In vitro evaluation of crosslinked bovine pericardium as potential scaffold for the oral cavity.

BACKGROUND: Bovine pericardium (BP) is a scaffold widely used in soft tissues regeneration; however, its calcification in contact with glutaraldehyde, represent an opportunity for its application in hard tissues, such as bone in the oral cavity. OBJECTIVE: To develop and to characterize decellularized and glutaraldehyde-crosslinked bovine pericardium (GC-BP) as a potential scaffold for guided bone regeneration GBR. METHODS: BP samples from healthy animals of the bovine zebu breed were decellularized and crosslinked by digestion with detergents and glutaraldehyde respectively. The resulting cell-free scaffold was physical, chemical, mechanical, and biologically characterized thought hematoxylin and eosin staining, DNA quantification, scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR), thermogravimetric analysis (TGA), differential scanning calorimetry (DSC), uniaxial tensile test, cell viability and live and dead assay in cultures of dental pulp stem cells (DPSCs). RESULTS: The decellularization and crosslinking of BP appeared to induce conformational changes of the CLG molecules, which led to lower mechanical properties at the GC-BP scaffold, at the same time that promoted cell adhesion and viability of DPSCs. CONCLUSION: This study suggests that the decellularized and GC-BP is a scaffold with the potential to be used promoting DPSCs recruitment, which has a great impact on the dental area.

Resin-based materials to control human dentin permeability under erosive conditions in vitro: A hydraulic conductance, confocal microscopy and FTIR study.

OBJECTIVES: To characterize the behavior of three different polymeric agents before and after an erosive challenge on dentin permeability, to analyze their degradation in both conditions, and to analyze their degree of conversion (DC). METHODS: The permeability of human dentin disks (1.0 ± 0.2 mm) was measured with smear layer, after its removal, after treatment (LpTreat) with Gluma Desensitizer, PRG Barrier Coat (PBC) or Icon infiltrant (n = 11/group) and after exposure to citric acid (LpEro) (6%, pH 2.1, 1 min). The specimens were analyzed under a Laser Scanning Confocal Microscope (n = 2/group) and the products' DC were calculated. Data were subjected to 2-way repeated measures ANOVA and post-hoc Bonferroni (permeability analysis), to paired t-test (for specimens treated with Icon) and to t-test (DC analysis) (α < 0.05). RESULTS: Icon showed the lowest LpTreat and LpEro values, while PBC and Gluma did not differ from each other under these conditions. Icon and PBC showed LpEro similar to a dentin with smear layer. Under the Laser Scanning Confocal Microscope, more deposits were noticeable on dentin after treating with PBC. Gluma presented the deepest penetration in dentin. The DC of PBC was the highest. SIGNIFICANCE: Icon caused the highest reduction on permeability values, followed by PBC and Gluma. PBC generated more deposits covering dentin and seemed to be more efficient after an erosive challenge. The association of a polymeric resin with inorganic ion-releasing fillers seem to be a great strategy to manage dentin hypersensitivity under erosive conditions.

Adventitial Collagen Cross-Linking by Glutaraldehyde Reinforcing Human Saphenous Vein - Implication for Coronary Artery Bypass Grafting.

INTRODUCTION: A weak venous wall is one of the major reasons contributing to vein graft failure after coronary artery bypass grafting (CABG). We investigated whether adventitial collagen cross-linking by glutaraldehyde reinforces venous wall, preserving the endothelium of veins during high-pressure distention. METHODS: Human saphenous veins (SVs) were collected from 40 patients undergoing CABG, and adventitia cross-linking was performed with 0.3% glutaraldehyde for five minutes. The cross-linked SVs were accessed by biodegradation assay, immunofluorescent staining, and tensile test. Native SVs and cross-linked SVs from another 20 patients received the 200 mmHg pressure distention for two minutes. Pressure-induced injury of SVs were accessed by immunohistochemistry and electron microscopy. RESULTS: Time to digestion was 97±13 minutes for native SVs and 720±0 minutes for cross-linked SVs (P<0.05). After adventitial cross-linking, the collagen I fibres of the vein remarkably presented with compact and nonporous arrangement. In the high-stretch region (stretch ratio 1.4-1.8), the Young's elastic modulus of stress-stretch ratio curve in cross-linked SVs was larger than that in native SVs (13.88 vs. 5.83, P<0.05). The cross-linked SVs had a lower extent of endothelial denudation without fibre fracture during high-pressure distension than native SVs. Comparing with the non-cross-linked SVs, the percentage of endothelial nitric oxide synthase staining length on the endothelium of cross-linked SVs was significantly preserved after high-pressure distension (85.2% vs. 64.7%, P<0.05). CONCLUSION: Adventitial collagen cross-linking by glutaraldehyde reinforced venous wall by increasing stiffness and decreasing extensibility of SVs and mitigated the endothelial damage under high-pressure distension.

The impact of bacterial diversity on resistance to biocides in oilfields.

Extreme conditions and the availability of determinate substrates in oil fields promote the growth of a specific microbiome. Sulfate-reducing bacteria (SRB) and acid-producing bacteria (APB) are usually found in these places and can harm important processes due to increases in corrosion rates, biofouling and reservoir biosouring. Biocides such as glutaraldehyde, dibromo-nitrilopropionamide (DBNPA), tetrakis (hydroxymethyl) phosphonium sulfate (THPS) and alkyl dimethyl benzyl ammonium chloride (ADBAC) are commonly used in oil fields to mitigate uncontrolled microbial growth. The aim of this work was to evaluate the differences among microbiome compositions and their resistance to standard biocides in four different Brazilian produced water samples, two from a Southeast Brazil offshore oil field and two from different Northeast Brazil onshore oil fields. Microbiome evaluations were carried out through 16S rRNA amplicon sequencing. To evaluate the biocidal resistance, the Minimum Inhibitory Concentration (MIC) of the standard biocides were analyzed using enriched consortia of SRB and APB from the produced water samples. The data showed important differences in terms of taxonomy but similar functional characterization, indicating the high diversity of the microbiomes. The APB and SRB consortia demonstrated varying resistance levels against the biocides. These results will help to customize biocidal treatments in oil fields.

In vivo evaluation of Vivere bovine pericardium valvular bioprosthesis with a new anti-calcifying treatment.

The objective of this study was to evaluate the effect of chemical treatment with glutamic acid to avoid calcification of biological cardiac valves. The bovine pericardium (BP) tissues were fixed with 0.5% glutaraldehyde (BP/GA), followed by treatment with glutamic acid (BP/GA + Glu) for neutralization of the free aldehyde groups. Microscopic analysis showed that the wavy structure of collagen fibrils was preserved, but changes in elastin's integrity occurred. However, the treatment did not promote undesirable changes in the thermal and mechanical properties of the modified BPs. These samples were systematically studied in rat subcutaneous tissue: control (BP/GA) and anticalcificant (BP/GA + Glu). After 60 days, both groups induced similar inflammatory reactions. In terms of calcification, BP/GA + Glu remained more stable with a lower index (3.1 ± 0.2 µg Ca2+ /mg dry tissue), whereas for BP/GA it was 5.7 ± 1.3 µg Ca2+ /mg dry tissue. Bioprostheses made from BP/GA + Glu were implanted in the pulmonary position in sheep, and in vivo echocardiographic analyses revealed maintenance of valvar function after 180 days, with low gradients and minimal valve insufficiency. The explanted tissues of the BP/GA + Glu group had a lower average calcium content 3.8 ± 3.0 µg Ca2+ /mg dry tissue. The results indicated high anticalcification efficiency of BP/GA + Glu in both subcutaneous implant in rats and in the experimental sheep model, which is an advantage that should encourage the industrial application of these materials for the manufacture of bioprostheses.

Combination of fixative agents and fixation times to visually differentiate the cortical from the medullary layer in bovine adrenal glands.

Chronic stress exposure commonly increases adrenals weight and changes their morphology. This study aimed to compare four methods to delimitate the cortical and medullary layers of adrenals glands in Nelore bulls. Fresh adrenals did not present differentiation between layers. Then, frozen adrenals were distributed in plastics bags with fixative Bouin (G1), 96ºGL ethylic alcohol (G2), 10% formaldehyde (G3), or 2.5% glutaraldehyde (G4). After 12 hours of fixation, the G1 adrenal glands did not show the entire cortical layer marked by Bouin's solution. For G2 and G3 there was a poor contrast, while for G4 there was a reasonable contrast. After 24 hours of fixation, G1 had an excellent contrast between layers, while G2 and G4 had a reasonable contrast and G3 a very bad contrast. After 48 hours it was difficult to differentiate cortical and medullar layers for G1; for Group 2 we get a reasonable contrast; and for G3 the contrast was bad. For G4 the contrast was not as sharp due to the medulla became dark. It was concluded that fixation of adrenals must be done in Bouin's solution for 24 hours to obtain an effective evaluation of the adrenals' morphometry.

Synergism between high hydrostatic pressure and glutaraldehyde for the inactivation of Staphylococcus aureus at moderate temperature.

The sterilization of transplant and medical devices should be effective but not detrimental to the structural properties of the materials used. In this study, we examined the effectiveness of chemical and physical agents for inactivating Staphylococcus aureus, a gram-positive bacterium and important cause of infections and biofilm production. The treatment conditions in this work were chosen to facilitate their subsequent use with sensitive materials. The effects of temperature, high hydrostatic pressure, and glutaraldehyde disinfectant on the growth of two strains of S. aureus (ATCC 25923 and BEC 9393) were investigated individually and/or in combinations. A low concentration of glutaraldehyde (0.5 mM), high hydrostatic pressure (300 MPa for 10 min), and moderate temperature (50 °C), when used in combination, significantly potentiated the inactivation of both bacterial strains by > 8 orders of magnitude. Transmission electron microscopy revealed structural damage and changes in area that correlated with the use of pressure in the presence of glutaraldehyde at room temperature in both strains. Biofilm from strain ATCC 25923 was particularly susceptible to inactivation. The conditions used here provided effective sterilization that can be applied to sensitive surgical devices and biomaterials, with negligible damage. The use of this experimental approach to investigate other pathogens could lead to the adoption of this procedure for sterilizing sensitive materials.

Comparison of Antimicrobial Activity between Chemical Disinfectants on Contaminated Orthodontic Pliers.

AIM: To compare the antimicrobial activity of the chemical substances--70% isopropyl alcohol, 2% glutaraldehyde (GTA) and 0.25% peracetic acid (PAA) in disinfecting orthodontic pliers contaminated in vitro with Streptococcui mutani, Staphylococci aureui and Candida albicani. MATERIALS AND METHODS: Distal end cutter pliers were divided into five groups: group 1 (negative control--sterilized pliers), group 2 (positive control--sterilized plier, subsequently contaminated), group 3 (disinfected with 70% isopropyl alcohol, friction method), group 4 (disinfected with 2% GTA, immersion method for 30 minutes), group 5 (disinfected with 0.25% peracetic acid (PAA), immersion method for 10 minutes). After the pliers were treated with one disinfectant and submitted to microbiological evaluation (by counting colony forming units), they were submitted to the same cleansing, sterilizing and contaminating processes, and were used in the following groups (crossover and washout study). The two-factor analysis of variance (ANOVA) test, followed by the Tukey test, was used to compare the groups. RESULTS: The results showed that there was no statistically significant difference between the three tested disinfectants. CONCLUSION: Although there were no statistically significant differences between the disinfectants, the chemical agents 2% glutaraldehyde and 0.25% PAA were effective in inhibiting the growth of the three microorganisms tested; however, 70% isopropyl alcohol was unable to completely eliminate S. aureui. CLINICAL SIGNIFICANCE: The chemical substances 2% glutaraldehyde and 0.25% PAA completely eliminated the microorganisms tested.

Analysis of a panel of rapidly growing mycobacteria for resistance to aldehyde-based disinfectants.

After several accounts across the globe of mycobacteria outbreaks associated with medical procedures and aldehyde disinfectants resistance, we undertook an analysis of mycobacteria isolated from patients seen in a hospital in the United States between 1994 and 2008 to determine prevalence of resistance to aldehyde-based disinfectants. Out of the 117 clinical isolates screened, 6 isolates belonging to the emerging Mycobacterium abscessus group were found to display significant levels of resistance to glutaraldehyde and ortho-phthalaldehyde.

Cytotoxicity of PVPAC-treated bovine pericardium: a potential replacement for glutaraldehyde in biological heart valves.

Acellular biological tissues, including bovine pericardium (BP), have been proposed as biomaterial for tissue engineering. BP is usually modified chemically to improve mechanical and biological properties using glutaraldehyde, the standard reagent for preservation of fresh bioprosthetic materials. Glutaraldehyde-fixed BP (Glut-BP), the most widely used material in heart valve manufacture, has been associated with calcification in vivo. In an attempt to reduce this issue and maintain its biocompatibility, this study assesses the physical properties and cytotoxicity of lyophilized BP treated with poly (vinylpyrrolidone-co-acrolein) (PVPAC-BP), a novel copolymer, as a substitute for glutaraldehyde. For that, PVPAC-BP surface ultrastructure, elastic function, water uptake and tissue calcification were evaluated. For the analysis of biocompatibility, fibroblasts (3T3-L1) and endothelial cells (HUVEC) were cultured on PVPAC-BP, Untreated-BP and Glut-BP. Nitric oxide (NO) release assay, fluorescence and SEM images of endothelial cells adhered on scaffolds were also performed. As results, the data show some advantages of PVPAC-BP over the Glut-BP. The PVPAC-BP maintains partially the original ultrastructure and elastic properties, improves scaffold hydration, and presents less calcium phosphate deposits. The cells demonstrated strong attachment, high proliferation rate, and formation of a monolayer on PVPAC-BP. Attached cells were also able to release NO de-monstrating regular metabolism. In conclusion, PVPAC may be considered as a promising alternative to BP treatment improving the efficiency of cell attachment and proliferation and also avoid immunogenicity.

Effect of hospital disinfectants on spores of clinical Brazilian Clostridium difficile strains.

The aim of this study was to evaluate the sporicidal activity of hospital disinfectants against spores of two Brazilian Clostridium difficile ribotypes and the BI/NAP1/027. Our results showed that CloroRio(®) and Cidex Opa(®) were the most efficient agents for eliminating spores of C difficile.

Stability and mechanical evaluation of bovine pericardium cross-linked with polyurethane prepolymer in aqueous medium.

The present study investigates the potential use of non-catalyzed water-soluble blocked polyurethane prepolymer (PUP) as a bifunctional cross-linker for collagenous scaffolds. The effect of concentration (5, 10, 15 and 20%), time (4, 6, 12 and 24 h), medium volume (50, 100, 200 and 300%) and pH (7.4, 8.2, 9 and 10) over stability, microstructure and tensile mechanical behavior of acellular pericardial matrix was studied. The cross-linking index increased up to 81% while the denaturation temperature increased up to 12 °C after PUP crosslinking. PUP-treated scaffold resisted the collagenase degradation (0.167±0.14 mmol/g of liberated amine groups vs. 598±60 mmol/g for non-cross-linked matrix). The collagen fiber network was coated with PUP while viscoelastic properties were altered after cross-linking. The treatment of the pericardial scaffold with PUP allows (i) different densities of cross-linking depending of the process parameters and (ii) tensile properties similar to glutaraldehyde method.

Estudo in vivo do comportamento de bioprótese liofilizada: seguimento de 3 meses em carneiros jovens / In vivo study of lyophilized bioprostheses: 3 month follow-up in young sheep

OBJETIVO: Para melhorar as propriedades mecânicas e imunogênicas, o glutaraldeído é utilizado no tratamento do pericárdio bovino que é utilizado em biopróteses. A liofilização do pericárdio bovino tratado com glutaraldeído diminui os radicais aldeído, com provável redução do potencial para calcificação. O objetivo deste estudo é avaliar os efeitos da liofilização em biopróteses valvares de pericárdio bovino como mecanismo protetor na diminuição da disfunção estrutural valvar. MÉTODOS: Foi realizado o implante de biopróteses de pericárdio bovino tratado com glutaraldeído, liofilizadas ou não, em carneiros de 6 meses de idade, sendo os animais eutanasiados com 3 meses de seguimento. As biopróteses foram implantadas em posição pulmonar, com auxílio de circulação extracorpórea. Um grupo controle e outro grupo liofilizado foram avaliados quanto ao gradiente ventrículo direito/artéria pulmonar (VD/AP) no implante e explante; análise quantitativa de cálcio; inflamação; trombose e pannus. O nível de significância estabelecido foi de 5%. RESULTADOS: O gradiente médio VD/AP, no grupo controle, no implante, foi 2,04 ± 1,56 mmHg e, no grupo de liofilização, foi 6,61 ± 4,03 mmHg. No explante, esse gradiente aumentou para 7,71 ± 3,92 mmHg e 8,24 ± 6,2 mmHg, respectivamente, nos grupos controle e liofilização. O teor de cálcio médio, após 3 meses, nas biopróteses do grupo controle foi 21,6 ± 39,12 µg Ca+2/mg de peso seco, em comparação com um teor médio de 41,19 ± 46,85 µg Ca+2/mg de peso seco no grupo liofilizado (P = 0,662). CONCLUSÃO: A liofilização de próteses valvares com pericárdio bovino tratado com glutaraldeído não demonstrou diminuição da calcificação neste experimento.

OBJECTIVE: Glutaraldehyde is currently used in bovine pericardium bioprosthesis to improve mechanical and immunogenic properties. Lyophilization is a process that may decrease aldehyde residues in the glutaraldehyde treated pericardium decreasing cytotoxicity and enhancing resistance to calcification. The aim of this study is to evaluate bioprosthetic heart valves calcification in adolescent sheep and to study the potential of lyophilization as a mechanism to protect calcification. METHODS: Two groups were evaluated: a control group in which a bovine pericardium prosthetic valve was implanted in pulmonary position and a lyophilized group in which the bovine pericardium prosthetic valve was lyophilized and further implanted. Sixteen sheeps 6 months old were submitted to the operation procedure. After 3 months the sheeps were euthanized under full anesthesia. RESULTS: Six animals of the control group reached 95.16 ± 3.55 days and six animals in the lyophilized group reached 91.66 ± 0.81 days of postoperative evolution. Two animals had endocarditis. Right ventricle/pulmonary artery (RV/PA) mean gradient, in the control group, at the implantation was 2.04 ± 1.56 mmHg, in the lyophilization group, the RV/PA mean gradient, at the implantation was 6.61 ± 4.03 mmHg. At the explantation it increased to 7.71 ± 3.92 mmHg and 8.24 ± 6.25 mmHg, respectively, in control and lyophilization group. The average calcium content, after 3 months, in the control group was 21.6 ± 39.12 µg Ca+2/mg dry weight, compared with an average content of 41.19 ± 46.85 µg Ca+2/mg dry weight in the lyophilization group (P=0.662). CONCLUSION: Freeze drying of the bovine pericardium prosthesis in the pulmonary position could not demonstrate calcification mitigation over a 3 month period although decreased inflammatory infiltration over the tissue.

Effect of SDS-based decelullarization in the prevention of calcification in glutaraldehyde-preserved bovine pericardium: study in rats.

OBJECTIVE: The aim of study was to investigate the SDS-based decellularization process as an anticalcification method in glutaraldehyde-preserved bovine pericardium in subcutaneous rat model. METHODS: Pericardium samples with 0.5 cm² area were divide in four groups: group GDA: 0.5% glutaraldehyde-preserved pericardium (GDA); group GDA-GL: GDA + 0.2% glutamic acid (GL); group D-GDA: decellularized (D) pericardium with 0.1% SDS + GDA and group D-GDA-GL: decellularized pericardium + GDA + 0.2% glutamic acid. After this samples were implanted in 18 rats in subcutaneous position till 90 days. Each animal received samples of the four groups. The explants were performed at 45 and 90 days. The explants were subjected to histology in glass slides stained with hematoxilin-eosin and alizarin red, morphometry evaluation and the calcium content was measured by flame atomic absorption spectrometry. RESULTS: The inflammatory infiltrate was the same in all groups, however more intense in GDA and GDA-GL groups in 45 days, increasing at 90 days. The calcium contents for 45 days were: 32.52 ± 3.19 µg/mg in GDA group; 22.12 ± 3.87 µg/ mg in GDA-GL group; 1.06 ± 0.38 µg/mg in D-GDA group and 3.99 ± 5.78 µg/mg in D-GDA-GL (P< 0.001). For 90 days were 65.91 ± 24.67 µg/mg in GDA group; 38.37 ± 13.79 µg/mg in GDA-GL group; 1.24 ± 0.99 µg/mg in D-GDA group and 30.54 ± 8.21 µg/mg in D-GDA-GL (P< 0.001). Only D-GDA did not show increase rates of calcium at 45 to 90 days (P=0.314). CONCLUSION: SDS-based decellularization process reduced the inflammatory intensity and calcification in bovine pericardium in subcutaneous rat model for 90 days.

Corneal crosslinking with genipin, comparison with UV-riboflavin in ex-vivo model.

PURPOSE: To investigate the efficacy and safety of Genipin and UV-riboflavin crosslinking (UV-CLX) in corneal crosslinking. METHODS: Porcine eyes were separated in groups for each crosslinker, genipin 0.25% UV-CLX (clinical crosslinking procedure), glutaraldehyde 0.1% (gold standard crosslinker), and control eyes. Intraocular pressure (IOP) was continuously monitored by a pressure sensor cannulated to the anterior chamber and the volume was changed. The changes in ocular pressure as a function of change of the ocular volume were evaluated. Ocular rigidity was calculated as the exponential of polynomial quadratic fit. Endothelial damage was evaluated in a viability assay with alizarin red staining as the changes in cell counts. RESULTS: Significant changes in IOP were observed in the globes were the cornea was stiffened with genipin and UV-CLX treatment (volume 200 µl: Genipin 19.4 mmHg, UVCRX 18.8 mmHg, glutaraldehide 23.9 mmHg, versus control 14.7 mmHg, and 400 µl genipin 31.5 mmHg, UV-CLX 26.0 mmHg, glutaraldehide 37.3 mmHg versus control 18.7 mmHg). The mean ocular ridigity coefficient was genipin 0.0078 mmHg/µl, UV-CLX 0.0065 mmHg/µl, glutaraldehide 0.0092 mmHg/µl, and 0.0046 mmHg/µl for control eyes. Endothelial cell damage was 5.9±1.8% (control), 10.3±1.7% (UV-CLX), 9.4±1.5% (Genipin 0.25%), and 40.1±6.2% (glutaraldehide). Some granules were observed in the UV-CLX group. Reduction of keratocites was observed in the UV CRX crosslinking. CONCLUSIONS: Corneal crosslinking was similar between UV-CLX and genipin with minimal toxicity to endothelial cells. Stiffened corneas by any method induced substancially higher IOP elevation when ocular volume is increased.

Efeito da descelularização com SDS na prevenção da calcificação em pericárdio bovino fixado em glutaraldeído: estudo em ratos / Effect of SDS-based decelullarization in the prevention of calcification in glutaraldehyde-preserved bovine pericardium: study in rats

OBJETIVO: Avaliar a descelularização com SDS como tratamento anticalcificante em pericárdio bovino fixado em glutaraldeído. MÉTODOS: Peças de 0,5 cm² foram implantadas em modelo subcutâneo de 18 ratos por até 90 dias. Foram formados quatro grupos: grupo GDA: pericárdio fixado em glutaraldeído 0,5% (GDA), grupo GDA-GL: pericárdio fixado em GDA + ácido glutâmico (GL) 0,2%, grupo D-GDA: pericárdio descelularizado (D) com SDS 0,1% e fixado em GDA e grupo D-GDA-GL: pericárdio descelularizado + GDA + ácido glutâmico 0,2%. Cada animal recebeu enxertos dos quatro grupos. Os explantes foram realizados com 45 e 90 dias. As avaliações foram: análise histológica com as colorações hematoxilina-eosina e alizarina-red, análise morfométrica e quantificação de cálcio por espectrometria de absorção atômica. RESULTADOS: O padrão de infiltrado inflamatório foi o mesmo nos quatro grupos, sendo mais intenso nos grupos GDA e GDA-GL aos 45 dias, ficando mais evidente aos 90 dias. O conteúdo de cálcio aos 45 dias foi de 32,52 ± 3,19 µg/ mg no grupo GDA; 22,12 ± 3,87 µg/mg no grupo GDA-GL; 1,06 ± 0,38 µg/mg no grupo D-GDA e 3,99 ± 5,78 µg/mg no grupo D-GDA-GL (P< 0,001). Aos 90 dias, foi de 65,91 ± 24,67 µg/mg no grupo GDA; 38,37 ± 13,79 µg/mg no grupo GDA-GL; 1,24 ± 0,99 µg/mg no grupo D-GDA e 30,54 ± 8,21 µg/mg no grupo D-GDA-GL (P< 0,001). O grupo D-GDA foi o único que não apresentou progressão da calcificação de 45 para 90 dias (P=0,314). CONCLUSÃO: A descelularização com SDS reduziu o processo inflamatório e inibiu a calcificação em pericárdio bovino implantado em modelo subcutâneo de ratos até 90 dias.

OBJECTIVE: The aim of study was to investigate the SDS-based decellularization process as an anticalcification method in glutaraldehyde-preserved bovine pericardium in subcutaneous rat model. METHODS: Pericardium samples with 0.5 cm² area were divide in four groups: group GDA: 0.5% glutaraldehydepreserved pericardium (GDA); group GDA-GL: GDA + 0.2% glutamic acid (GL); group D-GDA: decellularized (D) pericardium with 0.1% SDS + GDA and group D-GDA-GL: decellularized pericardium + GDA + 0.2% glutamic acid. After this samples were implanted in 18 rats in subcutaneous position till 90 days. Each animal received samples of the four groups. The explants were performed at 45 and 90 days. The explants were subjected to histology in glass slides stained with hematoxilin-eosin and alizarin red, morphometry evaluation and the calcium content was measured by flame atomic absorption spectrometry. RESULTS: The inflammatory infiltrate was the same in all groups, however more intense in GDA and GDA-GL groups in 45 days, increasing at 90 days. The calcium contents for 45 days were: 32.52 ± 3.19 µg/mg in GDA group; 22.12 ± 3.87 µg/ mg in GDA-GL group; 1.06 ± 0.38 µg/mg in D-GDA group and 3.99 ± 5.78 µg/mg in D-GDA-GL (P< 0.001). For 90 days were 65.91 ± 24.67 µg/mg in GDA group; 38.37 ± 13.79 µg/mg in GDA-GL group; 1.24 ± 0.99 µg/mg in D-GDA group and 30.54 ± 8.21 µg/mg in D-GDA-GL (P< 0.001). Only D-GDA did not show increase rates of calcium at 45 to 90 days (P=0.314). CONCLUSION: SDS-based decellularization process reduced the inflammatory intensity and calcification in bovine pericardium in subcutaneous rat model for 90 days.

In vivo study of lyophilized bioprostheses: 3 month follow-up in young sheep.

OBJECTIVE: Glutaraldehyde is currently used in bovine pericardium bioprosthesis to improve mechanical and immunogenic properties. Lyophilization is a process that may decrease aldehyde residues in the glutaraldehyde treated pericardium decreasing cytotoxicity and enhancing resistance to calcification. The aim of this study is to evaluate bioprosthetic heart valves calcification in adolescent sheep and to study the potential of lyophilization as a mechanism to protect calcification. METHODS: Two groups were evaluated: a control group in which a bovine pericardium prosthetic valve was implanted in pulmonary position and a lyophilized group in which the bovine pericardium prosthetic valve was lyophilized and further implanted. Sixteen sheep 6 months old were submitted to the operation procedure. After 3 months the sheep were euthanized under full anesthesia. RESULTS: Six animals of the control group reached 95.16 ± 3.55 days and six animals in the lyophilized group reached 91.66 ± 0.81 days of postoperative evolution. Two animals had endocarditis. Right ventricle/pulmonary artery (RV/PA) mean gradient, in the control group, at the implantation was 2.04 ± 1.56 mmHg, in the lyophilization group, the RV/PA mean gradient, at the implantation was 6.61 ± 4.03 mmHg. At the explantation it increased to 7.71 ± 3.92 mmHg and 8.24 ± 6.25 mmHg, respectively, in control and lyophilization group. The average calcium content, after 3 months, in the control group was 21.6 ± 39.12 µg Ca+2/mg dry weight, compared with an average content of 41.19 ± 46.85 µg Ca+2/mg dry weight in the lyophilization group (P=0.662). CONCLUSION: Freeze drying of the bovine pericardium prosthesis in the pulmonary position could not demonstrate calcification mitigation over a 3 month period although decreased inflammatory infiltration over the tissue.

Comparative study with two different enrichments in the culture media used in the disinfectant efficacy assay.

Recent changes in Brazilian legislation for commercial disinfectants have been published due to the recent epidemic of nosocomial infections caused by rapidly growing mycobacteria (RGM) in many states of Brazil over the last 8years. One of these documents requires that all the manufacturers provide evidence of efficacy of sterilizing and disinfectant products, used for semi critical medical devices, against the Mycobacterium bovis BCG Moreau and Mycobacterium abscessus subsp. bolletii INCQS 00594 strains by using the Confirmative in vitro Test for Determining Tuberculocidal Activity of Disinfectants recommended by the Association of Official Analytical Chemists. These changes have caused additional costs and increased problems for importation of enrichment products at national laboratories where disinfectant efficacy assay service is performed. Middlebrook ADC Enrichment (ADC) is provided by a unique manufacturer and used in the official protocol. The aim of the present study was to evaluate an alternative in house low-cost enrichment composed of fetal bovine serum and glucose (FBSG) with ADC for performance of disinfectant efficacy assay against mycobacteria. After obtaining the growth curves for M. abscessus ATCC 19977, M. abscessus subsp. bolletii INCQS 00594, Mycobacterium chelonae ATCC 35752, and Mycobacterium fortuitum ATCC 6841 by using ADC enrichment and FBSG in Kirchners and 7H9 culture media. Through statistical analysis via the Kruskal-Wallis test on the evaluation of microorganism growth rate, it was observed that there was no inhibition of RGM growth by any of the enrichments used. These results suggest that low-cost enrichment FBSG may be used as a potential substitute of ADC for composition of media for mycobacterial growth, including in disinfectant tests.

Bovine pericardium retail preserved in glutaraldehyde and used as a vascular patch.

BACKGROUND: In this study we evaluated the performance of bovine pericardium preserved in glutaraldehyde used as a vascular patch. METHODS: Fourteen young pigs, six females and eight males, weighting 10.3 - 18.4 kg were used in our study. We implanted three remnants in each pig, two in the abdominal aorta and one was juxtaposed to the peritoneum. The smooth face (SF) and rough face (RF) of each remnant were implanted turned to the vessel inner portion and one remnant was juxtaposed to the peritoneum. The animals were sacrificed in 4.5 - 8 months after surgery (75 - 109 kg). The remnants were assessed for aorta wall, fibroses formation in inner apposition and calcification related to the face turned to the vessel inner portion. RESULTS: The rough face showed a lower dilatation level compared to the face implanted in adjacent aorta. There was no difference between intensity and/or incidence of graft calcification when the superficies were compared. The bovine pericardium preserved in glutaraldehyde did not show alterations in its structure when implanted with different faces turned to the inner portion of vessel. CONCLUSION: When turned to the inner portion of the vessel, the rough face of the remnant presented a lower dilatation in relation to the adjacent aorta and a better quality of endothelium layer and did not show a difference between intensity and/or incidence of graft calcification.

Effect of repeated immersion solution cycles on the color stability of denture tooth acrylic resins

OBJECTIVE: Chemical solutions have been widely used for disinfection of dentures, but their effect on color stability of denture tooth acrylic resins after repeated procedures is still unclear. The aim of this in vitro study was to evaluate whether repeated cycles of chemical disinfectants affected the color stability of two denture tooth acrylic resins. MATERIAL AND METHODS: Sixty disc-shaped specimens (40 mm x 3 mm) were fabricated from two different brands (Artiplus and Trilux) of denture tooth acrylic resin. The specimens from each brand (n=30) were randomly divided into 6 groups (n=5) and immersed in the following solutions: distilled water (control group) and 5 disinfecting solutions (1 percent sodium hypochlorite, 2 percent sodium hypochlorite, 5.25 percent sodium hypochlorite, 2 percent glutaraldehyde, and 4 percent chlorhexidine gluconate). Tooth color measurements were made by spectrophotometry. Before disinfection, the initial color of each tooth was recorded. Further color measurements were determined after subjecting the specimens to 7, 21, 30, 45, 60, and 90 immersion cycles in each tested solution. Color differences (ΔE*) were determined using the CIE L*a*b* color system. Data were analyzed using two-way repeated measures analysis of variance (ANOVA) followed by Tukey tests. The significance level was set at 5 percent. RESULTS: There were statistically significant differences in ΔE* among the 5 disinfectants and water during the 90 cycles of immersion for both denture tooth acrylic resins. Distilled water promoted the greatest color change in both denture tooth acrylic resins, nevertheless none of tested disinfectants promoted ΔE* values higher than 1.0 on these acrylic materials during the 90 cycles of disinfection. CONCLUSIONS: Repeated immersion cycles in disinfecting solutions alter ΔE* values, however these values do not compromise the color of the tested denture tooth acrylic resins because they are imperceptible to the human eye.