No description available.

No description available.

Skin is the largest organ of the body. Its integrity plays a crucial role in maintaining physiological homeostasis, protects against mechanical forces and infections, fluid imbalance, and thermal dysregulation. Numerous pathological states, such as diabetes mellitus, peripheral vascular disease, thermal injuries, or degloving lead to inadequate wound healing, necessitating medical intervention. Established wound healing techniques such as autologous and allogeneic skin grafts are inefficient due to the limited availability of donor tissues or probable immunogenic reactions. Current research in the field of tissue engineering aims to facilitate wound healing and restore skin functionality, focusing on key aspects of wound healing, such as extracellular matrix (ECM) reorganization, cell growth, and collagen synthesis/deposition. The research aims at developing and characterizing an in-vitro fibrin gel culture model system that stimulates the process of wound healing. The specific goal of this research is to investigate how the varied chemical composition of fibrin hydrogels can enhance fibroblast proliferation and promote accelerated collagen matrix formation, which is a significant step in tissue repair and regeneration.The fibrin gels are optimized by modulating the primary gel constituents (i.e. the concentrations of fibrin and thrombin). The ensuing hydrogels are characterized using Scanning Electron Microscope and compression testing to test for fiber size, porosity, elasticity, and mechanical properties. Cultured fibroblasts are used to investigate the effects of varying fibrin concentrations on cell-biomaterial interactions, including cell proliferation, cellular infiltration, and network formation. Furthermore, matrix formation and maturation as a function of fibrinogen concentration as defined by collagen matrix deposition, are also studied. Increasing the fibrinogen concentration, lead to an increase in elasticity and Young’s modulus, while a decrease in thrombin concentration generated a stronger fiber structure. Additionally, a decrease in fibrinogen concentration resulted in an increased proliferation rate of fibroblast cells, suggesting better cell adhesion and network formation within the gel substrate. These results were consistent and confirmed by quantifying a mature collagen matrix deposited by fibroblasts when subjected to ascorbic acid. In summary, this research investigates how the varied chemical composition of fibrin hydrogels can enhance fibroblast proliferation and promote accelerated collagen matrix formation, which is a significant step in tissue repair and regeneration. / Bioengineering

京都大学 / 新制・課程博士 / 博士(医学) / 甲第24137号 / 医博第4877号 / 新制||医||1060(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 上野 英樹, 教授 森本 尚樹, 教授 寺田 智祐 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Angiogenesis is a central process to both physiological and pathological aspects of living organisms. Understanding the angiogenic system via the key mediator, vascular endothelial growth factor (VEGF), has led to the development of biophotonic models capable of monitoring how this process is programmed. The whole animal model tested here is based on the involvement of angiogenesis in a wound healing environment. This model proved to be functional as a system monitor but lacked the precision to yield significant results between the biological compounds tested (estrogen, methoxychlor, and relaxin). The in vitro model is based on angiogenesis in a cancer environment. This model proved to be both a valid and functional way of monitoring the biological compounds tested (CoCl2, epinephrine, and norepinephrine).

No description available.

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. / Marie Curie ... the CLaSSiC project

yes / There are various techniques to enhance tissue regeneration via the application of growth factors to the site of regeneration to induce cells to proliferate, differentiate and regenerate. Generally, direct application of growth factors has little effect [1] because the growth factor diffuses out from the site of regeneration very quickly. This is a problem that can be solved by a controlled release of growth factor at the site of action over a long period of time by use of a bioabsorbable scaffold. Growth factors are protein based molecules in the body which are produced by cells and attach to the cell surface. Growth factors bind to membrane receptors, which in turn activate an intracellular signalling pathway. This will activate or inhibit a gene causing either an up regulation or down regulation of a gene product, which then alters the cells behaviour.

Bone morphogenetic proteins (BMPs) and their receptors (BMPRs) coordinate tissue development and postnatal remodelling by regulating proliferation, differentiation and apoptosis. However, their role in wound healing remains unclear. To study this, transgenic mice overexpressing Smad1 (K14-caSmad1) or the BMP antagonist Noggin (K14-Noggin) were utilised, together with human and mouse ex vivo wound healing models and in vitro keratinocyte culture. In wild-type mice, transcripts for Bmpr-1A, Bmpr-II, Bmp ligands and Smad proteins were decreased following tissue injury, whilst Bmpr-1B expression was up-regulated. Furthermore, immunohistochemistry revealed a down-regulation of BMPR-1A in hair follicles adjacent to the wound in murine skin, whilst in murine and human wounds, BMPR-1B and phospho-Smad-1/5/8 expression was pronounced in the wound epithelial tongue. K14-caSmad1 mice displayed retarded wound healing, associated with reduced keratinocyte proliferation and increased apoptosis, whilst K14-Noggin mice exhibited accelerated wound healing. Furthermore, microarray analysis of K14-caSmad1 epidermis revealed decreased expression of distinct cytoskeletal and cell motility-associated genes including wound-associated keratins (Krt16, Krt17) and Myo5a versus controls. Human and mouse keratinocyte proliferation and migration were suppressed by BMP-4/7 both in vitro and ex vivo, whilst they were stimulated by Noggin. Additionally, K14-caSmad1 keratinocytes showed retarded migration compared to controls when studied in vitro. Furthermore, Bmpr-1B silencing accelerated migration and was associated with increased expression of Krt16, Krt17 and Myo5a versus controls. Thus, this study demonstrates that BMPs inhibit proliferation, migration and cytoskeletal re-organization in epidermal keratinocytes during wound healing, and raises a possibility that BMP antagonists may be used for the future management of chronic wounds.

No description available.