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<p>An essential stage of repair for a healing wound is the proliferation of cells in the damaged space. Cells such as fibroblasts, grow and migrate to aid in construction of new tissue and to close the wound. Current methods of studying fibroblast proliferation in wound healing include a 2D wound healing assay in which a cell monolayer is scratched, and the cells migrate into the pseudo-wound. However, this lacks the 3D architecture of a physiological wound. Current 3D models of wound healing often rely on the use of a preexisting matrix for structural assistance, however an isolated system of cell growth without requirement of structural aid may gather new insights on intercellular behavior and mechanical properties. Additionally, we to desire to fabricate a high through-put and easy to use 3D wound healing model than currently offered. Our engineering objective is to create a novel 3D model of wound healing.</p> <p><br></p> <p>This project aims to optimize fibroblast adhesion and proliferation for 3D microtissue fabrication by altering surface and extracellular matrix (ECM) properties to SU-8 scaffolding. Additionally, we consider the effect of different geometries on cell proliferation and cellular stresses/strains, fibronectin production as pseudo-wounds close, and make comparisons to intercellular cancer behavior. Our results show around a 66% decrease in overall culture time required for the microtissues to reach full confluency. Varying geometries in the tessellated design have revealed structural changes in the actin cytoskeleton formation of fibroblasts, and increased fibronectin production along edges of tensioned cells preparing to “close” the wound. When compared to human breast cancer cells, the cancer cells lack the ability to make critical cell to cell junctions that we observe in fibroblasts, noting the characteristic that cancer is like a wound that never heals.</p>

Chronic wounds have a detrimental impact on patient quality of life, a significant economic cost, and often lead to severe outcomes such as amputation, sepsis or death. The elaborate interplay between bacteria, cutaneous cells, immune cells, growth factors, and proteases in chronic wounds has complicated the development of new therapies that could improve outcomes for chronic wound patients. Existing in vitro models of chronic wounds do not appreciably mimic the complexity of the wound environment. In this work, tissue-engineered skin was developed with the goal of creating an in vitro platform appropriate for testing potential clinical therapies for chronic wounds. The Lumen-X, a digital light processing bioprinter, was used to create tissue-engineered skin from a 7.5% (w/v) gelatin methacryloyl hydrogel laden with primary dermal fibroblasts. This dermal layer was developed with an emphasis on providing a favourable microenvironment for the fibroblasts in order to mimic their in vivo phenotype. An epidermal layer of human keratinocytes was formed on the hydrogel surface and stratified through culture at the air-liquid-interface. The maturation of the epidermis was thoroughly characterized with histology, immunohistochemistry, and trans-epithelial electrical resistance analyses which showed a degree of maturation suitable for wound healing studies. To verify the suitability of this tissue-engineered skin for studying healing in vitro, sharp tweezers were used to create physical wounds in the epidermis which were then infected with Pseudomonas aeruginosa. Reepithelialisation, the production of the pro- inflammatory cytokine TNF-α, and the presence of bacteria were monitored over time, showing healing in wounds without infection and those treated with antibiotics, and potential biofilm formation in infected wounds. The tissue-engineered skin developed here is suitable for use as an in vitro model of the infected chronic wound environment. Future work includes developing better methods for creating the physical wound and characterizing the bacterial biofilm in order to improve the reproducibility and clarity of results. Such a model will then be well-poised to begin testing potential chronic wound therapies in vitro. / Graduate / 2023-07-26

Spinneret based Tunable Engineered Parameters (STEP) Platform is a recently reported pseudo-dry spinning and non-electrospinning technique that allows for the deposition of aligned polymeric nano-fibers with control on fiber diameters and orientation in single and multiple layers (diameter: sub 100nm micron, length: mm-cm), deposition (parallelism 2.5 degrees) and spacing (microns)). A wide range of polymers such as PLGA, PLA, PS, and PU have been utilized for their unique material properties in scaffold design. In this thesis two unique bioscaffolds are demonstrated for the measurement of group cell migration for wound closure and single cell contractility force for the study of force modulation. The wound healing assay bridges the gap between confluent reservoirs of NIH3T3 fibroblasts through arrangement of a suspended array of fibers guiding group cell migration along the fiber axis. This platform demonstrates that topographical and geometrical features of suspended fibers play a very important role in wound closure. Spacing, alignment and orientation were optimized to shown an increased rate of closure. In the second complementary assay, we report a fused-fiber network of suspended fibers capable of measuring single cell forces. Results from our experiments demonstrate that force behavior is dependent on mechanical properties such as stiffness and geometry of fiber networks. We also demonstrate changes in spatial and temporal organization of focal adhesion zyxin in response to single cell migration on these networks. / Master of Science

The objective of this study was to determine if topical antimicrobials silver sulfadiazine and povidone-iodine ointment increase rates of healing of equine distal limb wounds that heal by second intention. Second, to determine the effect of bandaging with these topical antimicrobials. Six healthy adult horses were used to create thirty-six, 2.5 cm2 standardized full-thickness metacarpal/tarsal skin wounds. Each wound was exposed to a single treatment: 1.0 % silver sulfadiazine cream bandaged (SSD-B), 1.0 % silver sulfadiazine slow release matrix bandaged (SDX-B), 1.0% silver sulfadiazine slow release matrix not bandaged (SDX-NB), povidone-iodine ointment bandaged (PI-B), untreated control bandaged (C-B) and untreated control not bandaged (C-NB) until healing. Wound area, granulation tissue area and perimeter were measured with planimetry software from digital images obtained at each observation. Exuberant granulation tissue was excised when present. The days until healing, rate of healing parameter, rates of contraction and epithelialization were compared among groups using pair-wise analysis of least square means. The healing parameters and mean days to healing did not statistically differ between groups. Analysis of percent wound contraction and rate of epithelialization between groups was similar. Mean number of days to healing ranged from 83 (PI-B and C-B) to101 (SSD-B). All bandaged wounds produced exuberant granulation tissue requiring excision compared to none of the unbandaged. The identified rates of epithelialization and wound contraction found insignificant differences between antimicrobial treated versus untreated wounds. Similarly, rates of epithelialization and wound contraction found insignificant differences between bandaged versus unbandaged wounds. Topical povidone-iodine and silver sulfadiazine did not increase rates of healing under bandage. The 1.0% silver sulfadiazine slow release matrix not bandaged (SDX-NB) adhered well to dry wounds. Silver sulfadiazine slow-release matrix provides does not impede wound healing and provides good adherence to dry wounds not amenable to bandaging. / Master of Science