Fractional Photothermolysis Laser Treatment of Male Pattern Hair Loss

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www.lutronic.com Fractional Photothermolysis Laser Treatment of Male Pattern Hair Loss Won Serk Kim, MD, Beom Joon Kim, MD,PHD Department of Dermatology, Kangbuk Samsung Hospital, Department of Dermatology, College of Medicine, Chung-Ang University, Seoul, Korea,

Fractional Photothermolysis Laser Treatment of Male Pattern Hair Loss Won Serk Kim, MD, Beom Joon Kim, MD,PHD Department of Dermatology, Kangbuk Samsung Hospital Department of Dermatology, College of Medicine, Chung-Ang University, Seoul, Korea Summary Background Various trials have been conducted for the management of male pattern hair loss. A variety of laser and light sources have been tried for MPHL. Objectives The study objectives were two folds: 1) To understand the effect of the 1550nm fractional erbium glass laser on the hair cycle in an alopecia mouse model, and 2) to study the clinical effect of the same laser applied to the treatment of MPHL. Materials and Methods In animal experiments, irradiation was applied to shaved skin of C3H/HeN mice with various energy and density settings as well as varied irradiation intervals. In a clinical pilot on human subjects, ten patients were treated on a five session basis, once a week, with a fractional photothermolysis laser at an energy of 5mJ, total density of 300 spots/cm 2. Results In the animal study, the hair stimulation effects were dependent upon energy level, density and irradiation interval. The anagen conversion of hair and increase of Wnt 5a, β catenin signals were observed. In the human pilot study, incremental provement of hair density and growth rate was observed. Conclusions The 1550nm fractional erbium glass laser may be an effective therapeutic tool to treat MPHL along with conventional treatment methods. Keywords : Male pattern hair loss, Fractional photothermolysis laser, WNT/β-catenin signal Introduction Various trials have been conducted for the management of male pattern hair loss (MPHL). Despite recent promising advances in skin immunology and hair biology, there are, at present, only disappointing alternatives to treat alopecia. There continues to be great interest in the potential role of laser and light treatments for hair loss. 1 A variety of laser and light sources have been tried for MPHL including the excimer laser, the He-Ne laser and PUVA and some success has been reported. Although the link between laser treatment and hair growth is not clear and the exact mechanism still unknown, there is evidence to support that laser irradiation might hold potential for the treatment of alopecia. Clinical examples of photo-induced hair growth include the paradoxical hair growth after laser hair removal. 2-5 Empirically, it has been observed that induction of some level of wound healing may be followed by hair growth even though the mechanism is not known. 6 These observations lead us to question what type of laser might be most suitable for proper wounding and hair growth. The objective for this study was to determine the effect of a 1550nm fractional erbium-glass laser on the hair cycle via an alopecia mouse model. Additionally, in half-split controlled human trials, we have induced fractional photothermolysis via the 1550nm wavelength to observe hair growth on male pattern hair loss. Materials and methods Animal study Materials 1. Animals C3H/HeN mice (Orient Bio, Sungnam, Korea) were used in animal experiments. The back skin of 7-week old mice was shaved with a clipper. The induced hair growth was studied in 7-week old C3H/HeN mice and hair follicles were synchronous ly matched in the telogen stage. 2. Devices 1550nm fractional erbium-glass laser (MOSAIC, Lutronic, Korea) irradiation was applied to shaved skin of C3H/HeN mice with various energy, density and irradiation interval. 2

Study design 1. Study 1: Laser irradiation with various energy levels and densities Six C3H HeN mice of 7 weeks age (telogen stage) were irradiated with various energy levels. Irradiated energy levels were 7mJ for mouse #1 and #2, 10mJ for mouse #3 and #4, and 15mJ for mouse #5 and #6. As noted by the arrow on figure 1a, each mouse irradiated with five different densities. Figure 1a. Six C3H HeN mice of 7 weeks age for Study 1 (left upper; 300spot/cm 2, left lower; 600spot/cm 2, right upper; 1000spot/cm 2, right lower; 1500spot/cm 2, upper center; 2000spot/cm 2 ) 2. Study 2: Laser irradiation of various intervals Six C3H HeN mice of 7 weeks age (telogen stage) were irradiat ed at various intervals. Irradiated every 4 weeks: mouse #1 and #2. Irradiated every 2 week: mouse #3 and #4. Irradiated every week: mouse #5 and #6. All six mice were irradiated at 7mJ energy with 800spot/cm 2 density on the left side of the back and 7mJ energy with 1500 spot/cm 2 density on the right side of the back. 3. Histopathologic studies Skin samples were obtained from irradiated mice and checked for hair changes. 4. Molecular studies Skin samples were obtained from irradiated mice. Molecular signals and growth factors known to be associated with hair cycle were checked. 1. Clinical photography Evaluation of degree of hair loss was performed before each treatment session and 1 month after the final treatment. Five standard digital photographs were taken (Canon, EOS 40D, 6.0 megapixels) before each treatment session. 2. Phototrichogram system For a more objective assessment, the phototrichogram system (Folliscope, Lead-M Corporation, Seoul, Korea) was used for the computer-aided evaluation of hair thickness, density and growth rate. Phototrichogram was taken at the same point on the parietal scalp before each session and 1 month after the last session. 3. Histopatologic studies In order to evaluate for histopathologic changes, four-millimeter punch biopsies from scalp were obtained from 3 patients under local anesthesia both before and 1 month after the last treatment. The tissue samples were prepared for light microscopic study by 10% formalin fixation. Paraffin-embedded tissue sections of 3 Jm thickness were processed for Haematoxylin and Eosin (H&E) stain. 4. Molecular studies Several molecular signals known to be associated with hair cycle were checked. These included semi-quantitative RT-PCR with wnt signal probes before and 24 hours after the fractional photothermolysis laser irradiation. Results In the animal study, we observed hair stimulation by the irradiation of 1550nm fractional erbium-glass laser, which was related to the change of hair cycle. The hair stimulation effects were dependent on energy level, density and irradiation interval. The animal study 1 showed that irradiation with high density can induce hair re-growth of telogen phase mouse hair. Hair regrowth was found in areas irradiated by high density irrespective of energy levels (Figure 1b). Pilot study of MPHL in human Treatment protocols Twenty patients with male pattern hair loss loss none of whom had previously been treated for hair growth were enrolled in this pilot study. As this study was a half-split study, the right side of frontal scalp was irradiated with the 1550 nm fractional photothermolysis laser with static operating mode; the left side of the scalp was untreated as a control. Patients were treated for eight-sessions, every 2 weeks, at an energy of 5mJ, total density of 300 spots/cm 2 (low energy and high density). During the course of the investigation, application of topical agent or administration of any medication was not permitted. Evaluation criteria A photograph and phototrichogram were taken of each patient before and 1 month after the last treatment. Biopsy was taken of five patients before treatment and 1 month after last treatment. Figure 1b. Hair re-growth was found in areas irradiated by high density irrespective of energy levels 3

The animal study 2 showed that hair re-growth was dependent on irradiation interval and density. Hair re-growth was more distinct in frequently irradiated mice and on the right side of back with high density irradiation (Figure 1c). The change of WNT signal and expression of β-catenin appears to be related to hair growth stimulated by fractional laser irradiation. From the results of the animal study, we have expanded our investigation of laser assisted hair growth in humans. In the human pilot study for MPHL, clinical improvement was observed in most of the patients (Figure 4). Figure 1c. Hair re-growth was more distinct in frequently irradiated mice and on the right side of back with high density irradiation. Low energy with high density, at a frequent irradiation interval of at least every 2 week s induced considerable hair stimulation. Histological findings revealed the anagen conversion of hair as shown on the figure 2. Figure 4. The human pilot study for MPHL Increments of hair density (Figure 5a, 5b) and growth rate improvements (Figure 5c, 5d) were observed after fractional photothermolysis laser treatment. Figure 2. Histological findings revealed the anagen conversion of hair Figure 5a. Increments of hair density Fractional laser irradiation can promote anagen hair growth and convert the telogen phase into the anagen phase. Molecular studies showed changes of molecular signals and cytokines associated with hair cycles. Wnt 5a, β-catenin signals were increased by laser irradiation (Figure 3). Figure 5b. Increments of hair density Figure 3. Molecular studies for changes of molecular signals and cytokines 4

Figure 7. Semi-quantitative RT-PCR studies with wnt-related primers Figure 5c. Growth rate improvements Adverse effect Breakage of the hair shaft was occasionally observed after irradiation associated with energy, density, and manual skills, especially at the high energy setting. Microscopic analysis by SEM showed blunt damage to the hair cuticle and cortex (Figure 8). Figure 5d. Growth rate improvements Histological findings revealed an increase in the number of an agen hair follicles on the lower dermis as shown on the figure 6. Figure 8. Microscopic analysis by SEM This was most noted when high energy and low density laser irradiation was used. Also, several patients complained about transient shedding one week after irradiation, a phenomenon that might be related to cutting of hair shaft. Side effects observed were pain during treatment and mild post-treatment erythema, pruritus, dryness and dandruff, all of which resolved within one week. Figure 6. Histological findings revealed an increase in the number of anagen hair follicles Semi-quantitative RT-PCR studies with wnt-related primers showed induction of Wnt10a signal 24 hours after the fractional photothermolysis laser irradiation (Figure 7). Discussion Androgenetic alopecia is defined as hereditary thinning of the hair induced by androgens in genetically susceptible men and women, also known as male-pattern hair loss in men and female-pattern hair loss in women. Pathophysiology of androgenetic alopecia is not fully understood. It is thought that progressive diminution of hair shaft diameter and length may be in response to systemic androgens. Miniaturization of follicles, decreased anagen, increased telogen, increased latency are observed in MPHL. Although vellus hairs on the pubic, axilla, male chest and beard convert to terminal hairs, terminal hairs change to vellus hairs on the scalp by site-specific action of androgens. 7 5

Treatment goals of androgenetic alopecia are those that increase hair coverage of the scalp and retard further hair thinning. Competitive inhibitors of type 2, 5-alpha reductase (Minoxidil, finasteride) and dutasteride, a competitive inhibitor of type 1 and 2 5-alpha reductase; as well as surgery are occasionally used for the treatment of male pattern hair loss. But, conventional medical treatments usually result in only mild satisfactory outcomes. Furthermore, hair transplantation has limitations in that it is invasive, expensive and not ideal for the early stage patient. To overcome the limitation of medical treatments for MPHL, there has been great interest in the potential role of laser/light treatments for male and female hair loss. 1 Observations some 50 years ago had indicated that, in mice, rabbits and humans, 8-10 some hair follicles develop anew after wounding. Ito et al 11 found that cells constituting the newly formed hair follicles are derived from inter-follicular epidermis, and not from existing hair bulges. If proper wounding can initiate hair growth, laser wounding for the treatment of alopecia will be possible. What laser is most suitable for proper wounding and subsequent hair growth? Several cases and studies on the treatment of alopecia areata have involved the use of the 308nm 12,13 excimer laser irradiation,the 904nm pulsed infrared and the 810nm diode laser irradiation for alopecia areata. 5,14 In this study, we have employed a fractional 1550 nm laser to induce micro coagulative wounds in the dermis. With the use of a fractional near infrared laser penetration depth and wound size can be easily regulated, making invisible small wounds without bleedings. 15 In spite of the energy loss by the reflection on the hair surface, fractional laser beam can penetrate the hairy scalp as compared with other laser systems. In this paper we have shown that both hair growth and modulation of the hair cycle in C3H mice are possible by using MOSAIC fractional photothermolysis laser treatment in the animal model. The study involved the use of a fractional photothermolysis laser treatment for the shaved back skin of eight C3H HeN mice in telogen stage (7 weeks). On the basis of C3H animal study, we applied MOSAIC fractional photothermolysis laser to ten human patients with male pattern hair loss. The second study was designed to evaluate the effect of fractional photothermolysis laser in the treatment of MPHL in humans. A number of possible physiological mechanism of laser irradiation may play a role in hair 16-18 growth improvement including: increased blood flow, promotion of anagen hair growth, enhanced conversion of the telogen phase into the anagen phase, and modulation of hair cycling. In the animal study, we have presented possible hair growth and modulation of hair cycle in the C3H mouse by using MOSAIC fractional photothermolysis laser. In the C3H animal study, we were able to identify that changing of WNT signal and expression of β-catenin might be related to hair growth induced by fractional laser irradiation. One of the earliest molecular pathways that positively regulates hair follicle initiation is the WNT/ β-catenin pathway. β-catenin is the downstream mediator of WNT signaling. And Wnt/β-catenin/Lef-1 signaling plats an important role in hair shaft formation. 19 Suggested mechanisms are following : Increased blood flow; Cytokines and growth factors such as PDGF, KGF, IGF and FGF associated with hair biology induced by laser wounding; Direct stem cell, bulge cells or dermal papilla cell stimulation; Unknown mechanisms. In the murine hair follicle model, many crucial Wnt molecules like Wnt5a, 10a, 10b, are expressed at specific morphogenetic epidermal placode areas. 20 In this human pilot study, mrna of unique Wnt10a was highly expressed 24 hrs after the fractional photothermolysis laser irradiation. As reported in the mouse model, wnt 10a is mainly expressed in the epithelial bulge area during anagen onset and could be expected to initiate the β- catenin activation of dermal papilla. As scalp tissue biopsies were taken 24 hrs after irradiation, initial WNT signal like Wnt10a was only elevated in mrna of tissue level. Other wound-related Wnt4, bulge-related Wnt3a, papillaproducing Wnt5a and tissue β-catenin expressions were not changed at the tissue sampling point. If additional time course tissues were available, it may be possible to identify additional anagen-related wnt molecules that play a role in laser induced hair growth. Animal studies and human trials suggest the usefulness of the fractional 1550 nm laser for the treatment of alopecia. MOSAIC fractional laser treatment might enhance hair density, growth rate, anagen/telogen ratio but not hair thickness. Proper energy level and density is necessary for effective stimulation of hair growth. Too frequent or high energy laser treatments may exacerbate alopecia. A high-energy protocol could increase incidence of breakage of the hair shaft by damage to hair cuticle and cortex; frequent treatment greater than two times a week might induce transient shedding. Therefore, low energy and high density protocol at 2 weeks interval was thought be the most suitable protocol for treatment of MPHL. However, further studies about exact mechanisms and controlled prospective human studies must be conducted to confirm the effectiveness of fractional laser. Additional studies would better confirm the most suitable parameters including energy and density, treatment interval as well as the best applicators for energy delivery to the hairy scalp. Finally, MOSAIC fractional laser treatment might be supportive care for MPHL but cannot replace current classical treatment. This can potentially create synergy with conventional. For example, fractional laser combined with topical agents or with anti-androgen agents might yield improved outcomes. References 1. Avram MR, Leonard RT Jr, Epstein ES, et al. The current role of laser/light sources in the treatment of male and female pattern hair loss. J Cosmet Laser Ther. 2007;9(1):27-8. 2. Bouzari N, Firooz AR. Lasers may induce terminal hair growth. Dermatol Surg. 2006;32:460. 3. Kontoes P, Vlachos S, Konstantinos M, et al. Hair induction after laser-assisted hair removal and its treatment. J Am Acad Dermatol. 2006;54:64.7. 4. Alajlan A, Shapiro J, Rivers JK, et al. Paradoxical hypertrichosis after laser epilation. J Am Acad Dermatol. 2005;53:85.8. 5. Bernstein EF. Hair growth induced by diode laser treatment. Dermatol Surg. 2005;31:584.6. 6. Levy V, Lindon C, Zheng Y, et al. Epidermal stem cells arise from the hair follicle after wounding. FASEB J. 2007;21(7):1358-66. 7. Paus R, Olsen EA, Messenger AG. Hair growth disorder, In: Wolff K, Goldsmith LA, Katz SI, Gilchrest BA, Paller AS, Leffell DJ, editors. Fitzpatrick s dermatology in general medicine. 7th ed. New York: McGraw-Hill, 2008:753-77. 6

8. Kligman AM, Strauss JS. The formation of vellus hair follicles from human adult epidermis. J Invest Dermatol. 1956;27:19-23 9. Billingham RE, Russell PS. Incomplete wound contracture and the phenomenon of hair neogenesis in rabbits' skin. Nature 1956;177:791-2. 10. Breedis C. Regeneration of hair follicles and sebaceous glands from the epithelium of scars in the rabbit. Cancer Res. 1954;14:575-9. 11. Ito M, Yang Z, Andl T, et al. Wnt-dependent de novo hair follicle regeneration in adult mouse skin after wounding. Nature. 2007;447:316.20. 12. NAWAF AM, FRCPC. 308-nm excimer laser for the treatment of alopecia areata. Dermatol Surg. 2007;33:1483.7. 13. Gundogan C, Greve B, Raulin C. Treatment of alopecia areata with the 308-nm xenon chloride excimer laser: case report of two successful treatments with the excimer laser. Lasers Surg Med. 2004;34(2):86-90. 14. Waiz M, Saleh AZ, Hayani R, Jubory SO. Use of the pulsed infrared diode laser (904 nm) in the treatment of alopecia areata. J Cosmet Laser Ther. 2006;8:27.30. 15. Cho SB, Lee JH, Choi MJ, et al. Efficacy of the fractional photothermolysis system with dynamic operating mode on acne scars and enlarged facial pores. Dermatol Surg. 2009;35(1):108-14. 16. Saygun I, Karacay S, Serdar M, et al. Effects of laser irradiation on the release of basic fibroblast growth factor (bfgf), insulin like growth factor-1 (IGF-1), and receptor of IGF-1 (IGFBP3) from gingival fibroblasts. Lasers Med Sci. 2008;23(2):211-5. 17. Zhou Z, Kawana S, Aoki E, et al. Dynamic changes in nerve growth factor and substance P in the murine hair cycle induced by depilation. J Dermatol. 2006;33(12):833-41. 18. Kuo YR, Wu WS, Jeng SF, et al. Suppressed TGF-β1 expression is correlated with up-regulation of matrix metalloproteinase-13 in keloid regression after flashlamp pulsed-dye laser treatment. Lasers Surg Med. 2005;36(1):38-42. 19. Cotsarelis G, Botchkarev V. Biology of hair follicles, In: Wolff K, Goldsmith LA, Katz SI, Gilchrest BA, Paller AS, Leffell DJ, editors. Fitzpatrick s dermatology in general medicine. 7th ed. New York: McGraw-Hill, 2008:739-48. 20. Reddy S, Andl T, Bagasra A, et al. Characterization of Wnt gene expression in developing and postnatal hair follicles and identification of Wnt5a as a target of Sonic hedgehog in hair follicle morphogenesis. Mech Dev. 2001;107(1-2):69-82. 7

Won-Serk Kim, MD Graduate School of Medicine, Seoul National University, South Korea Fellowship, Department of Dermatology, Samsung Medical Center Clinical Assistant Professor, Department of Dermatology, Samsung Medical Center Assistant Professor and Chairman, Department of Dermatology, Kangbuk Samsung Hospital, Sungkyunkwan University School of Medicine Director of the Korean Society for Aesthetic and Surgical Dermatology Beom Joon Kim, MD Ph.D, Dermatology, Chung-Ang University, Korea Assistant Professor, Department of Dermatology, College of Medicine, Chung-Ang University, Korea Assistant Professor, Department of Dermatology, Dong-Guk University International Hospital, Korea Secretary general, The 11th annual meeting of the SHSR(Society for -Hair Science Research, Japan) joint with KHRS(Korean Hair Research Society) Invited reviewer of British Journal of Dermatology, Dermatologic Surgery Editorial Board, The Journal of the American Academy of Dermatology Editorial Board, International Journal of Dermatology 8