<div>Vocal fold scarring is the fibrotic manifestation of most common pathological voice disorders. Voice disorders lead to direct healthcare costs of over $200 million annually and significantly reduce quality of life for patients. Despite advances in understanding the pathophysiology of vocal fold scarring, effective treatments for scarring and fibrosis remain elusive. The wound-healing cascade associated with vocal fold injury involves complex signaling interactions between cells and their extracellular matrix (ECM), which remain largely unexplored due to the lack of a physiologically relevant preclinical model to study them. Traditional preclinical models do not capture the complex 3D microenvironment of the vocal folds, and the use of stem cells or fibroblasts alone in models has resulted in poor reproducibility and predictability of in vitro models. Toward this end, this work describes the development of a preclinical model that strives to take into account cellular interactions between fibroblasts and epithelial cells and achieve a balance in the native vocal fold 3D environment to function as an in vitro model.</div><div><br></div><div>Since a major shortcoming of current in vitro models is the lack of a standardized epithelial fibroblast coculture, initial work focused on developing a coculture system between commercially available tracheal epithelial cells and vocal fold fibroblasts in an in vitro setting that would provide more accurate information about the disease pathophysiology and help design better targeted treatments. We designed a healthy and disease state coculture model that can be induced into a fibroplastic state to overexpress stress fibers using TGFβ1. We also demonstrated that both cell types maintained phenotype in the healthy and disease state coculture models.</div><div><br></div><div>To further transfer this model in a physiologically relevant 3D system, follow-up research characterized 3D matrices to mimic the native ECM of the vocal folds by using natural biomimetic materials found in the vocal folds such as hyaluronic acid, type I collagen, and type III collagen. We hypothesized that the ability to control the viscoelastic and structuralcharacteristics of the scaffold in combination with presenting relevant biological cues to cells will result in a better biomimetic scaffold. This research is expected to lay effective groundwork for developing a functional tissue engineered 3D coculture model that retains the reproducibility necessary to serve as a viable diagnostic and therapeutic screening platform.</div>

There are 5 distinct aspects to this study. (i) Two histological stains for collagen were compared against each other for the first time, namely Herovici's technique and picrosirius-polarization. (ii) Skin samples from embalmed cadaveric tissue from human cadavers were compared against samples taken from surgical patients. (iii) Skin samples were studied from different regions of the body to assess if dermal structure correlates with scarring potential. (iv) Skin samples were sectioned in a plane parallel to the epidermis to gain further insight into dermal structure. (v) A novel basement membrane stain was produced. Type I and type III collagen are important structural constituents of dermis and play a crucial role in wound healing. Only two traditional histological methods are thought to differentiate between them, so avoiding the need for antibodies. These were compared against each other for the first time in order to establish differences in image quality and discrimination between Type I and type III collagen. Neither technique requires antibodies, however picrosirius requires polarisation microscopy. to result in a clearer, consistently reproducible collagen staining pattern than the picrosirius method and more importantly did not require elaborate apparatus to analyze. Additionally other cellular elements were visible. Skin samples for research are often obtained from surgical excision. This clearly limits which tissues are available for comparative study to those areas operated on. Studying samples from embalmed medical school cadavers has the great advantage of studying areas of the body not routinely available from common surgical procedures. It was therefore desirable to assess whether embalmed cadaveric tissues exhibited different properties by virtue of their age and the embalming process compared to fresh surgical specimens, in order to give confidence that studies utilising the former would be equally valid. To test this, 58 skin samples from embalmed medical school cadavers were compared to skin samples from 38 fresh operative specimens. The levels of tissue preservation and processing artefacts were similar in both groups. Embalmed medical school cadavers clearly offer an opportunity to study tissue areas not routinely available during surgery. This is the first time such a comparison has been made. Many things will affect the final appearance of the scar, but the single most important determinant is the body region affected. The most common areas for unfavourable scarring, specifically keloid or hypertrophic scarring have been shown to be the ear, deltoid and sternal areas. To test the hypothesis that there is no difference in histological structure of skin that correlates to body region, comparative histology was undertaken exploring the regional variations of skin characteristics in 58 cadaveric samples. Closely comparable samples were taken from the deltoid (9), abdomen (13), sternum (10), post-auricular (5), earlobe (12) and eyelid (9). Epidermal thickness, epidermal appendage density and collagen fibre orientation were examined and qualitative structural differences were assessed for each region Skin samples were then grouped by both topographical location of the body and scarring potential. Skin samples exhibited qualitative and quantifiable regional variations in the characteristics studied. Epidermal thickness and appendage counts did not correlate with scarring potential. Both however were statistically significantly higher in skin sampled from the head compared to the trunk. Bundles of collagen fibres in the reticular dermis were grouped according to their orientation in relation to the coronal plane; either parallel, oblique or perpendicular. The ratio of oblique to parallel fibres was statistically significantly higher in body areas with poorer scarring prognosis. This corresponds to a more disorganised arrangement of collagen fibres in these areas. Further qualitative understanding of dermal collagen fibres came from perpendicular to conventional histological samples. This new method stained basement membranes purple, cytoplasm was stained greenish-brown and nuclei dark brown. Collagen fibres were either thin and blue or thick and green. This method was compared to PAS staining and although required more preparative steps allows greater identification of other cellular structures.

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The purpose of this study was to evaluate the effects of an antimicrobial photodynamic therapy (aPDT) employing the photosensitizer methylene blue dissolved in ethanol on the systemic oxidative status likewise on the gingival collagen content of rats with periodontitis. Male Wistar rats were randomly divided into two main groups: NC (negative control; no periodontitis) and the remaining animals were submitted to periodontitis induction group. In the last group, the cotton ligature was placed at the first right mandibular molar of each animal in a submarginal position to induce experimental periodontitis. The animals with periodontitis were subdivided into groups according to the periodontal treatment as follow: SRP group (scaling and root planing), aPDT I group (SRP+aPDT+MB dissolved in water), and aPDT II group (SRP+aPDT+MB dissolved in ethanol). After 7 days, the ligature was removed and periodontal treatments were performed. At 7, 15 and 30 days, rats were euthanized and gingival tissue was removed for morphometric analysis. The erythrocytes were used to evaluate systemic oxidative status. Besides that, it indicated a protective influence of aPDT II in erythrocytes already at 15 days observed by the elevation in levels of systemic antioxidant defense. The morphometric findings showed that aPDT II group was the only one that restored the percentage of total collagen area also in 15 days, as well as, aPDT II group recovered the type I collagen area at the same time-point. According to this study we could suggest that aPDT employed as an adjunct to the standard treatment of periodontitis (SRP) increases systemic protective response against oxidative stress periodontitis-induced facilitating and accelerating the periodontal healing particularly when methylene blue is dissolved in ethanol. / O objetivo deste estudo foi avaliar os efeitos da terapia fotodinâmica antimicrobiana (TFDa) usando o fotossensibilizador azul de metileno (AM) dissolvido em etanol sobre o status oxidativo sistêmico, bem como sobre o conteúdo de colágeno gengival de ratos com periodontite. Ratos machos Wistar foram divididos aleatoriamente em dois grupos principais: CN (controle negativo; sem periodontite) e os animais restantes foram o grupo submetido a indução de periodontite. No último grupo, a ligadura de algodão foi colocada no primeiro molar inferior direito de cada animal em uma posição subgengival para induzir a periodontite experimental. Os animais com periodontite foram subdivididos em grupos de acordo com o tratamento periodontal, como segue: grupo RAR (raspagem e alisamento radicular), TFDa I grupo (RAR + TFDa + AM dissolvido em água), e grupo TFDa II (RAR + TFDa + AM dissolvido em etanol). Após 7 dias, a ligadura foi removida e foram realizados os tratamentos periodontais. Aos 7, 15 e 30 dias, os ratos foram submetidos à eutanásia e foi removido o tecido gengival para análise morfométrica. Os eritrócitos foram usados para avaliar o status oxidativo sistêmico. O status oxidativo demostrou maiores níveis de peroxidação lipídica no grupo CP em 7, 15 e 30 dias, e indicou uma influência protetora da TFDa II, nos eritrócitos, já em 15 dias, observada a partir da elevação dos níveis de defesa antioxidante sistêmica. Os achados morfométricos mostraram que o grupo TFDa II restabeleceu o percentual de área total de colágeno também em 15 dias, bem como recuperou a área de colágeno tipo I no mesmo tempo. A partir deste estudo podemos sugerir que TFDa utilizada como um adjuvante ao tratamento padrão periodontal (RAR) aumenta a resposta protetora sistêmica contra o estresse oxidativo induzido pela periodontite, facilitando e acelerando a cicatrização periodontal, particularmente quando o azul de metileno é solubilizado em etanol.