Blood protein adsorption onto chitosan

Chitosan was recently indicated to enhance osteogenesis, improve wound healing but to activate the coagulation and the complement systems. In the present study approximately 10nm thick chitosan film were prepared on aminopropyltriethoxysilane (APTES) coated silicon. The surfaces were incubated in serum or plasma and subsequently in antibodies towards key complement and contact activation of coagulation proteins. The deposited amounts were compared with those on hydrophilic and hydrophobic silicon, APTES and IgG coated reference samples. Although large amounts of serum deposited to chitosan only a weak transient activation of the complement system and no activation of the intrinsic pathway was observed. Upon acetylation the chitosan layer became a strong activator of the alternative pathway of the complement. After incubation in human plasma anti-fibrinogen deposited onto chitosan but not onto the acetylated chitosan, a finding that may explain previous observations of procoagulant activity by chitosan

Analysing protein competition on self-assembled mono-layers studied with quartz crystal microbalance

The mechanisms by which proteins adsorb to surfaces of biomaterials have long been of interest. The present work started with the premise that small/hard and large/soft proteins will yield different sets of normalized frequency shift and dissipation signals when studied with a quartz crystal microbalance. The aim was to evaluate the usefulness of these raw data to study protein competition using protein incubations in sequence and from mixtures of albumin (BSA) and gamma-globulin (BGG) at various ratios. Increasing the concentration of BSA decreases the adsorption of subsequently incubated BGG. For BSA/ BGG mixtures the dissipation is similar for all logarithmic molar ratios BGG/BSA below 1 but soon decreases when the molar ratio of BSA/BGG (and opposite for the normalized frequency shift) is above 1, indicating preferential binding of BGG. Modelling indicated that differences in the film shear modulus and viscosity depend more on the properties of the self-assembling mono-layers (SAMs) than on the proteins. Films high in BSA tentatively differ in film shear modulus and viscosity from that of films high in BGG but only on the hydrophobic surfaces. The results were encouraging as the raw data were deemed to be able to point at protein adsorption competition.The authors thank the Portuguese National Science and Technology Foundation (FCT) for the Project Grants PTDC/FIS/68517/2006 and PTDC/FIS/68209/2006, and personal Grant BPD/39331/2007 for J.B

Fluorescence probe techniques to monitor protein adsorption-induced conformation changes on biodegradable polymers

The study of protein adsorption and any associated conformational changes on interaction with biomaterials is of great importance in the area of implants and tissue constructs. This study aimed to evaluate some fluorescent techniques to probe protein conformation on a selection of biodegradable polymers currently under investigation for biomedical applications. Because of the fluorescence emanating from the polymers, the use of monitoring intrinsic protein fluorescence was precluded. A highly solvatochromic fluorescent dye, Nile red, and a well-known protein label, fluorescein isothiocyanate, were employed to study the adsorption of serum albumin to polycaprolactone and to some extent also to two starch-containing polymer blends (SPCL and SEVA-C). A variety of fluorescence techniques, steady state, time resolved, and imaging were employed. Nile red was found to leach from the protein, while fluorescein isothiocyanate proved useful in elucidating a conformational change in the protein and the observation of protein aggregates adsorbed to the polymer surface. These effects were seen by making use of the phenomenon of energy migration between the fluorescent tags to monitor interprobe distance and the use of fluorescence lifetime imaging to ascertain the surface packing of the protein on polymer

Morphology and miscibility of chitosan/soy protein blended membranes

A physico-chemical characterization of blended membranes composed by chitosan and soy protein has been carried out in order to probe the interactions that allow membranes to be formed from these biopolymer mixtures. These membranes are developed aiming at applications in wound healing and skin tissue engineering scaffolding. The structural features of chitosan/soy blended membranes were investigated by means of solid state carbon nuclear magnetic resonance (NMR), infrared spectroscopy (FTIR), contact angle, and atomic force microscopy. FTIR investigations suggested that chitosan and soy may have participated in a specific intermolecular interaction. The proton spin–lattice relaxation experiments in the rotating frame on blended membranes indicated that independently of the preparation conditions, the blend components are not completely miscible possibly due to a weak polymer–protein interaction. It was also shown that the blended systems showed a rougher surface morphology which was dependent of soy content in the blend system

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

Surface engineered carboxymethylchitosan/poly(amidoamine) dendrimer nanoparticles for intracellular targeting

Novel highly branched biodegradable macromolecular systems have been developed by grafting carboxymethylchitosan (CMCht) onto low generation poly(amidoamine) (PAMAM) dendrimers. Such structures organize into sphere-like nanoparticles that are proposed to be used as carriers to deliver bioactive molecules aimed at controlling the behavior of stem cells, namely their proliferation and differentiation. The nanoparticles did not exhibit significant cytotoxicity in the range of concentrations below 1 mg mL"1, and fluorescent probe labeled nanoparticles were found to be internalized with highly efficiency by both human osteoblast-like cells and rat bone marrow stromal cells, under fluorescence-activated cell sorting and fluorescence microscopy analyses. Dexamethasone (Dex) has been incorporated into CMCht/PAMAM dendrimer nanoparticles and release rates were determined by high performance liquid chromatography. Moreover, the biochemical data demonstrates that the Dex-loaded CMCht/PAMAM dendrimer nanoparticles promote the osteogenic differentiation of rat bone marrow stromal cells, in vitro. The nanoparticles exhibit interesting physicochemical and biological properties and have great potential to be used in fundamental cell biology studies as well as in a variety of biomedical applications, including tissue engineering and regenerative medicine

Proteins and their peptide motifs in acellular apatite mineralization of scaffolds for tissue engineering

Many proteins in the inorganic=organic matrix of bone induce or modulate or inhibit mineralization of apatite in vivo. Many attempts have been made to mimic and understand this mechanism as part of bone formation, and ectopic mineralization and control thereof. Many attempts have also been made to use such proteins or protein fragments to harness their potential for improved mineralization. Such proteins and peptide motifs have also been the inspiration for attempts of making mimics of their structures and motifs using chemical or biological synthesis. The aim of this review is to highlight how proteins and (poly)peptides themselves impact mineralization in the human body, and how those could be used and have been used for improving apatite mineralization, for example, on or in materials that by themselves do not induce apatite mineralization but otherwise have interesting properties for use as bone tissue engineering scaffolds.J. Benesch wishes to acknowledge the financial support from FCT, postdoctoral fellowship scholarship SFRH/BPD/17584/2004. This work was carried out under the scope of the European Union NoE EXPERTISSUES (NMP3-CT-2004500283) and partially funded by the European Union FP6 STREP Project HIPPOCRATES (NMP3-CT-2003-505758) and FCT project ProteoLight (PTDC/FIS/68517/2006)

User-made immobilities: a transitions perspective

In this paper we aim to conceptualize the role of users in creating, expanding and stabilizing the automobility system. Drawing on transition studies we offer a typology of user roles including user-producers, user-legitimators, user-intermediaries, user-citizens and user-consumers, and explore it on the historical transition to the automobile regime in the USA. We find that users play an important role during the entire transition process, but some roles are more salient than others in particular phases. Another finding is that the success of the transition depends on the stabilization of the emerging regime that will trigger upscaling in terms of the numbers of adopters. The findings are used to reflect on potential crossovers between transitions and mobilities research

Quantification of adsorbed human serum albumin: A comparison between radioimmunoassay and simple null ellipsometry

Radioimmunoassay (RIA) and null ellipsometry are two common methods to quantify adsorbed proteins. However, the accuracy of null ellipsometry with a constant protein refractive index (n 1.465, k 0) at l 632.8 nm has this far not been explored. The present study compared the methods, and the degree of agreement between the simplified single wavelength null ellipsometry and RIA to quantify adsorbed proteins was explored on different surfaces. The quantification methods agreed well when A, ngstro¨m smooth hydrophilic or hydrophobic silicon surfaces, and freshly radio-labelled proteins were used. Some discrepancies were noted when either rough surfaces or stored and aged labelled proteins were used. The differences decreased when the aged protein solution was equilibrated with freshly dissolved proteins at room temperature (RT) for a few hours prior to the surface incubations. Significant differences were also noted between the methods when albumin was adsorbed at it’s iso-electric point (pH 4.8)