251 Photomedicine and Laser Surgery Volume 23, Number 3, 2005 © Mary Ann Liebert, Inc. Pp. 251–259 Biological Effects of Helium-Neon Laser Irradiation on Normal and Wounded Human Skin Fibroblasts D. HAWKINS and H. ABRAHAMSE ABSTRACT Objective: This study aimed to investigate a number of structural, cellular, and molecular responses to helium- neon (632.8 nm) laser irradiation following a single dose of 0.5, 2.5, 5, or 10 J/cm 2 on normal and wounded human skin fibroblasts. Background Data: Low-level laser therapy (LLLT) is a form of phototherapy, involv- ing the application of low-power monochromatic and coherent light to injuries and lesions to stimulate heal- ing. 1 This therapy has been successfully used for pain attenuation and to induce wound healing in nonhealing defects. 2 Methods: Changes in normal and wounded fibroblast cell morphology were evaluated by light mi- croscopy. Cellular parameters evaluated cell proliferation, cell viability, and cytotoxicity while molecular pa- rameters assessed the extent of DNA damage. Results: The results clearly demonstrate that LLLT has an effect on normal and wounded 3 human skin fibroblasts. The parameters showed that doses of 0.5, 2.5, 5, and 10 J/cm 2 were sufficient to produce measurable changes in fibroblast cells. Conclusion: A dose of 10 J/cm 2 ap- peared to produce a significant amount of cellular and molecular damage, which could be an important con- sideration for other therapies, such as photodynamic therapy. INTRODUCTION E NERGY DEPOSITED IN A TISSUE is the product of the power of the laser and the length of time that the beam is in contact with the tissue. The duration of exposure of the energy is an important variable in controlling desired and unwanted effects on tissue. 4 When a laser beam is irradiated onto tissue, a local or topical primary response occurs at the cellular level; this is followed to some degree over a greater area by a systemic sec- ondary response. Low-level laser therapy (LLLT) reactions are nonthermal since there is no immediate increase in the temper- ature of laser-irradiated tissue. 2 Experiments following LLLT have shown that immediate increase of heat in the target tissue is negligible (±1°C). As the law of Arndt-Schultz states, in a bi- ological system, weak stimuli will elicit strong reactions from the system, medium stimuli will cause moderate reactions, moderately strong stimuli will slightly inhibit the system, and very strong stimuli will completely retard it. 2 LLLT irradiation includes wavelengths from 500–1100 nm (visible to near infrared region of the spectrum). This type of radiation is a continuous wave of a constant beam of relatively low power or irradiance (0.04–50 J/cm 2 ), and the laser is di- rected at the target tissue using powers measured in milliwatts (mW). 2 A typical example of a laser used in LLLT is the he- lium-neon (He-Ne) laser, which emits red light at a wavelength of 632.8 nm. 5 Red light does not penetrate very effectively below the skin surface, but it is an excellent source of stimula- tion on a range of growth factors and is effective for wound healing or superficial condition.s 5 He-Ne laser light can penetrate as deep as 0.5 mm into freshly excised human skin and delivers the highest relative percentage of incident energy to a certain volume of tissue. A penetration depth of even a few microns can be regarded as sufficient, because most of the relevant target cells for the in- duction of wound healing by LLLT—namely, fibroblasts, ker- atinocytes, macrophages, and endothelial cells—are located within the epidermis and upper dermis. 4 The biostimulating effect of LLLT results in an increase in microcirculation, higher rates of ATP, RNA, and DNAsynthe- sis, and thus improved tissue oxygenation, nutrition, and re- generation. 6 The increased activity is generally followed by a period of decreased activity, usually after several irradiations. 2 Photons enter the tissue and are readily absorbed in the mito- chondria and at the cell membrane. The photonic energy is converted to chemical energy within the cell, in the form of ATP, which leads to normalization of cell function, pain relief, Faculty of Health Sciences, Technikon Witwatersrand, Doornfontein, Johannesburg, South Africa.