An investigation into the cellular mechanisms underlying photodynamic rejuvenation in human skin

Includes abstract.Includes bibliographical references.Photodynamic Rejuvenation (PDR) is a novel therapy used to treat the signs of skin ageing. It is a promising dermatological therapy due to its less severe side effects and superior results when compared to other chemical treatments. This therapy involves the topical application of a photosensitizing drug (PS) which is activated by a specific wavelength of light to react with oxygen and generate reactive oxygen species (ROS) in the skin. At low levels ROS are able to alter cell signalling and are thought to be the key mediators that reverse the signs of ageing. Dermatologists use this therapy to treat various characteristics of skin ageing such as fine/coarse wrinkles, mottled pigmentation, skin roughness and telangiectasia (small broken blood vessels near the surface of the skin). Initial clinical reports showed success; however, inconsistencies in patient outcomes provide impetus to improve characteristics of current treatment regimes including the PS, light sources, fluences and irradiances. As very little is known about the actual biological mechanism of PDR in human skin cells, the aim of this investigation was to first optimise a protocol using the PS, hypericin, activated with 3 different light sources. Hypericin, an extract from St Johns Wort, is a second generation PS that has many benefits such as low dark cytotoxicity, no carcinogenicity/mutagenicity, high quantum yield and can be activated by several wavelengths of light. We chose three light sources that emitted light within hypericin’s absorbance spectra: two lasers emitted light at 561nm and 632nm and lamps in a UVA transilluminator emitted a light range with a peak at 365nm. Cultured primary human fibroblasts were chosen as the cell model as they are an ideal representation of the dermal layer of the skin. Our results showed that low hypericin concentrations (0.25-0.5μM) at all three wavelengths caused an increase in cell viability. When this increase was investigated in relation to growth or cellular activity, growth curves showed that PDR with all 3 wavelengths had no effect on the cell proliferation rate. To confirm whether ROS was indeed occurring after the therapy, a ROS assay was performed. The yellow laser and UVA transilluminator, which emit light maximally absorbed by hypericin, were used. UVA served as the upper limit for ROS generation as this range of wavelengths is known to cause intracellular ROS. Yellow laseractivated hypericin resulted in a non-significant increase in intracellular ROS which was less than the levels in fibroblasts with UVA activated hypericin. This confirms that PDR using hypericin does generate ROS. As migration is considered inverse to collagen production and increased collagen is a main objective of skin rejuvenation, we studied fibroblast migration after PDR. To assess fibroblast migration in response to yellow laser light activation of hypericin, a scratch assay was used. This PDR protocol showed that migration was significantly slowed after treatment. Our proposal is that our PDR protocol with yellow laser light decreases migration diverting energy to producing collagen