Background/purpose: Stress induced premature senescence (SIPS) is defined as the long-term effect of subcytotoxic
stress on proliferative cell types. Cells in SIPS display differences at the level of protein expression which
affect energy metabolism, defense systems, redox potential, cell morphology and transduction pathways. This study
aimed to determine the effect of laser irradiation on second messengers in senescent cells and to establish if that
effect can be directly linked to changes in cellular function such as cell viability or proliferation. Materials and
Methods: Human keratinocyte cell cultures were modified to induce premature senescence using repeated sub-lethal
stresses of 200 uM H<sub>2</sub>O<sub>2</sub> or 5% OH every day for four days with two days recovery. SIPS was confirmed by
senescence-associated β-galactosidase staining. Control conditions included normal, repeated stress of 500 uM H<sub>2</sub>O<sub>2</sub>
to induce apoptosis and 200 uM PBN as an anti-oxidant or free radical scavenger. Cells were irradiated with
1.5 J/cm<sup>2</sup> on day 1 and 4 using a 648 nm diode laser (3.3 mW/cm<sup>2</sup>) and cellular responses were measured 1 h post
irradiation. The affect on second messengers was assessed by measuring cAMP, cGMP, nitric oxide and intracellular
calcium (Ca<sup>2+</sup>) while functional changes were assessed using cell morphology, ATP cell viability, LDH membrane
integrity and WST-1 cell proliferation. Results: Results indicate an increase in NO and a decrease in cGMP and
Ca<sup>2+</sup> in 200 uM H2O2 irradiated cells while PBN irradiated cells showed a decrease in cAMP and an increase in ATP
viability and cell proliferation. Conclusion: Laser irradiation influences cell signaling which ultimately changes the
biological function of senescent cells. If laser therapy can stimulate the biological function of senescent cells it may
be beneficial to conditions such as immune senescence, skin ageing, muscle atrophy, premature ageing of arteries in
patients with advanced heart disease, neurodegenerative disorders and chronic renal failure.
Phototherapy has become more popular and widely used in the treatment of a variety of medical conditions. To
ensure sound results as evidence of its effectiveness, well designed experiments must be conducted when
determining the effect of phototherapy. Cell culture models such as hypoxic, acidotic and wounded cell cultures
simulating different disease conditions including ischemic heart disease, diabetes and wound healing were used to
determine the effect of laser irradiation on the genetic integrity of the cell.
Even though phototherapy has been found to be beneficial in a wide spectrum of conditions, it has been shown to
induce DNA damage. However, this damage appears to be repairable. The risk lies in the fact that phototherapy
may help the medical condition initially but damage DNA at the same time leaving undetected damage that may
result in late onset, more severe, induced medical conditions including cancer.
Human skin fibroblasts were cultured and used to induce a wound (by the central scratch model), hypoxic (by
incubation in an anaerobic jar, 95% N<sub>2</sub> and 5% O<sub>2</sub>) and acidotic (reducing the pH of the media to 6.7) conditions.
Different models were irradiated using a Helium-Neon (632.8 nm) laser with a power density of 2.07 mW/cm2 and a
fluence of 5 J/cm<sup>2</sup> or 16 J/cm<sup>2</sup>. The effect of the irradiation was determined using the Comet assay 1 and 24 h after
irradiation. In addition, the Comet assay was performed with the addition of formamidopyrimidine glycosylase
(FPG) obviating strand brakes in oxidized bases at a high fluence of 16 J/cm<sup>2</sup>.
A significant increase in DNA damage was seen in all three injured models at both 1 and 24 h post-irradiation when
compared to the normal un-injured cells. However, when compared to non-irradiated controls the acidotic model
showed a significant decrease in DNA damage 24 h after irradiation indicating the possible induction of cellular
DNA repair mechanisms. When wounded cells were irradiated with higher fluences of 16 J/cm<sup>2</sup>, there was a
significant increase in DNA damage in irradiated cells with and without the addition of FPG. These results are
indicative of the importance of both cell injury model as well as fluence when assessing the effect of phototherapy
on DNA integrity.
<b>Background/purpose</b>: In vivo studies have demonstrated that phototherapy accelerates wound healing in the clinical
environment; however the exact mechanism is still not completely understood. The main focus of this study was to use
in vitro laboratory results to establish an effective treatment regimen that may be practical and applicable to the clinical
environment. This in vitro study aimed to compare the cellular responses of wounded fibroblasts following a single
exposure of 5 J/cm<sup>2</sup> or multiple exposures of low doses (2.5 J/cm<sup>2</sup> or 5 J/cm<sup>2</sup>) on one day of the week to a single
application of a higher dose (16 J/cm<sup>2</sup>) on day 1 and day 4. <b>Methodology</b>: Cellular responses to Helium-Neon
(632.8 nm) laser irradiation were evaluated by measuring changes in cell morphology, cell viability, cell proliferation,
membrane integrity and DNA damage. <b>Results</b>: Wounded cells exposed to 5 J/cm<sup>2</sup> on day 1 and day 4 showed an
increase in cell viability, increase in the release of bFGF, increase in cell density, decrease in ALP enzyme activity and
decrease in caspase 3/7 activity indicating a stimulatory effect. Wounded cells exposed to three doses of 5 J/cm<sup>2</sup> on day
1 showed a decrease in cell viability and cell proliferation and an increase in LDH cytotoxicity and DNA damage
indicating an inhibitory effect. <b>Conclusion</b>: Results indicate that cellular responses are influenced by the combination of
dose administered, number of exposures and time between exposures. Single doses administered with sufficient time
between exposures is more beneficial to restoring cell function than multiple doses within a short period. Although this
work confirms previous reports on the cumulative effect of laser irradiation it provides essential information for the
initiation of in vivo clinical studies.
An alternative treatment modality for diabetic wound healing includes low level laser therapy (LLLT). Biostimulation of such wounds may be of benefit to patients by reducing healing time. Structural, cellular and genetic events in diabetic wounded human skin fibroblasts (WS1) were evaluated after exposing cells in culture to a Helium-Neon (632.8nm), a Diode laser (830nm) and a Nd:YAG (Neodynium:Yttrium-Allumina-Gallium) laser (1064nm) at either 5J/cm<sup>2</sup> or 16J/cm<sup>2</sup>. Cells were exposed twice a week and left 24 hours post-irradiation prior to measuring effects. Structural changes were evaluated by assessing colony formation, haptotaxis and chemotaxis. Cellular changes were evaluated using cell viability, (adenosine-triphosphate, ATP production), and proliferation, (alkaline phosphatase, ALP and basic fibroblast growth factor, bFGF expression), while the Comet assay evaluated DNA damage and cytotoxicity was determined assessing membrane permeability for lactate dehydrogenase (LDH). Caspase 3/7 activity was used as an estimate of apoptosis as a result of irradiation. The irradiated diabetic wounded cells showed structural, cellular as well as molecular resilience comparable to that of unwounded normal skin fibroblast cells. With regards to fluence, 5J/cm<sup>2</sup> elicit positive cellular and structural responses while 16J/cm<sup>2</sup> increases cellular and genetic damage and cellular morphology is altered. Different wavelengths of LLLT influences the beneficial outcomes of diabetic wounded cells and although all three wavelengths elicit cellular effects, the penetration depth of 830nm plays a significant role in the healing of diabetic wounded human fibroblast cells. Results from this study validate the contribution of LLLT to wound healing and elucidate the biochemical effects at a cellular level while highlighting the role of different dosages and wavelengths in LLLT.
A variety of strategies have been utilised for prevention and treatment of chronic wounds such as leg ulcers, diabetic foot ulcers and pressure sores<sup>1</sup>. Low Level Laser Therapy (LLLT) has been reported to be an invaluable tool in the enhancement of wound healing through stimulating cell proliferation, accelerating collagen synthesis and increasing ATP synthesis in mitochondria to name but a few<sup>2</sup>. This study focused on an in-vitro analysis of the cellular responses induced by treatment with three different laser beam profiles namely, the Gaussian (G), Super Gaussian (SG) and
Truncated Gaussian (TG), on normal wounded irradiated (WI) and wounded non-irradiated (WNI) human skin fibroblast cells (WS1), to test their influence in wound healing at 632.8 nm using a helium neon (HeNe) laser. For each beam profile, measurements were made using average energy densities over the sample ranging from 0.2 to 1 J, with
single exposures on normal wounded cells. The cells were subjected to different post irradiation incubation periods, ranging from 0 to 24 hours to evaluate the duration (time) dependent effects resulting from laser irradiation. The promoted cellular alterations were measured by increase in cell viability, cell proliferation and cytotoxicity. The results obtained showed that treatment with the G compared to the SG and TG beams resulted in a marked increase in cell
viability and proliferation. The data also showed that when cells undergo laser irradiation some cellular processes are driven by the peak energy density rather than the energy of the laser beam. We show that there exist threshold values for damage, and suggest optimal operating regimes for laser based wound healing.