On the improvement of the process of synthesis of gelatin methacryloyl (GelMA) hydrogels and development of a hybrid nanoparticle-GelMA-based bioink for tissue engineering
Sánchez Rodríguez, Víctor Hugo
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Gelatin methacryloyl (GelMA) is a semisynthetic biomaterial that conserves some relevant advantages of native collagen such as the presence of cell-binding domains with protease-cleavage sites. GelMA-based hydrogels are currently used as biomaterials to develop cell-laden systems for several biomedical applications. GelMA provides the necessary physiological microenvironment and biocompatibility for tissue engineering studies. However, currently used methods to produce GelMA do not consider a strict control of the key parameters of the reaction process (i.e., mixing, location and rate of addition of methacrylic anhydride, and pH) which leads to batch to batch inconsistencies and low yields. In this work, a semi-automated process to synthesize and purify GelMA is presented. Briefly, the method considered (a) the use of a custom-made jacketed reactor with temperature, agitation, and pH control, (b) the addition of methacrylic anhydride (MA), the key reagent of the synthesis, by a controllable syringe pump, and (c) a continuous dialysis stage, carried through a set of peristaltic pumps, follows the procedure shortening effectively the time needed for methacrylic acid removal. Automated equipment and pH control reduce synthesis and purification times and enhance reactivity, leading to a higher degree of substitution. Using this synthesis and purification strategy, we conducted multiple sets of GelMA production and correlated reaction conditions to final yield and quality of the product. As a result of this process intensification, the required time of synthesis and purification was significantly reduced, and the amount of water required for the removal of cytotoxic residues (i.e., methacrylic acid) was minimized. While conventional protocols require a 2 weeks preparation time and result in an inconsistent quality of the final product, the methodologies presented here yield consistent quality GelMA in 5 days. Moreover, this work presents results on the rates of methacryloyl functionalization during the reaction stage and the rate of methacrylic acid removal during the purification stage. The degree of methacrylation (DoM), the exposure to UV light (intensity and time) during the photo-crosslinking time of GelMA hydrogels, as well as the gelatin and photoinitiator type and concentration have a strong and direct influence on the mechanical, physicochemical, properties of this biomaterial, which at the same time influence cellular behavior. Finally, we developed a proof-of-principle strategy to use our hydrogel as a nano-composite bio-ink for 3D printing. To fulfill the goal of proper rheology for GelMA extrusion, a variety of bioinks were prepared by mixing GelMA, gelatin, and halloysite-nanoparticles (HNT). The concentration of GelMA, gelatin, and nanoparticles was varied to create an executable 3D ink at room temperature taking advantage of a two-step thermal- / photo-crosslinking strategy. This approach demonstrated to obtain reproducible cell-laden scaffolds by using extrusion as a tool for 3D printing.
- Declaration of Autorshio p VHSR MNT.pdf
- Declaración de Autoría VHSR
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