Development of injectable hydrogels for application in tissue engineering

• Autor(a) principal: Ouro, Pedro Miguel da Silva
• Data de Publicação: 2022
• Tipo de documento: Dissertação
• Idioma: eng
• Título da fonte: Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
• Texto Completo: http://hdl.handle.net/10773/36688
• topic: Macromolecules; Chitosan; Hydrogel; Injectable system; Tissue engineering

Tissue engineering (TE) is an ever-growing and promising field in regenerative medicine, looking to develop biomimetic systems capable of replacing damaged tissues or organs. For that, biomimetic systems, such as hydrogels, based on natural macromolecules (e.g., chitosan), have been used. Currently, the most versatile hydrogel processing method is 3D bioprinting, a sophisticated method that allows the printing of different structures with excellent precision. However, the used system must gather injectability and printability features. Methacrylated chitosan (CHTMA)-based hydrogels have already been reported as injectable and cytocompatible systems. Still, these systems consisted of single crosslinked (covalent bonds) network, affording hydrogels with poor mechanical properties. As a way of improving upon this system, a new biomimetic, injectable, and cytocompatible dual-crosslinked (DC) CHTMA-Tricine-based hydrogel system, fully processed under physiological pH, was developed in this work. To do so, chitosan (CHT) was sequentially modified (in two reaction steps) with methacrylic acid (MA), and then, with tricine moieties affording CHTMA-Tricine in 74% overall yield. The methacrylate and tricine moieties’ insertion onto the CHT’s backbone was confirmed and quantified by spectroscopy technics, achieving 17% substitution degree (SD) for MA and 4% SD for tricine. DC CHTMA-Tricine-based hydrogels at 3%, 4% and 5% (w/v) concentrations were prepared in PBS, fully characterized (structurally and morphologically) and compared to their CHTMA-based counterparts. The injectability and printability capabilities of this new system were also evaluated. The system’s shear thinning properties were assessed and confirmed through rheological assays, so the system was considered injectable for all tested conditions. The 4% (w/v) concentration dispersion showed the best results and the system’s printability was also assessed and confirmed for this concentration. It was observed that the presence of non-covalent hydrogen bonds, established between tricine moieties, significantly improved the system’s mechanical properties when compared to its CHTMA counterpart, achieving, for instance, an increase of ~ 25 kPa (2.3 times higher) for the Young’s modulus and an increase of ~ 225.2 kJ.m־ᶟ (1.3 times higher) for the toughness, for 5% (w/v) concentration. 2D and 3D cytocompatibility was also conducted for the optimized CHTMA-Tricine-based system using MC3T3-E1 cells. 2D cytocompatibility tests showed no cytotoxicity for MC3T3 cells for all tested concentrations. Additionally, 3D cytocompatibility tests, at 4% (w/v) concentration, showed that MC3T3-E1 cells remain viable after 6 days encapsulated in the CHTMA-Tricine-based hydrogel system. However, these cells did not spread and a decrease in the metabolic activity and DNA content were observed. Overall, the results showed that the addition of tricine to the CHTMA-based system was an excellent option to improve upon its mechanical properties. Considering the lack of cell spreading, further studies should be carried out to overcome this limitation. Regardless, this novel injectable and printable DC CHTMA-Tricine-based hydrogel system has shown that it may be a promising option for future applications in tissue engineering and regenerative medicine.