Inhibition of calcification of bovine pericardium after treatment with biopolymers, E-beam irradiation and in vitro endothelization.

This work has investigated the in vitro calcification of bovine pericardium (BP) treated with chitosan (C), silk fibroin (SF) and electron beam irradiation after its endothelization in vitro. For this purpose, freeze-dried BP membranes treated with mixtures of C and SF (1:3, 1:1 and 3:1) and then irradiated by electron beam irradiation were seeded with human umbilical vein endothelial cells (HUVEC) in vitro. After 3 weeks of cultivation these membranes were submitted to in vitro calcification tests using simulated body fluid as the calcifying agent. Control membranes were also studied (without endothelial cells exposure). The results have shown that the membrane compatibility with HUVECs in vitro prevent such biomaterial from calcifying, showing a potential application in biomaterial area, such as cardiac valves and repair patches.

In vitro and in vivo evaluation of lyophilized bioprosthetic valve.

Freeze-drying of biological tissues allows for dry storage and gamma ray sterilization, which may improve their use as a medical prosthesis. The objective of this study was to evaluate the rehydration characteristics and hydrodynamic performance of prosthetic valves before and after lyophilization. Two size 23 bovine pericardium aortic valve prostheses from different manufacturers were evaluated in a Shelhigh (Union, NJ, USA) pulse duplicator (80 ppm, 5 L/min) before and after lyophilization. Flow and transvalvular pressure gradient were registered in vitro and in vivo, and images of opening and closing of the prosthesis were obtained in the pulse duplicator in a digital camera. Rehydration was evaluated by comparison of dry valve weight with valve weight after 15 min, and 1, 24, 48, and 72 h in saline solution, inside the pulse duplicator. In vivo performance was assessed by surgical implantation in Santa Inês young male sheep in the pulmonary position after 30 min rehydration with 0.9% saline. Transvalvular pressure gradient and flow measurements were obtained immediately after implantation and 3 months after surgery when valves were explanted. Captured images showed a change in the profile opening and closing of valve prosthesis after lyophilization. The gradient measured (in vitro) in two valves was 17.08 ± 0.57 and 18.76 ± 0.70 mm Hg before lyophilization, and 34.24 ± 0.59 and 30.40 ± 0.97 mm Hg after lyophilization. Rehydration of both lyophilized valves was approximately 82%. Drying changed the profile of the opening and closing of valve prostheses, and increased on average by 83% the gradient in vitro tests. The result of the in vivo tests suggests maintaining pressure levels of the animal with the lyophilized prostheses within acceptable levels.

Cytotoxicity and endothelial cell adhesion of lyophilized and irradiated bovine pericardium modified with silk fibroin and chitosan.

Grafts of biological tissues have been used since the 1960s as an alternative to the mechanical heart prostheses. Nowadays, the most consolidated treatment to bovine pericardial (BP) bioprostheses is the crosslinking with glutaraldehyde (GA), although GA may induce calcification in vivo. In previous work, our group demonstrated that electron beam irradiation applied to lyophilized BP in the absence of oxygen promoted crosslinks among collagen fibers of BP tissue. In this work, the incorporation of silk fibroin (SF) and chitosan (CHIT) in the BP not treated with GA was studied. The samples were irradiated and then analyzed for their cytotoxicity and the ability of adhesion and growth of endothelial cells. Initially, all samples showed cytotoxicity. However, after a few washing cycles, the cytotoxicity due to acetic acid and ethanol residues was removed from the biomaterial making it suitable for the biofunctional test. The samples modified with SF/CHIT and electron beam irradiated favored the adhesion and growth of endothelial cells throughout the tissue.

Preparation and characterization of ethanol-treated silk fibroin dense membranes for biomaterials application using waste silk fibers as raw material.

The possibility of producing valued devices from low cost natural resources is a subject of broad interest. The present study explores the preparation and characterization of silk fibroin dense membranes using waste silk fibers from textile processing. Morphology, crystallinity, thermal resistance and cytotoxicity of membranes as well as the changes on the secondary structure of silk fibroin were analyzed after undergoing treatment with ethanol. Membranes presented amorphous patterns as determined via X-ray diffraction. The secondary structure of silk fibroin on dense membranes was either random coil (silk I) or beta-sheet (silk II), before and after ethanol treatment, respectively. The sterilized membranes presented no cytotoxicity to endothelial cells during in vitro assays. This fact stresses the material potential to be used in the fabrication of biomaterials, as coatings of cardiovascular devices and as membranes for wound dressing or drug delivery systems.

Natural and prosthetic heart valve calcification: morphology and chemical composition characterization.

Calcification is the most common cause of damage and subsequent failure of heart valves. Although it is a common phenomenon, little is known about it, and less about the inorganic phase obtained from this type of calcification. This article describes the scanning electron microscopy (SEM)/energy dispersive X-ray spectroscopy and Ca K-edge X-ray absorption near edge structure (XANES) characterization performed in natural and bioprosthetic heart valves calcified in vivo (in comparison to in vitro-calcified valves). SEM micrographs indicated the presence of deposits of similar morphology, and XANES results indicate, at a molecular level, that the calcification mechanism of both types of valves are probably similar, resulting in formation of poorly crystalline hydroxyapatite deposits, with Ca/P ratios that increase with time, depending on the maturation state. These findings may contribute to the search for long-term efficient anticalcification treatments.

Effect of freeze-drying on the mechanical, physical and morphological properties of glutaraldehyde-treated bovine pericardium: evaluation of freeze-dried treated bovine pericardium properties.

PURPOSE: Biomaterials have been widely used in the field of regenerative medicine. Bovine pericardium tissue has been successfully used as a bioprosthetic material in manufacturing heart valves, but studies concerning the tissue are ongoing in order to improve its storage, preservation and transportation. This article provides an overview of the characteristics of bovine pericardium tissue chemically treated after the freeze-drying process. These characteristics are essential to evaluate the changes or damage to the tissue during the process. METHODS: The mechanical properties of the tissue were analyzed by three different methods due to its anisotropic characteristics. The physical properties were analyzed by a colorimetric method, while the morphological properties were evaluated by scanning electron microscopy (SEM). RESULTS: The freeze-dried bovine pericardium showed no significant change in its mechanical properties. There was no significant change in the elasticity of the tissue (p>0.05) and no color change. In addition, SEM analysis showed that the freeze-dried samples did not suffer structural collapse. CONCLUSIONS: It was concluded that glutaraldehyde-treated bovine pericardium tissue showed no significant change in its properties after the freeze-drying process.

Processing, characterization, and in vitro/in vivo evaluations of powder metallurgy processed Ti-13Nb-13Zr alloys.

This article presents details of processing, characterization and in vitro as well as in vivo evaluations of powder metallurgy processed Ti-13Nb-13Zr samples with different levels of porosity. Sintered samples were characterized for density, crystalline phases (XRD), and microstructure (SEM and EDX). Samples sintered at 1000 degrees C showed the highest porosity level ( approximately 30%), featuring open and interconnected pores ranging from 50 to 100 mum in diameter but incomplete densification. In contrast, samples sintered at 1300 and 1500 degrees C demonstrated high densification with 10% porosity level distributed in a homogeneous microstructure. The different sintering conditions used in this study demonstrated a coherent trend that is increase in temperature lead to higher sample densification, even though densification represents a drawback for bone ingrowth. Cytotoxicity tests did not reveal any toxic effects of the starting and processed materials on surviving cell percentage. After an 8-week healing period in rabbit tibias, the implants were retrieved, processed for nondecalcified histological evaluation, and then assessed by backscattered electron images (BSEI-SEM) and EDX. Bone growth into the microstructure was observed only in samples sintered at 1000 degrees C. Overall, a close relation between newly formed bone and all processed samples was observed.

Histological evaluation of biocompatibility of lyophilized bovine pericardium implanted subcutaneously in rats.

This article aims at investigating in vivo evaluation of lyophilization procedure on the biocompatibility of bovine pericardium treated with glutaraldehyde (GA). The bovine pericardium was fixed with 0.5% glutaraldehyde during 10 days and preserved in 4% formaldehyde (FA). Two groups of samples were prepared from treated membranes: Group 1, nonlyophilized samples and Group 2, lyophilized samples. Male Sprague-Dawley rats (4 weeks after birth) were anesthetized (pentobarbital sodium 25 mg/kg of body weight) and in each one were implanted subcutaneously in the dorsal region a sample from Group 1 and another from Group 2. These samples were explanted after 30 days for histological analysis. No intercurrences took place after the surgery. No differences (P > 0.05) in the calcification, granulomatous reaction, mononuclear infiltration, and granulation tissue development was observed between both groups. The implanted lyophilized samples presented a trend for a reduced inflammatory reaction. Lyophilization of the bovine pericardium does not seem to increase the above listed tissue reaction.

Cytotoxicity and genotoxicity of bovine pericardium preserved in glycerol.

Bovine pericardium is a widely utilized biomaterial. Usually, after harvesting, it is advantageous that the pericardium be immersed in glycerol to improve its shelf life. This can induce some degree of toxicity in the material. The studies were performed in compliance with the rules of ISO 10993 and OECD 487, in the biological evaluation of medical devices. The material was prepared without previous washing. After sterilization by gamma radiation the pericardium was immersed in RPMI 1640 culture medium to fulfill the extraction condition. The same extract was employed in the cytotoxic and genotoxic tests. The procedures were carried out with Chinese hamster ovary cell line and to determine the cytotoxicity, a colorimetric method with the tetrazolium compound MTS was used. For the genotoxicity, following the in vitro micronucleus assay, the test was developed with and without metabolic activation. The Cytotoxicity Index was graphically estimated at the extract concentration of 78%. In the genotoxicity test, the average value of cell proliferation index was found to be 1.62 +/- 0.02 with S9 metabolic activator and 1.91 +/- 0.01 without S9 metabolic activator. Both values are similar to the negative control value in the micronucleus assay. We observed that although the pericardium preserved in glycerol shows a certain level of cytotoxicity, it does not show any genotoxicity.

Lyophilized bovine pericardium treated with a phenethylamine-diepoxide as an alternative to preventing calcification of cardiovascular bioprosthesis: preliminary calcification results.

This study investigated the calcification process that occurred on chemically treated bovine pericardium substrata through tests with simulated body fluid solutions. The use of bovine pericardium bioprosthetic valves in heart valve surgery has a significant drawback due to the calcification processes. Thus, many routes such as chemical treatments in the substratum or the adoption of systemic therapies are considered in the literature with the intention to inhibit or to decelerate this process. The presented treatment using the two different phenetylamine-diepoxide solutions showed no effects on calcification experiments as showed by the tests. However, the lyophilized bovine pericardium samples, treated with both solutions, did not show any detectable phosphate deposits. The lyophilization of bovine pericardium before chemical treatments with cross-link agents as epoxy compounds may be an alternative to the conventional calcification prevention methods, but further investigations are recommended to check if the same behavior is found in all lyophilized systems.

Glutaraldehyde-treated bovine pericardium: effects of lyophilization on cytotoxicity and residual aldehydes.

This work assesses the effect of lyophilization on the cytotoxicity and residual aldehyde concentration of glutaraldehyde-treated and lyophilized bovine pericardium (group A), comparing it to conventional glutaraldehyde-treated bovine pericardium (group B). Cytotoxicity was measured by incubating a pericardium sample from each group in saline and assessing the eluant's influence on cellular growth. Residual aldehydes were measured by HPLC. Although both groups' eluants exhibited some cytotoxicity, the eluant from group A was less cytotoxic, with a cytotoxicity index (IC50(%)) of 41%. Group B eluants all had marked cytotoxic effects; cell growth was 24.15% of the negative control at the most dilute eluant concentration (6.25%). The mean residual glutaraldehyde level was less in group A than in group B (2.36 +/- 0.11 and 9.90 +/- 3.70 g/l, respectively; n=3, P < 0.05), but residual formaldehyde levels did not differ. These results demonstrate that compared with conventional glutaraldehyde-treated bovine pericardium, lyophilized pericardium is less cytotoxic, with fewer glutaraldehyde residues.

Citotoxicidade in vitro das membranas de hidrogel reticuladas por radiação ionizante / In vitro cytotoxicity of hydrogel membranes reticulate by ionizing radiations

Hidrogéis constituídos de poli N-vinil-2-porrolidona (PVP), ágar e polietilenoglicol (PEG) säo utilizados como membranas para uso tópico, no tratamento de lesöes de pele. Com o objetivo de melhorar algumas das propriedades mecânicas da membrana, como tensäo de ruptura e elongaçäo, substituiu-se o PEG pelo copolímero poli (dimetilsiloxano)-co-poli (óxido de etileno) (SEO). A biocompatibilidade "in vitro" das membranas de PVP-SEO com diferentes concetraçöes de SEO foi testada em cultura de células. O teste de citotoxicidade foi realizado pelo estudo comparativo de dois métodos: 1) difusäo em ágar, utilizando-se as linhagens celulares RC-IAL e NCTC Clone L-929 e 2) inibiçäo da formaçäo de colônias utilizando-se a linhagem celular CHO-K1. Os resultados obtidos nos dois métodos foram análogos e demonstraram que as membranas do PVP-SEO apresentaram toxicidade nas concentraçöes testadas e nas linhagens celulares utilizadas. Foi possível também concluir que a adiçäo de SEO ocasiona caráter citotóxico às membranas de PVP

Hydrogels composed by PVP, agar and PEG are used as wound dressings in the treatmentof skin ulcers. In order to improve the mechanical properties of the membrane, such as tensile strengthand elongation, PEG was replaced by copolymer poly (dimethilsiloxane)-co-polyoxiethylene (SEO).The "in vitro" biocompatibility evaluation of PVP-SEO membranes with different concentrations of SEOwas carried out in cell culture. A comparative study of cytotoxicity assays was performed by twomethods: 1) diffusion agar assay, using RC-IAL and NCTC Clone L-929 cells and 2) colony suppressionassay, using CHO-K1 cells. In the cytotoxic evaluation of PVP-SEO membranes by both methods,analogous results were obtained. It was also possible to conclude that the addition of SEO promotescytotoxicity in PVP membranes