Toughening robocast chitosan/biphasic calcium phosphate composite scaffolds with silk fibroin: Tuning printable inks and scaffold structure for bone regeneration.

The present work aims the production of composite bioceramic scaffolds by robocasting suppressing sintering as post printing process. To achieve this purpose, extrudable ink compositions containing a high concentration of bioceramic powders (hydroxyapatite and ß-tricalcium phosphate) embedded in aqueous polymeric solutions of chitosan and silk fibroin were fine-tuned. Polymeric solutions of chitosan/silk fibroin with different ratios were tested, maintaining the total amount of bioceramic solids at 30 vol%. The inks were characterized by rheological studies in viscometry and oscillatory modes, being the printable ones selected to produce scaffolds with different macropore sizes (300 µm and 500 µm). The scaffolds were characterized by mechanical properties (dry and wet conditions) and morphological features, as well as its degradability. In vitro studies were also evaluated in the scaffolds that presented the best structural performance. The addition of 2 wt% silk fibroin to a 5 wt% chitosan matrix allows to significantly improve the mechanical performance of the printed composite scaffolds, reflected in high values for Young's modulus and maximum compressive strength. This trend was continued in wet scaffolds with a concomitant reduction of mechanical properties. Regarding degradability, the scaffolds in general presented a weight loss in the range of 14-18% after 28 days incubation in HEPES solution at two different pH values at 37 °C, with an associated release of calcium and phosphorus ions. The scaffold with 300 µm porosity comprising the both polymers in its composition presented the less rate degradation when compared to the scaffolds with similar porosity and containing only chitosan as base matrix. Moreover, the combined natural polymers gave rise to a significant increase in the metabolic activity of human osteoblasts grown on the scaffolds with both macropore' size, being in line with the full cellular filling of their surfaces, demonstrated by SEM and confocal imaging. The advances presented in this work are a promising path in the ink's development for extrusion-based additive manufacturing techniques and subsequent biomaterials, encompassing suitable physical and chemical characteristics with high potential to be used as bone substitutes.

Effective production of multifunctional magnetic-sensitive biomaterial by an extrusion-based additive manufacturing technique.

A calcium phosphate (CaP)-based scaffold used as synthetic bone grafts, which smartly combines precise dimensions, controlled porosity and therapeutic functions, presents benefits beyond those offered by conventional practices, although its fabrication is still a challenge. The sintering step normally required to improve the strength of the ceramic scaffolds precludes the addition of any biomolecules or functional particles before this stage. This study presents a proof of concept of multifunctional CaP-based scaffolds, fabricated by additive manufacturing from an innovative ink composition, with potential for bone regeneration, cancer treatment by local magnetic hyperthermia and drug delivery platforms. Highly loaded inks comprising iron-doped hydroxyapatite and ß-tricalcium phosphate powders suspended in a chitosan-based solution, in the presence of levofloxacin (LEV) as model drug and magnetic nanoparticles (MNP), were developed. The sintering step was removed from the production process, and the integrity of the printed scaffolds was assured by the polymerization capacity of the ink composite, using genipin as a crosslinking agent. The effects of MNP and LEV on the inks' rheological properties, as well as on the mechanical and structural behaviour of non-doped and iron-doped scaffolds, were evaluated. Magnetic and magneto-thermal response, drug delivery and biological performance, such as cell proliferation in the absence and presence of an applied magnetic field, were also assessed. The addition of a constant amount of MNP in the iron-doped and non-doped CaP-based inks enhances their magnetic response and induction heating, with these effects more pronounced for the iron-doped CaP-based ink. These results suggest a synergistic effect between the iron-doped CaP-based powders and the MNP due to ferro/ferrimagnetic interactions. Furthermore, the iron presence enhances human mesenchymal stem cell metabolic activity and proliferation.

Tribological behaviour of unveneered and veneered lithium disilicate dental material.

The friction and wear behaviour of a lithium disilicate dental ceramic against natural dental enamel is studied, including the effect of the presence of a fluorapatite veneering upon the tribological properties of the material. The tribological behaviour was assessed using reciprocating pin-on-plate test configuration, at pH 3 and pH 7. The surface energy of the plates was determined, as well as the zeta potential of fluorapatite, lithium disilicate and enamel particles in artificial saliva. It was found that the friction and wear behaviour of the tested enamel/plate material tribocouples is less severe in unveneered plates. Initial surface roughness of the plate does not affect wear results. However the topography of the resulting wear track affects the corresponding wear loss: a smoother final wear track is associated with lower wear. The surface topography of the wear track, and thus the tribological performance of the tested materials, is very sensitive to the pH of the sliding solution. This is because the dissolution trend, wettability and surface charge of the used materials are pH dependent. Overall friction and wear are higher under basic pH conditions, especially when plates are veneered. A wear model is proposed that correlates the effect of the described parameters with the observed tribological behaviour at pH 7. Attained results show that fluorapatite coating of lithium disilicate dental crowns affects tooth/crown wear behaviour, resulting in increased wear of both the artificial crown and the opposing natural teeth. Coating should therefore be avoided in occlusal crown surfaces.

Synthesis, mechanical and biological characterization of ionic doped carbonated hydroxyapatite/ß-tricalcium phosphate mixtures.

The influence of ionic substituents in calcium phosphates intended for bone and tooth replacement biomedical applications is an important research topic, owing to the essential roles played by trace elements in biological processes. The present study investigates the mechanical and biological evaluation of ionic doped hydroxyapatite/ß-tricalcium phosphate mixtures which have been prepared by a simple aqueous precipitation method. Heat treating the resultant calcium phosphates in a carbonated atmosphere led to the formation of ionic doped carbonated hydroxyapatite/ß-tricalcium phosphate mixtures containing the essential ions of biological apatite. The structural analysis determined by Rietveld refinement confirmed the presence of hydroxyapatite as the main phase, together with a considerable amount of ß-tricalcium phosphate. Such phase assemblage is essentially due to the influence of substituted ions during synthesis. The results from mechanical tests proved that carbonate substitutions are detrimental for the mechanical properties of apatite-based ceramics. In vitro proliferation assays of osteoblastic-like cells (MC3T3-E1 cell line) to powders revealed that carbonate incorporation can either delay or accelerate MC3T3 proliferation, although reaching the same proliferation levels as control cells after 2 weeks in culture. Further, the powders enable pre-osteoblastic differentiation in a similar manner to control cells, as indirectly measured by ALP activity and Type-I collagen medium secretion.

Influence of setting liquid composition and liquid-to-powder ratio on properties of a Mg-substituted calcium phosphate cement.

The influence of four variables on various properties of a Mg-substituted calcium phosphate cement (CPC) was investigated. The variables were the heat treatment temperature of the precipitated powders, the composition of the setting liquid, the liquid-to-powder ratio (LPR), and the time over which hardened specimens were cured in air. The properties analysed were the phase composition of the starting powder, the initial setting time, the evolution of the storage shear modulus (G') and the loss shear modulus (G'') with the cement paste curing time (t), and the compressive strength. The presence of alpha-TCP in CPC facilitated the setting and hardening properties due to its progressive dissolution and the formation of brushite crystals. As far as the liquid composition is concerned, in cases where citric acid was used, adding a rheology modifier (10 wt.% polyethylene glycol or 0.5 wt.% hydroxyl propylmethylcellulose) to the acid led to an increase in the initial setting time, while an increase in the acid concentration led to a decrease in the initial setting time. The initial setting time showed to be very sensitive towards the LPR. The evolution of G' and G'' with curing time reflected the internal structural changes of cement pastes during the setting process. The compressive strength of the wet-hardened cement specimens with and without Mg increased with curing time increasing, being slightly higher in the case of Mg-substituted CPC. The results suggest that Mg-substituted CPC holds a promise for uses in orthopaedic and trauma surgery such as for filling bone defects.