Engineering a diabetic human 3D in vitro skin model to mimic foot ulcer

Diabetes Mellitus (DM) is a chronic disorder with global health impact, not only due to the mere pathology but also to its frequent association with various comorbidities and complications, such as cardiovascular diseases, obesity, neuropathy, and impaired wound healing. Given the increasing trend of overweight and obese diabetic patients, insulin resistance and the resulting biological cascades have gained significant research interest in recent years. Among the complications, foot ulcers are the most concerning type of chronic wounds in diabetic patients due to related comorbidities hindering healing and often leading to limb amputation. Despite the clinical relevance of DM, there is a remarkable lack of studies focused on diabetic skin and reliable easy-to-fabricate models to study the pathological phenomena and to test new treatments. In this frame, in vitro cell cultures are essential for understanding in vivo cellular mechanisms. Traditional 2D culture systems are widely used due to their ease of handling, low cost, and well-established analytical methods. However, they fail to replicate critical biochemical and physical cues present in tissue environments. On the other hand, animal models provide a comprehensive physiological environment, but face ethical concerns, high costs, and limitations in mimicking human-specific features. Scaffold-based 3D in vitro models that exploit extracellular matrix-like biomaterials represent an optimal balance between biological relevance, ease of use, and cost. In this context, my Master’s Degree Thesis project aimed to develop a 3D in vitro model that faithfully replicates diabetic skin, bridging the gap between traditional 2D cultures and animal models. Gelatin methacryloyl (GelMA) was used as biomaterial due to its tunable mechanical properties and biocompatibility. During this Thesis, GelMA has been synthesized, characterized and then used in the biofabrication of a 3D in vitro diabetic skin model. The final construct wanted to recapitulate epidermis (HaCaT keratinocytes) and dermis compartment (HFF-1 fibroblasts embedded in GelMA) in a diabetic scenario. Preliminary tests in 2D cell cultures were performed to define the hyperglycaemic environment, resulting in +25mM D-Glucose in the growth medium. Different concentrations of insulin and metformin were tested to define the optimal dose and assess the potential cytotoxicity of the treatment to obtain preliminary results on treatment strategies. Immunofluorescence was exploited to confirm morphological changes induced by the hyperglycaemic state, suggesting cellular senescence. After the 2D preliminary analysis, 3D in vitro skin models were biofabricated. Fibroblast-laden GelMa hydrogels were prepared and photopolymerized in 12 well-cell culture inserts and then keratinocytes were seeded on top of the hydrogels. The models were cultured in Air-to-Liquid Interface (ALI) for 4 weeks to promote the construct maturation. Then, samples were mechanically wounded and cells behaviour was assessed after 24 hours and 7 days post-injury. The diabetic model was characterized using multiple techniques such as ddPCR for RNA expression and immunofluorescence. The results demonstrated upregulated senescence biomarker expression, altered fibroblast matrix secretion, and reduced epidermis thickness. Future work will enhance the 3D model complexity incorporating immune cells and a vascular network to establish a more robust and representative model towards precision medicine.