京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第18178号 / 医博第3898号 / 新制||医||1004(附属図書館) / 31036 / 京都大学大学院医学研究科医学専攻 / (主査)教授 宮地 良樹, 教授 羽賀 博典, 教授 山下 潤 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DGAM

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Dermal fibroblasts positively contribute throughout the wounding response by secreting a profile of pro- and anti-inflammatory cytokines in the wound milieu. However, a chronically inflamed environment will, cause detrimental effects on the functional, secretory, and molecular properties of these cells. This study aims to understand how the effect of the pro-inflammatory cytokine TNF-α modulates both healthy and diabetic dermal fibroblast phenotype. To mimic a chronic inflammatory environment and assess whether fibroblasts respond similarly in different anatomical sites, donor-matched fibroblasts from face and scalp were pre-incubated for 3 days with different concentrations (2.5, 25 or 250 ng/ml) of TNF-α. All concentrations significantly impaired proliferation by day 14 in cells from both sites and stimulated (papillary) metabolic activity at day 14. However, this did not correlate with an increase in papillary cell senescence since this did not appear until passage 17, and then only at a supra pathophysiological concentration. Migration of dermal fibroblasts, assessed by the scratch assay. TNF-α significantly inhibited the cells migration, particularly in diabetic fibroblasts, suggesting they are more sensitive to TNF-α. Since TNF-α may stimulate the secretion of soluble paracrine factors by dermal fibroblasts, conditioned medium was collected to assess its effect on other dermal fibroblasts, however, this had no significant effect on migration. However, using gelatin zymography, it was found that TNF-α did stimulate the secretion of soluble paracrine factors that induce MMP activity in non-diabetic fibroblasts, mirroring previous observations that a pro-inflammatory environment can increase proteolytic activity, and indicating that diabetic fibroblasts were again more sensitive than healthy. No difference was observed with MMP-9 activity and nor did the results with dermal fibroblasts reach statistical significance, perhaps because of a relatively low n-number. The ability of TNF-α to modulate the expression of genes associated with the ECM (MMP-1, -2, -9, TIMP-1, and -2) and senescence (Sirt1 and 6) was investigated. There was no change in Sirt1 and Sirt6 expression and no evidence of paracrine effects (conditioned medium) on any of the genes. TNF-α significantly induced mRNA expression of MMP-1 in healthy non-scratched and scratched diabetic fibroblasts, and TIMP-1 in healthy non-scratched cells. There was also considerable donor variability that prevented statistical significance being achieved under the other conditions. The secretion of various cytokines associated with inflammation was compared in healthy and diabetic fibroblasts in the presence and absence of TNF-α. Seven cytokines were secreted, by healthy and diabetic male and female fibroblasts, although diabetic female fibroblasts did not secrete two of them. TNF-α stimulated secretion of cytokines in healthy and diabetic, male and female cells but the profiles of those released were different between the different groups. There was no TNF-α induced paracrine effect on cytokine secretion by healthy dermal fibroblasts. In conclusion, changes in the microenvironment and the influx of pro inflammatory cytokines may significantly alter the dermal fibroblast phenotype. Understanding these functional and molecular changes in response to inflammatory cytokines will give a better understanding of the differences between fibroblast activity in normal physiological wound healing and chronic or diabetic non-healing wounds.

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Assessing ecological risk to wildlife exposed to anthropogenic contamination in soil has traditionally been problematic. Attempts to standardize an approach to evaluate risk for various community types in North America have been challenging, given the variation in terrestrial communities and the values in which policy makers are bound to protect. This has resulted in vague, yet flexible guidance from the U.S. Environmental Protection Agency and other interested parties (e.g., the U.S. Army Corps of Engineers, and the Tri-Service Ecological Risk Assessment Working Group). Interpretation of these and other guidance has been variable, often resulting in conflicting opinions on how best to address the question of ecological risk to receptors that are exposed to xenobiotics in a soil matrix. This work reports the results of research designed to address the question of ecological risk to terrestrial vertebrates. Objective, ecologically-relevant criteria were used in the selection and development of models in this research. Several lines of logic were considered: 1) substance sensitivity, 2) ecological sensitivity (i.e., the species importance to the system; e.g., keystone species); and, 3) probability and extent of exposure. A primary soil contaminant at many U.S. Army installations is 2,4,6-trinitrotoluene (TNT). This was a result of the mass manufacturing, storing, and assembly of weapons from the early 1900's until the 1950s. The Army has reported soil concentrations of TNT ranging from 0.12 to 38,600 ug/g (Walsh and Jenkins 1992) and 0.08 to 64,000 ug/g (Hovatter et al. 1997). The chemical-physical properties of TNT result in a relatively unique compound, not easily amenable to current modeling techniques to estimate exposure to terrestrial wildlife. Moreover, there are few data describing the effects of exposure to TNT in other than mammals, fish, and specific invertebrates. In this research, the pathways of exposure and selected potential toxic effects from TNT exposure were investigated in a terrestrial salamander: Ambystoma tigrinum (tiger salamanders). A. tigrinum was chosen since they are exclusively carnivorous, relatively long-lived, have a thin integument, and are large enough to investigate individual effects. These investigations were designed to mimic natural conditions as closely as possible, though maintain a degree of homogeneity in a laboratory environment. All studies exposed salamanders to soil and food (earthworms) in identical preparations. As such, these exposures were considered complete, eliminating assumptions made regarding daily food consumption, systemic dermal dose, etc. The first study examined the relative contribution of dermal or oral exposures to the whole-body burdens of TNT and primary metabolites. A poly-chlorinated biphenyl (PCB) mixture (Aroclor7 1260) was used with TNT to simultaneously to assist in the evaluation of each pathway, since the fate and transport of PCBs are well characterized. Tiger salamanders were exposed 28 days in situ. The dermal route of exposure contributed the most to the final burdens of TNT in salamanders, with the primary reduction products, 2-amino-4,6-dinitrotoluene and 4-amino, 2,6-dinitrotoulene reaching higher concentrations than of parent compound. Other TNT metabolites were found in insignificant quantities. The concentrations of PCBs were higher in the oral treatment, as expected. These results were corroborated in a subsequent study using Ambystoma maculatum (spotted salamanders). The second series of investigations evaluated the potential toxic effects from TNT exposure. Two treatments consisting of TNT and a control were used to evaluate these effects to A. tigrinum. The salamanders were exposed in situ for 14 days to TNT in soil and food (earthworms of which were exposed to TNT in the soil in similar preparations). Non-specific immune effects were evaluated through the characterization of splenic phagocytes in their ability to: 1) phagocytize foreign particles, and 2) digest (through oxygen radicals) phagocytized material. This was conducted using fluorescent microspheres and a fluorescent chemical probe specific to hydrogen peroxide, measured per each cell using flow cytometry. Other data collected included histological examination (e.g., liver, kidney, and other miscellaneous organs), blood differentials, weight changes over time, organ/ body weight comparisons, and an analysis of organ-specific metabolism. No significant effects were noted in salamanders exposed to these conditions. Coordinated with the preceding study included a search for biomarkers of exposure and an investigation of the metabolites of TNT in situ. Biotransformation products of TNT were found including primary (e.g., 2-amino-4,6-dinitrotoluene) and secondary (e.g., 2,4-diamino-6-nitrotoluene) in relative concentrations in skin, liver, and kidney. Biomarkers of exposure included an analysis of cytochrome p450, b5, and the glutathione antioxidant enzymes in liver, kidney, skin, lung, and serum, respectively. Traces of parent compound were found in the skin and liver only. Levels of 2,4-diamino-6-nitrotoluene were found only in the liver and kidney, suggesting that TNT is reduced primarily in or on the skin. Levels of p450 were higher in TNT exposed salamanders than controls. Glutathione and related enzyme levels are reported. This work suggests that salamanders have levels of detoxification enzymes capable of the biotransformation of anthropogenic substances in soil rivaling that of mammals. Another investigation evaluated these same immunological parameters in white-footed mice (Peromyscus leucopus). This species was chosen based on the relative importance of small mammals to the community structure in many North American ecosystems. Mice were exposed to TNT in the feed at 0.264, 0.066, 0.033, and 0.017%, where actual daily dose estimates for males were 604, 275, 109, and 65; and for females was 544, 282, 143, and 70 mg/kg/d. An investigation to evaluate the specificity of commercially-available monoclonal antibodies specific to cell surface markers for thymocytes and splenocytes in inbred mice was unsuccessful. These results suggest the recognition epitopes of monoclonal antibodies prepared against Old-World mice are not conserved into Peromyscus, a New-World species. However, high dose males and females had larger spleens consistent with the hemolytic effects previously reported for mammals exposed to TNT. Further, males exposed at all levels had reduced phagocytic activity of splenocytes, and reduced hydrogen peroxide production associated with the two highest doses relative to controls. Females showed no response relative to treatment. This research has shown the feasibility for these types of investigations, and provides toxicity information valuable for modeling estimates of ecological risk. Further, the in situ exposures have provided media concentrations that are or are not toxic for species of concern. This type of information reduces the uncertainty associated with ingestion modeling estimates, dermal exposure estimates, and other factors not traditionally considered in toxicity studies. / Ph. D.

A silent pandemic, chronic, non-healing wounds are a major cause of morbidity, with treatment and management representing significant health burdens. The opportunistic pathogens Staphylococcus aureus and Pseudomonas aeruginosa are the most common species isolated from chronic wounds. Polydimethylsiloxane (PDMS), a biocompatible and, inexpensive to fabricate polymer, can undergo various modifications. The ability of the produced polymers to attract S. aureus and P. aeruginosa, either from the planktonic state, or while sessile in biofilms on ex vivo skin, was investigated using flat (FL) or patterned (PT) PDMS with or without 1% or 10% triclosan Patterned PDMS + 10% triclosan (PT 10%) attracted significantly more live S. aureus and P. aeruginosa, as determined using Colony Forming Unit (CFU) analysis (*p<0.01), Scanning Electron Microscopy (SEM) (*p<0.01) and Confocal Scanning Laser Microscopy (CSLM) (*p<0.01). The released triclosan was not cytotoxic against either bacteria or primary cultures of human dermal fibroblasts using Water Soluble Tetrazolium Salts (WST-1) assay. High performance liquid chromatography analysis highlights low level of triclosan release from the PDMS. Bacterial infection in co-culture using the Boyden chamber assay increased fibroblast viability in the presence of PDMS (*p<0.05). PT 10% demonstrated superior biofilm transfer from epidermis (*p<0.05), in comparison to all other analysed polymers. In summary, the unique topography of PDMS combined with triclosan attracted bacteria most efficiently. This promising data suggests potential for engineering a patterned polymer to physically transfer biofilms from wounds, and importantly lacks bactericidal properties which is vital in the quest to combat antimicrobial resistance.

This study investigates the bioactivity of bioprinted scaffolds cultured with adult human dermal fibroblast cells (aHDF) used for tissue regeneration by employing Lumen X+ 3D bioprinter. Polyethylene glycol diacrylate (PEGDA) is the widely used bioink in these Lumen bioprinters. Although PEGDA is meritorious for its photopolymerization capabilities and biological and biomedical applications, it has very minimal inherent cell adhesion and proliferation properties. Hence in order to improvise this challenge we demonstrated the development of a novel kind of tissue-specific hybrid bioinks utilizing PEGDA that has been altered by incorporating short, self-assembling dipeptides that are based on FF or Phe-Phe hydrogels. To leverage the bioactivity of FF peptides, we first encapsulated adult human dermal fibroblasts (aHDF) in Fmoc-FF hydrogel and investigated how the HDF cells interacted to the microenvironment of the peptide in terms of adhesion and material-cell interactions. Further, we fabricated three different sets of honeycomb lumen lattice constructs: (i) Matrigel coating on lumen lattice, (ii) peptide coating on lumen lattice, and (iii) peptide infused lumen lattice lumen was printed by combing PEGDA bioinks. We investigated the potential ability of these scaffolds to enhance cell attachment rate, proliferation, and expansion of human dermal fibroblast cells and drug-delivering abilities.