Cellularized methacryloyl gelatin bioinks for microextrusion bioprinting of in vitro wound models

Chronic wounds (CWs) are a leading public health concern worldwide representing an economic and social burden and affecting patient’s quality of life. Several smart therapeutic strategies have been developed over the years, but the 90% of them fail in the clinical phase. The main issues responsible for their unsuccessful clinical translation are strictly related to the adopted preclinical validation tools that do not accurately reproduce CW complexity. In this perspective, three dimensional (3D) in vitro wound models are promising tools to preclinically test advanced therapies/wound dressings in a more relevant and ethically sustainable scenario. In this context, this work aimed at engineering a multifunctional bioink for the fabrication of 3D cellularized constructs via microextrusion bioprinting as potential in vitro wound models. To this purpose, gelatin (Gel) was first reacted with different amounts of methacrylic anhydride (MA) under environmental-friendly conditions leading to a differently derivatized methacryloyl gelatin (GelMA) platform. Infrared spectroscopy only proved the absence of functionalization-induced degradation phenomena. Reaction success was indirectly and directly assessed by quantifying GelMA residual free amines and acrylates through colorimetric assays and proton nuclear magnetic resonance (1H NMR, peak at 5.6 ppm) spectroscopy, respectively. Results demonstrated degree of methacryloylation (DoM) tunability (range: 55-93%) by controlling the amount of MA and validated the consistency of colorimetric assays as alternative methods to verify the reaction success. Then, GelMA hydrogels (DoM: 55-93%, polymeric concentration: 5%-12.5% w/V) were optimized for their use as thermo- and Vis light-responsive bioinks. Temperature ramp tests showed a reduction in the onset (Tonset) and gelation (Tgel) temperatures upon DoM increase (e.g., Tonset = 25.1°C vs 21.4°C and Tgel = 30.1°C vs 29.0°C for GelMA with 55% and 93% DoM, respectively). On the other hand, faster crosslinking kinetics and higher storage modulus values (G’) were measured upon Vis light exposure (60s, 80k Lux) by DoM increase from 55% to 93% (i.e., G’ = 160 vs 1140 Pa). Based on these results, GelMA with the highest DoM (i.e., GelMA93) was selected for further investigations. By varying the polymeric concentration between 5% and 12.5% w/V, Tgel measured for GelMA93 hydrogels was approx. 4°C lower compared to Gel as proved through the tube inverting test. Moreover, prolonged irradiation time (i.e., 30-90s, 80k Lux) resulted in improved crosslinking kinetics. Hydrogels proved to be able to absorb external fluids in physiological-mimicking conditions while preserving their stability up to 14d. All tested formulations were cytocompatible according to the ISO 10993-5 (NIH-3T3 cell viability higher than 70%) as assessed through CellTiter Blue, CytoTox-One and Live&Dead assays. Moreover, immunofluorescence staining revealed that hydrogel extract-treated cells showed similar morphology to the control. Furthermore, NIH-3T3 murine fibroblasts loaded in the bioink were able to spread and proliferate up to 7d in normal culture conditions. Lastly, a proof-of-concept of hydrogel printability was carried out by defining a printability window. Hence, the implementation of green functionalization procedures, the easy processability of GelMA hydrogels and their good biological response open the way for the engineering of smart bioinks with promising features for the fabrication of 3D in vitro wound models.