(A-2) Development of a Mouse Model for Evaluation of Wound Healing Implants and Devices

Introduction:: In vivo models represent the gold standard in evaluating physiological and pathological processes, and a large number of models are able to investigate the wound healing capacity of dermal tissue. Dermal tissue is essential in preventing infections, and, hence, engineered tissue capable of regenerating a functional epithelium in animal models is required. This tissue can be achieved by providing basement membrane biomimetic surfaces favorable to the migration and polarization of epithelial cells. Current models used to evaluate engineered dermal tissue, including the splinted full thickness model, can be used to evaluate processes that take place under different conditions. Most models, however, focus on understanding a specific parameter of wound healing and some of the advances that take place throughout the healing process, disregarding other vital parameters and the role they play in healing. A minimal investigation of outcome parameter can lead to poor understanding of the components of wound healing and harder transferability of data from animals to humans. Consequently, models that better recapitulate native process and relevant evaluation are required. To this end, we fabricate biomimetic basement membranes that are able to recruit host epithelial cells and stimulate epithelium formation at the replacements-environment interface. This was made possible after further expanding the humanized dermal wound model to incorporate the ability to 1) test the efficacy of different implant materials, and 2) to use a well-rounded approach in evaluating angiogenic, epithelial and immune response parameters related to the overall wound healing process.


Materials and Methods:: We fabricated thin fiber layers that could adsorb a protein mixture resembling basement membrane properties. Fibers were prepared by electrospinning, and a protein mixture was adsorbed to the surface. Following in vitro migration, proliferation and differentiation studies, the response to the implant was studied in an in vivo murine model of excisional wound healing. We tested different implant and splint sizes as well as different methods of implant suturing and implant coverages. Implants were prepared to range in diameter from 5 to 8 mm and were sutured to the surrounding skin using purse-string suturing and interrupted suturing. Here, three different electrospun implants were placed after creating skin punches in the backs of mice and the response to the different implants was studied in a model of excisional wound healing (Figure 1A). Wound photos were taken every other day and the wound areas were measured. Additionally, immunofluorescent staining to detect the presence of angiogenic, proliferative and epithelial markers were carried out after 3 weeks. A semiquantitative scoring system was developed to score implants according to the percentage of tissue areas infiltrated with inflammatory cells.


Results, Conclusions, and Discussions:: Migration studies showed an increase in cell numbers migrating to fiber layers with proteins. This is vital since native basement membranes act as substrates for the adhesion and migration of epithelial cells. Cell differentiation experiments showed that fibers enhanced the expression of several mucous secreting and ciliated cell genes. Upon assessing methods to incorporate implants and silicone splints, suturing 6 mm diameter implants with interrupted 8-0 sutures was found to best secure the implants to the surrounding tissue, while 6-0 suture were needed to secure the splints following skin adhesion. Securing the covered implants with a bandage was essential to prevent the exposure of wound areas to the outside environment. Wound areas (Figure 1B) decreased over the study time-period and an epithelium layer was regenerated over the surface of the implants. This layer was more intact for some implants over others as was further confirmed in H&E (Figure 1C), E-cadherin and pan-cytokeratin staining. Furthermore, angiogenic markers CD31 and α-SMA indicated the presence of mature blood vessels around implants, while staining for cell proliferation Ki67 denoted around 10% positively stained cells (Figure 1D). Immune studies revealed the compatibility of implants. Together, these studies confirm progression of the healing process and wound area closure upon the incorporation of the fabricated electrospun implants.

The presentation of basement membrane proteins using fiber layers increases cell adherence and migration to fibers, stimulates the differentiation of basal cells and promotes epithelialization in an in vivo dermal wound model by mimicking the composition of native membranes. Specific methods used to incorporate implants to the humanized splinted model are crucial to the success of the model. This model allowed the evaluation of implants in a manner that captures multiple parameters of wound healing including angiogenesis, proliferation, epithelialization and immune response testing. Thus, the utilization of the developed electrospun implants for use in promoting epithelialization as well as the use of the model to investigate the healing capacity of implants made of different materials, porosities, degradability will ultimately confirm the translation of this model for a wide range of studies and are the current next steps we are exploring.


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