A microgel-based approach for optimized wound healing
Date
2025
Authors
Moretti de Andrade, Thiago Antonio
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Abstract
Skin tissue engineering strategies that leverage the properties of biomaterials for in vitro wound healing investigation have emerged as an effective approach to creating more realistic models providing ethically and scientifically preferable models to animal experimentation. Granular material with spherical-shape and rod-shape have stood out in this scenario, creating a biocompatible interface for cell proliferation and migration. Gelatin Methacryloyl (GelMA) is a pivotal crosslinkable hydrogel in the fabrication of granular biomaterials due to its versatile properties in enhancing the mechanical strength, and biocompatibility. Therefore, the combination of GelMA’s cell-friendly properties and the enhanced porosity generated among its particles in microgel mimicking the extracellular matrix underscores the novelty of this study in the investigation of the keratinocytes’ viability and migration in GelMA microgel-based to optimize the wound healing process in a more effective and realistic method than the regular 2D methods. To address this investigation, it was strategically designed and printed (by Anycubic 4K Mono printer - DLP-based) one structure to be used as a mold of the 7.5% GelMA with keratinocytes (1.0 × 106 / mL of GelMA) in the 12-well plates after its crosslinking by 405 nm LEDs. The middle of this mold was designed to form one vat in the middle of the 7.5% GelMA that is intended to be placed the microgel (10% GelMA spherical-shape with 2.5% GelMA between the particles to crosslink all microgel in the 7.5% GelMA ring) with no keratinocytes. As the microgel was surrounded by keratinocytes in 7.5% GelMA (from its bottom and around), it was possible to investigate the viability and migration of the keratinocytes from the GelMA to the microgel layer naturally and by themselves, with neither stimulation nor chemotaxis. The control group was the 10% GelMA bulk (non-microgel, no droplets) placed in the vat, in the same conditions as the experimental group (10% GelMA spherical-shape droplets). Live/dead was performed to qualitatively evaluate the keratinocytes’ viability and migration; DAPI stain analyses (from the 3D construct samples and from the 2D cryostat-slices’ samples) were performed to qualitatively confirm keratinocytes’ migration. All analysis were investigated on days 1, 3 and 7. Overall, the model has been shown to be feasible, more realistic and promising for studying cell migration, taking advantage the own GelMA’s porosity and the additional GelMA microgel’s porosity, making suitable for short-term in vitro wound healing investigation with no vascularization. The model established here was more effective and realistic to investigate keratinocyte migration from the bottom and across surrounding the microgel area, providing a more relevant system than traditional 2D cultures, such as scratch assays or Transwells. With the second mold design, the most suitable method has been established, and the optimal GelMA bioink concentration (2.5% of GelMA bioink) for promoting keratinocyte migration has been identified. Beyond this, the established method offers a foundation for future studies and broader applications in wound healing and regenerative medicine, for example, investigating the potential effects of the microgel in the wound healing in vitro and in vivo, associated or not with pathologies, such as diabetes, that can impair the wound healing process.
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Keywords
Microgel, GelMA, Keratinocyte, Proliferation, Wound Healing