| Resumo: | Tissue engineering research attempts to satisfy the needs of support, reinforcement and in some cases organization of the regenerating tissue with a controlled supply of bioactive substances that might positively influence the behaviour of incorporated or ingrowing cells. As demonstrated by the recent advances on biomaterials, the ideal scaffold for tissue regeneration should offer a 3D interconnected porous structure behaving as a template to promote cells adhesion and proliferation and vascularisation as well thus stimulating the new tissue ingrowth. A special interest has been focused on chitosan (CH - the partially deacetylated derivative of chitin) scaffolds for bone regeneration due to its biological and physical properties, in spite of some drawbacks regarding its lack of mechanical strength and bioactivity. The incorporation of bioactive calcium phosphates materials in the polymer matrix is expected to reinforce chitosan scaffolds improving their mechanical performance and osteoconductivity. In the present work, chitosan based scaffolds were produced by freeze-drying CH solutions containing calcium phosphate (CaP) particles, either as fibers of hydroxyapatite (HA), platelets of monetite or a mixture of both. CaP particles were prepared by a wet precipitation method. The calcium phosphate precipitation was monitored by taking a number of samples during 3-days. Evolution of the morphology and crystal phase composition of the precipitated particles were followed by scanning electron microscopy (SEM), N2 adsorption using the BET isotherm (BET), and X-ray diffraction (XRD). It was observed that the increase of refluxing temperature allowed a faster transformation of octacalcium phosphate fibers into HA fibers, hence shortening the precipitation time required for obtaining HA fibers, Chitosan based scaffolds suspensions at two different pH values were frozen at three different temperatures before freeze-drying (thermally induced phase separation-TIPS). SEM, XRD, microcomputed tomography (μ-CT) and Fourier transformed infrared spectroscopy (FTIR) were used to analyze the physical and chemical properties of the composite scaffolds. Compressive mechanical tests were also undertaken to characterize the materials. Bioactivity studies were performed in simulated body fluid (SBF) solutions by monitoring the Ca and P concentration variations of SBF solutions. Highly interconnected macroporous scaffolds with a pore size ranging from of 50 to 250μm, interconnectivity around 91-98.5%, and porosity higher than 80% were obtained. The freezing temperature and the pH of chitosan solution/suspension revealed to play a significant influence in the pore structure. The higher pH (pH=5) and the higher freezing temperature (T=0ºC) were found as the most favourable conditions for ice crystal growth which resulted in larger pores. It was also observed that CaP particles incorporation in the CH matrix increased the scaffold mechanical strength which was also conditioned by the pore size and by the reinforcing particle morphology. The bioactivity studies revealed the CaP contribution for the scaffold bioactivity. The composite scaffolds having brushite and HA (obtained at pH=2) exhibited enhanced bioactivity as compared to composite CH/HA scaffolds based. CH based scaffolds were also prepared by incorporating HA granules loaded with dexamethasone (DEX), a drug model, in CH solution. The granules were obtained by spray drying HA nanosized particles suspended in DEX solution. The drug release profiles of DEX were determined in phosphate-buffered solution (PBS) by DEX concentration evaluation in the releasing medium by Ultraviolet (UV) spectroscopy at the wavelength of 242 nm. Among the different DEX release patterns corresponding to the various DEX loading methodologies which were tested, an adequate release profile could be selected: it showed that the release of 80% of the DEX loaded amount could be ensured during ~30 days, thus enabling a prolonged and slowest DEX release as compared to literature reports. It is thus found that the CH scaffolds engineered with a calcium phosphate based drug delivery system (DDS) provides the desirable association of a bioactive and osteoconductive matrix with an in situ controlled release of a therapeutic agent. These results point out an additional potential of the composite CH/HA scaffolds for behaving as a controlled drug release system (DDS).
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