Wound healing in focus: establishment of a porcine wound model, strategies to improve the healing outcome using bacterial cellulose and development of a novel cell-based therapy

Many are the threats capable to impose a challenge to the skin structure, generating a disturbance to its once cohesive barrier. The healing process is the mechanism the body uses to repair the damage and to reestablish tissue integrity. This process demands a well-orchestrated cascade of events generated by a multitude of cells coming from different tissues. Any misinterpreted cue or delayed event could lead to an unsatisfactory outcome. The study of such intricate mechanism requires a reliable and well-established model. Additionally, to support the remaining tissue during the healing process, appropriate treatments using dressings capable of meeting some of the demands of the resident cells are needed (Fig. 1). The aim of this thesis is, therefore, to: 1 - introduce a standardized in vivo wound model capable of offering reproducible results to aid researcher groups to investigate burn wounds through an excisional wound; 2 - explore the supportive effect of a bacterial nanocellulose (BNC) dressing available in the market and expand its possible applications; and lastly 3 - present the development of a novel wound dressing composed of freeze-dried keratinocytes and examine its healing support. The development of the porcine wound model will be presented in the Chapter II - Part I, where an extensive analysis was performed to investigate the standardization of the created wounds and morphometric results of the tissue, including: the consistency of the wound size, the effect of several sampling processes, the consistency of the results throughout the entire wound area and influence of the wound position on the dorsal region of the animal. Additionally, using the morphometrical data generated by the first animals, an automated software was trained to perform further morphological measurements automatically. Data generated by this technology was also considered during the morphometric analysis. Furthermore, the development of a second model, bearing additional wounds was proposed and investigated. An interesting analysis of the effect of the residual dermis under the wound bed was performed, indicating that some wound healing parameters were in a certain matter affected by this factor. Using the generated data and considering the morphological differences for the dermis of the animals, maps were created to help future groups to obtain more reproducible and reliable results, ultimately avoiding the need for more animal tests. Using the models established in Part I, a thorough analysis of BNCs was performed, including: 1 - its efficacy to support the healing process; 2 - the influence of a combination of this dressing and different secondary wound dressings; 3 - its capacity as a delivery platform for antiseptics and the challenges to prepare the dressing to allow a delivery of hypochlorous solutions; and 4 - a modification to transform the dressing to become pH-responsive, allowing a full-time monitoring of the wound bed (Chapter II -Part II). Firstly, physicochemical characteristics of three BNC dressing available in the market were investigated and compared. Subsequently, using a human ex vivo burn wound model, the cooling effect and the benefit of an immediate treatment using one of the dressings were analyzed. Using the in vivo models previously presented, various healing parameters were measured through histological assessments. The supportive effect offered by the BNC dressing was then studied via these assessments. By a combination of this BNC and secondary dressings, one more permissive to evaporation and one that retain the moisture in the wound bed, an interesting effect on the repair process of the skin tissues was obtained. Furthermore, the combination of this BNC dressing with antiseptic solutions were explored in order to test a sustained delivery of the active molecules (Polyhexamethyl biguanide; Octenidine; and Polyvinylpirrolidone). Moreover, the diffusion of fluorescent molecules of different sizes was also tested to better understand the kinetic involved in the loading and the releasing processes. Afterwards, the uptake and delivery of hypochlorite solutions was in focus. Due to the challenges observed for this process, a novel method was developed to enable the BNC dressing to be combined with this reactive molecule. This new procedure is in process to be patented. Demonstrating the versatility of this dressing, a modification to the cellulose structure of the BNC was carried, allowing the dressing to be responsive to pH variations, improving its applications in the medical field. Finally, in the Chapter II -Part III, a completely new dressing was proposed and developed. This dressing uses freeze-dried keratinocyte and is aimed to enhance the healing response of the residual tissue to achieve better wound repair outcome. The effect of the freeze-drying process on the healing capacity of a commercially available tissue engineered skin substitute (TESS) was assessed to verify the maintenance of its supportive attribute. Later, the healing support characteristic of the newly developed dressing was analyzed using the in vivo wound models previously presented. The Chapter III presents a summarization of the obtained results, their limitations and how the developed knowledge address some of the present needs of the medical field and how they can further improve the quality of the treatment offered to the patients.