collagen remained in the DPB after the SCCO2 process was confirmed by Masson trichrome staining. The physicochemical characteristics of DPB were investigated by scanning electron microscopy and x-ray diffraction. The cytotoxicity and biocompatibility tests according to ISO10993 and its efficacy for bone regeneration in osteochondral defects in rabbits were evaluated. The rabbit pyrogen test confirmed DPB was non-toxic. In vitro and in vivo biocompatibility tests of the DPB did not show any toxic or mutagenic effects. The bone regeneration potential of the DPB presented no significant histological differences compared to commercially available deproteinized bovine bone. In conclusion, DPB produced by SCCO2 exhibited similar chemical characteristics to human bone, no toxicity, good biocompatibility, and enhanced bone regeneration in rabbits comparable to that of deproteinized bovine bone. Results from this study could shed light on the potential application of the SCCO2 extraction technique to generate a native decellularized scaffold for bone tissue regeneration in human clinical trials.

A Novel Polyurethane Expandable Root Canal Sealer.

INTRODUCTION: Endodontic sealers play a vital role in the obturation of root canal space. The aim of this study was to evaluate the utility of a recently developed polyurethane expandable sealer (PES), along with its cytotoxicity and dimensional changes. METHODS: L929 fibroblasts and an cell viability assay (MTS assay) were used to determine the cytotoxicity of dental sealers (AH Plus [Dentsply Maillefer, Ballaigues, Switzerland], Sure-Seal Root [Sure Dent Corporation, Gyeonggi-do, South Korea], and the PES) at 24, 48, 72, and 96 hours. An advanced choroidal neovascularization model was used to assess the effect of these sealers on angiogenesis. Thirty-six extracted single-rooted human teeth were prepared and randomly divided into 3 groups (n = 12). Obturation was performed with gutta-percha and a sealer using lateral compaction as follows: group 1, AH Plus; group 2, Sure-Seal; and group 3, PES. The average depth of sealer penetration into dentinal tubules was measured with a scanning electron microscope. Data were analyzed using 1-way analysis of variance and post hoc Tukey tests (level of significance, P < .05). RESULTS: The values of MTS, choroidal neovascularization, and the penetration depth of PES were significantly higher than in other experimental groups (P < .05). The lowest values were noted in specimens of AH Plus, whereas the highest were detected in the PES group. CONCLUSIONS: PES showed promising results in terms of biocompatibility and dentinal tubule adaptation and penetration.

Mineralization-inducing potentials of calcium silicate-based pulp capping materials in human dental pulp cells.

BACKGROUND: To provide a long-term bacterial seal through the formation of reparative dentin bridge, calcium silicate-based pulp capping materials have been used at sites of pulpal exposure. The aim of this study was to evaluate the mineralization-inducing potentials of calcium silicate-based pulp capping materials (ProRoot MTA [PR], Biodentine [BD], and TheraCal LC [TC]) in human dental pulp cells (HDPCs). METHODS: Specimens of test materials were placed in deionized water for various incubation times to measure the pH variation and the concentration of calcium released. The morphology of HDPCs cultured on the specimens was examined using a confocal laser scanning microscope (CLSM). Alizarin red S staining and alkaline phosphatase assays were used to evaluate mineralization-inducing potentials of the capping materials. RESULTS: BD showed the highest calcium release in all test periods, followed by PR and TC. (p<0.05). All experimental groups showed high alkalinity after 1 day, except at 14 days. BD showed the highest cell viability compared with PR and TC after 1 and 3 days, while TC showed the lowest value (p<0.05). The CLSM analysis showed that cells were well adhered and expressed actin filaments for all pulp capping materials. Mineralization by PR and BD groups was higher than that by TC group based on alizarin red S staining. BD showed significantly higher alkaline phosphatase activity than PR and TC, while TC showed the lowest value (p<0.05). CONCLUSION: Within the limitations of the in vitro study, BD had higher mineralization-inducing potential than PR and TC.

Compilation of International Standards and Regulatory Guidance Documents for Evaluation of Biomaterials, Medical Devices, and 3-D Printed and Regenerative Medicine Products.

The development of biomaterials, medical device components, finished medical products, and 3-D printed and regenerative medicine products is governed by a variety of international and country-specific standards and guidelines. Of greatest importance to planning, executing, and reporting biocompatibility, safety and efficacy studies for most biomaterials and medical components or products are the International Organization for Standardization guidelines, U.S. Pharmacopeial Convention, ASTM International, and Conformité Européenne (European Conformity) marking. The International Medical Device Regulators Forum publishes harmonized standards similar to the International Council for Harmonization. Good Laboratory Practices are applicable and guidance documents for the development of drugs and biologics can also be relevant to biomaterials, medical device components, and medical products and more recently to products produced by 3-D printing or additive manufacturing. Regenerative products may have medical device-based scaffolding and may be treated as biologics, reflecting the cell and tissue components. This compilation of international standards and guidelines provides toxicologic pathologists, toxicologists, bioengineers, and allied professionals with an overview of and source for important regulatory documents that may apply to the nonclinical development of their products.

Biocompatibility and Cytotoxic Evaluation of New Sorbent Cartridges for Blood Hemoperfusion.

BACKGROUND/AIMS: The use of adsorption cartridges for hemoperfusion (HP) is rapidly evolving. For these devices, the potential induced cytotoxicity is an important issue. The aim of this study was to investigate potential in vitro cytotoxic effects of different sorbent cartridges, HA130, HA230, HA330, HA380 (Jafron, China), on U937 monocytes. METHODS: Monocytes were exposed to the sorbent material in static and dynamic manners. In static test, cell medium samples were collected after 24 h of incubation in the cartridges. In dynamic test, HP modality has been carried out and samples at 30, 60, 90, and 120 min were collected. RESULTS: Compared to control samples, there was no evidence of increased necrosis or apoptosis in monocytes exposed to the cartridges both in the static and dynamic tests. CONCLUSION: Our in vitro testing suggests that HA cartridges carry an optimal level of biocompatibility and their use in HP is not associated with adverse reactions or signs of cytotoxicity.

Human bone graft cytocompatibility with mesenchymal stromal cells is comparable after thermal sterilization and washing followed by γ-irradiation: an in vitro study.

Human bone allografts present a better alternative to autografts in terms of minimization of the harvesting procedure complications. Prior to the use in clinical applications, they require sterilization which aims to reduce bioburden. This often comes at the expense of their biological properties as carriers of cells. In this study, we evaluated the cytocompatibility of human bone allografts processed and sterilized by three different methods with mesenchymal stromal cells. Bone morphology, biological and biochemical properties of the extracted bone-conditioned medium and viability of cells were assessed. We found that chemical sterilization had a strong negative effect on cell viability, whereas thermal sterilization and washing with subsequent γ-irradiation both resulted in a bone graft compatible with the progenitor cells. Moreover, washing of the bone prior to sterilization allowed solid removal of cell debris and other bone marrow components. Taken together, our findings demonstrate the importance of a proper choice of the bone graft processing method for the production of the biomaterial suitable for tissue engineering.

In vitro and in vivo biocompatibility of reoss® in powder and putty configurations

Article This study evaluated comparatively two configurations (powder and putty) of a composite biomaterial based on PLGA (Poly(lactide-co-glycolide)/nanoescale hydroxyapatite (ReOss®, Intra-Lock International) through microscopic morphology, in vitro cytotoxicity, biocompatibility and in vivo response as a bone substitute. SEM and EDS characterized the biomaterials before/after grafting. Cytocompatibility was assessed with murine pre-osteoblasts. Osteoconductivity and biocompatibility were evaluated in White New Zealand rabbits. Both configurations were implanted in the calvaria of eighteen animals in non-critical size defects, with blood clot as the control group. After 30, 60 and 90 days, the animals were euthanized and the fragments containing the biomaterials and controls were harvested. Bone blocks were embedded in paraffin (n=15) aiming at histological and histomorphometric analysis, and in resin (n=3) aiming at SEM and EDS. Before implantation, the putty configuration showed both a porous and a fibrous morphological phase. Powder revealed porous particles with variable granulometry. EDS showed calcium, carbon, and oxygen in putty configuration, while powder also showed phosphorus. After implantation EDS revealed calcium, carbon, and oxygen in both configurations. The materials were considered cytotoxic by the XTT test. Histological analysis showed new bone formation and no inflammatory reaction at implant sites. However, the histomorphometric analysis indicated that the amount of newly formed bone was not statistically different between experimental groups. Although both materials presented in vitro cytotoxicity, they were biocompatible and osteoconductive. The configuration of ReOss® affected morphological characteristics and the in vitro cytocompatibility but did not impact on the in vivo biological response, as measured by the present model.

Resumo Este estudo avaliou comparativamente duas configurações (pó e massa) de um biomaterial composto com base de PLGA (Poli(láctico-co-glicólico)/hidroxiapatita em nanoescala (ReOss®, Intra-Lock International) através da morfologia microscópica, citotoxicidade in vitro, biocompatibilidade e resposta in vivo como substituto ósseo. MEV e EDS caracterizaram os biomateriais antes/após o enxerto. A citocompatibilidade foi avaliada em pré-osteoblastos murinos. A osteocondutividade e a biocompatibilidade foram avaliadas em coelhos Branco da Nova Zelândia. Ambas as configurações foram implantadas na calvária de dezoito animais em defeitos não-críticos, com coágulo sanguíneo como grupo controle. Após 30, 60 e 90 dias, os animais foram eutanasiados e os fragmentos contendo os biomateriais e controles coletados. Blocos ósseos foram embebidos em parafina (n=15) destinados às análises histológica e histomorfométrica, e em resina (n=3) destinadas à MEV e EDS. Antes da implantação, a configuração massa mostrou ambas fases morfológicas porosa e fibrosa. O pó revelou partículas porosas com granulometria variável. EDS mostrou cálcio, carbono e oxigênio na configuração massa, enquanto o pó mostrou também fósforo. Após a implantação a EDS revelou cálcio, carbono e oxigênio em ambas configurações. Os materiais foram considerados citotóxicos pelo teste XTT. A análise histológica mostrou nova formação óssea e nenhuma reação inflamatória nos sítios de implante. Entretanto, a análise histomorfométrica indicou que a quantidade de osso neoformado não foi estatisticamente diferente entre os grupos experimentais. Embora ambos os materiais tenham apresentado citotoxicidade in vitro, foram biocompatíveis e osteocondutores. A configuração do ReOss® afetou as características morfológicas e a citocompatibilidade in vitro, porém não impactou a resposta biológica in vivo, como medido pelo presente modelo.

pH influences the biocompatibility of methylene blue solutions.

OBJECTIVE: The aim of this study was to investigate the biocompatibility of methylene blue at different pH levels through the method of implantation in subcutaneous tissue. MATERIALS AND METHODS: Eighty-four sterilized polyethylene tubes were allocated in the subcutaneous tissue of 28 rats, each one receiving four tubes, set into four groups: group tube (G-T)-empty tube, fibrin group (G-F)-tube filled with fibrin sponge, group methylene blue pH 7 (G-MB/pH 7)-tube filled with fibrin sponge soaked by methylene blue (100 µg/ml) at pH 7.0, and group methylene blue pH 1 (G-MB/pH 1)-tube filled with fibrin sponge and soaked by methylene blue (100 µg/ml) at pH 1.0. After 7, 15, and 30 days, seven animals from each group were euthanized, and the tubes involved by the surrounding tissue were removed and fixed with 4% buffered formaldehyde solution. The collected pieces were processed and histological sections (4 µm) were stained with hematoxylin and eosin and analyzed by light microscopy. Scores were assigned to analysis of histopathologic parameters. The results were statistically analyzed by the Kruskal-Wallis test (p ≤ 0.05). RESULTS: At 7 and 30 days, the G-MB/pH 1 group showed no significant difference in the G-T control group, while G-MB/pH 7 had a significant increase on tissue reaction, also when compared to G-T. At 15 days, there was no statistical difference between the groups. CONCLUSION: Within the limits of this study, it is concluded that methylene blue at pH 1.0 provides better biocompatibility than at pH 7.0.

Biocompatibilidade da resina acrílica de prótese ocular: análise in vitro e in vivo / Biocompatibility of ocular prosthesis acrylic resin: in vitro and in vivo analysis

O objetivo desse estudo foi avaliar a influência de diferentes ciclos e métodos de polimerização da resina acrílica (RA) branca de próteses oculares sobre a biocompatibilidade de células da conjuntiva humana e resposta inflamatória do tecido subcutâneo de ratos. Para isso, foram confeccionados corpos de prova em RA termopolimerizados em água aquecida (RNAA), por energia de microondas (RNTM) e quimicamente ativados (RNQA). Para a análise in vivo, a resposta inflamatória desses 3 grupos (n=20/grupo) foi avaliada no tecido subcutâneo de 20 ratos Wistar por 7, 15, 30 e 60 dias (d). Células inflamatórias foram contadas no tecido adjacente ao corpo de prova após coloração com hematoxilina e eosina. A análise imunohistoquímica foi realizada para a detecção de IL-1ß, IL-6, TNFα, IL-17 e CCL20. Para a análise in vitro, diferentes ciclos de polimerização para cada método citado foram avaliados, totalizando 11 grupos (n=8/grupo). Foram realizadas análises de grau de conversão (GC), MTT, Alamar Blue, ELISA, RT-PCR em tempo real e dupla marcação de Anexina V e iodeto de propídio. Dados quantitativos foram submetidos à Análise de Variância e ao teste de Tukey com significância de 5%. Os resultados qualitativos foram comparados visualmente. Na análise in vivo, houve infiltrado inflamatório moderado para os grupos RNTM e RNQA e leve para o grupo RNAA após 7 d. O infiltrado inflamatório e a imunomarcação dos alvos testados diminuiu gradativamente ao longo dos 60 d. O grupo RNTM exibiu mais células inflamatórias, com exceção do grupo RNAA, que apresentou mais eosinófilos e linfócitos após 15 d, e do grupo RNQA, onde foi observado mais macrófagos em 15 d e neutrófilos em 60 d. Os grupos RNAA e RNQA apresentaram maior imunomarcação de IL-1ß após 7 d. O grupo RNQA apresentou maior imunomarcação de IL-1ß (15 e 30 d), IL-6 (30 e 60 d), IL-17 (15 e 30 d) e TNF-α (7 d). Os grupos RNAA e RNTM apresentaram maior imunomarcação de TNF-α nos períodos de 15 e 30 d, enquanto o grupo RNTM, aos 60 d. Na análise in vitro, todos os grupos apresentaram proliferação celular maior que 75%. O ciclo longo de polimerização em microondas apresentou menor GC e percentual de proliferação celular no MTT e resultou em grande liberação de IL-2. No ensaio de Alamar Blue, esse grupo apresentou baixo percentual de proliferação celular, assim como o grupo que recebeu ciclo longo de polimerização em água aquecida e grupos submetidos à ativação química. Maior liberação de IL-6 foi observada nos grupos submetidos à ativação química e de IL-23 para o ciclo curto de polimerização em microondas. Maior expressão gênica de TGF ß1 ocorreu para o grupo que recebeu ciclo longo de polimerização em água aquecida seguido de 30 min de armazenamento em água. Maior expressão gênica de CASP9 ocorreu para o grupo ativado quimicamente sobre a bancada. Pode-se concluir que os métodos de polimerização por meio de energia de microondas (ciclos longo e curto) e por ativação química desencadearam uma resposta inflamatória mais intensa. Dentre os métodos de polimerização recomendados pelo fabricante, a polimerização em água aquecida apresentou resultados mais satisfatórios(AU)

The aim of this study was to evaluate the influence of different cycles and methods of white color acrylic resin (AR) for ocular prosthesis on the biocompatibility of human conjunctival cells and on the inflammatory response of rat subcutaneous tissue. For this, AR specimens were prepared in water bath (NRWB), by microwave energy (NRME), and chemically activated (ANR). For in vivo analysis, the inflammatory response of these 3 groups (n=20/group) was assessed in subcutaneous tissue of 20 Wistar rats at 7, 15, 30 and 60 days (d). Inflammatory cells were counted in the tissue adjacent to specimen after staining with hematoxylin and eosin. The immunohistochemical analysis was performed for the detection of IL-1ß, IL-6, TNFα, IL-17, and CCL20. For in vitro analysis, different cycles of polymerization for each method were evaluated, with a total of 11 groups (n=8/group). The degree of conversion (DC), MTT, ELISA, real-time RT-PCR and Annexin V and propidium iodide assays were performed. Quantitative data were submitted to Analysis of Variance and Tukey test with a 5% significance. Qualitative data were submitted to visual comparison. In in vivo analysis, there was a moderate inflammatory infiltrate for groups NRME and ANR, and a light infiltrate for the group NRWB after 7 d. The inflammatory infiltrate and the immunolabeling of tested targets decreased gradually during the 60 d. The group NRME exhibited the highest number of inflammatory cells, except for the group NRWB, which presented a higher number of eosinophils and lymphocytes after 15 d, and for the group ANR, where a higher number of macrophages and neutrophils were observed at 15 d and at 60 d, respectively. Groups NRWB and ANR showed higher IL-1ß immunolabeling after 7 d. The group ANR had the highest immunolabeling of IL-1ß (15 and 30 d), IL-6 (30 and 60 d), IL-17 (15 and 30 d), and TNF-α (7 d). Groups NRWB and NRME showed greater immunolabeling in the periods of 15 and 30 d, while the group NRME had also high results at 60 d. In in vitro analysis, all groups showed cell proliferation higher than 75%. The long cycle of polymerization using microwave energy resulted in lower DC and lower percentage of cell proliferation in the MTT assay and in large release of IL-2. In the Alamar Blue assay, this group had a low percentage of cell proliferation, as well as the group that received a long cycle of polymerization in water bath and groups submitted to chemical activation. A higher release of IL-6 was observed in groups submitted to chemical activation and of IL-23, for the short cycle of polymerization in microwave. Higher TGF ß1 gene expression occurred for the group that received long cycle of polymerization in water bath followed by 30 min of storage in water. Higher CASP 9 gene expression occurred for the chemically activated group on bench. It can be concluded that the polymerization by microwave energy (long and short cycles) and by chemical activation resulted in higher inflammatory response. Among methods recommended by the manufacturer, the water bath polymerization showed more satisfactory results(AU)

Cytocompatibility of a self-adhesive gutta-percha root-filling material.

CONTEXT: A novel root-filling material based on the incorporation of ultrafine alkaline bioactive glass particles (bioactive gutta-percha, [BGP]) was developed to work without sealer. AIM: In the present study, the objective was to verify the in vitro biological response to this material by assessing its cytocompatibility. MATERIALS AND METHODS: Prototypes of BGP were compared to conventional gutta-percha (GP), dense polystyrene beads as a negative control and fragments of latex as a positive control. Extracts of each material were prepared according to ISO 10993-5:2009, and human osteoblast-like cells in primary culture were exposed to all extracts for 24 h. Cell viability was assayed sequentially for three different parameters: mitochondrial activity, membrane integrity, and cell density. STATISTICAL ANALYSIS USED: Nonparametric analysis (using Kruskal-Wallis test combined with post hoc Dunn's test) was performed for comparison among groups, with significance established at 5%. RESULTS: BGP reduced mitochondrial activity to 62% of control, but presented no toxicity on membrane integrity and proliferation assays. BGP effect on metabolism was dose-dependent and reduced to acceptable levels with dilution. CONCLUSION: The novel GP material presented slight dose-dependent effects on cell metabolism but did not affect cell survival.

Elemental ion release and cytotoxicity of antimicrobial acrylic resins incorporated with nanomaterial.

OBJECTIVES: This study evaluated the release of ions and the cytotoxicity of acrylic resins incorporated with silver vanadate decorated with silver nanoparticles (AgVO3 ). BACKGROUND: The inhibition of the accumulation of microorganisms on the resins is critical in preventing diseases. However, the hypothesis is that the release of ions from the incorporation of AgVO3 may be important in biocompatibility. MATERIALS AND METHODS: Specimens of autopolymerising (AP) and heat-polymerising resin (HP) with AgVO3 were prepared and immersed in culture medium. The release of silver ions (Ag) and vanadium (V) was evaluated by mass spectrometry with inductively coupled plasma (ICP-MS) (n=9) and the cell viability of fibroblasts L929 by MTT (3-[4,5-dimethylthiazol- 2yl]-2,5-diphenyltetrazolium bromide) (n=12). The results were evaluated with analysis of variance (ANOVA), Tukey and Pearson correlation test (α=.05). RESULTS: The groups containing AgVO3 presented a difference in relation to the control (0%) regarding the release of Ag and V (P<.0001). All groups showed a reduction in L929 viability when compared with the cellular control (100%) (P<.0001). In comparison with the control resins for HP, a reduction in the metabolism of cells occurred starting at 2.5% and for AP at 5% (P<.0001). A positive correlation was found between the concentration of AgVO3 and the ion release, and a negative between the ion release and the cell viability. CONCLUSIONS: Significant numbers of Ag and V ions were released from resins with higher concentrations of AgVO3 , presenting cytotoxicity for cells, suggesting that the use of low concentrations is indicated to avoid risks to patients.

A cytotoxicity study of silicon oxycarbide nanowires as cell scaffold for biomedical applications.

GOAL: Nanowires are promising biomaterials in multiple clinical applications. The goal of this study was to investigate the cytotoxicity of carbon-doped silica nanowires (SiOxCy NWs) on a fibroblastic cell line in vitro. MATERIALS AND METHODS: SiOxCy NWs were grown on Si substrates by CVD process. Murine L929 fibroblasts were cultured in complete DMEM and indirect and direct cytotoxicity tests were performed in agreement with ISO 19003-5, by quantitating cell viability at MTT and chemiluminescent assay. Cell cultures were investigated at Scanning Electron Microscope (SEM) and immunocytochemistry to observe their morphology and investigate cell-NWs interactions. Furthermore, hemocompatibility with Platelet-rich Plasma was assayed at SEM and by ELISA assay. RESULTS: SiOxCy NWs proved biocompatible and did not impair cell proliferation at contact assays. L929 were able to attach on NWs and proliferate. Most interestingly, L929 reorganised the NW scaffold by displacing the nanostructure and creating tunnels within the NW network. NWs moreover did not impair platelet activation and behaved similarly to flat SiO2. CONCLUSIONS: Our data show that SiOxCy NWs did not release cytotoxic species and acted as a viable and adaptable scaffold for fibroblastic cells, thus representing a promising platform for implantable devices.

Enhanced vascular biocompatibility of decellularized xeno-/allogeneic matrices in a rodent model.

Glutaraldehyde preservation is the gold standard for cardiovascular biological prosthesis. However, secondary calcifications and the absence of tissue growth remain major limitations. Our study assessed in vitro and in vivo the biocompatibility of human (fascia lata, pericardium) and porcine tissues (pericardium, peritoneum) treated with a physicochemical procedure for decellularization and non-conventional pathogens inactivation. Biopsies were performed before and after treatment to assess decellularization (HE/Dapi staining/DNA quantification/MHC I/alpha gal immunostaining) and mechanical integrity. Forty-five rats received an abdominal aortic patch of native cryopreserved tissues (n = 20), treated tissues (n = 20) or glutaraldehyde-preserved bovine pericardium (GBP, control, n = 5). Grafts were explanted at 4 weeks and processed for HE/von Kossa staining and immunohistochemistries for lymphocytes (CD3)/macrophages (CD68) histomorphometry. 95% of decellularization was obtained for all tissues except for fascia lata (75%). Mechanical properties were slightly altered. In the in vivo model, a significant increase of CD3 and CD68 infiltrations was found in native and control implants in comparison with decellularized tissues (p < 0.05). Calcifications were found in 3 controls. Decellularized tissues were recolonized. GBP showed the most inflammatory response. This physicochemical treatment improves the biocompatibility of selected xeno/allogeneic tissues in comparison with their respective native cryopreserved tissues and with GBP. Incomplete decellularization is associated with a significantly higher inflammatory response. Our treatment is a promising tool in the field of tissue decellularization and tissue banking.