Effect of crosslinking in chitosan/aloe vera-based membranes for biomedical applications

The  positive  interaction  between  polysaccharides  with  active  phytochemicals found  in  medicinal  plants  may  represent  a  strategy  to  create  active  wound dressing  materials  useful  for  skin  repair.  In  the  present  work,  blended membranes  composed  of  chitosan  (Cht)  and  Aloe  vera  gel  were  prepared through the solvent casting, and were crosslinked with genipin to improve their properties. Topography, swelling, wettability, mechanical properties and in vitro cellular response of the membranes were investigated. With the incorporation of aloe  vera  gel  into  chitosan  solution,  the  developed  chitosan/aloe-based membranes  displayed  increased  roughness  and  wettability;  while  the  genipin crosslinking  promoted  the  formation  of  stiffer  membranes  in  comparison  to those  of  the  non-modified  membranes.  Moreover, in  vitro cell  culture  studies evidenced that the L929 cells have high cell viability, confirmed by MTS test and calcein-AM staining. The findings suggested that both blend compositions and crosslinking  affected  the  physico-chemical  properties  and  cellular  behavior  of the developed membranes.The authors acknowledge financial support from Portuguese Foundation for Science and Technology - FCT (Grant SFRH/BPD/45307/2008; SFRH/BD/64601/2009), "Fundo Social Europeu" - FSE, and "Programa Diferencial de Potencial Humano - POPH". This work was partially supported by the FEDER through POCTEP 0330_IBEROMARE_1_P

The use of ionic liquids in the processing of chitosan/silk hydrogels for biomedical applications

Natural polymers are adequate renewable resources for the processability of well-defined architectures for several applications. Combinations of polysaccharides and proteins may mimic the naturally occurring environment of certain tissues. The main goal of this work renders the application of green chemistry principles, namely the use of ionic liquids (ILs) and biorenewable sources, such as chitosan (CHT) and silkfibroin (SF), to process new hydrogel-based constructs. Although the solubilization of both materials in ILs has been studied individually, this work reports, for the first time, the role of ILs as solvent, for the production of hydrogels from blends of chitosan and silkfibroin (CSF). These systems offer the advantage of being homogeneous and presenting easy and short dissolution time of both biomacromolecules. Moreover, the use of chitosan obtained fromα- andβ-chitin allowed the production of blended hydrogels with distinct physical–chemical properties.In vitroassays demonstrated that these hydrogels supported the adhesion and growth of primary human dermalfibroblasts. Taken these properties together, the CSF hydrogels might be promising biomaterials to be explored for skin tissue engineering approaches.Fundação para a Ciência e a Tecnologia FCT - SFRH/BPD/45307/2008, SFRH/BPD/ 34704/2007, SFRH/BD/64601/2009, PTDC/QUI/68804/2006FEDER - POCTEP 0330_IBEROMARE_1_P

Hybrid biodegradable membranes of silane-treated chitosan/soy protein for biomedical applications

In recent years, progress in the field of hybrid materials has been accelerated through use of the sol–gel process for creating materials and devices, which benefit from the incorporation of both inorganic and organic components. In this work, organic–inorganic hybrid membranes were prepared from tetraethoxysilane and a blend system composed of chitosan and soy protein. By introducing a small amount of siloxane bond into the chitosan/soy protein system, the chitosan/soy protein hybrid membranes were improved in terms of structure, topography and mechanical properties. It appears that the chitosan/soy protein hybrid membranes were formed by discrete inorganic moieties entrapped in the chitosan/soy protein blend, which improved the stability and mechanical performance assessed by the dynamic mechanical analysis as compared to chitosan/soy protein membrane. Also, in vitro cell culture studies evidenced that the chitosan/soy protein hybrid membranes are non-cytotoxic over a mouse fibroblast-like cell line. The hybrid membranes of silane-treated chitosan/soy protein developed in this work have potential in biomedical applications, including tissue engineering.This work was financially supported by the Portuguese Foundation for Science and Technology - FCT (Grant SFRH/BPD/45307/2008, SFRH/BPD/21786/2009, SFRH/BPD/39331/2007 and SFRH/BD/64601/2009), 'Fundo Social Europeu' - FSE and 'Programa Diferencial de Potencial Humano - POPH' and was partially supported by the FEDER through POCTEP 0330_IBEROMARE_1_P

A combined approach for comparative exoproteome analysis of Corynebacterium pseudotuberculosis

Background: Bacterial exported proteins represent key components of the host-pathogen interplay. Hence, we sought to implement a combined approach for characterizing the entire exoproteome of the pathogenic bacterium Corynebacterium pseudotuberculosis, the etiological agent of caseous lymphadenitis (CLA) in sheep and goats. Results: An optimized protocol of three-phase partitioning (TPP) was used to obtain the C. pseudotuberculosis exoproteins, and a newly introduced method of data-independent MS acquisition (LC-MSE) was employed for protein identification and label-free quantification. Additionally, the recently developed tool SurfG+ was used for in silico prediction of sub-cellular localization of the identified proteins. In total, 93 different extracellular proteins of C. pseudotuberculosis were identified with high confidence by this strategy; 44 proteins were commonly identified in two different strains, isolated from distinct hosts, then composing a core C. pseudotuberculosis exoproteome. Analysis with the SurfG+ tool showed that more than 75% (70/93) of the identified proteins could be predicted as containing signals for active exportation. Moreover, evidence could be found for probable non-classical export of most of the remaining proteins. Conclusions: Comparative analyses of the exoproteomes of two C. pseudotuberculosis strains, in addition to comparison with other experimentally determined corynebacterial exoproteomes, were helpful to gain novel insights into the contribution of the exported proteins in the virulence of this bacterium. The results presented here compose the most comprehensive coverage of the exoproteome of a corynebacterial species so far

In vitro evaluation of the behaviour of human polymorphonuclear neutrophils in direct contact with chitosan-based membranes

Several novel biodegradable materials have been proposed for wound healing applications in the past few years. Taking into consideration the biocompatibility of chitosan-based biomaterials, and that they promote adequate cell adhesion, this work aims at investigating the effect of chitosanbased membranes, over the activation of human polymorphonuclear neutrophils (PMNs). The recruitment and activation of polymorphonuclear neutrophils (PMNs) reflects a primary reaction to foreign bodies. Activation of neutrophils results in the production of reactive oxygen species (ROS) such as O2 − and HO− and the release of hydrolytic enzymes which are determinant factors in the inflammatory process, playing an essential role in the healing mechanisms. PMNs isolated from human peripheral blood of healthy volunteers were cultured in the presence of chitosan or chitosan/soy newly developed membranes. The effect of the biomaterials on the activation of PMNs was assessed by the quantification of lysozyme and ROS. The results showed that PMNs, in the presence of the chitosan-based membranes secrete similar lysozyme amounts, as compared to controls (PMNs without materials) and also showed that the materials do not stimulate the production of either O2 − or HO−. Moreover, PMNs incubated with the biomaterials when stimulated with phorbol 12-myristate 13-acetate (PMA) or formyl-methionyl-leucyl-phenylalanine (fMLP) showed a chemiluminescence profile with a slightly lower intensity, to that observed for positive controls (cells without materials and stimulated with PMA), which reflects the maintenance of their stimulation capacity. Our data suggests that the new biomaterials studied herein do not elicit activation of PMNs, as assessed by the low lysozyme activity and by the minor detection of ROS by chemiluminescence. These findings reinforce previous statements supporting the suitability of chitosan-based materials for wound healing applications

Helminth Coinfection Does Not Affect Therapeutic Effect of a DNA Vaccine in Mice Harboring Tuberculosis

From 14 diseases considered by WHO as Neglected Tropical Diseases, four involve helminth infections, such as schistosomiasis and soil-transmitted helminthiasis. Toxocariasis is a soil-transmitted worm highly prevalent in many developing countries, while schistosomiasis causes an annual mortality of 14,000 deaths per year, with 200–300 million infected people and 10% at risk of infection worldwide. Additionally, tuberculosis (TB) remains one of the leading causes of morbidity and mortality in many settings, particularly in the world's poorest countries. Mycobacteria and helminths are co-endemic and induce opposing patterns of immune responses in the host, recognized as Th1 and Th2 respectively. These co-existing patterns could be associated with the failure of TB vaccines. In this sense, we investigated the inflammatory and immune response in a coinfection model with T. canis or S. mansoni and M. tuberculosis analyzing the effects of an immunotherapy that has previously shown efficacy in experimental TB. This immunotherapy is based on a DNA vaccine that codifies a mycobacterial heat shock protein (hsp65), which can prevent TB in a prophylactic and also therapeutic setting. In this work, we show that helminth coinfection does not abrogate the therapeutic effects of DNAhsp65 vaccine against TB

Chitosan improves the biological performance of soy-based biomaterials

Soybean protein has been proposed for distinct applications within nutritional, pharmaceutical, and cosmetic industries among others. More recently, soy-based biomaterials have also demonstrated promising properties for biomedical applications.However, althoughmany reports within other fields exist, the inflammatory/immunogenic potential of those materials is still poorly understood and therefore can hardly be controlled. On the contrary, chitosan (Cht) has been well explored in the biomedical field, either by itself or combined with synthetic or other natural-based polymers. Therefore, the combination of chitosan with soybean protein is foreseen as a suitable approach to control the biological behavior of soy-based biomaterials. Under this context this work was designed to try to understand the influence of chitosan in the host response elicited by soy-based biomaterials. Soybean protein isolate powder (SI-P) and Cht powder (Cht-P) were injected as suspension into the intraperitoneal cavity of rats. SI-P induced the recruitment of higher numbers of leukocytes compared to the Cht-P during the entire observation period. In this sense, SI-P elicited a considerable reaction from the host comparing to the Cht-P, which elicited leukocyte recruitment similar to the negative control.After subcutaneous implantation of the soybean and denatured membranes, (SI-M and dSI-M) a severe host inflammatory reaction was observed. Conversely, Cht/soy-based membranes (Cht/soy-based membranes) showed the induction of a normal host response after subcutaneous implantation in rats, which allowed concluding that the addition of chitosan to the soy-based membranes improved their in vivo performance. Thus, the presented results assert the improvement of the host response, considering inflammatory cells recruitment, and overall inflammatory reaction,when chitosan is combined to soybean. Together with previous results that reported their promising physicochemical characteristics and their inability to activate human polymorphonuclear neutrophils in vitro, the herein presented conclusions reinforce the usefulness of theCht/ soy-based membranes and justify the pursue for a specific application within the biomedical field.The author Tircia C. Santos acknowledges the Marie Curie European Program for a short-term scholarship in the Alea Jacta EST project (MEST-CT-2004-008104). This work was developed under the scope of the European Network of Excellence EXPERTISSUES (NMP3-CT-2004-5000283)

Secondary metabolites from the sponges Aplysina fistularis and Dysidea sp. and the antituberculosis activity of 11-ketofistularin-3

The present investigation reports the isolation of aeroplysinin-2, 2-(3,5-dibromo-4-methoxyphenyl)-N,N,N-trimethyletanamonium, 7,9-dibromo-10-hydroxy-8-methoxy-1-oxa-2-azaspiro[4.5]deca-2,6,8-trien-3-carboxylic acid and its methyl ester, 11-oxoaerothionin, aerothionin, 11-keto-12-hydroxyaerothionin, 11-ketofistularin-3 and fistularin-3 from Aplysina fistularis, as well as of furodysinin lactone and 9α,11α-epoxicholest-7-en-3β,5α,6α,10-tetrol-6-acetate from Dysidea sp. Although the extracts of both sponges displayed antituberculosis activity, only 11-ketofistularin-3 isolated from A. fistularis displayed antimycobacterial activity against Mycobacterium tuberculosis H34Rv, with MIC at 16 μg/mL and SI of 40, a result that reinforce that fistularin-3 derivatives are interesting leads for the development of antituberculosis drugs.American Society of Pharmacognosy FoundationFundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)BIOTA/FAPESP - BIOprospecTAUSP - PIBIC/CNPqFAPES

In vivo performance of chitosan/soy-based membranes as wound dressing devices for acute skin wounds

Wound management represents a major clinical challenge on what concerns healing enhancement and pain control. The selection of an appropriate dressing plays an important role in both recovery and esthetic ap- pearance of the regenerated tissue. Despite the wide range of available dressings, the progress in the wound care market relies on the increasing interest in using natural-based biomedical products. Herein, a rat wound- dressing model of partial-thickness skin wounds was used to study newly developed chitosan/soy (cht/soy)- based membranes as wound-dressing materials. Healing and repair of nondressed, cht/soy membrane-dressed, and Epigard -dressed wounds were followed macroscopically and histologically for 1 and 2 weeks. cht/soy membranes performed better than the controls, promoting a faster wound repair. Re-epithelialization, ob- served 1 week after wounding, was followed by cornification of the outermost epidermal layer at the second week of dressing, indicating repair of the wounded tissue. The use of this rodent model, although in impaired healing conditions, may enclose some drawbacks regarding the inevitable wound contraction. Moreover, being the main purpose the evaluation of cht/soy-based membranes’ performance in the absence of growth factors, the choice of a clinically relevant positive control was limited to a polymeric mesh, without any growth factor influencing skin healing/repair, Epigard. These new cht/soy membranes possess the desired features regarding healing/repair stimulation, ease of handling, and final esthetic appearance-thus, valuable prop- erties for wound dressings.The author Tircia C. Santos acknowledges the Portuguese Foundation for Science and Technology (FCT) for her PhD grant (SFRH/BD/40861/2007). This work was developed under the scope of the European Network of Excellence EXPERTISSUES (NMP3-CT-2004-5000283)