Engineering Skin Wound Models for the Validation of Wound Dressings: a look inside the in vitro induction of oxidative and inflammatory processes

Chronic wounds (CW) are one of the leading causes of illness worldwide, impacting around 40 million people globally. Several advanced therapies have been developed over the years, although often being ineffective due to the lack of patient-specificity and reliable testing conditions. Consequently, there is a growing demand for developing skin tissue models which can adequately mimic the complexity of CW environment and work as reliable tools for testing/validating wound dressings. In this context, this thesis aims at designing a 3D in vitro ulcer model by integrating extrusion-based bioprinting and murine fibroblasts (NIH-3T3). To this purpose, methacryloyl gelatine (GelMA) was first synthetized by reacting gelatine with methacrylic anhydride to expose acrylate groups along the polymer chains, thus obtaining a thermo- and photo-sensitive polymer. Infrared spectroscopy only verified the preservation of polymer structural integrity upon functionalisation, while synthesis success was assessed through the colorimetric Ninhydrin and 2,4,6-Trinitrobenzene Sulfonic Acid assays giving a degree of methacryloylation equal to 85.4±1.1% and 88.8±2.3%, respectively. Subsequently, GelMA hydrogels were prepared by dissolving the polymer in culture medium at different concentrations (5% w/v, 7.5% w/v, 10% w/v) and adding Eosin Y (0.05 mM), Triethanolammine (0.2% v/v) and N-Vinylcaprolactam (0.3% w/V) as photo-initiator, co-photoinitiator and co-monomer, respectively. Thermo-sensitivity was evaluated through tube inverting test showing a decrease in the sol-gel temperature as the polymeric concentration decreased (i.e., Tgel=25 °C, 24 °C and 22 °C for GelMA at 10% w/v, 7.5% w/v, and 5% w/v, respectively). Then, hydrogels photo-sensitivity was studied by exposing the formulations to Visible light (400-700 nm) for different time intervals (0s-60s). 60 s of irradiation were selected as the optimal exposure time, since the obtained samples appeared sufficiently stable at 37 °C. Simultaneously, hydrogels were able to absorb external fluids up to 14 days, but only samples at 7.5% and 10% w/v were stable in physiological conditions (T=37 °C, pH=7.4). All tested formulations were cytocompatible according to the ISO 10993-5 (i.e., NIH-3T3 cell viability>70%), as assessed by CellTiter-Blue® assay. Moreover, the extrusion-based fabrication of a 3D in vitro non-cellularized model was optimized. Subsequently, pathological oxidative stress and inflammation conditions were first optimized on 2D NIH-3T3 cultures. Oxidative stress was first induced using hydrogen peroxide (H2O2, 100–750 µM) before treatment with Gallic Acid (GA, 1.6–100 µg/mL) as antioxidant agent. ROS quantification and fluorescence images evidenced a reduction in the oxidative stress levels with values comparable to the control after 24h of cell treatment with GA at 18.75 µg/mL. Differently, persistent inflammation was first induced through Lipopolysaccharides/interferon-ϒ and then, inflamed cells were treated with Ibuprofen as anti-inflammatory drug. The concentrations of pro-inflammatory TNF-α and Interleukin-10 were monitored through ELISA assays. To conclude, results proved the successful (i) implementation of green procedures for polymer functionalization and processing in the form of 3D skin ulcers and, (ii) optimization of protocols for the induction of pathological conditions in 2D cultures paving the way for the development of 3D skin ulcers models as pre-clinical validation tool for patient personalized wound dressings.