Rozprawy doktorskie na temat „Wound healing Physiology”

Chronic wounds represent an under-acknowledged socioeconomic epidemic, affecting 1.8 million new patients per year and costing the US health care system upwards of $25 billion annually. This substantial cost is rapidly growing due to a disproportionate occurrence in the ever-aging population. Key features associated with age-related impairment of wound healing include limited energy and nutrient exchange, unremitting inflammations, increased reactive oxygen species (ROS), and diminished blood flow. Most chronic wound therapies target specific molecular mechanisms; however, there are often multiple mitigating factors that prevent normal wound closure. This is likely one reason most wound therapies are minimally effective. In the standard American diet, carbohydrates are broken down for fuel (glucose). While fasting, starvation, and calorie or carbohydrate restriction, beta-oxidation of stored fats in the liver produces ketone bodies (primarily acetoacetate (AcAc) and β-hydroxybutyrate (βHB) to serve as energy metabolites for extra-hepatic tissues. In addition to enhancing metabolic physiology, ketone bodies have recently been discovered to have signaling properties that are independent of their function as energy metabolites. Here we present the evidence for a novel method of inducing therapeutic ketosis via exogenous ketone supplementation to promote enhanced ischemic wound healing in young and aged Fischer 344 rats. Preliminary mechanistic studies demonstrated that exogenous ketone supplementation enhanced wound healing via increasing proliferation and migration, decreasing lactate production, and decreasing ROS production as well as affecting inflammatory cytokines and growth factors. We conclude that exogenous ketone supplementation will be an effective, cost efficient, low toxicity therapy to promote enhancement of wound healing in an aged population.

The birth of complexity research derives from the logical progression of advancement in the scientific field afforded by reductionist theory. We present in silico models of two complex physiological processes, wound healing and coagulation/fibrinolysis based on two common tools in the study of complex physiology: ordinary differential equations (ODE) and Agent Based Modeling (ABM). The strengths of these two approaches are well-suited in the analysis of clinical paradigms such as wound healing and coagulation. The complex interactions that characterize acute wound healing have stymied the development of effective therapeutic modalities. The use of computational models holds the promise to improve our basic approach to understanding the process. We have modified an existing ordinary differential equation model by 1) evolving from a systemic model to a local model, 2) the incorporation of fibroblast activity, and3) including the effects of tissue oxygenation. Possible therapeutic targets, such as fibroblast death rate and rate of fibroblast recruitment have been identified by computational analysis. This model is a step toward constructing an integrative systems biology model of human wound healing. The coagulation and fibrinolytic systems are complex, inter-connected biological systems with major physiological roles. We present an Agent Based Modeling and Simulation (ABMS) approach to these complex interactions. This ABMS method successfully reproduces the initiation, propagation, and termination of blood clot formation and its lysis in vitro due to the activation of either the intrinsic or extrinsic pathways. Furthermore, the ABMS was able to simulate the pharmacological effects of two clinically used anticoagulants, warfarin and heparin, as well as the physiological effects of enzyme deficiency/dysfunction, i.e., hemophilia and antithrombin III-heparin binding impairment, on the coagulation system. The results of the model compare favorably with in vitro experimental data under both physiologic and pathophysiologic conditions. Our computational systems biology approach integrates reductionist experimental data into a cohesive model that allows rapid evaluation of the effects of multiple variables. Our ODE and AMBS models offer the ability to generate non-linear responses based on known relationships among variables and in silico modeling of mechanistic biological rules on computer software, respectively. Simulations of normal and disease states as well as effects of therapeutic intervention demonstrate the potential uses of computer simulation. Specifically, models may be applied to hypothesis generation and biological advances, discovery of new diagnostic and therapeutic options, platforms to test novel therapies, and opportunities to predict adverse events during drug development. The ultimate aim of such models is creation of bedside simulators that allow personalized, individual medicine; however, a myriad of opportunities for scientific advancement are opened through in silico experimentation.

A characteristic feature of chronic wounds is a prolonged inflammatory response as well as susceptibility to infection. Studies have shown that during the inflammatory response, there is a significant increase in the levels of neutrophil-derived enzymes. The purpose of this work was to determine whether the anionic macromolecule polystyrene sulfonate (PSS) and five of its salt forms, namely PSS-calcium, PSS-chlorhexidine, PSS-doxycycline, PSS-glutathione and PSS-silver are able to inhibit the activity of three of the enzymes whose levels are elevated in chronic wounds: elastase, cathepsin G and myeloperoxidase. In addition to the enzyme inhibition study, the various formulations’ antimicrobial properties were analyzed by evaluating their ability to inhibit the growth of three common clinical isolates: Staphylococcus aureus, Pseudomonas aeruginosa and Acinetobacter baumanii. It is worthy to note that the structure of PSS makes it a very flexible platform to which other molecules can be added in order to address a variety of “targets” as well as tailor quantitative strength. The results from this project showed that purified PSS and the various salt derivatives were able to inhibit elastase and cathepsin G activity. In addition, three of the therapeutic cations attached to PSS: silver, doxycycline and chlorhexidine retained their intrinsic antimicrobial properties without having an adverse effect on healthy tissue. In summary, this study demonstrated that PSS possessed an intrinsic ability to inhibit a number of proteases and that it could also be used as a delivery vehicle for other compounds with potential therapeutic value.

Includes bibliography (leaves 191-219). Examines the levels of insulin-like growth factor and the presence of IGF binding proteins in human wound fluid. Tests the potency of IGF-1 and 2 analogues in in vitro models of fibroblast activity and their effect on healing in normal and diabetic rodent wounds. Shows that IGF-1, IGF-2 and their binding proteins are present in fluid from a partial thickness cutaneous wound; that the binding proteins negatively modulate the activity of insulin-like growth factors in vitro, but that the IGFs do not necessarily show enhanced activity in vivo at the wound site if binding protein affinity is decreased. Discusses possible roles of these binding proteins in wound repair.

The growing rates of problematic wounds in the population and the subsequent increase in morbidity and mortality warrant further understanding of wound healing and the development of therapeutic agents targeted to alleviate these devastating concerns. The complement system is composed of bactericidal and hemolytic proteins that increase capillary leakage and inflammatory cell migration. It allows for an anaphylactic reaction and the recruitment of inflammatory cells. Fibroblast recruitment and subsequent collagen deposition in wounds is responsible for wound healing and is regulated by inflammatory cells. However, little is known about role the complement system may have on wound healing strength. This work investigates the effects of the topical application of collagen, complements C3 and C5 in varied formulations on the paired surgical skin incision rat model. These potential findings may help further enhance the understanding of wound healing and allow for a novel therapeutic approach for the treatment of patients.<br>Les plaies aigues et chroniques sont associées à des taux de morbidité et mortalité importantes, et c'est pourquoi il est important de réaliser des études approfondies qui permettraient de développer des agents thérapeutiques qui stimulent la guérison de plaie. Le lien entre le système de complément et la guérison de plaies est encore méconnu. Le système de complément est composé de protéines bactéricides et hémolytiques qui augmentent la fuite capillaire tout en stimulant la migration de cellules. Il permet une réaction anaphylactique en recrutant des cellules inflammatoires telles que les fibroblastes suivis par la déposition de collagène au site de la plaie; ensemble celles-ci participent activement à la guérison et sont par la suite modulées par d'autres cellules inflammatoires. Cette étude analyse les effets de l'application topique de collagène, de compléments C3 et C5 sous diverses formes, au site de la plaie. Le modèle expérimental sélectionné a été réalisé par l'application d'une incision chirurgicale sur la peau dorsale du rat. Ces futurs résultats amélioreront notre compréhension de la guérison de plaies tout en permettant le développement d'une approche thérapeutique originale pour le traitement des patients atteints de plaies aigues et chroniques.

The introduction of necrophagous fly larvae (maggots) into chronic wounds for the purpose of inducing healing is an ancient practice that has recently undergone a renaissance in Western medicine. Through clinical observations, maggots are broadly recognised to debride the wound of necrotic tissue, cleanse the wound of infection and promote granulation tissue formation. Despite such recognition, little research at the biological level has been undertaken to identify the mechanisms by which maggots accomplish such feats. The dermal fibroblast is a major cellular component of granulation tissue and as such, its migration into the wound plays a vital role in new tissue growth. Fibroblast migration is directed by the composition of the extracellular matrix. Maggot secretions contain proteolytic enzymes that are active against a variety of extracellular matrix proteins which are present at the wound site. Hence, this thesis focused upon the effects of maggot secretions on human dermal fibroblast adhesion and migration in the presence of common extracellular matrix proteins. This was with the aim of elucidating the mechanisms by which maggots stimulate tissue formation within the wound and from there, developing new products that may be used to promote wound healing. Experiments showed that maggot secretions modulated fibroblast adhesion to tissue culture plastic surfaces and to surfaces coated with collagen and particularly fibronectin. Modification of the protein-coated surface by enzymes present within the secretion appeared to play a role. Fibroblast migration upon a fibronectin-coated surface was enhanced in the presence of maggot secretions. The same also occurred in the presence of a higher concentration of secretions when the cells were located within a three-dimensional environment comprising collagen gel and fibronectin. Evidence suggested that this may have been associated with enhanced matrix re-modelling.

The cytoskeleton is an intrinsic aspect of all cells, and is essential for many cellular events including cell motility, endocytosis, cell division and wound healing. Remodeling of the cytoskeleton in response to these cellular activities leads to significant alterations in the morphology of the cell. One such alteration is the formation of an actomyosin contractile array required for cytokinesis, wound healing and embryonic development.<br>Cellular structure and shape depends upon tensional prestress brought about by the organization of cytoskeletal components. Using the Xenopus laevis oocyte wound healing model, it is first described how diminished cellular tension affects the balance of the Rho family of GTPases, and subsequently prevents the formation of actomyosin contractile arrays. This suggests that cellular tension in the cell is not created at the level of the cytoskeletal elements but rather via the upstream signaling molecules: RhoA and Cdc42.<br>The role of N-WASP (Neural-Wiscott Aldrich Syndrome Protein), a mediator of Arp2/3 based actin polymerization, is next examined for its putative role in cellular wound healing. Xenopus laevis oocytes injected with mutant N-WASP constructs reveals in vivo evidence that functional N-WASP is required for appropriate contractile array formation and wound closure.<br>Lastly, it is revealed that the cellular structures involved with single cell wound healing in other model systems are also important for the initial repair of severed muscle cells. Actin, non-muscle myosin-II, microtubules, sarcomeric myosin and Cdc42 are all recruited and reorganized at the edge of damaged C2C12 myotubes. This data promotes the possibility that an actomyosin array may be established in injured muscle cells as well.

The positive effect of photobiomodulation in wound healing has previously been reported, however there is a considerable lack of knowledge regarding the molecular mechanisms involved, and no consensus on light parameters. Cytochrome c oxidase (CCO) is established as the main photoreceptor in cells, but light also induces nitric oxide (NO), production of reactive oxygen species (ROS) and activation of ion channels. Emerging new molecular targets include the GPCRs opsins (OPNs) and the circadian clock transcription factors, cryptochromes (CRYs). Localisation of OPN1-SW, OPN3, OPN5, CRY1 and CRY2 was seen in female facial and abdominal human skin. Furthermore, expression of these photoreceptors was retained in primary epidermal keratinocytes and dermal fibroblasts in culture; both cell types expressed OPN1-SW, OPN3, CRY1 and CRY2, at the mRNA and protein level. OPN2 was only expressed in cultured dermal fibroblasts, while in line with in situ expression, OPN5 was only expressed in cultured keratinocytes. The photoreceptor-expressing cultured epidermal keratinocytes demonstrated a dose- and wavelength- dependent response in both metabolic activity and cell migration in a scratch-wound assay. Specifically, low dose (2 J/cm2) blue light (447 nm) increased metabolic activity, but it did not impact keratinocyte migration. In contrast, high dose (30 J/cm2) blue light had no effect on metabolism, but inhibited migration of epidermal keratinocytes. Red light (655 nm) at 30 J/cm2 stimulated metabolic activity but did not modulate migration, while a higher dose of 60 J/cm2 had no effect on keratinocyte metabolic activity. In order to study OPN3 and CRY1 function, they were silenced in keratinocytes using siRNA; additionally 8 μM KL001 was used to stabilize CRY1. KL001 inhibited migration, and induced KRT1 and KRT10, an effect which was abrogated by knockdown of OPN3. Interestingly, knockdown of OPN3 upregulated CRY1 expression, while KL001 upregulated OPN3 expression, indicating a regulation by OPN3 of the molecular epidermal clock. Low levels of blue light increased early differentiation of epidermal keratinocytes, which was mediated by OPN3 and circadian clock mechanisms. However, low levels of blue light decreased keratinocyte DNA synthesis, which was mediated by circadian clock independently of OPN3. Translation of parameters ex vivo showed increasing re-epithelialisation and induction of OPN3 and CRY1 expression following exposure to 2 J/cm2 of blue light; however high doses of blue light inhibited re-epithelialisation. Red light, also increased re-epithelialisation, but had no effect on OPN3 or CRY1 expression. In conclusion, photoreceptors are expressed in human skin and they mediate DNA synthesis, migration and differentiation of epidermal keratinocytes. Furthermore, low dose of blue light interacts with OPN3 to induce epidermal differentiation, through the regulation of the circadian clock. A better understanding of the molecular mechanisms behind the photobiomodulation response in vitro will help to develop light based therapies for human wound healing. Interestingly, selected light parameters translated to human ex vivo skin showed a beneficial effect of low doses of blue (2 J/cm2) and red (30 J/cm2) light in re-epithelialisation.<br>Marie Curie ... the CLaSSiC project

Tissue engineered scaffolds were constructed to mimic the native extracellular matrix (ECM) and promote cell migration of keratinocytes and fibroblasts. Electrospinning technology was used to fabricate these nano-scale matrices that consist of varying compositions and fiber diameters. The purpose of this study was to examine how average fiber diameter and scaffold composition regulate cell migration. Odyssey infrared scanning evaluated this on a macroscopic level, whereas confocal microscopy focused on a more microscopic approach. The expression of proteases released into the culture media was also examined. The results from this study suggest that fiber diameter increases as a function of electrospinning starting concentration. Altering the composition by adding a basement membrane-like material, Matrigel, does not statistically affect the average fiber diameter. Fibroblast migration is greater on collagen scaffolds than gelatin scaffolds based on surface area measurements. Confocal images illustrate a distinct cell polarity and various cell morphologies of fibroblasts on electrospun collagen scaffolds. Cell-matrix interactions are more prominent on intermediate to large scale fibers. However, cell-cell contacts are more prevalent at the smallest fiber diameters, suggesting that this scaffold acts like or as a two-dimensional surface. The expression of matrix metalloproteases (MMPs), specifically MMP-2 and MMP-9, by fibroblasts during in vivo cell migration assays, suggests that the greatest amount of matrix remodeling is at the two extremes of fiber diameters.

Les cellules peuvent migrer sous différentes conditions qui dépendent de l'environnement biochimique ou mécanique. Connaître les mécanismes de la migration, les protéines impliquées et leur régulation est essentiel pour comprendre les processus de morphogénèse ou certaines situations pathologiques. Dans ce contexte, la migration collective des cellules est un processus clé qui intervient pendant le développement ainsi que dans la vie adulte. Elle joue un rôle très important pour la formation et l'entretien des couches épithéliales, notamment au cours du développement embryonnaire et pendant la cicatrisation des trous épithéliaux résultant, par exemple, d'une blessure. Lorsque l'épithélium présente une discontinuité, des mécanismes actifs qui impliquent une migration coordonnée des cellules sont nécessaires pour préserver l'intégrité des tissus. Dans ce travail, nous avons étudié les mécanismes impliqués dans la fermeture des trous dans un épithélium. Pour des blessures de faible taille, le mode de fermeture dit de purse string est souvent évoqué, impliquant la contraction d'un anneau contractile d'acto-myosine qui ferme la blessure. Pour des blessures de tailles plus importantes, il est courant d'observer un mécanisme différent conduisant { la migration active des cellules du bord qui couvrent la surface "libre".Pour étudier ces aspects de manière quantitative et reproductible, nous avons développé une nouvelle méthode basée sur des techniques de microfabrication et de lithographie dite " molle " qui permet de faire une étude quantitative de la fermeture des trous épithéliaux. Nous avons fabriqué des substrats de micropiliers de diamètre et de forme variés dans les quels les cellules sont libres de pousser entre les microstructures. Lorsqu'elles sont parvenues à confluence, on retire le substrat qui laisse apparaître des trous contrôlés.De cette manière, nous avons observé que les cellules épithéliales forment des lamellipodes pour la fermeture de ces trous. Le mécanisme de fermeture dépend de la taille des trous et nous avons pu observer différents régimes en fonction de diamètre des piliers. Les trous petits (de la taille d'une seule cellule) sont fermés par un mécanisme passif alors que la fermeture de trous plus larges nécessite un mécanisme actif de migration conduisant à la formation de lamellipodes et à des modes de migration collective. Par la suite, nous nous sommes intéressés à l'aspect mécanique de la fermeture des trous épithéliaux. Pour cela, nous avons utilisé un système d'ablation laser pour rompre quelques cellules dans une monocouche épithéliale. Nous avons alors mesuré les forces de traction que les cellules exercent au substrat et leur évolution temporelle et spatiale. Nous avons pu mettre en évidence différents modes de traction: au début, les cellules exercent des forces de traction importantes sur leur substrat pour laisser place à des contraintes mécaniques qui sont davantage issues d'un processus collectif au travers de la formation d'un câble multicellulaire qui les relie les cellules de bord entre elles. En conclusion, ce travail nous a permis d'obtenir des informations sur les mécanismes dynamiques de fermeture des tissus épithéliaux qui sont évidemment impliqués dans la cicatrisation des blessures mais aussi dans certains problèmes de malformations congénitales lors l'embryogenèse.

"July, 2001"<br>Addendum inserted in back.<br>Includes bibliographical references (leaves 250-284)<br>xxiii, 284 leaves : ill. (some col.), plates (some col.) ; 30 cm.<br>Title page, contents and abstract only. The complete thesis in print form is available from the University Library.<br>Thesis (Ph.D.)--University of Adelaide, Dept. of Surgery, 2001

"July, 2001"<br>Addendum inserted in back.<br>Includes bibliographical references (leaves 250-284)<br>xxiii, 284 leaves : ill. (some col.), plates (some col.) ; 30 cm.<br>Thesis (Ph.D.)--University of Adelaide, Dept. of Surgery, 2001

Introduction Keloids are formed by the excessive production of scar tissue, which extends beyond the margins of the original injury, often resulting in lesions of grotesque dimensions. Keloids present a major dilemma to surgeons because of the high recurrence rate with recurrent growth often larger than the original keloid. The high recurrence rate and the poor response of keloids to therapy present a great challenge to surgeons. The numerous therapeutic regimens demonstrate that to date there is no single therapy that is absolutely successful. Therefore, it is necessary to comprehensively establish the pathology of keloids and to determine the aetiopathogenesis of the lesion in order to eventually provide unfailing specific effective treatment and to better understand the mechanisms regulating fibrosis in various fibroproliferative diseases. Aim To evaluate the pathology and aetiopathogenesis of keloid formation. Methods The research protocol for the study was approved by the Nelson R Mandela Faculty of Medicine Ethics Committee. Informed consent was obtained before the biopsies were taken. Keloid and non-lesional skin biopsies were obtained from thirty two patients who had multiple lesions in various locations, bringing the total number of keloids and apparently normal skin biopsies processed and examined to fifty eight. The biopsied specimens were processed for paraffin wax embedment and routine haematoxylin and eosin, differential and immunocytochemical staining. Sections were scrupulously examined using the Olympus BH-2 microscope; features pertinent to the study were photographed with the Olympus DP 10 microscope digital camera system. The stored images were studied, using the Camedia graphics processing programme. Results The results of the study showed that keloids comprise many distinct regions categorized as: the zone of hyalinising collagen bundles, fine fibrous areas, areas of inflammation, zone of dense regular connective tissue, nodular fibrous area and area of angiogenesis. Fibroblastic phenotypes present ranged from spindle, fibrohistiocytic, epitheloid, elongated flattened condensed fibroblastic cells to few wavy, fuzzy, polygonal and atrophic cell types. Immunocytochemically these cells were vimentin-positive and actin- and desmin-negative. Few myofibroblastic phenotypes were also identified and these were vimentin- and alpha smooth muscle actin-positive and desmin-negative. The fibroblastic and myofibroblastic phenotypes were in proliferative or degenerative stages and pathological features exhibited were the presence of vesicular, degenerate or calcified nuclei; nuclear and plasma membrane damage; cytoplasmic and nucleoplasmic clearing; atrophy, pyknosis and swelling. Severe, moderate to mild paravascular inflammation was observed around the microvessels of the sub-papillary plexus and within the keloid. There was compression and occlusion of small blood vessels, coagulation necrosis and dissolution of mural cells of small blood vessels and small peripheral nerves. Also present in keloids were oedematous areas, disorganised and hyalinised connective tissue fibres and increased numbers of degranulated and degranulating mast cells. Elastic fibres in keloids were minimal or absent whereas at the border of keloids there was an increase.Discussion Degenerate, occluded and compressed microvessels were a widespread pathological feature in keloids. This resulted in impaired vascular supply to each of the keloid regions which impacted directly on the pathology of keloids where degeneration and necrosis, manifesting the lack of nutrients and oxygen to tissue, were found throughout the keloid. The vascular supply was impaired because of the chronic inflammatory destruction of the microvessels and the elevated stress within keloids. Factors contributing to increased intrinsic stress were: 1) the lack of elastic fibres in keloids which decreased the elastic limit, leading to effects of excessive deformational force which were compression and stiffening of tissue; 2) the high tension skin covering keloid prone areas had low stretch and a low elastic modulus; 3). protruding hard connective tissue such as bony prominences or cartilage into the dermis of keloid prone skin; 4) contractile forces exerted by wound healing fibroblastic cells; and 5) external forces. Compression and occlusion of blood vessels induced ischaemic and reperfusion tissue injury. During the reperfusion phase blood rich in growth factors returned to tissue stimulating tissue growth. Tissue growth was also promoted by elevated internal stress which stimulated increasing levels of gene expression, collagen synthesis and mitotic activity. All these growth promoting effects resulted in keloid formation.<br>Thesis (Ph.D.)-University of KwaZulu-Natal, Durban, 2013.

Includes bibliography (leaves 191-219).<br>x, 219 leaves<br>Examines the levels of insulin-like growth factor and the presence of IGF binding proteins in human wound fluid. Tests the potency of IGF-1 and 2 analogues in in vitro models of fibroblast activity and their effect on healing in normal and diabetic rodent wounds. Shows that IGF-1, IGF-2 and their binding proteins are present in fluid from a partial thickness cutaneous wound; that the binding proteins negatively modulate the activity of insulin-like growth factors in vitro, but that the IGFs do not necessarily show enhanced activity in vivo at the wound site if binding protein affinity is decreased. Discusses possible roles of these binding proteins in wound repair.<br>Thesis (M.D.) -- University of Adelaide, Dept. of Surgery, 2001?

The effect of absorbable packing on the healing of nasal respiratory epthelium after endoscopic sinus surgery (ESS) was examined in a diseased sheep model. Full thickness injuries were created on the lateral nasal wall of sheep infested with Oestrus ovi. Sites of injury were packed on one side with hyaluronic acid (HA) packing or hyaluronic acid packing impregnated with insulin-like growth factor- 1 (HA+IGF1) in a randomized fashion. The opposite side was left unpacked as a control. Biopsies were obtained for light microscopy, scanning electron microscopy, and ciliary beat frequency (CBF) analysis over a period of 16 weeks. Statistical analysis of results was performed in order to determine if any intervention had any impact on healing and to determine if there was any correlation between extent of regeneration as assessed by electron microscopy and CBF. Furthermore assessment of the effect of isotonic and hypertonic saline on ciliary beat frequency was performed in healthy human volunteers. Reepithelialization was increased in the HA+IGF1 group compared to the HA group and controls at eight weeks after injury but not at later time points. Cilial regeneration was improved in the HA+IGF1 group compared to the HA group and controls at 16 weeks. CBF was noted to be worse at the eight week time point with the HA+IGF1 group compared to the HA group and controls, but no other statistically significant effects on CBF were noted. This most likely represents a spurious finding. Wide distributions of CBF results were noted, reflecting numerous missing data points due to methodological difficulties. There was a trend noted toward increased CBF with improved grades of reciliation, although this correlation was not statistically significant. However this trend was supported by the finding of statistically significant differences between individual and combined grades of reciliation. Hypertonic saline was found to have a ciliostimulatory effect when compared to normal saline at 5 minutes after administration in healthy human subjects. This effect had disappeared by 60 minutes after administration. It is suggested that the presence of insulin-like growth factor- 1 at the time of mucosal injury improves epithelial regeneration in the short term, but is not sufficient for this effect to be sustained. This improved early epithelial regeneration forms a foundation for cilial regeneration, as is reflected in an improved grade of reciliation at 16 weeks. Our interventions had no effect on CBF, and various experimental problems made it difficult to provide further comment on CBF results. There is evidence that CBF improves as the grade of cilial regeneration improves following ESS. Furthermore, hypertonic saline appears to also have a positive impact on CBF, which is likely to reflect changes in the rheological properties of mucous. A number of possible avenues of enquiry are delineated and recommendations for future research are outlined.<br>Thesis (M.S.)--Department of Surgery, 2005.

Introduction: Endoscopic sinus surgery (ESS) is at present the gold standard therapeutic modality for chronic rhinosinusitis (CRS) resistant to medical therapy. Whilst results from ESS for CRS are generally good, postoperative bleeding and impaired wound healing with adhesion formation remains a concern. Due to patient discomfort and the detrimental effects on wound healing caused by most packing materials, many surgeons no longer routinely use nasal packing. Surgeons have in the past sought agents which would provide post-operative haemostasis without detrimentally affecting wound healing. Antifibrinolytics have been available for many years, however, their topical application has only been explored in the last few years. Recently different forms of chitosan have separately shown significant promise as powerful haemostatic and anti-adhesion agents. The aim of this thesis was to explore the progressive understanding of the interaction between haemostasis and wound healing with possible development of a novel agent. Methods: The first step to scientifically assess bleeding after sinus surgery was to develop a standardised method of video endoscopy and grading the surgical field during ESS. This was done as a multinational collaborative trial. Once this assessment tool was validated a randomised controlled trial evaluating the effect of two antifibrinolytics (epsilon aminocaproic acid and tranexamic acid) was conducted. Further evaluation was then conducted on other possible hemostatic and antiadhesion substances. This included various combinations of a novel chitosan gel. These gels were trialled in vitro to determine their effect on human nasal fibroblasts derived from CRS patients. Fibroblast adhesion and proliferation as well as closure of standardised wounds were studied. The most promising of these gels was then used in an in vivo sheep model. Once effectiveness of the chitosan-dextran gel was shown in the laboratory, this was evaluated against a number of currently available hemostatic and anti-adhesion substances in a standardised model of wound healing in sheep with CRS. This model had been previously extensively validated in our department. Full thickness mucosal injuries were created on the lateral nasal wall and ethmoids of twenty sheep and recombinant tissue factor (rTF), SprayGel or Chitosan-Dextran derivative gel applied topically in a randomized fashion. Adhesion formation and severity as well as microscopic wound healing and ciliary function were analysed at day 28, 56, 84 and 112 post initial surgery. A further sheep study was conducted applying chitosan dextran gel to standardised mucosal injuries and comparing its effect on the control of bleeding to control. Bleeding time and grade were recorded and wound healing monitored via serial videoendoscopy over two weeks and objectively measured. Results: a) Assessment of the bleeding scales showed that inter and intra observer reliability for both scales tested were significantly improved by employing a standardized video-endoscopy technique. The Wormald scale proved to be more reliable and sensitive to changes in the most common surgical fields encountered in ESS. b) Tranexamic acid showed a modest but clinically significant improvement in the surgical field at 2, 4 and 6 minutes after application. Epsilon aminocaproic acid did not effectively improve the surgical field. c) Nasal fibroblast adhesion and proliferation were significantly impaired with dextran and chitosan. The most effective ratio that delayed but did not prevent wound closure were 5 % chitosan: 5 % dextran gel. d) In a standardised sheep model of mucosal wound healing the chitosan gel significantly decreased lateral nasal wall and ethmoidal adhesions at all time points. The chitosan group had a significantly greater percentage of re-epithelialisation and reciliation than control and rTF. In addition the mean cilial grade in the chitosan group was significantly better than control. e) The chitosan dextran gel was significantly more haemostatic at 2,4, and 6 minutes after injury with no significant difference noted in wound healing. Conclusions: Standardised methods of videoendoscopy and grading the surgical field in ESS are valuable tools for further research. Tranexamic acid significantly improved the surgical field to a moderate degree in ESS compared to control. Chitosan gel is a promising new powerful haemostatic bio-polymer which has a mild inhibitory effect on fibroblast attachment and proliferation. This may partially explain the significant improvement in microscopic wound healing and reduction in adhesion formation seen in a sheep model of chronic sinusitis. Future work evaluating this gel in the setting of a human trial is currently underway.<br>http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1375402<br>Thesis (Ph.D.) -- University of Adelaide, School of Medicine, 2009

The skin is a multifunctional organ and the primal frontier to the external environment. It is the first line of defence against external aggressors and any injuries inflicted in the vertebrate skin are rapidly repaired to re-establish immune defence and integument homeostasis. In mammals the outcome of skin injury is repair and scaring but in other vertebrates such as amphibians and fishes’ regeneration of the skin occurs and the disrupted tissue is replaced by skin of the same architecture and functionality as the original. Skin regeneration in vertebrates has been poorly explore and comparisons of the healing process in animals that heal scar free with mammalian wounds that scar will provide novel insights on the skin repair program and identify novel drug targets for mammalian skin disorders. The aim of this thesis was to identify key factors involved in skin homeostasis and repair and to generate a simple model for skin repair integrating metabolic, endocrine and immune considerations. The model species of this study was the gilthead sea bream (Sparus aurata) and using morphological and gene expression analysis the processes involved in wound healing in the regenerating fish skin in response to superficial damage caused by scale removal are described. Two gene families related to tissue repair in mammals (Angiopoietin-like family, ANGPTLs and Osteoglycin, OGN) were studied in detailed and compared and the effect of the diet supplement alpha-ketoglutarate (AKG), an inducer of collagen synthesis, in the integumentary system of adult sea bream explored. In overall the results obtained contribute to improve the current state of the art on the morphology and physiology of adult teleost skin and its regeneration after damaged and highlights for the importance of fish skin as a comparative model to study cutaneous repair in vertebrates.<br>O integumento é composto pela pele e os seus apêndices (pêlos, glândulas e escamas) e constitui a primeira linha de defesa do organismo a agressões externas e agentes patogénicos em vertebrados. A pele é o maior órgão do corpo e o primeiro a ser formado durante o desenvolvimento embrionário. É um órgão de estrutura simples, no entanto, biologicamente complexo e essencial à sobrevivência, e para além do seu papel na imunidade inata, é também um importante órgão sensorial e neuro-endócrino. Devido à sua importância, a pele quando danificada, recupera rapidamente para restabelecer as defesas imunitárias e a homeostasia do integumento. Em mamíferos, o processo de reparação cutânea leva à formação de uma cicatriz com consequente perda de funcionalidade do tecido afetado, mas noutros vertebrados, tais como anfíbios e peixes, cuja estrutura da pele é semelhante aos mamíferos existe a capacidade de regeneração tecidular e a pele danificada é reconstruída com igual topologia e funcionalidade. Em peixes teleósteos, o processo de regeneração da pele é pouco estudado sendo, no entanto, relevante uma vez que uma melhor compreensão sobre este processo permitirá estabelecer comparações do processo de reparação da pele em indivíduos adultos que têm mecanismos de regeneração como a de mamíferos, que perdem essa capacidade ainda na vida fetal e formam cicatrizes. Isto proporcionará um melhor entendimento dos processos regenerativos em vertebrados e permitirá a descoberta de novas moléculas que podem ajudar na compreensão das desordens da pele em mamíferos. O objetivo desta tese consistiu em identificar os fatores chave envolvidos na homeostasia e reparação da pele, e estabelecer um modelo simples de reparação da pele em vertebrados integrando considerações metabólicas, endócrinas e imunitárias. O organismo experimental utilizado neste estudo foi a dourada (Sparus aurata), um peixe teleósteo marinho de elevado valor económico e comercial na Europa, para qual já existem vários estudos fisiológicos e moleculares sobre o crescimento, metabolismo e homeostasia da energia e função imune. Recorrendo a várias análises morfológicas e moleculares esta tese descreve o processo de reparação da pele em dourada após uma agressão superficial ao integumento provocada pela remoção das escamas. Uma análise de dados de microarray de pele da dourada a 3 e 7 dias após a remoção das escamas (disponíveis no grupo) permitiu caracterizar os processos biológicos sub-expressos durante a regeneração da pele (capitulo 2). Neste estudo, verificou-se que no modelo utilizado a inflamação é independente da reepitelização do tecido, e que componentes da imunidade inata e adquirida estão suprimidos nas fases iniciais de reparação e só depois da função de barreira da pele ser reestabelecida é que o integumento amadurece e adquire a sua funcionalidade original. Duas famílias de genes associadas à reparação tecidular em mamíferos e possivelmente envolvidas na regeneração da pele em teleósteos foram selecionadas com base em estudos anteriores realizados pelo grupo de investigação, e ainda pouco caracterizadas em peixes, foram estudadas em detalhe: os membros da família das proteínas tipo angiopoietinas – (ANGPTLs) (capitulo 3) e o membro da família dos small leucine-rich proteoglycan o proteoglicano osteoglicina (OGN) (capitulo 4) e a sua evolução e papel no processo de regeneração tecidular em dourada comparada com os homólogos em tetrápodes. As ANGPTLs são uma família numerosa em peixes (10-13 membros) com vários genes homólogos de mamíferos duplicados. A analise evolutiva e comparativa identificou um novo membro desta família (angptl9) em peixes e outros tetrápodes (anfíbios e aves) mas não em mamíferos e que o gene dos mamíferos ANGPTL8 está ausente em outros vertebrados. Vários genes candidatos expressos na pele de peixes foram identificados, no entanto, o angptl1b, angptl2b e angptl7 parecem ter um papel importante no processo de regeneração pois são diferencialmente expressos durante as fases iniciais do processo de regeneração em dourada em pele intacta e regenerada. A existência e expressão de alguns membros da ANGPTL na pele de peixes e mamíferos, a sua ligação com a reparação do integumento, especificamente na dourada e a sua expressão associada a processos essenciais no programa de reparação sugere que o seu papel na regeneração e homeostasia da pele de vertebrados é conservada. Analises in silico identificaram dois genes para a OGN em peixes teleósteos (ogn1 e ogn2), e estudos de expressão tecidular e em culturas de células primárias in vitro de osso e mióticos de dourada sugerem que estes poderão estar envolvidos em múltiplas funções em peixes e que após duplicação diferenciaram-se e possivelmente adquiriram funções especificas. No entanto, no modelo de regeneração em estudo ambas as cópias duplicadas possuem funções conservadas na reparação da matriz da pele de peixes, tal como em mamíferos. O efeito do suplemento alimentar ácido alfa-cetoglutarato (AKG), estimulador da síntese de colagénio em mamíferos e aves, foi estudado no integumento e na regeneração da pele em douradas adultas (capitulo 5). A presença desta molécula quando na dieta dos peixes, estimula a biomineralização das escamas, acelera o crescimento das escamas em regeneração e promove a proliferação das células da epiderme, sugerindo que têm um papel importante na homeostasia da pele dos peixes e que tal como em mamíferos parece acelerar o processo de reepitelização após uma agressão. Os resultados obtidos nesta tese, contribuíram para um aumento da informação previamente disponível sobre a morfologia e a fisiologia da pele em peixes teleósteos adultos, e sobre a sua regeneração após uma agressão. Novos genes e moléculas que partilham a mesma origem evolutiva, motivos funcionais e papel biológico conservado no processo de regeneração com a dos mamíferos foram identificadas em peixes. Este trabalho realça a importância da pele dos peixes como um importante modelo comparativo para o estudo da reparação cutânea em vertebrados.

Dans mon projet de doctorat, j’ai étudié des fonctions primordiales de l’épithélium respiratoire telles que la régulation du transport ionique, la clairance liquidienne et la réparation épithéliale. J’ai particulièrement mis l’emphase sur le rôle des canaux potassiques qui interviennent dans ces trois fonctions de l’épithélium respiratoire. J’ai tout d’abord prouvé que la modulation des canaux potassiques régulait l’activité du promoteur de αENaC, en partie via la voie de signalisation ERK1/2, dans des cellules alvéolaires. Cette régulation entraîne une variation de l’expression génique et protéique du canal ENaC. Physiologiquement, il en résulte une augmentation du phénomène de clairance liquidienne suite à l’activation des canaux K+, tandis que l’inhibition de ces canaux la diminue sévèrement. J’ai aussi pu démontrer que l’absence de canal KvLQT1 entraînait une diminution du courant (ENaC) sensible à l’amiloride, dans les cellules de trachée en culture primaire, isolées de souris KO pour kcnq1. Dans la seconde partie de mon étude, j’ai évalué l’impact de l’hyperglycémie sur la capacité de transport ionique et de réparation de cellules épithéliales bronchiques saines ou Fibrose Kystique. Mes résultats montrent que l’hyperglycémie diminue le transport transépithélial de chlore et le transport basolatéral de potassium. Des études préalables du laboratoire ayant montré que les canaux K+ et Cl- contrôlent les processus de réparation, j’ai donc évalué si ceux-ci étaient modifiés par l’hyperglycémie. Et en effet, l’hyperglycémie ralentit la vitesse de réparation des cellules issues des voies aériennes (CFBE-wt et CFBE-ΔF508). J’ai donc démontré que le transport de potassium intervenait dans des fonctions clés de l’épithélium respiratoire, comme dans la régulation génique de canaux ioniques, le contrôle de la clairance liquidienne alvéolaire, et que l’hyperglycémie diminuait le transport ionique (K+ et Cl-) et la réparation épithéliale.<br>During my Ph.D. training, I studied 3 important functions of respiratory epithelium : regulation of ion transport, liquid clearance and epithelial repair. I focused on potassium channels, because they control these three respiratory epithelial functions. First, I proved that αENaC promoter activity was regulated following K+ channel modulation, in alveolar cells. This regulation of αENaC promoter which might be through a modification of ERK1/2 phosphorylation, was followed by ENaC mRNA and protein expression regulation. I then showed that activation of KvLQT1 and KATP channels increased alveolar liquid clearance, whereas inhibition of these K+ channels decreased the alveolar clearance. I showed that the absence of KvLQT1 channel inhibited the amiloride-sensitive current (ENaC), in tracheal epithelial cells isolated from KvLQT1-KO mice. In the second part of my Ph.D. project, I studied the impact of hyperglycemia on Cystic Fibrosis (CF) and non-CF epithelial cells. I first observed that K+ and Cl- currents were reduced by hyperglycemia. Because we have previously shown that wound-healing process was dependant on K+ and Cl- channels, I then evaluated the impact of hyperglycemia on wound-healing. As expected, hyperglycemia slowed the repair rate of non-CF (CFBE-wt) and CF (CFBE-ΔF508) cell monolayers.

The Lhx2 transcription factor plays essential roles in morphogenesis and patterning of ectodermal derivatives as well as in controlling stem cell activity. Here, we show that during murine skin morphogenesis, Lhx2 is expressed in the hair follicle (HF) buds, whereas in postnatal telogen HFs Lhx2(+) cells reside in the stem cell-enriched epithelial compartments (bulge, secondary hair germ) and co-express selected stem cell markers (Sox9, Tcf4 and Lgr5). Remarkably, Lhx2(+) cells represent the vast majority of cells in the bulge and secondary hair germ that proliferate in response to skin injury. This is functionally important, as wound re-epithelization is significantly retarded in heterozygous Lhx2 knockout (+/-) mice, whereas anagen onset in the HFs located closely to the wound is accelerated compared with wild-type mice. Cell proliferation in the bulge and the number of Sox9(+) and Tcf4(+) cells in the HFs closely adjacent to the wound in Lhx2(+/-) mice are decreased in comparison with wild-type controls, whereas expression of Lgr5 and cell proliferation in the secondary hair germ are increased. Furthermore, acceleration of wound-induced anagen development in Lhx2(+/-) mice is inhibited by administration of Lgr5 siRNA. Finally, Chip-on-chip/ChIP-qPCR and reporter assay analyses identified Sox9, Tcf4 and Lgr5 as direct Lhx2 targets in keratinocytes. These data strongly suggest that Lhx2 positively regulates Sox9 and Tcf4 in the bulge cells, and promotes wound re-epithelization, whereas it simultaneously negatively regulates Lgr5 in the secondary hair germ and inhibits HF cycling. Thus, Lhx2 operates as an important regulator of epithelial stem cell activity in the skin response to injury.

Indiana University-Purdue University Indianapolis (IUPUI)<br>Tendinopathy is a common degenerative pathology that is characterized by activity related pain, focal tendon tenderness, intratendinous imaging changes, and typically results in changes in the histological, mechanical, and molecular properties of the tendon. Tendinopathy is difficult to study in humans, which has contributed to limited knowledge of the pathology, and thus a lack of appropriate treatment options. However, most believe that the pathology is degenerative as a result of a combination of both extrinsic and intrinsic factors. In order to gain understanding of this pathology, animal models are required. Because each tendon is naturally exposed to different conditions, a universal model is not feasible; therefore, an appropriate animal model must be established for each tendon susceptible to degenerative changes. While acceptable models have been developed for several tendons, a reliable model for the Achilles tendon remains elusive. The purpose of this dissertation was to develop an animal model of Achilles tendinopathy by investigating the individual and combined effects of an intrinsic and extrinsic factor on the rodent Achilles tendon. Rats selectively bred for high capacity running and Sprague Dawley rats underwent uphill treadmill running (an extrinsic factor) to mechanically overload the Achilles tendon or served as cage controls. Collagenase (intrinsic factor) was injected into one Achilles tendon in each animal to intrinsically break down the tendon. There were no interactions between uphill running and collagenase injection, indicating that the influence of the two factors was independent. Uphill treadmill running alone failed to produce any pathological changes in the histological or mechanical characteristics of the Achilles tendon, but did modify molecular activity. Intratendinous collagenase injection had negative effects on the histological, mechanical, and molecular properties of the tendon. The results of this dissertation demonstrated that the combined introduction of uphill treadmill running and collagenase injection did not lead to degenerative changes consistent with human Achilles tendinopathy. Intratendiouns collagenase injection negatively influenced the tendon; however, these changes were generally transient and not influenced by mechanical overload. Future studies should consider combinations of other intrinsic and extrinsic factors in an effort to develop an animal model that replicates human Achilles tendinopathy.