Bovine osteoblasts cultured on polyanionic collagen scaffolds: an ultrastructural and immunocytochemical study.

Collagen is the most abundant protein in the body and is also the most important component of the extracellular matrix. Collagen has several advantages as a biomaterial such as lack of toxicity, biocompatibility, biodegradability, and easy reabsorption. In this study, we examined bovine osteoblasts cultured on native or anionic collagen scaffolds prepared from bovine pericardium after selective hydrolysis of glutamine and asparagine side chain amides for periods from 24 (BP24) and 48 h (BP48). The cells were cultured in control and mineralization medium at 37 °C in the presence of 5% CO(2). Transmission and scanning electron microscopy, energy dispersive spectroscopy, and an immunocytochemical marker were used for analysis. Cells with an irregular morphology forming a confluent multilayer were observed on matrices kept in control medium. Most of these cells presented a polygonal or elongated flattened morphology. Several spherical deposits of calcium crystal associated with phosphorus were observed on the native and BP48 matrices. Similar results were observed in samples kept in control medium except with lower calcium/phosphorus ratio. Vesicles actively expelled from the cell membrane were also seen (do this vesicles corresponds to calcium/phosphorus deposits). Osteocalcin was clearly visible on matrices kept in mineralization medium and was more expression on the surface of BP48 matrices. The results showed that anionic collagen is able to support osteoblastic differentiation, regardless of the medium used. Finally, the BP48 matrix promoted better osteoblast differentiation than the native matrix.

Effects of different sterilization methods on the morphology, mechanical properties, and cytotoxicity of chitosan membranes used as wound dressings.

The aim of this work was to compare the effects induced by two different sterilization methods (exposure to gamma radiation or ethylene oxide) and an antiseptic technique (immersion in 70% ethanol aqueous solution) on the morphology, tensile strength, percentage of strain at break, and in vitro cytotoxicity to Vero cells on chitosan membranes designed for wound healing. Four distinct membrane compositions were evaluated, with chitosan, glycerol, and chitin used as components. Gamma radiation, in spite of being one of the most commonly employed sterilizing agents, negatively affected the morphology of membranes composed solely by chitosan as well as the percentage of strain at break of the chitosan-membranes containing glycerol on their composition. Moreover, its use affected the color of the chitosan membranes. The use of 70% ethanol aqueous solution did not change the chitosan-membrane characteristics significantly, but its use has limitations concerning the process scale up. With ethylene oxide (EtO), chitosan-membrane morphology, percentage of strain at break, and in vitro cytotoxicity to Vero cells were not significantly affected. The tensile strength of the membranes containing chitin were reduced after the treatment with ethylene oxide; however, the obtained values were comprised in the range verified for normal human skin. Therefore, because the final characteristics of the membranes treated with ethylene oxide are appropriate when considering their use as wound healing devices, and because this sterilization process is easily adjusted to use on an industrial scale, EtO can be considered the most adequate sterilizing agent for chitosan membranes. However, it should be considered that this chemical is associated with toxicity, flammability, and environmental risks, as well as with possible material contamination with ethylene oxide residues.