Thesis (doctoral)--University of Göteborg, 1993. / Added t.p. with thesis statement inserted. Includes bibliographical references.

Thesis (M.D.S.)--University of Hong Kong, 2001. / Includes bibliographical references (leaves 86-107). Also available in print.

Thesis (Ph.D.)--Virginia Commonwealth University, 2010. / Prepared for: Dept. of Biochemistry. Title from title-page of electronic thesis. Bibliography: leaves 137-141.

The skin acts as a protective barrier, is crucial for thermoregulation and also forms part of the sensory, immunological and endocrine system. Therefore skin preservation is paramount to preserving life. The loss of skin homeostasis, through injury, initiates the wound healing process where the final outcome is the formation of a scar. Scar treatment remains a challenge, despite a plethora of treatments, resulting in a poor outcome and sub-optimal response to existing therapies. Photodynamic therapy (PDT) has been used to treat oncologic conditions affecting the skin. Its action depends on a photosensitiser and a specific light source. Aminolevolinic acid (5ALA) and its methyl ester (MALA) are commonly used pro-drugs of the photosensitiser protoporphyrin IX (PpIX), which in combination with red light produces reactive oxygen species (ROS). ROS will cause different responses such as cell death and tissue destruction. There is limited clinical evidence emerging for the use of PDT in treating wound healing and pathological skin scarring. For this reason, further investigations are required to better understand the role of PDT in adult human skin wound healing and skin scarring. The aim of this investigation was to evaluate the accumulation of PpIX after exposure to 5ALA or MALA, phototoxicity of red light arrengment, citotoxicity, cell death inducction, ROS generation and a gene related analysis post-PDT in keloid fibroblasts compared to normal skin fibroblasts. Optimization of a wound healing organ culture (WHOC) model and evaluation of re-epithelialization, cell death, proliferation, extracellular matrix arreangment (ECM) and a related gene analysis after 5ALA-PDT ex vivo. General histology, cell death, proliferation, ECM rearrengment and a gene related analysis after PDT in skin scarring ex vivo. This investigation found PpIX accumulation higher with MALA compared to 5ALA. Phototoxicity and cytotoxicity was site specific within the lesion and increased proportionately to fluence rates. ROS generation leads to the decrease of cytoproliferation and increased apoptosis and necrotic cell death, COLI, COLIII an HSP70 were found down-regualted. Ex vivo wound geometry, system of support and growth media were optimized in a human wound healing organ culture (WHOC). WHOCs treated with 5ALA-PDT (20 J/cm2), showed an advancing re-epithelialization tongue 3.5 folds longer, which were highly proliferative, showing increased CK14 and p16 levels. The neo-epidermis was fully differentiated and neo-collagen was present. PCNA, p16, COLI, COLIII, MMP3, MMP19 and alpha-SMA were significantly more expressed in the dermis. MALA/5ALA-PDT (40 J/cm2) applied to striae alba, fine line, hypertrophic and keloid scars ex vivo coused an increased of apoptosis while proliferation decreased, matrix components were found to be re-organised, both according to the severity of the scar. COLI and COLIII genetic expression decreased while MMP3 and tropoelastin increased significantly. However, no statistically significant difference was observed between 5ALA and MALA-PDT treatments. In conclusion, this thesis shows that cytotoxicity post-PDT in KD fibroblasts is dependent on the lesional site within the scar, a precursor of intracellular photosensitiser and fluence. PDT in wound healing ex vivo shows increased re-epithelialization and ECM reconstruction and remodelling. Finally, in dermal fibrosis morphological and cellular effects of the application of PDT correlate with the degree and severity of dermal fibrosis. In view of this, PDT may be ideal for treating abnormal skin scarring and improving human cutaneous wound healing.

M. Tech. / Phototobiomodulation (PBM), also known as low level laser therapy (LLLT) or photobiostimulation, is a non-invasive form of therapy that utilizes low intensity laser light or irradiation to provide healing. However, in order for healing to be successful certain laser parameters need to be taken into consideration i.e. fluence (dosage), wavelength and power density. Laser therapy has been used for various medical applications and fields. Multiple cytokines and growth factors are involved in wound healing including Interleukin (IL)-1, IL-6 and Tumour Necrosis Factor alpha (TNF- a). In diseased state(s) such as diabetes mellitus (DM) or psoriasis, these growth factors or cytokines are either found elevated or decreased depending on various factors and for abnormally prolonged periods. However, inflammatory cytokines are usually elevated. Phototherapy has been reported to accelerate wound healing, attenuate pain and cease inflammation. However, the effect of phototherapy on cytokine modulation has not been explored extensively, especially under various stress mechanisms. Furthermore, the pathway that laser irradiation induces on modulated pro-inflammatory cytokines has not been clearly elucidated as scientists typically report on the up- or down-regulated expression of cytokines. Numerous authors have reported on the efficacy of laser irradiation to enhance the rate of wound healing and proliferation in normal and diabetic cells or tissue; however, literature that has demonstrated the latter on hypoxic insulted cells is inadequate. In this study hypoxic insult was induced as it is one of the factors that usually prolong the healing process in diabetic wounds. Prior to commencing with the main study, a pilot study was done to exclude the effect of osmotic pressure on cells grown in media containing additional glucose, and thus simulating a diabetic model iv in vitro. Mannitol was used as a control since it is not absorbed by the cells. The study involved four groups namely: normal, normal wounded, mannitol wounded and diabetic wounded cells with each group having a non-irradiated control. Mannitol wounded and diabetic wounded cells had a final concentration of 30 mM mannitol and glucose respectively. A wavelength of 636 nm at a fluence of 5 J/cm2 was used on day 1; experiments were repeated four times and all tests were done in duplicate. Cellular responses (Trypan Blue, adenosine triphosphate (ATP) and lactate dehydrogenase (LDH)) and morphological changes were assessed after 1 h incubation post-irradiation in both irradiated and non-irradiated cultures.

Infection of thermal injuries by bacteria is a growing concern in the healthcare community, leading to increased rates of morbidity and mortality. P. aeruginosa, a rod-shaped, Gram-negative bacteria is one of the bacterial species most commonly found in infected burns. Detecting infections in burns is still a somewhat archaic process involving visual inspection, in which dressings have to be removed (also causing more pain and discomfort to patients) before samples are sent to a laboratory for analysis. Timely in situ detection systems, which limit disturbances to the wound area, could drastically improve patient comfort and healing outcomes. While established infections, with fully developed biofilms, are difficult to treat, loose bacteria early on in an infection and biofilm formation are more likely to fall easy prey to antibiotics, if the appropriate drugs are administered in a timely manner. In this thesis a smart wound management system, geared towards detecting and eliminating P. aeruginosa infections in burns is presented. Both non-functionalized general purpose electrodes, paired with an affordable open source potentiostat, for electrochemical analysis, and on demand drug releasing elements were developed by layering conductive materials onto everyday cotton threads. The sensing elements were thoroughly characterized with the detection of a P. aeruginosa biomarker over a range of physiologically relevant concentrations and conditions. The ability of the thread based sensors to detect live bacteria and be integrated in textile wound dressings was demonstrated. Controlled drug release was also demonstrated through the development of several drug release profiles. The presented technology has the potential to greatly improve patient outcomes in burn wards and provides a platform for tackling other infectious organisms with the further development of more thread based tools. / Graduate / 2021-08-25

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In humans, the epidermis is the main site for the synthesis of Vitamin D3 (cholecalciferol) from 7-dehydrocholesterol. Cholecalciferol undergoes further hydroxylation in the liver and kidney to produce the active form of the circulating hormone 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3). In target cells, 1,25(OH)2D3 interacts with the specific intracellular vitamin D receptor (VDR), a member of the nuclear receptor superfamily. However, epidermal keratinocytes, in addition to being target cells, have enzymes required for autocrine production of 1,25(OH)2D3. They can convert cholecalciferol to 1,25(OH)2D3 via 25-hydroxylase (CYP2R1) and 1α-hydroxylase (CYP27B1). Another enzyme, 24-hydroxylase (CYP24A1), regulates local levels by inactivating 1,25(OH)2D3. While recent studies have shown that absence of VDR or 1,25(OH)2D3 impairs formation of granulation tissue during wound healing in mice, little is known about the autocrine and paracrine regulation of biologically active vitamin D3 by human dermal fibroblasts during cutaneous wound healing. Primary cultures of human keratinocytes and fibroblasts expressed VDR and all the cytochrome enzymes necessary for autocrine production of vitamin D. The relative expression of VDR mRNA was higher in dermal fibroblasts than donor-matched keratinocytes. In contrast, epidermal keratinocytes had a higher mRNA expression of vitamin D3 metabolising enzymes. A scratch wound assay confirmed that 1,25(OH)2D3 stimulated keratinocyte migration, but paradoxically inhibited fibroblast migration as early as 4h, yet neither cholecalciferol nor 25-hydroxyvitamin D3 had any effect. VDR knockdown using small interfering RNA (siRNA) abolished the inhibitory effect of 1,25(OH)2D3 on fibroblast migration, demonstrating the requirement for the VDR in this response. Immunofluorescent staining revealed that 1,25(OH)2D3 increased nuclear VDR protein expression, without a corresponding increase in VDR mRNA transcription only in mechanically wounded dermal fibroblasts, indicating activation of the receptors. Incubation with either 1,25(OH)2D3, cholecalciferol or 25(OH)D3 up-regulated CYP24A1 transcription. This response was most pronounced with 1,25(OH)2D3, suggesting a tightly regulated feedback control on 1,25(OH)2D3 bioavailability within the dermis. In addition, cholecalciferol also increased CYP2R1 and CYP27B1 mRNA expression in scratched dermal fibroblasts, providing evidence for autocrine regulation of 1,25(OH)2D3 by dermal fibroblasts. Expression of α-SMA protein was up-regulated in cultured dermal fibroblasts following scratching, which was down-regulated in the presence of 1,25(OH)2D3. These observations suggest that 1,25(OH)2D3 may restrict differentiation of wounded dermal fibroblasts into pro-fibrotic myofibroblasts. 1,25(OH)2D3 also down-regulated MMP-2 secretion and collagen type I to III ratio in scratched dermal fibroblasts. Using a human ex vivo wound healing model, it was demonstrated that 1,25(OH)2D3, but not cholecalciferol, stimulated the rate of wound closure. In summary, this study has confirmed that human dermal fibroblasts express the transcriptional machinery for autocrine production of 1,25(OH)2D3, and a higher VDR expression suggests they are more responsive than keratinocytes. Changes in CYP and VDR expression in the presence of cholecalciferol, 25-hydroxyvitamin D3 or 1,25(OH)2D3 indicate fine-tuning of the bioavailability of vitamin D in the dermis after wounding. Down-regulation of α-SMA, MMP-2 secretion and the collagen type I to III ratio by 1,25(OH)2D3 highlight an important role for 1,25(OH)2D3 in modulating wound healing and the scarring process.

The purpose was to objectively measure the rate of healing of equine distal limb wounds when a 10% Natural Proprietary Compound (NPC) was compared to a topical antibacterial cream (1% silver sulfadiazine- SSD). Five horses had two wounds measuring 6.25cm2 created on the dorsomedial aspect of each limb. Two contralateral limbs were randomly chosen to be bandaged and the other two limbs were un-bandaged – with one limb of each group being treated with either NPC or SSD. On each limb the most proximal wound was left without topical treatment to act as a control. There was no significant difference between SSD and the compound evaluated in this study when either perimeter or area was assessed. Control wounds were significantly smaller than those treated with an ointment regardless of other variables, giving rise to a proposed location effect. Bandaging by day was significant for the time period approximating 2-5 weeks.