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Type 1 Diabetes Mellitus (T1DM) is an autoimmune condition resulting in the destruction of insulin-producing beta cells, leading to chronic hyperglycemia and accumulation of advanced glycation end products (AGEs). These factors contribute to serious complications such as diabetic foot ulcers (DFUs) (1-2), which result from chronic inflammation, vascular damage, and neuropathy. Despite their impact, diabetic skin alterations remain underexplored. Improved understanding of these mechanisms could aid in identifying key pathways underlying DFUs and in developing targeted therapies (3).
In this study, a 3D in vitro model of diabetic skin was developed to replicate key features of the disease, and to develop a dynamic in vitro model of diabetic skin that recapitulates biological and mechanical features of the pathology. As a preliminary step, human immortalized keratinocytes (HaCaT) and human foreskin fibroblasts (HFF-1) were cultured under normoglycemic (NG, 25 mM glucose) and hyperglycemic (HG, 50 mM glucose) conditions in both 2D and 3D settings. Fibroblasts were embedded in gelatin methacryloyl (GelMA) hydrogels formulated either in with NG or HG medium, with results indicating a loss in metabolic activity under hyperglycemic conditions.
Subsequently, full-thickness 3D skin models were fabricated. Fibroblasts were encapsulated in normo- or hyper-glycemic GelMA and seeded onto PET inserts, following by UV photopolymerization of the dermal layer. Subsequently, keratinocytes were seeded on top of the dermal layer. After 3 days of submerged culture, constructs were exposed to air-liquid interface (ALI) conditions for 28 days to promote epidermal differentiation. Morphological and molecular analyses were performed via immunofluorescence staining and droplet digital PCR (ddPCR).
Immunofluorescence staining revealed reduced epidermal thickness and morphological changes in keratinocytes and fibroblast under hyperglycemia, consistent with features observed in diabetic skin (Figure 1-2, Table 1) . Gene expression analysis (Figure 3) showed decreased COL1A1 expression, indicative of impaired extracellular matrix remodeling, while COL3A1 expression was less affected. Increased IL8 levels in hyperglycemic conditions reflected the diabetic inflammatory profile. Moreover, elevated CDKN1A expression indicated enhanced cellular senescence, hallmark of diabetic skin aging. Finally, VEGF expression was reduced under hyperglycemic conditions, mirroring diabetic impaired angiogenesis and delayed wound healing.
These results validate the engineered model’s ability to replicate critical features of diabetic skin, providing a promising tool for studying diabetic skin pathophysiology and screening treatments.
Future work will introduce standardized wounds using a custom-designed and 3D printed wounding device, enabling the assessment of healing responses under untreated and treated conditions using therapies such as insulin and metformin. To increase physiological relevance, hypodermal and vascular layers will be integrated, along with macrophages to simulate chronic inflammation. This will enable the creation of a robust and translationally relevant diabetic foot ulcer model for drug testing and disease research.
References:
1) Rhee, Kim. The role of advanced glycation end products in diabetic vascular complications. Diabetes Metab J. 2018;42(3):188–95.
2) Phang, Arumugam. A review of diabetic wound models—Novel insights into diabetic foot ulcer. J Tissue Eng Regen Med. 2021;
3) De Macedo, Skin disorders in diabetes mellitus: An epidemiology and physiopathology review. Diabetol Metab Syndr. 2016;8(1):1–8.
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