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.