Light-Emitting Diode–Generated Red Light Inhibits Keloid Fibroblast Proliferation Andrew Mamalis, BS, MS,*† and Jared Jagdeo, MD, MS*†‡
BACKGROUND Red light is part of the visible light spectrum that does not generate DNA adducts associated with skin cancer and photoaging and may represent a safer therapeutic modality for treatment of keloid scars and other fibrotic skin diseases. Our laboratory previously demonstrated that light-emitting diode–generated red light (LED-RL) inhibits proliferation of skin fibroblasts. The effects of LED-RL on keloidal skin are not well characterized. OBJECTIVE
To determine the effect of LED-RL on keloid-derived fibroblast proliferation and viability in vitro.
METHODS Irradiation of primary keloid–derived human skin fibroblasts using LED-RL panels was performed in vitro, and modulation of proliferation and viability was quantified using trypan blue dye exclusion assay. Statistical analysis was performed using analysis of variance to compare treatment arms and the Student t-test to compare each treatment arm with the paired bench control arm. RESULTS Keloid fibroblasts treated with LED-RL 240, 320, and 480 J/cm2 demonstrated statistically significant dose-dependent decreases in relative proliferation rate of 12.4%, 16.5%, and 28.9%, respectively, compared with matched nonirradiated controls (p < .05) and did not significantly alter viability relative to the matched nonirradiated controls. CONCLUSION Light-emitting diode–generated red light can inhibit keloid fibroblast proliferation in a dosedependent manner without altering viability. Light-emitting diode–generated red light has the potential to contribute to the treatment of keloids and other fibrotic skin diseases and is worthy of further translational and clinical investigation. The authors have indicated no significant interest with commercial supporters.
T
he biological effects and clinical uses of visible light are not well characterized. Although ultraviolet (UV) light is used to treat skin diseases, it causes DNA adducts that are associated with increased rates of skin cancers and premature photoaging.1,2 Light in the visible spectrum (400–760 nm) does not cause the deleterious effects of UV and therefore presents a potentially safer treatment modality.3 Noncoherent light-emitting diode–generated red light (LED-RL) (633 6 15 nm) is part of the visible light spectrum. Currently, LED phototherapy is clinically used in
dermatology primarily for photorejuvenation and acne and likely has potential for treatment of other skin diseases.4,5 Visible light phototherapy is mechanistically based on the photobiomodulatory effects caused by light in this spectrum. Light-emitting diode–generated red light phototherapy is hypothesized to function through photostimulation of the heme–copper group within the cytochrome C component of the electron transport chain that results in mitochondrial upregulation.6 In addition, LED phototherapy has been shown
*Department of Dermatology, University of California at Davis, Sacramento, California; †Dermatology Service, Sacramento VA Medical Center, Mather, California; ‡Department of Dermatology, SUNY Downstate Medical Center, Brooklyn, New York Photo Therapeutics provided the device used in this study.
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© 2014 by the American Society for Dermatologic Surgery, Inc. Published by Lippincott Williams & Wilkins ISSN: 1076-0512 Dermatol Surg 2015;41:35–39 DOI: 10.1097/01.DSS.0000452650.06765.51
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·
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Copyright © American Society for Dermatologic Surgery. Unauthorized reproduction of this article is prohibited.
LED-RL INHIBITION OF KELOID FIBROBLAST PROLIFERATION
to lead to downstream protein transcriptional changes leading to modulation in the levels of cytokines, growth factors, inflammatory mediators, and reactive oxygen species generation.6–8 Keloid scars, also termed keloids, are a significant clinical burden worldwide that result from an overgrowth of fibrotic tissue outside the original boundaries of an injury and occur secondary to defective wound healing.9–11 Keloids often lead to a functional, aesthetic, or psychosocial impact on patients as highlighted by quality-of-life studies.12 Treatment of keloids is challenging and even with combination therapy, recurrence is common.9 Advances in laser and light-based technology have introduced new ways to manage keloids that may result in improved aesthetic and symptomatic outcomes and decreased keloid recurrence.13 Based on our previously published data demonstrating that LED-RL decreases the proliferation rate of normal human dermal fibroblasts, we hypothesized that LEDRL would be capable of decreasing the proliferation rate of human keloid-derived fibroblast cells and investigated the same doses (240, 320, and 480 J/cm2) in this study that were shown to inhibit normal fibroblast proliferation without affecting viability. We believe that LED-RL phototherapy has the potential to be used as a noninvasive treatment for keloid scars. Because of the safety, portability, and cost-effectiveness of LEDs, LED-RL phototherapy holds potential benefits over other modalities used to treat keloids such as surgery, intralesional steroids, and ablative and nonablative laser treatment. Furthermore, LED-RL can be used in combination as adjunctive therapy with other devices and drugs used to treat keloids. The exact pathogenesis of keloids has not been elucidated, and the lack of adequate animal models that recapitulate the properties of keloids has hindered keloid research.14 It has been hypothesized that animals do not form keloids and other scars because of the lack of sebaceous glands.15 Some animal scar models exist that exploit the presence of pseudosebaceous glands, such as the rabbit ear hypertrophic scarring model.16 Few animal models of keloids exist, such as the xenograft model and the poly-L-lactic acid
36
scaffold model.17–19 However, in vitro cultures of keloid-derived fibroblasts demonstrate increased proliferation, increased collagen production, increased migration speed, differential apoptosis, and increased production of growth factors.20–23 Because of the lack of an adequate animal model, we opted to study the effects of LED-RL using in vitro cultures of keloid-derived fibroblasts, as in vitro keloid-derived fibroblasts have been shown to maintain their phenotype in culture.20–23 We measured proliferation rate and viability changes as in vitro surrogates to keloid growth and therapeutic toxicity. The study of keloidderived fibroblast modulation, function, proliferation, and other cellular properties represents our laboratory’s current effort to increase understanding of the photobiomodulatory effects of LED-RL on keloid-derived fibroblast cultures in vitro. This research builds on our previously reported findings that LED-RL can decrease normal human skin fibroblast proliferation in vitro.24,25 Herein, we report the effects of LED-RL on keloid-derived human skin fibroblast proliferation and viability in vitro. Methods Briefly, monolayers of primary keloid–derived human skin fibroblasts, previously isolated from excised patient keloid tissue under an institutional review board–approved protocol, were cultured in Dulbecco modified Eagle medium (Gibco–Invitrogen, Carlsbad, CA) with 10% bovine calf serum (Gibco) and 1% penicillin, streptomycin, and neomycin antibiotic mixture (Gibco). Cultured keloid-derived fibroblasts have been shown to maintain their phenotype in culture.20 The cell cultures were maintained in incubators at 37C with 5% carbon dioxide. Fibroblasts seeded at 2 · 104 cells per 35-mm dish were irradiated 24 hours after seeding, using a 633-nm LED array (Omnilux New-U; Photo Therapeutics, Carlsbad, CA) at a power density of 872.6 W/m2 at room temperature. Media temperatures measured throughout irradiation remained constant at 32 to 34C. By remaining within physiologic temperatures, heat stress was not induced. After 48 hours of additional incubation to allow the cells to proliferate, the cells were harvested, counted, and assessed for viability according to trypan blue exclusion assay as
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MAMALIS AND JAGDEO
previously described.19 Briefly, the number of cells in suspension is measured by sampling a known volume (10 mL) and measuring the cellular concentration with a hemocytometer examined using light microscopy. Dead cells have permeable cell membranes allowing uptake of the trypan blue dye (seen as blue cells using light microscopy). Therefore, the trypan blue dye allows clear discrimination of live from dead cells because the dye is excluded from live cells with intact cell membranes. Viability can then be calculated based on the ratio of live (total minus dead) to total cells. All experiments were repeated to verify data reproducibility and accuracy, and similar trends were demonstrated in a second keloid-derived human skin fibroblast culture isolated from a different patient (data not shown). Each experimental plate receiving LED-RL treatment was randomly matched with a bench control plate (BCP) to ensure that the measured effect was a result of LED-RL treatment and not because of ambient light or environment. Bench control plates were derived from the same stock of cell suspension, taken out of the incubator at the same time as their matched treatment pairs, and left on the bench for the same amount of time and in the same ambient environment as the treatment plates. Bench control plates were protected from the LED-RL source, placed on a digital warming block, and media temperatures measured throughout time on the bench remained 32 to 34C. Treatment plates and BCPs were then returned to the incubator and processed according to the same protocol. Cell counts included all cells in the sample, including media change, trypsinization products, and washes. Mean percentage proliferation and viability (live cells/total [live + dead] cells) relative to nonirradiated controls are reported as means and standard errors of the mean. Statistical analysis was performed using analysis of variance to compare treatment arms and the Student t-test to compare each treatment arm with the paired bench control arm.
Results Our experimental results demonstrate that treatment of primary keloid–derived human skin fibroblasts
using LED-RL (633 nm) inhibits proliferation in a dose-dependent manner without causing significant effect on viability at fluences of 240, 320, and 480 J/cm2 (Figures 1 and 2). Statistically significant decreases in cell proliferation are noted at the following fluences (time): 240 (45 minutes, 50 seconds), 320 (61 minutes, 7 seconds), and 480 J/cm2 (91 minutes, 41 seconds) (Figure 1). Plates treated with 240, 320, and 480 J/cm2 all demonstrated a statistically significant decrease in relative proliferation rate of 12.4%, 16.5%, and 28.9%, respectively, compared with matched BCPs (p < .05). Treatment at the 240 (98.8% 6 3.8%), 320 (100.4% 6 2.2%), and 480 J/cm2 (100.0% 6 2.6%) doses did not significantly alter viability relative to the nonirradiated controls (Figure 2).
Discussion Our data demonstrate that LED-RL can decrease the proliferation of keloid-derived fibroblasts in vitro in a dose-dependent manner (Figure 1). Treatment with fluences of 240, 320, and 480 J/cm2 resulted in statistically significant decreases in cell number without a significant decrease in viability as compared with BCPs (Figure 2). We recently reported similar findings using LED-generated infrared and red light to treat normal human skin fibroblasts.24,25 To our knowledge, this is the first report of the antiproliferative effect of LED-RL on keloid-derived skin fibroblasts in vitro. We believe that our findings serve as “proof of principle” and may establish the foundation for the translation of these “bench” findings to clinical studies to determine the antifibrotic effects of LED-RL for the treatment of keloids and other cutaneous scarring conditions. Attention to bias is necessary when attempting to demonstrate light-induced effects on human skin fibroblasts. Specifically, the observed effects may be attributed to variation in proliferation patterns between the experimental and control plates. The effects may also be attributed to various environmental factors other than the light energy that the LED device generated. We controlled for environmental conditions by using bench controls, monitored to confirm matching
41:1:JANUARY 2015
37
Copyright © American Society for Dermatologic Surgery. Unauthorized reproduction of this article is prohibited.
LED-RL INHIBITION OF KELOID FIBROBLAST PROLIFERATION
Figure 1. Light-emitting diode–generated red light (633 nm) decreases relative cell proliferation in a dose-dependent manner in vitro. A statistically significant inhibition of proliferation was seen at 240 J/cm2 = 87.6% 6 3.8%, p = .015; 320 J/cm2 = 83.5% 6 2.2%, p = .003; and 480 J/cm2 = 71.1% 6 8.0%, p = .007. Nonirradiated cells, 0 J/cm2 = 100.0% 6 7.6%. Forty-eight hours after irradiation, cells were assessed by trypan blue exclusion assay. Proliferation is presented as percent of the matched BCPs. Error bars represent standard error of the mean. *Statistical significance, p < .05.
temperatures, and repeated all experiments 3 times to account for variation in cellular proliferation patterns. Further studies are needed to demonstrate these findings in vivo.
Light-emitting diode–generated red light phototherapy may represent a safe and cost-effective modality to improve patient outcomes with keloids or other fibrotic skin disorders and therefore warrants further study.
Figure 2. In keloid fibroblasts treated with LED-generated red light (633 nm) cellular viability is maintained in vitro at fluences of 0 J/cm2 = 100.0% 6 2.6%, 240 J/cm2 = 98.8% 6 3.8%, 320 J/cm2 = 100.4% 6 2.2%, and 480 J/cm2 = 100.0% 6 2.6%. Forty-eight hours after irradiation, cells were assessed by trypan blue assay. Relative viability is presented as percent of the control. Error bars represent standard error of the mean. No statistically significant differences were found between these groups when comparing viability.
38
DERMATOLOGIC SURGERY
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MAMALIS AND JAGDEO
Further in vitro studies are needed to elucidate the mechanism underlying LED-RL phototherapy and to investigate its efficacy to mitigate key pathogenic processes related to skin fibrosis, such as collagen production. Given that LED-RL phototherapy decreases keloid-derived fibroblast proliferation without decreasing cellular viability, we hypothesize that LEDRL phototherapy may mechanistically decrease proliferation rate through cell cycle modulation. Future studies are aimed at investigating the effects of LED-RL on cell cycle modulation and other pathways. In addition to decreasing proliferation, we propose that LED-RL phototherapy may be able to modulate additional keloid-derived fibroblasts phenotypic differences associated with skin fibrosis. These data may serve as the in vitro foundation for the investigation of LED-RL phototherapy for the treatment of keloids and other forms of skin scarring and fibrosis. Keloid scarring and other forms of skin fibrosis occur in the dermis, and it is well established that red light penetrates into the mid-to-deep dermis.21 Therefore, we hypothesize that our findings will translate to clinical therapy of keloid and other forms of fibrotic disease. Future research will focus on establishing dosing regimens, elucidation of mechanism of action through interrogation of the transforming growth factor-beta pathway and molecular signaling pathways linked to skin fibrosis, and translation of findings to clinical studies to improve the lives of patients with cosmetically and functionally impairing keloids and other skin scarring conditions.
References 1. Uitto J. The role of elastin and collagen in cutaneous aging: intrinsic aging versus photoexposure. J Drugs Dermatol 2008;7:s12–6.
7. Kolbe L. How much sun protection is needed? Are we on the way to fullspectrum protection? J Invest Dermatol 2012;132:1756–7. 8. Mahmoud BH, Ruvolo E, Hexsel CL, Liu Y, et al. Impact of longwavelength UVA and visible light on melanocompetent skin. J Invest Dermatol 2010;130:2092–7. 9. Bayat A, McGrouther DA, Ferguson MW. Skin scarring. BMJ 2003; 326:88–92. 10. Yang YC, Cheng YW, Lai CS, Chen W. Prevalence of childhood acne, ephelides, warts, atopic dermatitis, psoriasis, alopecia areata and keloid in Kaohsiung County, Taiwan: a community-based clinical survey. J Eur Acad Dermatol Venereol 2007;21:643–9. 11. Chen GY, Cheng YW, Wang CY, Hsu TJ, et al. Prevalence of skin diseases among schoolchildren in Magong, Penghu, Taiwan: a community-based clinical survey. J Formos Med Assoc 2008;107:21–9. 12. Bock O, Schmid-Ott G, Malewski P, Mrowietz U. Quality of life of patients with keloid and hypertrophic scarring. Arch Dermatol Res 2006;297:433–8. 13. Mamalis AD, Lev-Tov H, Nguyen DH, Jagdeo JR. Laser and light-based treatment of keloids—a review. J Eur Acad Dermatol Venereol 2013. 14. van den Broek LJ, Niessen FB, Scheper RJ, Gibbs S. Development, validation and testing of a human tissue engineered hypertrophic scar model. ALTEX 2012;29:389–402. 15. Al-Attar A, Mess S, Thomassen JM, Kauffman CL, et al. Keloid pathogenesis and treatment. Plast Reconstr Surg 2006;117:286–300. 16. Ramos ML, Gragnani A, Ferreira LM. Is there an ideal animal model to study hypertrophic scarring? J Burn Care Res 2008;29:363–8. 17. Chung HJ, Steplewski A, Chung KY, Uitto J, et al. Collagen fibril formation. A new target to limit fibrosis. J Biol Chem 2008;283:25879–86. 18. Shetlar MR, Shetlar CL, Kischer CW, Pindur J. Implants of keloid and hypertrophic scars into the athymic nude mouse: changes in the glycosaminoglycans of the implants. Connect Tissue Res 1991;26:23–36. 19. Yagi Y, Muroga E, Naitoh M, Isogai Z, et al. An ex vivo model employing keloid-derived cell-seeded collagen sponges for therapy development. J Invest Dermatol 2013;133:386–93. 20. Fujiwara M, Muragaki Y, Ooshima A. Keloid-derived fibroblasts show increased secretion of factors involved in collagen turnover and depend on matrix metalloproteinase for migration. Br J Dermatol 2005;153:295–300. 21. Koch RJ, Goode RL, Simpson GT. Serum-free keloid fibroblast cell culture: an in vitro model for the study of aberrant wound healing. Plast Reconstr Surg 1997;99:1094–8. 22. Syed F, Ahmadi E, Iqbal SA, Singh S, et al. Fibroblasts from the growing margin of keloid scars produce higher levels of collagen I and III compared with intralesional and extralesional sites: clinical implications for lesional site-directed therapy. Br J Dermatol 2011;164:83–96.
2. Marrot L, Meunier JR. Skin DNA photodamage and its biological consequences. J Am Acad Dermatol 2008;58:S139–48.
23. Wu CS, Wu PH, Fang AH, Lan CC. FK506 inhibits the enhancing effects of transforming growth factor (TGF)-b1 on collagen expression and TGF-b/Smad signalling in keloid fibroblasts: implication for new therapeutic approach. Br J Dermatol 2012;167:532–41.
3. Liebel F, Kaur S, Ruvolo E, Kollias N, et al. Irradiation of skin with visible light induces reactive oxygen species and matrix-degrading enzymes. J Invest Dermatol 2012;132:1901–7.
24. Lev-Tov H, Brody N, Siegel D, Jagdeo J. Inhibition of fibroblast proliferation in vitro using low-level infrared light-emitting diodes. Dermatol Surg 2013;39:422–5.
4. Morton CA, Scholefield RD, Whitehurst C, Birch J. An open study to determine the efficacy of blue light in the treatment of mild to moderate acne. J Dermatolog Treat 2005;16:219–23.
25. Lev-Tov H, Mamalis A, Brody N, Siegel D, et al. Inhibition of fibroblast proliferation in vitro using red light-emitting diodes. Dermatol Surg 2013;39: 1167–70.
5. Russell BA, Kellett N, Reilly LR. A study to determine the efficacy of combination LED light therapy (633 nm and 830 nm) in facial skin rejuvenation. J Cosmet Laser Ther 2005;7:196–200. 6. Huang YY, Sharma SK, Carroll J, Hamblin MR. Biphasic dose response in low level light therapy—an update. Dose Response 2011;9:602–18.
Address correspondence and reprint requests to: Jared Jagdeo, MD, MS, 3301 C Street, Sacramento, CA 95816; or e-mail: [email protected]
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Copyright © American Society for Dermatologic Surgery. Unauthorized reproduction of this article is prohibited.
BACKGROUND Red light is part of the visible light spectrum that does not generate DNA adducts associated with skin cancer and photoaging and may represent a safer therapeutic modality for treatment of keloid scars and other fibrotic skin diseases. Our laboratory previously demonstrated that light-emitting diode–generated red light (LED-RL) inhibits proliferation of skin fibroblasts. The effects of LED-RL on keloidal skin are not well characterized. OBJECTIVE
To determine the effect of LED-RL on keloid-derived fibroblast proliferation and viability in vitro.
METHODS Irradiation of primary keloid–derived human skin fibroblasts using LED-RL panels was performed in vitro, and modulation of proliferation and viability was quantified using trypan blue dye exclusion assay. Statistical analysis was performed using analysis of variance to compare treatment arms and the Student t-test to compare each treatment arm with the paired bench control arm. RESULTS Keloid fibroblasts treated with LED-RL 240, 320, and 480 J/cm2 demonstrated statistically significant dose-dependent decreases in relative proliferation rate of 12.4%, 16.5%, and 28.9%, respectively, compared with matched nonirradiated controls (p < .05) and did not significantly alter viability relative to the matched nonirradiated controls. CONCLUSION Light-emitting diode–generated red light can inhibit keloid fibroblast proliferation in a dosedependent manner without altering viability. Light-emitting diode–generated red light has the potential to contribute to the treatment of keloids and other fibrotic skin diseases and is worthy of further translational and clinical investigation. The authors have indicated no significant interest with commercial supporters.
T
he biological effects and clinical uses of visible light are not well characterized. Although ultraviolet (UV) light is used to treat skin diseases, it causes DNA adducts that are associated with increased rates of skin cancers and premature photoaging.1,2 Light in the visible spectrum (400–760 nm) does not cause the deleterious effects of UV and therefore presents a potentially safer treatment modality.3 Noncoherent light-emitting diode–generated red light (LED-RL) (633 6 15 nm) is part of the visible light spectrum. Currently, LED phototherapy is clinically used in
dermatology primarily for photorejuvenation and acne and likely has potential for treatment of other skin diseases.4,5 Visible light phototherapy is mechanistically based on the photobiomodulatory effects caused by light in this spectrum. Light-emitting diode–generated red light phototherapy is hypothesized to function through photostimulation of the heme–copper group within the cytochrome C component of the electron transport chain that results in mitochondrial upregulation.6 In addition, LED phototherapy has been shown
*Department of Dermatology, University of California at Davis, Sacramento, California; †Dermatology Service, Sacramento VA Medical Center, Mather, California; ‡Department of Dermatology, SUNY Downstate Medical Center, Brooklyn, New York Photo Therapeutics provided the device used in this study.
·
© 2014 by the American Society for Dermatologic Surgery, Inc. Published by Lippincott Williams & Wilkins ISSN: 1076-0512 Dermatol Surg 2015;41:35–39 DOI: 10.1097/01.DSS.0000452650.06765.51
·
·
· 35
Copyright © American Society for Dermatologic Surgery. Unauthorized reproduction of this article is prohibited.
LED-RL INHIBITION OF KELOID FIBROBLAST PROLIFERATION
to lead to downstream protein transcriptional changes leading to modulation in the levels of cytokines, growth factors, inflammatory mediators, and reactive oxygen species generation.6–8 Keloid scars, also termed keloids, are a significant clinical burden worldwide that result from an overgrowth of fibrotic tissue outside the original boundaries of an injury and occur secondary to defective wound healing.9–11 Keloids often lead to a functional, aesthetic, or psychosocial impact on patients as highlighted by quality-of-life studies.12 Treatment of keloids is challenging and even with combination therapy, recurrence is common.9 Advances in laser and light-based technology have introduced new ways to manage keloids that may result in improved aesthetic and symptomatic outcomes and decreased keloid recurrence.13 Based on our previously published data demonstrating that LED-RL decreases the proliferation rate of normal human dermal fibroblasts, we hypothesized that LEDRL would be capable of decreasing the proliferation rate of human keloid-derived fibroblast cells and investigated the same doses (240, 320, and 480 J/cm2) in this study that were shown to inhibit normal fibroblast proliferation without affecting viability. We believe that LED-RL phototherapy has the potential to be used as a noninvasive treatment for keloid scars. Because of the safety, portability, and cost-effectiveness of LEDs, LED-RL phototherapy holds potential benefits over other modalities used to treat keloids such as surgery, intralesional steroids, and ablative and nonablative laser treatment. Furthermore, LED-RL can be used in combination as adjunctive therapy with other devices and drugs used to treat keloids. The exact pathogenesis of keloids has not been elucidated, and the lack of adequate animal models that recapitulate the properties of keloids has hindered keloid research.14 It has been hypothesized that animals do not form keloids and other scars because of the lack of sebaceous glands.15 Some animal scar models exist that exploit the presence of pseudosebaceous glands, such as the rabbit ear hypertrophic scarring model.16 Few animal models of keloids exist, such as the xenograft model and the poly-L-lactic acid
36
scaffold model.17–19 However, in vitro cultures of keloid-derived fibroblasts demonstrate increased proliferation, increased collagen production, increased migration speed, differential apoptosis, and increased production of growth factors.20–23 Because of the lack of an adequate animal model, we opted to study the effects of LED-RL using in vitro cultures of keloid-derived fibroblasts, as in vitro keloid-derived fibroblasts have been shown to maintain their phenotype in culture.20–23 We measured proliferation rate and viability changes as in vitro surrogates to keloid growth and therapeutic toxicity. The study of keloidderived fibroblast modulation, function, proliferation, and other cellular properties represents our laboratory’s current effort to increase understanding of the photobiomodulatory effects of LED-RL on keloid-derived fibroblast cultures in vitro. This research builds on our previously reported findings that LED-RL can decrease normal human skin fibroblast proliferation in vitro.24,25 Herein, we report the effects of LED-RL on keloid-derived human skin fibroblast proliferation and viability in vitro. Methods Briefly, monolayers of primary keloid–derived human skin fibroblasts, previously isolated from excised patient keloid tissue under an institutional review board–approved protocol, were cultured in Dulbecco modified Eagle medium (Gibco–Invitrogen, Carlsbad, CA) with 10% bovine calf serum (Gibco) and 1% penicillin, streptomycin, and neomycin antibiotic mixture (Gibco). Cultured keloid-derived fibroblasts have been shown to maintain their phenotype in culture.20 The cell cultures were maintained in incubators at 37C with 5% carbon dioxide. Fibroblasts seeded at 2 · 104 cells per 35-mm dish were irradiated 24 hours after seeding, using a 633-nm LED array (Omnilux New-U; Photo Therapeutics, Carlsbad, CA) at a power density of 872.6 W/m2 at room temperature. Media temperatures measured throughout irradiation remained constant at 32 to 34C. By remaining within physiologic temperatures, heat stress was not induced. After 48 hours of additional incubation to allow the cells to proliferate, the cells were harvested, counted, and assessed for viability according to trypan blue exclusion assay as
DERMATOLOGIC SURGERY
Copyright © American Society for Dermatologic Surgery. Unauthorized reproduction of this article is prohibited.
MAMALIS AND JAGDEO
previously described.19 Briefly, the number of cells in suspension is measured by sampling a known volume (10 mL) and measuring the cellular concentration with a hemocytometer examined using light microscopy. Dead cells have permeable cell membranes allowing uptake of the trypan blue dye (seen as blue cells using light microscopy). Therefore, the trypan blue dye allows clear discrimination of live from dead cells because the dye is excluded from live cells with intact cell membranes. Viability can then be calculated based on the ratio of live (total minus dead) to total cells. All experiments were repeated to verify data reproducibility and accuracy, and similar trends were demonstrated in a second keloid-derived human skin fibroblast culture isolated from a different patient (data not shown). Each experimental plate receiving LED-RL treatment was randomly matched with a bench control plate (BCP) to ensure that the measured effect was a result of LED-RL treatment and not because of ambient light or environment. Bench control plates were derived from the same stock of cell suspension, taken out of the incubator at the same time as their matched treatment pairs, and left on the bench for the same amount of time and in the same ambient environment as the treatment plates. Bench control plates were protected from the LED-RL source, placed on a digital warming block, and media temperatures measured throughout time on the bench remained 32 to 34C. Treatment plates and BCPs were then returned to the incubator and processed according to the same protocol. Cell counts included all cells in the sample, including media change, trypsinization products, and washes. Mean percentage proliferation and viability (live cells/total [live + dead] cells) relative to nonirradiated controls are reported as means and standard errors of the mean. Statistical analysis was performed using analysis of variance to compare treatment arms and the Student t-test to compare each treatment arm with the paired bench control arm.
Results Our experimental results demonstrate that treatment of primary keloid–derived human skin fibroblasts
using LED-RL (633 nm) inhibits proliferation in a dose-dependent manner without causing significant effect on viability at fluences of 240, 320, and 480 J/cm2 (Figures 1 and 2). Statistically significant decreases in cell proliferation are noted at the following fluences (time): 240 (45 minutes, 50 seconds), 320 (61 minutes, 7 seconds), and 480 J/cm2 (91 minutes, 41 seconds) (Figure 1). Plates treated with 240, 320, and 480 J/cm2 all demonstrated a statistically significant decrease in relative proliferation rate of 12.4%, 16.5%, and 28.9%, respectively, compared with matched BCPs (p < .05). Treatment at the 240 (98.8% 6 3.8%), 320 (100.4% 6 2.2%), and 480 J/cm2 (100.0% 6 2.6%) doses did not significantly alter viability relative to the nonirradiated controls (Figure 2).
Discussion Our data demonstrate that LED-RL can decrease the proliferation of keloid-derived fibroblasts in vitro in a dose-dependent manner (Figure 1). Treatment with fluences of 240, 320, and 480 J/cm2 resulted in statistically significant decreases in cell number without a significant decrease in viability as compared with BCPs (Figure 2). We recently reported similar findings using LED-generated infrared and red light to treat normal human skin fibroblasts.24,25 To our knowledge, this is the first report of the antiproliferative effect of LED-RL on keloid-derived skin fibroblasts in vitro. We believe that our findings serve as “proof of principle” and may establish the foundation for the translation of these “bench” findings to clinical studies to determine the antifibrotic effects of LED-RL for the treatment of keloids and other cutaneous scarring conditions. Attention to bias is necessary when attempting to demonstrate light-induced effects on human skin fibroblasts. Specifically, the observed effects may be attributed to variation in proliferation patterns between the experimental and control plates. The effects may also be attributed to various environmental factors other than the light energy that the LED device generated. We controlled for environmental conditions by using bench controls, monitored to confirm matching
41:1:JANUARY 2015
37
Copyright © American Society for Dermatologic Surgery. Unauthorized reproduction of this article is prohibited.
LED-RL INHIBITION OF KELOID FIBROBLAST PROLIFERATION
Figure 1. Light-emitting diode–generated red light (633 nm) decreases relative cell proliferation in a dose-dependent manner in vitro. A statistically significant inhibition of proliferation was seen at 240 J/cm2 = 87.6% 6 3.8%, p = .015; 320 J/cm2 = 83.5% 6 2.2%, p = .003; and 480 J/cm2 = 71.1% 6 8.0%, p = .007. Nonirradiated cells, 0 J/cm2 = 100.0% 6 7.6%. Forty-eight hours after irradiation, cells were assessed by trypan blue exclusion assay. Proliferation is presented as percent of the matched BCPs. Error bars represent standard error of the mean. *Statistical significance, p < .05.
temperatures, and repeated all experiments 3 times to account for variation in cellular proliferation patterns. Further studies are needed to demonstrate these findings in vivo.
Light-emitting diode–generated red light phototherapy may represent a safe and cost-effective modality to improve patient outcomes with keloids or other fibrotic skin disorders and therefore warrants further study.
Figure 2. In keloid fibroblasts treated with LED-generated red light (633 nm) cellular viability is maintained in vitro at fluences of 0 J/cm2 = 100.0% 6 2.6%, 240 J/cm2 = 98.8% 6 3.8%, 320 J/cm2 = 100.4% 6 2.2%, and 480 J/cm2 = 100.0% 6 2.6%. Forty-eight hours after irradiation, cells were assessed by trypan blue assay. Relative viability is presented as percent of the control. Error bars represent standard error of the mean. No statistically significant differences were found between these groups when comparing viability.
38
DERMATOLOGIC SURGERY
Copyright © American Society for Dermatologic Surgery. Unauthorized reproduction of this article is prohibited.
MAMALIS AND JAGDEO
Further in vitro studies are needed to elucidate the mechanism underlying LED-RL phototherapy and to investigate its efficacy to mitigate key pathogenic processes related to skin fibrosis, such as collagen production. Given that LED-RL phototherapy decreases keloid-derived fibroblast proliferation without decreasing cellular viability, we hypothesize that LEDRL phototherapy may mechanistically decrease proliferation rate through cell cycle modulation. Future studies are aimed at investigating the effects of LED-RL on cell cycle modulation and other pathways. In addition to decreasing proliferation, we propose that LED-RL phototherapy may be able to modulate additional keloid-derived fibroblasts phenotypic differences associated with skin fibrosis. These data may serve as the in vitro foundation for the investigation of LED-RL phototherapy for the treatment of keloids and other forms of skin scarring and fibrosis. Keloid scarring and other forms of skin fibrosis occur in the dermis, and it is well established that red light penetrates into the mid-to-deep dermis.21 Therefore, we hypothesize that our findings will translate to clinical therapy of keloid and other forms of fibrotic disease. Future research will focus on establishing dosing regimens, elucidation of mechanism of action through interrogation of the transforming growth factor-beta pathway and molecular signaling pathways linked to skin fibrosis, and translation of findings to clinical studies to improve the lives of patients with cosmetically and functionally impairing keloids and other skin scarring conditions.
References 1. Uitto J. The role of elastin and collagen in cutaneous aging: intrinsic aging versus photoexposure. J Drugs Dermatol 2008;7:s12–6.
7. Kolbe L. How much sun protection is needed? Are we on the way to fullspectrum protection? J Invest Dermatol 2012;132:1756–7. 8. Mahmoud BH, Ruvolo E, Hexsel CL, Liu Y, et al. Impact of longwavelength UVA and visible light on melanocompetent skin. J Invest Dermatol 2010;130:2092–7. 9. Bayat A, McGrouther DA, Ferguson MW. Skin scarring. BMJ 2003; 326:88–92. 10. Yang YC, Cheng YW, Lai CS, Chen W. Prevalence of childhood acne, ephelides, warts, atopic dermatitis, psoriasis, alopecia areata and keloid in Kaohsiung County, Taiwan: a community-based clinical survey. J Eur Acad Dermatol Venereol 2007;21:643–9. 11. Chen GY, Cheng YW, Wang CY, Hsu TJ, et al. Prevalence of skin diseases among schoolchildren in Magong, Penghu, Taiwan: a community-based clinical survey. J Formos Med Assoc 2008;107:21–9. 12. Bock O, Schmid-Ott G, Malewski P, Mrowietz U. Quality of life of patients with keloid and hypertrophic scarring. Arch Dermatol Res 2006;297:433–8. 13. Mamalis AD, Lev-Tov H, Nguyen DH, Jagdeo JR. Laser and light-based treatment of keloids—a review. J Eur Acad Dermatol Venereol 2013. 14. van den Broek LJ, Niessen FB, Scheper RJ, Gibbs S. Development, validation and testing of a human tissue engineered hypertrophic scar model. ALTEX 2012;29:389–402. 15. Al-Attar A, Mess S, Thomassen JM, Kauffman CL, et al. Keloid pathogenesis and treatment. Plast Reconstr Surg 2006;117:286–300. 16. Ramos ML, Gragnani A, Ferreira LM. Is there an ideal animal model to study hypertrophic scarring? J Burn Care Res 2008;29:363–8. 17. Chung HJ, Steplewski A, Chung KY, Uitto J, et al. Collagen fibril formation. A new target to limit fibrosis. J Biol Chem 2008;283:25879–86. 18. Shetlar MR, Shetlar CL, Kischer CW, Pindur J. Implants of keloid and hypertrophic scars into the athymic nude mouse: changes in the glycosaminoglycans of the implants. Connect Tissue Res 1991;26:23–36. 19. Yagi Y, Muroga E, Naitoh M, Isogai Z, et al. An ex vivo model employing keloid-derived cell-seeded collagen sponges for therapy development. J Invest Dermatol 2013;133:386–93. 20. Fujiwara M, Muragaki Y, Ooshima A. Keloid-derived fibroblasts show increased secretion of factors involved in collagen turnover and depend on matrix metalloproteinase for migration. Br J Dermatol 2005;153:295–300. 21. Koch RJ, Goode RL, Simpson GT. Serum-free keloid fibroblast cell culture: an in vitro model for the study of aberrant wound healing. Plast Reconstr Surg 1997;99:1094–8. 22. Syed F, Ahmadi E, Iqbal SA, Singh S, et al. Fibroblasts from the growing margin of keloid scars produce higher levels of collagen I and III compared with intralesional and extralesional sites: clinical implications for lesional site-directed therapy. Br J Dermatol 2011;164:83–96.
2. Marrot L, Meunier JR. Skin DNA photodamage and its biological consequences. J Am Acad Dermatol 2008;58:S139–48.
23. Wu CS, Wu PH, Fang AH, Lan CC. FK506 inhibits the enhancing effects of transforming growth factor (TGF)-b1 on collagen expression and TGF-b/Smad signalling in keloid fibroblasts: implication for new therapeutic approach. Br J Dermatol 2012;167:532–41.
3. Liebel F, Kaur S, Ruvolo E, Kollias N, et al. Irradiation of skin with visible light induces reactive oxygen species and matrix-degrading enzymes. J Invest Dermatol 2012;132:1901–7.
24. Lev-Tov H, Brody N, Siegel D, Jagdeo J. Inhibition of fibroblast proliferation in vitro using low-level infrared light-emitting diodes. Dermatol Surg 2013;39:422–5.
4. Morton CA, Scholefield RD, Whitehurst C, Birch J. An open study to determine the efficacy of blue light in the treatment of mild to moderate acne. J Dermatolog Treat 2005;16:219–23.
25. Lev-Tov H, Mamalis A, Brody N, Siegel D, et al. Inhibition of fibroblast proliferation in vitro using red light-emitting diodes. Dermatol Surg 2013;39: 1167–70.
5. Russell BA, Kellett N, Reilly LR. A study to determine the efficacy of combination LED light therapy (633 nm and 830 nm) in facial skin rejuvenation. J Cosmet Laser Ther 2005;7:196–200. 6. Huang YY, Sharma SK, Carroll J, Hamblin MR. Biphasic dose response in low level light therapy—an update. Dose Response 2011;9:602–18.
Address correspondence and reprint requests to: Jared Jagdeo, MD, MS, 3301 C Street, Sacramento, CA 95816; or e-mail: [email protected]
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