Production and characterization of alginate bilayer membranes for releasing simvastatin to treat wounds.

This study aims to produce and characterize alginate bilayer membranes composed of single membranes with varying cross-linking degrees to modulate simvastatin release, with potential to be used for wound-dressing. The single-layer and bilayer membranes were characterized by weight, thickness, surface pH, equilibrium-humidity, swelling degree, solubility, infrared spectroscopy (attenuated total reflectance Fourier-transform infrared), scanning electron microscopy, and water vapor transmission. Simvastatin diffusion and release rates were analyzed using Franz's cells; its indirect cytotoxicity was analyzed using human keratinocyte cells. The difference in the cross-linking degree (bottom and top layers) influenced the morphology of the membrane, and consequently its physical barrier properties. An in vitro release study demonstrated that the bilayer membrane could sustain drug-release for longer time as compared to the single-layer membrane, which could be potentially beneficial for long-term treatment of chronic wounds. A cell viability assay showed that simvastatin-loaded alginate membranes could be characterized as noncytotoxic, demonstrating their potential for use in wound-dressing applications.

Inhalation of Bacterial Cellulose Nanofibrils Triggers an Inflammatory Response and Changes Lung Tissue Morphology of Mice.

In view of the growing industrial use of Bacterial cellulose (BC), and taking into account that it might become airborne and be inhaled after industrial processing, assessing its potential pulmonary toxic effects assumes high relevance. In this work, the murine model was used to assess the effects of exposure to respirable BC nanofibrils (nBC), obtained by disintegration of BC produced by Komagataeibacter hansenii. Murine bone marrow-derived macrophages (BMMΦ) were treated with different doses of nBC (0.02 and 0.2 mg/mL, respectively 1 and 10 µg of fibrils) in absence or presence of 0.2% Carboxymethyl Cellulose (nBCMC). Furthermore, mice were instilled intratracheally with nBC or nBCMC at different concentrations and at different time-points and analyzed up to 6 months after treatments. Microcrystaline Avicel-plus® CM 2159, a plant-derived cellulose, was used for comparison. Markers of cellular damage (lactate dehydrogenase release and total protein) and oxidative stress (hydrogen peroxidase, reduced glutathione, lipid peroxidation and glutathione peroxidase activity) as well presence of inflammatory cells were evaluated in brochoalveolar lavage (BAL) fluids. Histological analysis of lungs, heart and liver tissues was also performed. BAL analysis showed that exposure to nBCMC or CMC did not induce major alterations in the assessed markers of cell damage, oxidative stress or inflammatory cell numbers in BAL fluid over time, even following cumulative treatments. Avicel-plus® CM 2159 significantly increased LDH release, detected 3 months after 4 weekly administrations. However, histological results revealed a chronic inflammatory response and tissue alterations, being hypertrophy of pulmonary arteries (observed 3 months after nBCMC treatment) of particular concern. These histological alterations remained after 6 months in animals treated with nBC, possibly due to foreign body reaction and the organism's inability to remove the fibers. Overall, despite being a safe and biocompatible biomaterial, BC-derived nanofibrils inhalation may lead to lung pathology and pose significant health risks.

Development of a strategy to functionalize a dextrin-based hydrogel for animal cell cultures using a starch-binding module fused to RGD sequence.

BACKGROUND: Several approaches can be used to functionalize biomaterials, such as hydrogels, for biomedical applications. One of the molecules often used to improve cells adhesion is the peptide Arg-Gly-Asp (RGD). The RGD sequence, present in several proteins from the extra-cellular matrix (ECM), is a ligand for integrin-mediated cell adhesion; this sequence was recognized as a major functional group responsible for cellular adhesion. In this work a bi-functional recombinant protein, containing a starch binding module (SBM) and RGD sequence was used to functionalize a dextrin-based hydrogel. The SBM, which belongs to an alpha-amylase from Bacillus sp. TS-23, has starch (and dextrin, depolymerized starch) affinity, acting as a binding molecule to adsorb the RGD sequence to the hydrogel surface. RESULTS: The recombinant proteins SBM and RGD-SBM were cloned, expressed, purified and tested in in vitro assays. The evaluation of cell attachment, spreading and proliferation on the dextrin-based hydrogel surface activated with recombinant proteins were performed using mouse embryo fibroblasts 3T3. A polystyrene cell culture plate was used as control. The results showed that the RGD-SBM recombinant protein improved, by more than 30%, the adhesion of fibroblasts to dextrin-based hydrogel. In fact, cell spreading on the hydrogel surface was observed only in the presence of the RGD-SBM. CONCLUSION: The fusion protein RGD-SBM provides an efficient way to functionalize the dextrin-based hydrogel. Many proteins in nature that hold a RGD sequence are not cell adhesive, probably due to the conformation/accessibility of the peptide. We therefore emphasise the successful expression of a bi-functional protein with potential for different applications.

Studies on the hemocompatibility of bacterial cellulose.

Among the strategies to improve a material's hemocompatibility, pre-coating with the tripeptide Arg-Gly-Asp (RGD) is used to favor endothelialization thus lowering thrombogenicity. The blood compatibility of native and RGD-modified bacterial cellulose (BC) was studied in this work for the first time. The plasma recalcification time and whole blood clotting results demonstrate the hemocompatibility of BC. A significant amount of plasma protein adsorb to BC fibres, however, according to analysis by intrinsic tryptophan fluorescence techniques when albumin, γ-globulin, and fibrinogen from pure protein solutions adsorb to BC do not undergo detectable conformational modifications. Human microvascular endothelial cells cultured on RGD-modified BC readily form a confluent cell layer, inhibiting the adhesion of platelets. As a general conclusion, both native and RGD-modified BCs may be classified as hemocompatible materials.

Improving bacterial cellulose for blood vessel replacement: Functionalization with a chimeric protein containing a cellulose-binding module and an adhesion peptide.

Chimeric proteins containing a cellulose-binding module (CBM) and an adhesion peptide (RGD or GRGDY) were produced and used to improve the adhesion of human microvascular endothelial cells (HMEC) to bacterial cellulose (BC). The effect of these proteins on the HMEC-BC interaction was studied. The results obtained demonstrated that recombinant proteins containing adhesion sequences were able to significantly increase the attachment of HMEC to BC surfaces, especially the RGD sequence. The images obtained by scanning electron microscopy showed that the cells on the RGD-treated BC present a more elongated morphology 48h after cell seeding. The results also showed that RGD decreased the in-growth of HMEC cells through the BC and stimulated the early formation of cord-like structures by these endothelial cells. Thus, the use of recombinant proteins containing a CBM domain, with high affinity and specificity for cellulose surfaces allows control of the interaction of this material with cells. CBM may be combined with virtually any biologically active protein for the modification of cellulose-based materials, for in vitro or in vivo applications.