Influence of freezing temperature and deacetylation degree on the performance of freeze-dried chitosan scaffolds towards cartilage tissue engineering

Chitosan-based porous structures have been significantly studied across the world as potential tissue engineering scaffolds. Despite the differences in chitosan produced from squid pens or crustacean shells, with the former being more reactive and easily available with a higher degree of deacetylation (DD), most of the studies report the use of crab or shrimp chitosan as they are readily available commercial sources. The aim of this work was to highlight the great potential of chitosan produced from squid pens for biomedical application. From freeze-dried scaffolds for soft tissue engineering, we investigated the influence of the DD of chitosan and the freezing temperature during processing on their performance. Chitosan was obtained by deacetylation of β-chitin previously isolated from endoskeleton of giant squid Dosidicus gigas (DD 91.2%) and compared with a commercially available batch obtained from crab shells (DD 76.6%). Chitosan solutions were frozen at â 80° C or â 196° C and further freeze-dried to obtain 3D porous structures (scaffolds). Scaffolds prepared at â 196° C have a compact structure with smaller pores, while those prepared at â 80° C showed a lamellar structure with larger pores. The compressive modulus varied from 0.7 up to 8.8 MPa. Both types of scaffolds were stable on PBS, including in the presence of lysozyme, up to 4 weeks. Furthermore, the squid chitosan scaffolds processed at â 80° C promoted ATDC5 chondrocyte-like cells adhesion and proliferation. The results suggest that the developed squid chitosan scaffolds might be further exploited for ap- plications in cartilage tissue engineering.This work was partially funded by ERDF through POCTEP Projects 0330_IBEROMARE_1_P and 0687_NOVOMAR_1_P, Atlantic Area Project 2011-1/164 MARMED and by European Union through European Research Council – Project ComplexiTE (ERC-2012-ADG 20120216-321266). Portuguese Foundation for Science and Technology is gratefully acknowledged for post-doc grants of R.P. Pirraco (SFRH/BPD/101886/2014) and S.S. Silva (SFRH/BPD/112140/2015) and PhD grant of Lara L. Reys (SFRH/BD/112139/2015). The authors would also like to acknowledge to Dr. Julio Maroto, from Fundación CETMAR (Spain) and Roi Vilela, from PESCANOVA S.A. (Spain), for the kind offer of squid pens.info:eu-repo/semantics/publishedVersio

Cartilage regeneration approach based on squid chitosan scaffolds : in-vitro assessment

During the past decades, marine organisms have been the focus of considerable attention as potential source of valuable materials. For instance, chitosan is a biopolymer with high potential in the biomedical field and can be produced from crustacean shells and squid pens [1]. In this sense, we propose the use of chitosan to produce scaffolds for regenerative medicine purposes. An alkaline solution was used to deproteinize squid pens and isolate β- chitin (Chaussard 2004), which was further converted into chitosan through a deacetylation reaction. Chitosan was then processed into porous structures by freeze-drying [3], where chitosan solutions (4%) were submitted to different freezing temperature of -80ºC and - 196ºC. The produced structures were further submitted to neutralization methods with 4% NaHO, including in some cases a pre-washing step using ethanol/water solutions (100:0; 90:10, 80:20; 70:30 and 50:50) [4]. The morphology of scaffolds produced using either squid or commercial chitosan revealed a lamellar structure, independent of the source and/or freezing temperature. All chitosan scaffolds produced exhibited no-cytotoxic behaviour over L929 cells. To test the in vitro functionality of the scaffolds, cells from the mouse chondrogenic cell line ATDC-5 were seeded in the scaffolds and cultured for different time periods. Scaffolds made from squid chitosan were shown to promote better cell adhesion than commercial chitosan scaffolds and comparable or better cell proliferation. This demonstrates that squid chitosan is a valuable alternative to produce scaffolds for different applications in regenerative medicine, namely the regeneration of cartilage

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

Protein crystallization in a microfluidic contactor with nafion®117 membranes

UIDB/50006/2020 UIDP/50006/2020 “Erasmus Mundus Doctorate in Membrane Engineering”–EUDIME (FPA 2011–2014, http://www.eudime.unical.itProtein crystallization still remains mostly an empirical science, as the production of crystals with the required quality for X-ray analysis is dependent on the intensive screening of the best protein crystallization and crystal’s derivatization conditions. Herein, this demanding step was addressed by the development of a high-throughput and low-budget microfluidic platform consisting of an ion exchange membrane (117 Nafion® membrane) sandwiched between a channel layer (stripping phase compartment) and a wells layer (feed phase compartment) forming 75 independent microcontactors. This microfluidic device allows for a simultaneous and independent screening of multiple protein crystallization and crystal derivatization conditions, using Hen EggWhite Lysozyme (HEWL) as the model protein and Hg2+ as the derivatizing agent. This microdevice offers well-regulated crystallization and subsequent crystal derivatization processes based on the controlled transport of water and ions provided by the 117 Nafion® membrane. Diffusion coefficients of water and the derivatizing agent (Hg2+) were evaluated, showing the positive influence of the protein drop volume on the number of crystals and crystal size. This microfluidic system allowed for crystals with good structural stability and high X-ray diffraction quality and, thus, it is regarded as an efficient tool that may contribute to the enhancement of the proteins’ crystals structural resolution.publishersversionpublishe

What makes a task a problem in early childhood education?

This article begins with a theoretical discussion of the characteristics that a task should feature to be regarded as a mathematics problem suitable for pre-primary students. Those considerations are followed by a report of a classroom experience in which three problems involving quotative or partitive division were posed to pre-primary school pupils to determine the presence of otherwise of the respective characteristics. The findings show that the characteristics of pre-primary education problems depend on two factors: mathematical activity that engages pupils and a structure that favours both their understanding of the problem and the application and verification of the solutions

Nonparametric Regression Density Estimation Using Smoothly Varying Normal Mixtures

We model a regression density nonparametrically so that at each value of the covariates the density is a mixture of normals with the means, variances and mixture probabilities of the components changing smoothly as a function of the covariates. The model extends existing models in two important ways. First, the components are allowed to be heteroscedastic regressions as the standard model with homoscedastic regressions can give a poor fit to heteroscedastic data, especially when the number of covariates is large. Furthermore, we typically need a lot fewer heteroscedastic components, which makes it easier to interpret the model and speeds up the computation. The second main extension is to introduce a novel variable selection prior into all the components of the model. The variable selection prior acts as a self adjusting mechanism that prevents overfitting and makes it feasible to fit high dimensional nonparametric surfaces. We use Bayesian inference and Markov Chain Monte Carlo methods to estimate the model. Simulated and real examples are used to show that the full generality of our model is required to fit a large class of densities

Divergent Priors and Well Behaved Bayes Factors

Divergent priors are improper when defined on unbounded supports. Bartlett's paradox has been taken to imply that using improper priors results in ill-defined Bayes factors, preventing model comparison by posterior probabilities. However many improper priors have attractive properties that econometricians may wish to access and at the same time conduct model comparison. We present a method of computing well defined Bayes factors with divergent priors by setting rules on the rate of diffusion of prior certainty. The method is exact; no approximations are used. As a further result, we demonstrate that exceptions to Bartlett's paradox exist. That is, we show it is possible to construct improper priors that result in well defined Bayes factors. One important improper prior, the Shrinkage prior due to Stein (1956), is one such example. This example highlights pathologies with the resulting Bayes factors in such cases, and a simple solution is presented to this problem. A simple Monte Carlo experiment demonstrates the applicability of the approach developed in this paper

Induction of proteasome expression in skeletal muscle is attenuated by inhibitors of NF-κB activation

The potential for inhibitors of nuclear factor-κB (NF-κB) activation to act as inhibitors of muscle protein degradation in cancer cachexia has been evaluated both in vitro and in vivo. Activation of NF-κB is important in the induction of proteasome expression and protein degradation by the tumour factor, proteolysis-inducing factor (PIF), since the cell permeable NF-κB inhibitor SN50 (18 μM) attenuated the expression of 205 proteasome α-subunits, two subunits of the 195 regulator MSSI and p42, and the ubiquitin-conjugating enzyme, E214k, as well as the decrease in myosin expression in murine myotubes. To assess the potential therapeutic benefit of NF-κB inhibitors on muscle atrophy in cancer cachexia, two potential inhibitors were employed; curcumin (50 μM) and resveratrol (30 μM). Both agents completely attenuated total protein degradation in murine myotubes at all concentrations of PIF, and attenuated the PIF-induced increase in expression of the ubiquitin-proteasome proteolytic pathway, as determined by the 'chymotrypsin-like' enzyme activity, proteasome subunits and E2 14k. However, curcumin (150 and 300 mg kg-1) was ineffective in preventing weight loss and muscle protein degradation in mice bearing the MAC16 tumour, whereas resveratrol (1 mg kg-1) significantly attenuated weight loss and protein degradation in skeletal muscle, and produced a significant reduction in NF-κB DNA-binding activity. The inactivity of curcumin was probably due to a low bioavailability. These results suggest that agents which inhibit nuclear translocation of NF-κB may prove useful for the treatment of muscle wasting in cancer cachexia