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Tissue Engineering

Tissue Engineering - Poster Session E

Poster BB14 - Towards re-epithelization in a human skin equivalent wound model with implications for diabetic foot ulcers

Saturday, October 26, 2024
10:00 AM – 11:00 AM EST
Location: Exhibit Hall E, F & G

Introduction:  Recent advancements in tissue engineering have demonstrated the feasibility and utility of developing 3D-bioprinted wounded skin models. These models exhibit greater physiological relevance with human skin and facilitate improved exploration of the physiological mechanisms and processes of wound healing. However, no such models exist of diabetic skin ulcers, the most common cause of morbidity in diabetic patients. The goal of this study was to develop and standardize a 3D-bioprinted skin model that recapitulates the morphology and pathology of a diabetic skin ulcer in a simple, low-cost procedure.

Materials and

Methods:  The methodology was separated into four components: creating a diabetic phenotype in fibroblast cells to mimic diabetic conditions, creating a healthy bioprinted skin, optimizing a wounding process, and evaluation of the healing process.

An alginate/gelatin bioink was used to print a dermal construct with fibroblasts and subsequently wounded. To probe differences in wound healing between diabetic and nondiabetic conditions, the wounded skin models were evaluated in vitro under hypoxic and normoxic conditions, using dermal cells tolerized to physiologically-relevant hyperglycemic and normoglycemic conditions. To characterize the proliferative and migratory potential of the tolerized NIH 3T3 fibroblast cells a standard 2D scratch assay was performed under diabetic (high glucose media, hypoxia) and normal (low glucose media, normoxia) conditions over 48 hours.

The processes for in vitro cellular tolerization and ex vivo wounding of cell-laden 3D-bioprinted skin constructs were optimized. Artificial wounds of different sizes, depths, and shapes were created by a 1 mm, 2 mm, and 4 mm punch biopsy tool and with a sterile knife.

Constructs were harvested for analysis just prior to wounding and on days 4, 7, and 14 after wounding to visualize the wound morphology and changes in the structure of the extracellular matrix over healing. Cell viability in each bioink formulation with and without the addition of fibrinogen was evaluated using a Calcein AM cell viability assay in which cells stain green if alive and red if apoptotic at days 1, 4, and 7.

Results, Conclusions, and Discussions: Low glucose environments are shown to increase the wound healing capabilities of diabetic cells. However, there was no significant difference in the percentage of wound area closed between these conditions at normoxia. As hypoxia is known to be another contributing factor to the dysfunction diabetic fibroblasts experience during endothelial healing, additional 2D scratch assays were prepared to mimic the combination of the hypoxic and hyperglycemic environmental conditions of diabetic wounds. When compared to normoxic and normoglycemic environmental conditions there was a significant difference.

In diabetic patients, the size of a DFU is often the first analysis for its severity. Consequently, only the 1 mm and 2 mm punch biopsies were selected as they were the only tools that generated wounds below the DFU threshold, corresponding to smaller Grade 1-4 wounds. This finding was conserved in the cell-laden bioprinted constructs following wounding at day 14.

In the formulation of the extrusion-based bioprinting procedure, the crosslinking parameters that required optimization were time of crosslinking and temperature of crosslinking during bioprinting. Creating wounds that could spontaneously heal due to their small size was prioritized during this initial phase of model development.

Although more studies will have to be completed, this study showed that a functional diabetic phenotype in NIH 3T3 fibroblast cells was able to be grown with an optimized tolerization procedure. A viable skin construct using a low-cost bioink formulation was able to be grown for a month in culture as well as 3D bioprinted healthy skin models that were adapted to diabetic conditions. These newly established procedures will support future development of 3D-bioprinted wound models and aid in the evaluation of how varied wound parameters alter re-epithelialization and remodeling. More broadly, this work presents preliminary data that can be used in the future for replicable manufacturing of 3D diabetic wound models for use as drug screening tools.

Acknowledgements (Optional):