Skin wound healing presents a challenging and expensive clinical problem with nearly 20 million wounds requiring intervention leading to an annual cost of more than $8 million. Tissue engineered skin substitutes are valuable not only as a clinical therapy for chronic wounds and severe traumas, but also as in vitro 3D model systems to investigate wound healing and skin pathogenesis. However, these substitutes are limited by a lack of topography at the dermal-epidermal junction (DEJ). In contrast, the native DEJ is characterized by a series of dermal papillae which project upward into the epidermal layer and create physical topographic microniches that support keratinocyte stem cell clustering. In this thesis, we created novel 3D skin model systems to investigate the role of microtopography in regulating keratinocyte function and cell fate using scaffolds containing precisely engineered topographic features. We hypothesized that the microtopography of the DEJ creates distinct keratinocyte microniches that promote epidermal morphogenesis and modulate keratinocyte stem cell clustering which can be harnessed to create a more robust skin substitute that expedites wound closure. Using photolithographic techniques, we created micropatterned DEJ analogs and micropatterned dermal-epidermal regeneration matrices (µDERM) which couple a dermal support matrix to a micropatterned DEJ analog. We found that the incorporation of microtopography into our in vitro skin model resulted in a thicker, more robust epidermal layer. Additionally, we identified three distinct functional keratinocyte niches: the proliferative niche in narrow channels, the synthetic niche in wide channels and the keratinocyte stem cell niche in narrow channels and corner topographies. Ultimately, incorporation of both narrow and wide channels on a single construct allowed us to recreate native keratinocyte stem cell patterning in vitro. These model systems will allow us to investigate the role of cellular microniches in regulating cellular function and epidermal disease pathogenesis as well as to identify topographic cues that enhance the rate of wound healing.

To date, the reconstructive approach addressing chronic non-healing wounds, deep tissue damage, and severe wound defects relies upon avascular dermal grafts and autologous flap techniques. Such flaps are limited by donor site availability and morbidity, while current dermal grafts rely upon host cellular invasion for neovascularization and incorporation. These products fail to include an inherent vascular network and the supporting cells necessary to ensure adequate incorporation and graft survival beyond the most optimal wound beds. Herein, we fabricate a pre-vascularized full-thickness cellularized skin equivalent containing a three-dimensional vascularized network of interconnected macro and microchannels lined with vascular cells, within a collagen neodermis populated with fibroblasts, and an epidermis comprised of human keratinocytes capable of providing whole tissue perfusion. Previously, our lab has employed a sacrificial microfiber technique to develop tissue-engineered scaffolds with an inherent hierarchical network of microvessels, which recapitulates the organization of an arteriole, venule, and capillary bed. Utilizing a type-I collagen hydrogel matrix, vascular cells were seeded within pre-fabricated channels and allowed to proliferate to generate an endothelialized microvasculature. These collagen scaffolds were subsequently anastomosed into rat models to demonstrate the clinical feasibility of such approach. The present study aims to more closely recapitulate the in vivo structure of human skin via the incorporation of vital epidermal and dermal components of native skin into a biocompatible construct containing a complex hierarchical vasculature, which may be anastomosed using standard microsurgical techniques and immediately perfused. Pluronic F127 was used as the sacrificial material: 1.5 mm diameter “U” shaped macrofibers and 100-500 µm-interwoven microfibers were heat extruded and then embedded within type-I collagen into which Cyan Fluorescent Protein (CFP)-tagged human placental pericytes and human foreskin fibroblasts (HFF1) had been encapsulated. Following pluronic sacrifice, resultant channels were intraluminally seeded with Red Fluorescent Protein (RFP)-tagged human aortic smooth muscle cells, Green Fluorescent Protein (GFP)-tagged human umbilical vein endothelial cells, and topically seeded with human epidermal keratinocytes (HEK). Construct microstructure was analyzed using multiphoton microscopy (MPM) after 7, 14 and 28 days of culture. Additionally, after 14 and 28 days of culture, endothelial cells were extracted from the construct using collagenase digestion and Real Time (RT)-qPCR performed to analyze expression of markers of angiogenesis and maturation of the vascular network. MPM demonstrated a hierarchical vascular network containing macro and microvessels lined by endothelial and smooth muscle cells, supported by perivascular pericytes, all in appropriate microanatomic arrangement. Neodermal HFF1 proliferated throughout the observation period and the HEK neoepidermis developed into a stratified epidermis along the superior aspect of the construct. Angiogenic sprouting from the nascent vascular network into neovessel like structures was noted. RT- qPCR revealed relative expression of Jagged1, Dll4, Ve-Cadherin, and CD31. We have successfully fabricated a novel tissue-engineered pre-vascularized full thickness skin flap, which recapitulates the inherent hierarchical vasculature found within human skin and is suitable for in vivo perfusion. We provide the platform for an on- demand, geometrically tunable tissue engineered skin equivalent with an anastomosable vascular network. This tissue-engineered skin flap holds the potential to transform reconstructive surgical practice by eliminating the consequences of donor site morbidity, and enabling rationally designed, patient-specific flaps for each unique wound environment and anatomic location. / 2017-06-16T00:00:00Z

Melanoma skin cancer is the abnormal growth of the melanocytes – the pigmented cells located in the epidermis. The current gold standard diagnostic technique for determining whether a lesion is cancerous involves subjectively examining suspicious lesions and performing an invasive biopsy to confirm melanoma. This method may neglect some lesions or cause scarring from biopsies that turn out to be benign. Thus, impedance-based detection using a multi-electrode device was investigated as a noninvasive technique to diagnose melanoma skin cancer. The multi-electrode device was designed with 8 equally spaced Ag/AgCl electrodes surrounding one central electrode at a 5 mm radius. The electrodes were held in place by a vice-like mechanism using three circular Delrin sections. The electrodes were interfaced to an 8:1 multiplexer and National Instruments Educational Laboratory Virtual Instrumentation Suite (ELVIS) for measurement control and impedance analysis. The ELVIS system, multiplexer, and electrode device were validated for accuracy with various values of resistors and capacitors. Raw and cooked chicken thigh meat and skin were tested to evaluate the capabilities of the electrode device to discern different tissue types and tissue moisture contents by impedance measurements. EpidermTM and Melanoma tissue-engineered skin analogues, provided by MatTek Corporation, were tested to mimic the in situ disease state. The electrode device was found to produce reliable measurements for known electrical components with resistances between 10 ohms and 100 k-ohms and capacitances between 10 nF and 10 uF. The measurements from the chicken tissues and tissue-engineered skin constructs – excluding cooked chicken skin data – fell within the reliable range of the electrode device and were thus considered reliable as well. All analyses concluded that a statistical difference between the impedances of raw meat and raw skin, cooked meat and raw meat, and EpidermTM and Melanoma existed. Therefore, the hypothesis that a multi-electrode device could differentiate between melanoma and healthy skin tissues based on impedance measurements was satisfied.

Wound infection is a major clinical burden that can significantly impede the healing process and cause severe pain. Prolonged wound infection can lead to long-term hospitalization or death. Pre-clinical research to evaluate new drugs or treatment strategies relies on animal studies. However, animal studies have several challenges including interspecies variations, cost, and, ethics question the success of these models. Recent advances in tissue engineering have enabled the development of in vitro human skin models for wound infection modeling and drug testing. The existing skin models are mostly representative of the healthy human skin and its normal functions. However, to study the wound healing process and the response of skin to the infection, there is still a need to develop a skin model mimicking the wound infection. This work presents a simplified functional infected epidermis model, fabricated with enzymatically crosslinked gelatin hydrogel. The immortalized human keratinocytes, HaCaT cells, was successfully cultured and differentiated to a multilayer epidermis structure at the air-liquid interface, and expressed terminal differentiation marker, filaggrin, in the outer layer. The barrier function of the epidermis model was studied by measuring the electrical resistance and tissue permeability across the layer. The results showed that the developed epidermis model offered a higher electrical resistance and a lower drug permeability compared to the cell monolayer on gelatin and cell-free gelatin. To show the capability of the developed epidermis model in wound modeling and drug, the model was infected with Escherichia coli and the inflammatory response of keratinocytes was studied by measuring the level of proinflammatory cytokines, including IL-1β and TNF-α. The results demonstrated the proinflammatory response of the epidermis model to infection by producing a higher level of TNF-α and IL-1β compared to the control group. While treating with antibiotic ciprofloxacin terminated the proinflammatory response and reduced the level of TNF-α and IL-1β. The robust fabrication procedure and functionality of this model suggest that this model has great potential for wound modeling and high throughput drug testing. / Graduate