Fibrotic diseases account for an estimated 45% of the total number of deaths in the developed world (Wynn 2007). Tendons are an excellent model for studying the dysregulated response which leads to fibrosis, as tendons have an organized, parallel matrix, in which tissue defects could easily be distinguished. Wong et al. (2006b) demonstrated the presence of a bell-shaped region around sutures in tendons that was devoid of cells in histological sections. The mechanisms of the formation of this acellular zone, that was also noted in cornea and cartilage (Matsuda et al. 1999; Hunziker and Stähli 2008), were unknown. It was hypothesized that the acellular zone was formed by cell death and that suturing caused alterations to the extracellular matrix of sutured regions of tendon, which made the acellular zone refractory to cellular re-population. The acellular zone was tracked in sutured tendons for up to a year to determine the temporal properties of the acellular zone. Electron microscopic and time lapse studies were carried out to determine if the acellular zone formed by cell migration or cell death. Microarray analysis was conduced to confirm this and to reveal potential molecular targets for future studies. The extracellular matrix of sutured tendons was studied by electron, atomic, scanning and polarized light microscopy and mechanical measurements were obtained using nanoindentation. It was concluded that the acellular zone formed within 24 hours and persisted for up to a year. Tension and size of the suture's grasp were also shown to be important for acellular zone formation. Cell death was the main effector of acellular zone formation. Microarray analysis showed evidence of upregulation of inflammatory mediators and programmed necrosis pathways. The sutured extracellular matrix was denser, more disorganized and had a lower Young's modulus than unsutured regions of the same tendon. These differences in the properties of the extracellular matrix of sutured tendons may be the cause of the persistence of the acellular zone.

Over the past decade the concept that the hair follicle plays an important role in cutaneous wound repair has been established. Several elegant lineage tracing studies have demonstrated that hair follicle derived cells contribute to the long term maintenance of the epidermis following repair, while an absence of hair follicles is known to delay repair. The exact mechanisms surrounding hair follicle derived repair are unknown. Moreover, while multiple stem cell niches are present within the hair follicle, their relative importance during wound repair is still unclear. The hair follicle is also a regenerative mini-organ, undergoing regular cycles of growth and regression throughout life, yet surprisingly this has not been previously investigated with respect to wound repair. Data presented in this thesis reveals an unappreciated, yet fundamental link between the independent processes of hair cycle and wound repair, with a substantial acceleration in the rate of repair (~50%) observed in anagen phase. Importantly, the hair follicle appears to play a global role in repair, with differences in the contribution of multiple cell types to wound repair. In addition, this thesis addresses the early kinetics of hair follicle wound response for the first time. Anagen hair follicles are found predisposed to a more rapid and extensive response to injury, suggesting a higher overall percentage of repair derived from the hair follicle in anagen phase. Surprisingly, the bulge stem cell region, while critical for hair cycle appears to play little role in the events immediately following injury, and is not required for initiation of re-epithelialisation. Gene expression profiling reveals numerous genes associated with anagen accelerated repair, and identifies altered modulation of the immune system as a key mechanism. Further, anagen wounds are associated with an upregulation of developmental transcription factors, which may imply a more regenerative healing phenotype. These data reveal numerous targets with the potential to accelerate repair, which now require validation for their therapeutic potential. These targets could be of importance in promoting the repair of chronic wounds, an area of unmet clinical need. More generally, this thesis has established hair cycle as an important experimental variable, which must be controlled for in all future in vivo murine wounding studies.

The purpose of this study was to determine the effects of varying periods of immobilization on ligamentous healing and strength in a rat experimental model. Sixty-one mature male Wistar rats were used. The left knee medial collateral ligament was surgically exposed, divided, and repaired. The rats were randomly placed into one of four groups: Group A, no immobilization, Group B, 2 weeks' immobilization, Group C, 6 weeks' immobilization, and Group D, 10 weeks' immobilization of the operated limb. The right knee served as a control. The ligaments were studied histologically and biomechanically at 2 weeks, 6 weeks, 10 weeks and 20 weeks post-operatively. Histologic samples were objectively evaluated with the light microscope using a Maturity Index Score and Scale that were devised based on the numbers and orientation of the fibroblasts and the amount and orientation of the collagen fibres. Ligament-bone preparations were studied using an Instron material testing machine to determine the biomechanical properties of the ligament until failure. Utilizing the Maturity Index Score and Scale, it was shown that Group A, with no immobilization, matured more rapidly than the other groups, and achieved full maturity at 20 weeks post-operatively. The other groups all showed a retarded rate of healing while immobilized. The electron microscopic study supported this data by demonstrating the level of metabolic activity of the fibroblasts which decreased with increasing maturity and by demonstrating that the size, amount and orientation of the collagen fibers increased with mobilization. The biomechanical testing showed that at 2 weeks post-operative, Group A had achieved a strength which was 46% of controls while Group B was only 29% of controls (p = 0.055). At 6 weeks Group A was 65% of controls, Group B was 56% of controls and Group C was 39% of controls (p = 0.0004). At 20 weeks Group A was 83% of controls, Group B was 71% of controls, Group C was 66% of controls and Group D was 48% of controls (p = 0.0005). Group A was 71% stronger than Group D at this time, indicating that the healing medial collateral ligament attained a greater strength and histologically matured more rapidly if mobilization is begun immediately. / Science, Faculty of / Botany, Department of / Zoology, Department of / Graduate

No description available.

Sea bass (Lateolabrax maculatus) has been used for dietary therapy practice in China. In traditional Chinese medicinal books, it has been indicated that sea bass can be applied for managing many inflammation associated conditions. However, the studies on the pharmacological mechanisms of anti-inflammation of sea bass remain scarce. Hence, this study aims to illustrate the pharmacological and chemical basis for the folk use of sea bass in managing inflammation- associated conditions. For in vivo studies, dietary effect of sea bass on inflammation-associated conditions in ulcerative colitis, skin wounds, and intestinal dysbiosis were evaluated. A series of inflammatory mediators associated with wound healing and ulcerative colitis, and the proliferation effects of fibroblasts upon treatments were studied via Western blotting, enzyme-linked immunosorbent assay (ELISA), hematological parameters, histopathological and Immunofluorescence analysis, using cutaneous wound model and DSS induced colitis model, respectively. β-diversity analysis and species variance statistics were conducted to evaluate the effect of ASB on the microbial communities with colitis and discovered the high dimensional biomarkers. Results showed that ASB could significantly ameliorate several pathophysiological and morphological features in DSS induced colitis. ASB has a potential in accelerating the proliferation phase of wound healing via well-organized abundant collagen deposition, angiogenesis, strengthening the skin contraction and skin organ maturation in wounds. Moreover, the study also found that ASB could significantly down-regulated the expression levels of inflammatory associated mediators in colitis and skin wound. Additionally, principal coordinate analysis (PCoA) and relative abundance at phylum level among groups were indicated that ASB possess a potential amelioration on intestinal dysbiosis in colitis. Histogram of linear discriminant analysis (LDA) scores and Cladogram as the results of LEfSe analysis identified that Christensenellaceae might be treated as the the biomarker for treating colitis. For in vivo studies, macrophages and fibroblasts were used for further evaluation. Result showed that ASB could significantly inhibited the production of pro-inflammatory mediators in lipopolysaccharide (LPS) induced macrophages. The mRNA and protein expression level of inflammatory associated mediators were significantly down-regulated upon ASB treatment. Moreover, results also suggested that ASB treatment has a closely link to accelerate the wound healing through migration and proliferation enhancement. Furthermore, the characterization of the aqueous extract of sea bass (ASB) was conducted. Six kinds of peptides and two protein identified from fraction F1 by LC-QE-HF-MS might be responsible for anti-inflammatory activity. It confirmed that Fraction F1 could be treated as the main component for contributing the potential anti-inflammatory activities to ASB. Current results illustrated that fraction F1 (kDa) is a kind of nanoparticles with stability separated from ASB. It can be treated as a promising candidate for treating inflammation associated conditions, providing the chemical basis for the folk use of sea bass in managing inflammation-associated conditions. Current studies established a pharmacological and chemical basis for the folk use of sea bass in managing inflammation-associated conditions. A further justification for the clinical application of sea bass in treating inflammation associated conditions is necessary. Keywords: sea bass; inflammation; ulcerative colitis; wound healing; gut microbiota; peptides

This diploma thesis is focused on the study of bioactive wound dressings. During the thesis, hydrogel, lyophilized and nanofiber wound dressings were prepared. Hydrogel and lyophilized wound dressings were prepared on basis of two polysaccharides – alginate and chitosan. Nanofiber wound dressings were prepared by spinning polyhydroxybutyrate. All prepared wound dressings were enriched with bioactive substances, which represented analgesics (ibuprofen), antibiotics (ampicillin) and enzymes (collagenase). Into hydrogel and lyophilized wound dressings were all the mentioned active substances incorporated, whereas nanofiber wound dressings were only with ibuprofen and ampicillin prepared. The theoretical part deals with the anatomy and function of human skin. There was explained the process of wound healing and also there were introduced available modern wound dressings. The next chapter of the theoretical part deals with materials for preparing wound dressings (alginate, chitosan, polyhydroxybutyrate) and with active substances, which were used during the experimental part of this thesis. In the theoretical part, the methods of preparation of nanofiber wound dressings and also the methods of cytotoxicity testing used in this work were presented. The first part of the experimental part of this thesis was focused on preparing already mentioned wound dressings. Then, their morphological changes over time and also the gradual release of incorporated active substances into the model environment were monitored. The gradual release of ampicillin was monitored not only spectrophotometrically, but also by ultra-high-performance chromatography. In wound dressings, in which collagenase was incorporated, was also the final proteolytic activity of this enzyme monitored. The effect of the active substances was observed on three selected microorganisms: Escherichia coli, Staphylococcus epidermidis and Candida glabrata. The cytotoxic effect of the active substances on the human keratinocyte cell line was monitored by MTT test and LDH test. A test for monitoring the rate of wound healing – a scratch test – was also performed.

Epithelial wound healing requires the coordination of cells to migrate as a unit over the basement membrane after injury. An excellent model tissue is the corneal epithelium, which is an avascular stratified squamous tissue that responds to growth factors and nucleotides when the epithelial barrier is damaged. One signal that has a ubiquitous response in epithelial wound healing is the cellular release of the nucleotide ATP, which may occur because of mechanics forces and/or change in cell shape. Within milliseconds to seconds after injury, extracellular ATP binds to purinoreceptors and triggers a transient Ca2+ wave, which is used by cells to transduce mechanical signals into chemical signals and alter signaling pathways. To understand the process of this coordinated movement, it is critical to study the dynamics of cell-cell communication. In this study we developed a novel method to identify and characterize the degree of cell-cell communication that occurs through sustained Ca2+ mobilizations after injury, which are concentrated along the epithelial wound edge and reduced in cells distal to the injury. Using MATLAB analyses, we generated profiles of the sustained Ca2+ mobilizations, and demonstrated that the Ca2+ response was replicated in ex vivo organ culture models. The sustained Ca2+ mobilizations were present also after stimulation with either BzATP or UTP, which are agonists of P2X7 and P2Y2 respectively. The probability that cells would communicate was greater in response to BzATP compared to UTP. The specificity of these ligands was demonstrated using competitive inhibitors of P2Y2 and P2X7 receptors, AR-C 118925XX and A438079, respectively. An inhibitor of pannexin-1, 10Panx, attenuated both wound closure and BzATP agonist-initiated response. These sustained mobilizations are correlated with changes in cellular morphology and motility, which were prominent in cells at the leading edge of the wound during cell migration. Together, our results demonstrate that the sustained Ca2+ mobilizations mediated by purinoreceptors and pannexins are a vital component in regulating the long-term response to injury, as studied in organ culture.

In the vast majority of cases, appropriate identification and mitigation of risk factors can prevent or minimize pressure ulcer (PU) formation. However, some PUs are unavoidable. Based on the importance of this topic and the lack of literature focused on PU unavoidability, the National Pressure Ulcer Advisory Panel hosted a multidisciplinary conference in 2014 to explore the issue of PU unavoidability within an organ system framework, which considered the complexities of nonmodifiable intrinsic and extrinsic risk factors. Prior to the conference, an extensive literature review was conducted to analyze and summarize the state of the science in the area of unavoidable PU development and items were developed. An interactive process was used to gain consensus based on these items among stakeholders of various organizations and audience members. Consensus was reached when 80% agreement was obtained. The group reached consensus that unavoidable PUs do occur. Consensus was also obtained in areas related to cardiopulmonary status, hemodynamic stability, impact of head-of-bed elevation, septic shock, body edema, burns, immobility, medical devices, spinal cord injury, terminal illness, and nutrition.

Glucan is a natural product immunomodulator that has been reported to enhance early wound repair. The mechanism of glucan-stimulated wound repair was thought to be indirect via macrophage release of wound growth factors. However, recent data indicate that there are glucan-specific receptors on human fibroblasts that can modulate cellular function following interaction with the glucan ligand. In this study we examined the effect of glucan on activation of the transcription factors activator protein-1 (AP-1) and specificity protein-1 (Sp1) in normal human dermal fibroblasts. AP-1 and Sp1 are involved in the regulation of cytokine and procollagen genes. In addition, we evaluated the effect of glucan on wound growth factor and vascular endothelial growth factor (VEGF) mRNA expression in primary cultures of normal human dermal fibroblasts. Glucan (1 μg/ml) stimulated fibroblast AP-1 and Sp1 activation in a time-dependent manner, although the temporal kinetics varied between the two transcription factors. AP-1 binding activity was increased (p<0.05) at early time intervals (1, 2, 4, 8 and 12 h), while Sp1 nuclear binding activity was increased (p<0.05) at later time intervals (12, 24, 36 and 48 h). Glucan (1 μg/ml) stimulated fibroblast expression of neurotrophin 3 (NT-3), platelet derived growth factor A (PDGF-A), platelet derived growth factor B (PDGF-B), fibroblast growth factor acidic (aFGF), fibroblast growth factor basic (bFGF), transforming growth factor alpha (TGFα), transforming growth factor beta (TGFβ) and VEGF mRNA at 8 h.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第18851号 / 医博第3962号 / 新制||医||1007(附属図書館) / 31802 / 京都大学大学院医学研究科医学専攻 / (主査)教授 別所 和久, 教授 鈴木 茂彦, 教授 瀬原 淳子 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM