Papers by Author: Rui L. Reis

Abstract: Chitosan membranes were subjected to a pre-treatment in a double diffusion system, with a calcium solution in one chamber and a phosphate solution in the other chamber. Both chambers were separated by the chitosan membrane and subject to three mineralization periods (5, 10 and 15 minutes). After this pre-treatment the bioactivity of the different calcium phosphate coatings formed was tested for different periods of immersion time, 7, 14 and 21 days at room temperature and 37°C, in acellular simulated body fluid (1.0x). The results obtained demonstrated that the calcium phosphate coatings formed during the pre-treatment process are bioactive. It was found that the calcification is effective just in the side of the membrane exposed to the calcium solution chamber. This enabled to develop membranes with asymmetric osteoinductive properties that can be useful in different orthopedic applications.

Abstract: Bone marrow cells are a potential source to induce different lineage cells which can be used to rebuild or replace damaged tissues using a Tissue Engineering (TE) approach. However, TE strategies usually require the use of a material to support the development of a biological tissue. Beta-polyvinylidene fluoride (β-PVDF) is a biocompatible, thermoplastic with piezo-electrical properties that has been shown to provide a good cellular attachment and therefore might present advantageous properties as a scaffold material for cell seeding/culturing. The present study describes the characterization of β-PVDF membranes as a support material for growth and differentiation of goat marrow cells (GMCs) into osteoblasts, leading to the formation of substitutes for tissue regeneration. The obtained results suggest that β-PVDF piezoelectric properties influence cellular behavior. β- PVDF membranes not only enhance GMCs adherence and proliferation but also improve differentiation towards the osteogenic phenotype both in static and dynamic culture conditions. Furthermore, β-PVDF membranes exhibit very promising properties, suggesting that this material provides adequate support for the seeding and the development of undifferentiated cells towards a desired phenotype.

Abstract: In this study we explore the possibility of incorporating Sr into nano-apatite coatings prepared by a solution-derived biomimetic methodology for coating titanium based implants. The way this element is incorporated in the apatite structure and its effects on the stereochemistry and morphology of the resulting apatite layers were investigated, as well as the resulting mineralization kinetics. The presence of Sr in solution induced an inhibitory effect on mineralization, leading to a decrease in the thickness of the mineral layers. This ion was incorporated in the apatite structure through a substitution mechanism by replacing Ca in the crystal lattice. The obtained Sr-substituted biomimetic coatings are expected to enhance bone formation and osteointegration.

Abstract: Articular cartilage has an inadequate natural rebuilding capacity. Tissue engineering has shown to have potential to provide an effective alternative to engineer the damaged cartilage. In this study, an integrated porous bi-layered scaffold was developed aiming to mimic the requirements of cartilage and underlying subchondral bone. The osteochondral approach explored in this work was to include a common polymeric component in both cartilage and bone components, which maximised the integration at the interface by mean of a melt-based processing route. A blend of starch and poly(Llactic acid),PLLA, was used in the cartilage side, which was found to possess an adequate water uptake capability. For the bone region, to induce bioactivity, PLLA had been reinforced with hydroxyapatite (HA) and bioactive glass (BG). The surfaces of the constructs were investigated as a function of soaking time in a simulated body (SBF) fluid using scanning electron microscopy (SEM) and FTIR. The SEM – FTIR indicated a bone-like apatite formation and the surface coverage by apatite layer increased with increasing soaking time, whereas the cartilage-layer did not exhibit the formation of any apatite like layer.

Abstract: In this study, it is shown that it is possible to develop 3D-porous bilayer hydroxyapatite/chitosan scaffolds by means of combining a sintering and a freeze-drying technique. Scanning electron microscopy (SEM/EDS) studies revealed that the scaffolds possess a well-defined orientation and anisotropic porosity, with pore size ranging between 50-350 µm. Fourier transform infrared spectroscopy (FTIR) and X-ray diffraction (XRD) patterns evidenced the formation of crystalline hydroxyapatite. Moreover, the compression tests revealed that these scaffolds have adequate mechanical properties for being used in tissue engineering of osteochondral defects.

Abstract: Current tissue engineering strategies involve the use of scaffold materials with properties specific for the target tissue. When the tissue being treated is bone, properties such as bone bonding behaviour and excellent biological performance are very desirable. Many strategies involve the creation in vitro of a suitable hybrid construct (i.e., comprising a scaffold material and cells). These scaffolds are then inserted into the defect site, thus achieving faster integration and hence, repair. Herein, we describe the synthesis and characterization of starch-based microparticles for bone tissue engineering. This study describes the properties of two types of starch-based microparticles: their bioactivity in vitro (when processed with Bioactive Glass 45S5), the good biological performance and also the ability to be used as controlled release vehicles of bioactive molecules, such as steroids and growth factors.

Abstract: This work aims to study the effect of incorporating proteins with different isoelectric points (pI) on the structure, composition and morphology of biomimetic calcium-phosphate (Ca-P) coatings. For that, bovine serum albumin (BSA) and lysozyme, having respectively acidic and basic pIs, were used as model proteins. It was observed that the incorporation of positively charged proteins, such as lysozyme, was able to significantly change the structure of the coatings, possibly due to the preferential interactions between the protein and negatively charged phosphate ions. These results indicated that proteins with different characteristics can be incorporated into biomimetic Ca-P coatings in order to obtain a hybrid coating and at the same tailoring their properties.

Abstract: Bioactive polymeric microspheres can be produced by pre-coating them with a calcium silicate solution and the subsequent soaking in a simulated body fluid (SBF). Such combination should allow for the development of bioactive microspheres for several applications in the medical field including tissue engineering. In this work, three types of polymeric microspheres with different sizes were used: (i) ethylene-vinyl alcohol co-polymer (20-30 'm), (ii) polyamide 12 (10-30 'm) and (iii) polyamide 12 (300 'm). These microspheres were soaked in a calcium silicate solution at 36.5°C for different periods of time under several conditions. Afterwards, they were dried in air at 100°C for 24 hrs. Then, the samples were soaked in SBF for 1, 3 and 7 days. Fourier transformed infrared spectroscopy, thin-film X-ray diffraction, and scanning electron microscopy showed that after the calcium silicate treatment and the subsequent soaking in SBF, the microspheres successfully formed a bonelike apatite layer on their surfaces in SBF within 7 days due to the formation of silanol (Si-OH) groups that are quite effective for apatite formation.

Abstract: The molecular weight and crystallinity of systems based on poly(L-lactic acid) PLLA is an important issue as it can influence, besides the general physical properties of the polymer, the patterns of cell adhesion, proliferation and cell morphology. The objective of the present study was to evaluate how crystallinity and molecular weight of PLLA influence the referred parameters. Four conditions were tested: low molecular weight amorphous and semi-crystalline PLLA disks, and high molecular weight amorphous and semi-crystalline PLLA disks, obtained from hot press. The thermal properties of the studied materials were accessed by differential scanning calorimetry. For the cell culture studies a human osteosarcoma cell line (SaOS-2) was chosen. Disks were immersed in a cell suspension containing 5x104 cells/ml and kept in culture for periods up to two weeks. Cell viability and proliferation of SaOS-2 cells was assessed by MTS test and a total protein assay, respectively. The adhesion and morphology of SaOS-2 cells on PLLA disks was assessed by scanning electronic microscopy. Results showed that cell viability was not affected by the different tested conditions. However, cell proliferation was increased in the high molecular weight amorphous samples and cells seemed to have higher adhesion patterns on semi-crystalline samples. This is probably happening due to different rates of integrin interaction with the substrate leading to different patterns of focal adhesion points formation.

Abstract: N-Carboxymethyl chitosan (NCMC) is a water soluble derivative of chitosan. The NCMC beads were prepared by using ionotropic gelation process with the counter polyanion tripolyphoshate at pH 4.0 and characterized by scanning electron microscopy. The swelling behavior of the beads at different time intervals was monitored at different pH conditions. The in vitro drug release behavior in various pH solutions was studied using indomethacin as a model drug with two different concentrations (0.3 and 0.6% w/w). The release percent of indomethacin from NCMC beads was found to increase with increasing of pH in phosphate buffer solution medium due to the ionization of carboxymethyl group and high solubility of indomethacin in alkaline medium. These results indicated that the NCMC beads are useful for controlled drug delivery systems through oral administration by avoiding the drug release in the highly acidic gastric fluid region of the stomach.