Papers by Author: Rui L. Reis

Abstract: In this paper we report a new type of cross-linked porous structure based on a chitosansoy protein blend system developed by means of combining a sol-gel process with the freeze-drying technique. The final structure was investigated by Fourier transform infrared spectroscopy with attenuated total reflectance (FTIR-ATR), contact angle measurements and the morphology by scanning electron microscopy (SEM). The water uptake capability and the weight loss were measured up to 14 days and their mechanical properties were assessed with compression tests. Results showed that the addition of tetraethyl orthosilicate (TEOS) to the chitosan-soy protein blend system provide specific interactions at the interface between the two polymers allowing to tailor the size and distribution as well as the degradation rate of the hybrids. Finally, TEOS incorporation induces an increase of the surface energy that influences the final physicochemical properties of the materials.

Abstract: Phosphorylated chitosan (P-chitosan) was synthesized by means of graft copolymerization technique. The conjugate membranes were prepared from oxidised starch and Pchitosan using reductive alkylation crosslinking. The synthesized membranes were characterised by FT-IR. In order to characterize degradation behaviour of this conjugated system, the membranes were incubated in enzyme solutions of alpha-amylase and lysozyme as well as a physiological saline solution (PBS) used as control solution. In PBS, low starch containing membranes (0.16-0.38 weight (starch)/weight (P-chitosan), (ws/wc)) and control membranes have not showed significant change in their weight during two months of incubation. High starch containing membranes (0.73-1.04 ws/wc) indicated less than 20 % weight loss after this period. After α-amylase incubation, a distinct degradation behaviour was observed from starch-P-chitosan membranes. The degradation of the conjugate membranes was found to be fast with increasing starch content. Weight losses between 20 to 55 % were detected for the lowest (0.16 ws/wc) starch and highest (1.04 ws/wc) starch containing membranes, respectively. In the lysozyme degradation study, the conjugate membranes were not degraded by enzymatic activity and the weights of membranes were seen to be increased about 20 % because of swelling. The control membranes showed gradual weight loss in enzyme solutions. These results indicated the lysozyme degradation of starch-free P-chitosan membranes and inhibition of degradation P-chitosan by highly conjugated starch molecules.

Abstract: Fosfosal, a phosphate derivative of salicylic acid, which presents both analgesic and antiinflammatory properties, was used as a model drug to study the potential of recently developed chitosan membranes (with different crosslinking degrees) to be used as drug release rate-controlling membranes. The fosfosal permeation across these membranes was studied using an in-house built developed diffusion cell with online automatic monitoring. Experiments were performed using phosphate buffer saline (PBS) solution at 37°C. Different flow properties of the detection set up were determined in order to estimate the errors introduced by the automatic online monitoring system. For increasing crosslinking degrees the permeability initially decreased, and then increased, likely as a consequence of the crosslinking influence on a variety of properties like crystallinity and hydrophilicity that have opposite influence on permeability. In summary, it was possible to control the drug release profile by means of changing the degree of crosslinking of chitosan membranes and to follow the respective release kinetics by means of using the developed diffusion cell.

Abstract: This study aims to investigate the effect of culturing conditions (static and flow perfusion) on the proliferation and osteogenic differentiation of rat bone marrow (RBM) stromal cells seeded on two starch based three-dimensional scaffolds exhibiting distinct porous structures. For this purpose, it was selected: i) a scaffold based on SEVA-C (a blend of starch with ethylene vinyl alcohol) obtained by extrusion with a blowing agent and ii) a scaffold based on SPCL (a blend of starch with polycaprolactone) obtained by a fiber bonding process. The obtained results suggest that flow perfusion culture enhances the osteogenic differentiation of RBM cells and improves their distribution in 3-D starch-based scaffolds, by improving nutrients delivery in the interior of the scaffolds and simultaneously by stimulating the seeded cells by exposing them to fluid shear forces. They also indicate that scaffold architecture and pore interconnectivity affect the homogeneity of the formed tissue.

Abstract: Sulfonic groups (-SO3H) were covalently attached on different polymeric surfaces enabling them to induce apatite nucleation, for developing bioactive apatite-polymer composites with a bonelike 3-dimensional structure. High molecular weight polyethylene (HMWPE) and ethylene-co-vinyl alcohol co-polymer (EVOH) were used. The polymers were soaked in two types of sulphate-containing solutions with different concentrations, sulphuric acid (H2SO4) and chlorosulfonic acid (ClSO3H). To incorporate calcium ions into to the sulfonated polymers, the samples were soaked in a saturated Ca(OH)2 solution for 24 hours. After soaking of the samples in a simulated body fluid (SBF), formation of an apatite layer on both surfaces was observed. The results obtained prove the validity of the proposed concept and show that the -SO3H groups are effective on inducing apatite nucleation on the surface of these polymers.

Abstract: In the present work we describe the synthesis of starch-based/BG 45S5 particles and their in vitro bioactivity behaviour by means of immersion in a simulated body fluid. The composite particles have shown to form a layer of Ca-P at their surface, whose nature was confirmed by chemical and morphological analysis. In order to evaluate the ability of these particles to be used as carriers for cell culture, undifferentiated rat cells were selected and parameters like cell adhesion, proliferation and expression of osteoblastic markers, were evaluated. The starch-based micro-particles have shown to support cellular activity, allowing cells to attach, proliferate and express specific markers while cultured in the particles surface. The final goal is to be able to use these particles as carriers for cells and simultaneously as in-situ forming constructs for tissue engineering and regenerative medicine applications.

Abstract: In this study, it is shown that it is possible to prepare carboxymethyl-chitosan/Ca-P hybrids using an innovative “auto-catalytic” co-precipitation method, namely by using an acid and an oxidant bath. The X-ray diffraction (XRD) patterns evidenced the formation of crystalline calcium-phosphate precipitates when using an acid bath, while amorphous ones were obtained for those produced in the oxidant bath. The Fourier Transform Infrared spectroscopy (FTIR) and Scanning Electron Microscopy (SEM/EDS) studies revealed that the extent of the polymer precipitation and formation of calcium-phosphates is directly dependent on the pH and composition of the baths. Furthermore, by conducting bioactivity tests in a simulated body fluid (SBF) followed by the SEM/EDS analysis it was possible to detect the formation of an apatite layer with a cauliflower-like morphology on the surface of hybrids prepared by the acid bath, after 7 days of immersion. These results are quite promising because they can allow for the production of bioactive and biodegradable 3D porous scaffolds to be used in bone tissue engineering applications.

Abstract: The aim of this research was to develop a new methodology to obtain bioactive coatings on bioinert and biodegradable polymers that are not intrinsically bioactive. In this study, three types of materials were used as substrates: (i) high molecular weight polyethylene (HMWPE) and two different types of starch based blends (ii) starch/ethylene vinyl alcohol blends, SEVA-C, and (iii)starch/cellulose acetate blends, SCA. These materials were obtained by injection moulding and by extrusion with blowing agents in order to obtain compact/porous 3D architectures. Three types of baths were developed in order to produce the newly proposed auto-catalytic Ca-P coatings: (i)alkaline, (ii) acid, and (iii) oxidant bath. The obtained results indicated that it was possible to coat the materials surfaces with calcium phosphate (Ca-P) layer with only 60 min of immersion in the different types of auto-catalytic solutions. These innovative auto-catalytic electroless route allows for the production of an adherent bioactive film on the polymeric surfaces. Furthermore, it was possible observe by SEM/EDS the clear bioactive nature of the Ca-P coatings after different immersion periods, in a simulated body fluid (SBF).

Abstract: In the present study sodium clodronate, a well known therapeutic agent from the family of bisphosphonates (BP) was incorporated in an apatite coating, previously formed on the surface of a starch based biomaterial by a sodium silicate methodology, as a strategy to develop a site-specific drug delivery system for bone tissue regeneration. The effects on the resulting apatite coatings were evaluated in terms of morphology, chemistry and structure. As a preliminary approach, this first study aimed at evaluating also the effects of this BP on the viability, growth and function of a human osteoblast cell line, since there is still little information available on the interaction between BPs and this type of cells.

Abstract: A bioactive polyethylene polymer substrate can be produced by incorporation of sulfonic functional groups (-SO3H ) on its surface. Variation of the surface potential of the polyethylene modified with -SO3H groups with soaking in SBF were investigated using a laser electrophoresis zeta-potential analyzer. To complement the study using the laser electrophoresis, the surface was examined by X-ray photoelectron spectroscopy (XPS) and scanning electron microscopy with an attached energy dispersive electron probe X-ray analyser (FE-SEM/EDS). It was found that the surface potential of the polyethylene was highly negative charged after soaking in SBF for 0.5 h, increased for higher soaking times (up to 48 h), and then decreased. The negative charge of the polymer at soaking time of 0.5 h is attributed to the presence of –SO3H groups on the surface. The initial increase in the surface potential was attributed to the incorporation of positively charged calcium ions to form a calcium sulphate, and then the subsequent decrease was assigned to the incorporation of negatively charged phosphate ions to form an amorphous calcium phosphate, which eventually transformed into apatite.