Chitosan membranes exhibiting shape memory capability by the action of controlled hydration

Chitosan membranes can undergo a glass transition at room temperaturetriggered by hydration. The mechanical properties of the membranes were followed by atension test and dynamic mechanical analysis (DMA), with the sample in wet conditionsafter being immersed in varying compositions of water/ethanol mixtures. Results show thatwith the increasing of water content, the Young’s and storage modulus decreasesystematically. For water contents of ca. 35 vol%, chitosan (CHT) exhibits a glasstransition, showing an elastomeric plateau in the elastic modulus above this hydration leveland the occurrence of a peak in the loss factor. Due to the semi-crystalline nature of CHT,membranes of this biomaterial present a shape memory capability induced by water uptake.By fixation of the permanent shape by further covalent cross-linking, the membranes canhave different permanent shapes appropriate for different applications, including in thebiomedical area.This work was supported by the Portuguese Foundation for Science and Technology Foundation (FCT) through project PTDC/FIS/115048/2009

Cell adhesion in free-standing multilayer films made of chitosan and alginate

The method for preparing multilayer ultrathin films by the consecu- tive deposition of oppositely charged polyelectrolytes has gained tre- mendous recognition due the user friendly preparation, capability of incorporating high loads of different types of biomolecules in the films, fine control over the materials’ structure, and robustness of the products under ambient and physiological conditions. However the preparation of such films needs the assembly on a substrate and, sometimes, cannot be detached from it, which has limited the appli- cation of such films in areas as tissue engineering and regenerative medicine (TERM).Thus, the production of free-standing films is of extreme importance once it allows the direct experimental determi- nation of many physical properties of fundamental significance such as ion permeation and mechanical properties that can be tuned for real-world applications. In this work, we investigated the elaboration of free-standing multilayer films made of chitosan (CHI) and alginate (ALG), by detaching a polyelectrolyte multilayer film from its under- lying substrate without any postprocessing step. The conditions for optimized film growth were investigated. The adhesion of C2C12 myoblast cells on the CHI/ALG membrane was assessed by cytoskele- tal and nuclear staining. A good cell adhesion and spreading was observed all over the surface. The results demonstrate the potential of such biocompatible free standing membranes made of CHI and ALG for applications in TERM

Biomineralization in chitosan/Bioglass® composite membranes under different dynamic mechanical conditions

Fundamental aspects of biomineralization may be important in order to understand and improve calcification onto the surface of biomaterials. The biomineralization process is mainly followed in vitro by assessing the evolution of the apatite layer that is formed upon immersion of the material in Simulated Body Fluid (SBF). In this work we propose an innovative methodology to monitor apatite deposition by looking at the evolution of the mechanical/viscoelastic properties of the sample while immersed in SBF, using non-conventional dynamic mechanical analysis (DMA) performed under distinct displacement amplitudes (d). The biomimetic biomineralization process in composite membranes of chitosan (CTS) with Bioglass® (BG) was followed by measuring the change of the storage modulus, E′, and the loss factor, tan δ, at 37 °C and in SBF, both online (d = 10 μm and d = 30 μm) and offline (d = 0 μm). The online experiments revealed that the E′ decreased continuously up in the first hours of immersion in SBF that should be related to the dissolution of BG particles. After that, an increase of the stiffness was verified due to the apatite deposition. SEM/EDS observations upon 24 h of immersion in SBF showed higher development of apatite deposition with increasing displacement amplitude.This work was financially supported by Foundation for Science and Technology (FCT) by the projects PTDC/QUI/69263/2006, PTDC/CTM-BPC/112774/2009 and, through the scholarship SFRH/BD/64601/2009 granted to Sofia G. Caridade

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

Processing of novel bioactive polymeric matrixes for tissue engineering using supercritical fluid technology

The aim of this study was to develop a new process for the production of bioactive 3D scaffolds using a clean and environmentally friendly technology. The possibility of preparing composite scaffolds of Bioglass® and a polymeric blend of starch and poly(L-lactic acid) (SPLA50) was evaluated. Supercritical phase-inversion technique was used to prepare inorganic particles loaded starch-based porous composite matrixes in a onestep process for bone tissue engineering purposes. Due to their osteoconductive properties some glasses and ceramics are interesting materials to be used for bone tissue engineering purposes; however their poor mechanical properties create the need of a polymeric support where the inorganic fraction can be dispersed. Samples impregnated with different concentrations of Bioglass® (10 and 15% wt/wt polymer) were prepared at 200 bar and 55 °C. The presence of Bioglass® did not affect the porosity or interconnectivity of the polymeric matrixes. Dynamic mechanical analysis has proven that the modulus of the SPLA50 scaffolds increases when glass particles are impregnated within the matrix. In vitro bioactivity studies were carried out using simulated body fluid and the results show that a calciumphosphate layer started to be formed after only 1 day of immersion. Chemical analysis of the apatite layer formed on the surface of the scaffold was performed by different techniques, namely EDS and FTIR spectroscopy and X-ray diffraction (XRD). The ion concentration in the simulated body fluid was also carried out by ICP analysis. Results suggest that a bone-like apatite layer was formed. This study reports the feasibility of using supercritical fluid technology to process, in one step, a porous matrix loaded with a bioactive material for tissue engineering purposes.Ana Rita C. Duarte is grateful for financial support from Funda go para a Ciencia e Tecnologia through the grant SFRH/BPD/34994/2007. The authors also acknowledge the financial support from FCT through the project PTDC/QUI/69263/2006

Effect of solvent-dependent viscoelastic properties of chitosan membranes on the permeation of low molecular weight drugs

Chitosan (CTS), a widely used biopolymer in different biomedical applications, is a derivative of chitin, the most abundant polysaccharide found in the marine world. This work aimed at providing relevant information about the use of CTS membranes in separation applications or in sustained release systems of therapeutic molecules. Moreover, the mechanical characterization may be also very important in such kind of applications, especially if the materials are tested in adequate physiological conditions. Chitosan membranes, both non-crosslinked and crosslinked with genipin, were characterized by dynamic mechanical analysis, swelling and permeability experiments using a model molecule. The membranes were tested immersed in different mixtures of water/ethanol. The swelling equilibrium varied linearly with the volumetric composition of the solvent mixture. The mechanical properties of CTS increased with the enhancement of the crosslinking density. A peak of the loss factor appeared at 24.5% of water attributed to the α-relaxation of chitosan and simultaneously a reduction of the storage modulus was observed. This was the first time that the glass transition (Tg) dynamics was monitored in a polymer in immersion conditions, where the plasticizer composition in the bath changed in a controlled way. Permeability decreased sharply until it reached very small values around the Tg. We hypothesise that conformational mobility of the polymeric chains may play an important role in the diffusion properties of molecules through polymer matrices. Results may elucidate some aspects regarding to relationships between glass transition and transport properties that may be important in the use of CTS in TE strategies.info:eu-repo/semantics/publishedVersio

Effect of solvent-dependent viscoelastic properties of chitosan membranes on the permeation of 2-phenylethanol

The viscoelastic behaviour of chitosan was followed by dynamic mechanical analysis (DMA) while the sample was immersed in gradient compositions of water/ethanol mixtures. The swelling equilibrium of chitosan membranes, both crosslinked with genipin or not, increased linearly with the water content. Increasing the water content, it was simultaneously observed a peak in the loss factor (around 25 vol.%) and a reduction of the storage modulus, which was attributed to the a-relaxation of chitosan. This was the first time that the glass transition dynamics in a polymer was monitored in immersion conditions where the composition of the plasticizer in the bath is changed in a controlled way. The water content at which tan d presented a maximum increased with both increasing frequency and increasing crosslinking density. The permeability decreased steadily with the ethanol content, reaching very low values around the glass transition. Therefore we hypothesize that conformational mobility of the polymeric chains may play an important role in the diffusion properties of molecules trough polymeric matrices.This work was partially supported by the Portuguese Foundation for Science and Technology (FCT), through funds from the POCTI and/or FEDER programmes and through the scholarship SFRH/BPD/34545/2007 granted to Ricardo M.P. da Silva. This work was carried out under the scope of the European NoE EXPERTISSUES (NMP3-CT-2004-500283) and also partially supported by the EU funded projects HIPPOCRATES (STREP - NMP3-CT-2003-505758) and PROTEUS (INTERREG III A - SP1.P151/03). The technical assistance of Ana Pinheiro in the execution of the DMA experiments is also acknowledged

Evaluation of different formulations of gellan gum-based hydrogels for tissue engineering of intervertebral disc

Gellan gum based-hydrogels present advantageous features for application as acellular and cellular nucleus pulposus (NP) substitutes due to the possibility of fine-tuning its physico-chemical and biological properties. In this study, ionic-crosslinked hydrogel discs were produced by means of mixing a raw and chemically modified material, i.e., high acyl gellan gum (HAGG) and methacrylated low acyl gellan gum (GG-MA), respectively. The hydrogel discs were characterized in terms of its mechanical properties and degradation/swelling ability. The biocompatibility of the different hydrogel formulations was assessed in vitro using NP rabbit cells isolated from the intervertebral disc. The biological performance of the developed gellan gum-based hydrogels formulations was evaluated by: (i) culturing of NP cells in the presence of the hydrogel leachables, and (ii) seeding or encapsulation of the NP cells within the hydrogels. The present work demonstrated that as HAGG content increases, the modulus of the hydrogels decreases. Moreover, the increase of the HAGG content induces a higher weight loss of the GG-MA/HA-GG formulation as compared to GG-MA hydrogel. The in vitro study revealed that hydrogels are non-cytotoxic and support the encapsulation of rabbit NP cells. The methacrylated gellan gum and formulations possessing high acyl gellan gum present tunable properties that may be interesting for application as NP substitutes

Three-dimensional plotted scaffolds with controlled pore size gradients : effect of scaffold geometry on mechanical performance and cell seeding efficiency

Scaffolds produced by rapid prototyping (RP) techniques have proved their value for tissue engineering applications, due to their ability to produce predetermined forms and structures featuring fully interconnected pore architectures. Nevertheless, low cell seeding efficiency and non-uniform distribution of cells remain major limitations when using such types of scaffold. This can be mainly attributed to the inadequate pore architecture of scaffolds produced by RP and the limited efficiency of cell seeding techniques normally adopted. In this study we aimed at producing scaffolds with pore size gradients to enhance cell seeding efficiency and control the spatial organization of cells within the scaffold. Scaffolds based on blends of starch with poly(e-caprolactone) featuring both homogeneously spaced pores (based on pore sizes of 0.75 and 0.1 mm) and pore size gradients (based on pore sizes of 0.1–0.75–0.1 and 0.75–0.1– 0.75 mm) were designed and produced by three-dimensional plotting. The mechanical performance of the scaffolds was characterized using dynamic mechanical analysis (DMA) and conventional compression testing under wet conditions and subsequently characterized using scanning electron microscopy and micro-computed tomography. Osteoblast-like cells were seeded onto such scaffolds to investigate cell seeding efficiency and the ability to control the zonal distribution of cells upon seeding. Scaffolds featuring continuous pore size gradients were originally produced. These scaffolds were shown to have intermediate mechanical and morphological properties compared with homogenous pore size scaffolds. The pore size gradient scaffolds improved seeding efficiency from !35% in homogeneous scaffolds to !70% under static culture conditions. Fluorescence images of cross-sections of the scaffolds revealed that scaffolds with pore size gradients induce a more homogeneous distribution of cells within the scaffold.This work was supported by the European NoE EXPERTISSUES (NMP3-CT-2004-500283)

Chitosan membranes containing micro or nano-size bioactive glass particles : evolution of biomineralization followed by in-situ dynamic mechanical analysis

A newfamilyofbiodegradablepolymer/bioactiveglass(BG)compositematerialshas emergedbasedontheavailabilityofnano-sizedbioactiveparticles.Suchnovelbiocompo- sites canhaveenhancedperformance,intermsofmechanicalpropertiesandbioactivity, and theycanbedesignedtobeusedinboneregenerationapproaches. In thiswork,membranesofchitosan(CTS)andchitosanwithbioactiveglass(BG)both micron andnanosizedparticles(CTS/mBG,CTS/nBG,respectively)werepreparedby solvent casting.Microstructuralandmechanicalpropertieswereevaluatedinorderto compare theeffectsoftheincorporationofmicro(mBG) andnano(nBG)particlesinthe chitosan matrix. In vitro bioactivity testswereperformedtocharacterizetheapatitelayer that isformedonthesurfaceofthematerialafterbeingimmersedinsimulatedbodyfluid (SBF). Thebiomineralizationprocessonthebiomaterialswasalsofollowedusingnon- conventionaldynamicmechanicalanalysis(DMA),bothonlineandoffline.InsuchDMA experiments,thechangeinthestoragemodulus, E0, andthelossfactor,tan d, were measured asafunctionoftheimmersiontimeinSBF.TheresultsdemonstratedthatCTS/ nBG membranespossessenhancedmechanicalpropertiesandhigherbioactivityin comparisonwiththeCTS/mBG membranes.SuchresultssuggestthepotentialofnBGfor the developmentofbioactivecompositesforboneregenerationapplications.This work was financially supported by Foundation for Science and Technology (FCT) by the projects PTDC/QUI/69263/2006, PTDC/CTM-BPC/112774/2009 and, through the scholarship SFRH/BD/64601/2009 granted to Sofia G. Caridade. The authors acknowledge Dr D. Mohn and Prof. W. Stark (Em Zurich) for providing the nBG particles used