Mineralization of Recombinant Collagen-Like Peptide to Design Hybrid Scaffolds for Bone Tissue Regeneration

The present PhD Thesis is focused on the fabrication of hybrid scaffolds for bone regeneration through biomimetic mineralization of recombinant collagen peptide (RCP) in the presence of magnesium (Mg) to closely mimic ionic composition of bone apatite. RCP, produced by FUJIFILM Manufacturing Europe B.V. under tradename of CellnestTM, is based on human collagen type I and it is enriched in arginine-glycine-aspartic acid sequence (RGD), that acts as a cell binding site. Biomimetic mineralization of RCP has been adapted from the bioinspired mineralization protocol of natural collagen carried out previously by our group to obtain bone-like scaffolds. Inspired in nanocomposite nature of bone, hybrid scaffolds were developed through a bottom-up approach that started on the evaluation of apatite mineralization mechanism in the presence of RCP and Mg and how it can affect the nanocrystal morphology and composition. Then, three-dimensional prototype scaffolds were developed through freeze-drying of mineralized slurry to determine optimum mineral content. Furthermore, the interfacial properties of mineralized matrices in the presence and in the absence of Mg were evaluated at nanoscopic level. Since RCP is water-soluble molecule, the use of crosslinking technologies (genipin and dehydrothermal treatment) to ensure the stability under physiological conditions as well as the resistance to enzymatic degradation was investigated. The subsequent step was the design of 3D isotropic scaffolds with suitable pore size, porosity and permeability by modifying several freezing parameters during freeze-drying of mineralized RCP slurry. Finally, in vitro tests were carried out to evaluate how biochemical signals (i.e., surface chemistry and ion release from scaffold) together with biophysical signals (e.g., surface nano-topography) conferred via biomimetic mineralization can persuade and guide Mesenchymal Stem Cells (MSCs) interaction and fate. The in vitro studies of designed scaffolds were performed in static as well as under dynamic conditions in a bioreactor of direct perfusion.