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Lasers in Surgery and Medicine 40:520–528 (2008)

Temporary Removal by Low Fluence Photoepilation: Histological Study on Biopsies and Cultured Human Hair Follicles

1 2 3 4 Guido F. Roosen, MSc, Gillian E. Westgate, PhD, Mike Philpott, PhD, Paul J.M. Berretty, MD, PhD, 5 6 Tom (A.M.) Nuijs, PhD, * and Peter Bjerring, MD, PhD 1Philips Electronics Nederland, 1077 XV Amsterdam, The Netherlands 2Westgate Consultancy Ltd., Bedford MK 43 7QT, UK 3Queen Mary’s School of Medicine and Dentistry, London E1 2AT, UK 4Catharina Hospital, 5602 ZA Eindhoven, The Netherlands 5Philips Research, 5656 AE Eindhoven, The Netherlands 6Molholm Hospital, DK-7100 Vejle, Denmark

Background and Objectives: We have recently shown INTRODUCTION that repeated low fluence photoepilation (LFP) with Clinical results of photoepilation treatments reported in (IPL) leads to effective , the literature in general show variability in hair reduction which is fully reversible. Contrary to permanent hair effectiveness, both in rate and duration of clearance. Based removal treatments, LFP does not induce severe damage to on ‘‘selective photothermolysis’’ as the proposed mecha- the . The purpose of the current study is to nism of action [1], this variability can partly be explained by investigate the impact of LFP on the structure and the the broad range of applied parameters such as fluence, physiology of the hair follicle. pulse width and spectrum of the light. Similarly, variability Study Design/Materials and Methods: Single pulses of between subjects such as type, hair color, and hair 2 IPL with a fluence of 9 J/cm and duration of 15 milliseconds follicle (HF) geometry also contributes to these differences were applied to one lower leg of 12 female subjects, followed [2–4]. In general, however, clinical results observed for by taking a single biopsy per person, either immediately, or photoepilation can be classified into temporary effects and after 3 or 7 days. Additionally, we present a novel approach permanent effects. to examine the effects of LFP, in which ex vivo hairy human Permanent hair reduction as defined by the FDA is skin was exposed to IPL pulses with the same ‘‘the long-term, stable reduction in the number of parameters as above, followed by isolation and culturing re-growing after a treatment regime, which may include of the hair follicles over several days. Samples were several sessions. The number of hairs re-growing must examined histologically and morphologically. be stable over a time period greater than the duration of Results: The majority of the cultured follicles that had the complete growth cycle of HFs, which varies from 4 to been exposed to LFP treatment showed a marked treat- 12 months according to body location’’.1 The hair cycle has ment effect. The containing part of the hair follicle four stages, active growth (anagen), regression (catagen), bulb was the target and a catagen-like transformation was resting with an anchored club hair (telogen) and exogen observed demonstrating that hair formation had ceased. (when the hair is shed) [5]. Anagen is deemed to be the The other follicles that had been exposed to LFP showed a stage of the cycle most susceptible to photoepilation less strong or no response. The skin biopsies also revealed treatment [6]. that the melanin-rich region of the hair follicle bulb matrix Histological studies show that permanent effects are was targeted; other parts of the follicle and the skin often the result of extensive immediate post-treatment remained unaffected. Catagen/telogen hair follicles were damage in the HFs leading to miniaturization of coarse visible with unusual melanin clumping, indicating this terminal hairs to fine vellus-like hairs or even complete cycle phase was induced by the IPL treatment. degeneration of the follicles [2,7]. The reported types of Conclusions: Low fluence photoepilation targets the pigmented matrix area of the anagen hair follicle bulb, 1http://www.fda.gov/cdrh/consumer/laserfacts.html (September 22, causing a highly localized but mild trauma that interrupts 2007). the hair cycle, induces a catagen-like state and eventually leads to temporary loss of the hair. Lasers Surg. Med. 40: *Correspondence to: Tom (A.M.) Nuijs, PhD, Philips Research, 520–528, 2008. ß 2008 Wiley-Liss, Inc. High Tech Campus 34, Mailbox 7.071, 5656 AE Eindhoven, The Netherlands. E-mail: [email protected] Key words: temporary hair removal; low fluence; photo- Accepted 22 July 2008 Published online in Wiley InterScience epilation; biopsies; histology; human hair follicle; intense (www.interscience.wiley.com). pulsed light (IPL) DOI 10.1002/lsm.20679

ß 2008 Wiley-Liss, Inc. TEMPORARY HAIR REMOVAL BY LFP 521 immediate damage include severe damage to the hair at the bulge region, but not to the hair bulb, although they shafts, the follicle inner (IRS) and outer root only investigated the acute effects. In a white paper, sheath (ORS), including thermal coagulation and vapor- Manstein et al. present research on ex vivo HFs from scalp ization injury [8–13]. It has also been shown that effective skin showing that exclusive bulb damage to the HFs is hair reduction can be obtained even though the hair shaft possible without damaging other parts of the follicle.2 remains in place for several days before being shed [14]. It is Despite the relatively low fluencies applied in these studies, hypothesized that permanent HF damage is obtained if the the reported damage still extends over a large part of the stem cells of the follicle in the ‘‘bulge’’ area are damaged, HF, similar to that reported for higher fluencies [26], but disabling the processes of hair follicle re-growth [1,2,15,16]. without necrotic damage. Trelles et al. [27] showed by skin However, in various so-called permanent photoepilation histology that the use of a filterless flashlamp-based system studies also a temporary effect is reported [7,17–20]. This delivering 7.5 J/cm2 (SpaTouch) leads to partial degener- effect is often independent of the degree of permanent hair ation of the HFs and coagulation changes in the adjacent reduction (as determined at least four months after the tissue. Also clinically this device leads to relative strong treatment). This temporary effect consists of a strong hair treatment effects including side effects like edema, crusting clearance 2–3 weeks after the treatment which is main- and even blistering [12]. These strong effects are most tained for approximately 12 weeks, followed by partial or probably the result of the ‘‘Light and Heat Energy’’ complete re-growth [18,21]. Apparently, most of the technology3 by which also a substantial amount of heat affected HFs go through a non-hair producing period, energy is delivered to the skin. without losing the ability to develop a hair. Histological Based on our clinical results [23] and the above- studies reported in the literature suggest this temporary mentioned types of damage to hair follicles, we hypothesize effect is a treatment-induced disturbance of the hair cycle, that our parameters lead to an effective induction of consistent with a catagen-like transition of the HF [8,18,21] catagen and telogen with no permanent damage to the without destruction of the stem cell region. For this hair follicle. The aim of this study was to validate this catagen-inducing effect, it has been proposed that it is hypothesis. The study approach was twofold. In the first sufficient to damage the matrix cells of anagen HFs, place we tracked treatment responses in individual HFs coagulate blood vessels of the follicle papilla or partially over time as a result of LFP treatment. We designed a novel destroy the ORS [22]. approach of illuminating ex vivo followed by isolation of HFs through micro-dissection and maintenance of the hair follicles in vitro for at least five days during Low Fluence Photoepilation which a detailed morphological analysis was conducted. In the search for a reversible treatment regime, we Secondly, to investigate clinical effects in skin and to showed that low fluence photoepilation (LFP) can result in compare in vitro with in vivo data, biopsies were taken effective temporary hair removal [23]. Our method consists from subjects over a time-course following a single LFP of a light treatment using intense pulsed light (IPL) using treatment. fluencies that are significantly lower than those generally used for permanent hair reduction. The fluencies we MATERIALS AND METHODS applied for LFP are in the 10 J/cm2 range, while permanent hair reduction is usually achieved with fluencies in the IPL Systems 2 range of 20–60 J/cm [20]. Our clinical studies [23] show In this study, excised human scalp skin from cosmetic that LFP results in extremely effective hair removal with operations was exposed to IPL treatment. To illuminate up to 95% hair reduction after two treatments in 4 weeks. these ex vivo skin samples, a bespoke non-commercial IPL Subsequent treatment every 2 weeks for a period of device was built in our workshop that delivered 9 J/cm2 12 weeks maintained the site hair free. However, complete during a pulse of 15 milliseconds with exponential decay. hair re-growth was observed 12 weeks after the last treat- The spectrum ranged from 600 to 1,100 nm and is displayed ment. Low fluence photoepilation, therefore, presents an in Figure 1. The optical coupling was accomplished by a option to achieve high clearance rates without permanent sapphire window sized 1050 mm2 and an optical gel. Each damage to the HF. footprint area received a single pulse. The literature includes only a few reports of studies in Treatment of subjects was performed with an Ellipse which low fluencies were applied in a clinical setting. Flex IPL system (Danish Dermatologic Development). A Drosner et al. [24] report effective hair reduction in the hair bearing area on the lower leg was chosen as suitable 2 10 J/cm range using an alexandrite laser and suggest this location for a biopsy. After and application of an approach could lead to long lasting results. McCoy et al. [16] optical gel, this area was exposed to a single pulse of 9 J/cm2 2 applied fluencies from 10 to 40 J/cm using a 3 milliseconds and 15 milliseconds. ruby laser. At a fluence of 10 J/cm2 they found damage to the hair shaft consisting of homogenization (disruption of the 2Manstein, D. and Pourshagh, M. Effects of fluence and pulse cellular structure by thermal damage) and after 4 weeks duration for flashlamp exposure on hair follicles, 2006. http:// follicles appeared to have moved to late catagen and telogen www.palomarmedical.com/FileUploads/Altshuler-EffectsFluence PulseLight.pdf (September 22, 2007). phases. Liew et al. [25] used a single ruby laser treatment 3http://www.radiancy.com/int/lhescience.htm (September 22, on ex vivo scalp skin at 14 and 20 J/cm2 and did find damage 2007). 522 ROOSEN ET AL.

* Each HF was placed free-floating in 1 ml serum-free culture medium in a well of a 24-well plate. Medium used was Williams E medium supplemented with Glutamine (2 mM), Insulin (10 mg/ml), hydrocortisone (100 ng/ml), antibiotics (Pen/Strep). Glutamine, Insulin and hydrocortisone were previously defined as the only supplements required for serum free maintenance of human hair follicles in vitro [30]. HFs were incubated in a humidified atmosphere of 5% CO2/95% air.

During the days of culturing, gross morphology analysis was carried out on all individual HFs at 24–48-hour intervals using an inverted phase contrast microscope. Following 24 hours in vitro maintenance a set of two treated and two control HFs were stained with Propidium iodide (PI) and were analyzed using a fluorescence microscope. Fig. 1. Light spectrum of the IPL system used for the LFP PI is not absorbed by viable cells but can penetrate cell treatment of the human scalp skin for the in vitro model. membranes of dying or dead cells, thus acting as a marker for cell death. In Vitro Model Biopsies Human scalp skin was obtained from Caucasian females aged 35–60 years undergoing facelift surgery. After gently Approval for the study was obtained through the medical washing the skin samples using phosphate buffered saline ethics committee (Catharina Hospital Eindhoven). A total (PBS), hair bearing parts were trimmed and were warmed of 10 Caucasian females consented to having one 3 mm up to 378C in a humidified CO incubator. A layer of optical punch biopsy taken. The skin of the of the subjects 2 2 coupling gel was applied to the surface of the skin samples was shaved and the LFP treatment was applied (9 J/cm , which were divided randomly into a control and a treat- 15 milliseconds). The treatment was well tolerated by all ment group; the latter group was given a single treatment subjects. Skin types ranged from Fitzpatrick type 1 to VI, using the previously described IPL system applying 9 J/cm2 age varied from 19 to 50 years, (mean 37 years); hair color in a 15 milliseconds pulse. During the treatment the skin ranged from mid-blonde to black and density was 2 samples were positioned on a precision hotplate set to a variable ranging from 2 to 20 hairs/cm with the mean 2 temperature of 378C (LaboTech DHP 15). The control skin being 9 hairs per cm . samples underwent the same procedure but without the Biopsies were taken from the exposed area either actual IPL exposure. After this procedure the HFs were immediately after treatment (within 1–2 hr, n ¼ 4), 3 days isolated and maintained in vitro as previously described after treatment (n ¼ 4) or 7 days after treatment (n ¼ 2). by Philpott et al. [28–30]. This procedure included the Each subject had only one biopsy taken and only at one following steps: point in time. For each biopsy a location was selected accommodating at least one HF. The area to be biopsied was * All skin samples were cut into small blocks of approx- anesthetized using chloraethyl spray and then the biopsy imately 5 mm wide by 15–20 mm long. This was done in tool was punched into skin in the direction of hair growth, to such a way that the orientation of the long side of the also include the lower part of the HFs. A total of 10 biopsies blocks was aligned with the growth direction of the were obtained during the study. They were fixed in hairs, in order to reduce the number of damaged HF paraformaldehyde (4%, phosphate buffer) and processed bulbs as much as possible. into wax and sectioned, either in the transverse direction * The blocks were then cut at the dermal-subcutaneous (perpendicular to the long axis of the follicle) or in the fat interface. In this way the subcutaneous fat portions longitudinal direction, (parallel to the long axis of the hair were obtained containing follicular bulbs, which were follicle). All sections were de-waxed and stained with then only slightly anchored to the surrounding fatty hematoxylin–eosin for light microscopy. tissue. These portions were placed in serum-free isolation medium (Williams E medium, Sigma-Aldrich RESULTS Chemie B.V., Zwijndrecht, The Netherlands) in a Petri dish and put under a dissection microscope. Using an In Vitro Maintenance of Hair Follicles Isolated angled watchmakers forceps, the cut end of the HF was From LFP Treated and Control Scalp Skin gently grasped and withdrawn from the subcutaneous In total 89 HFs were cultured, originating from three fat portion while applying a gentle force. different patients undergoing facelift surgery. Of these * Once isolated, the HFs were stored in a warm isolation follicles, 44 were harvested from skin samples exposed to medium. By using the dissection microscope, the HFs IPL treatment, the other 45 were dissected from the skin were visually checked for signs of damage caused by the samples that were not treated with light. All HFs were isolation procedure. Damaged HFs were discarded. tracked individually each 24–48 hour via light microscopy. TEMPORARY HAIR REMOVAL BY LFP 523

Fig. 3. Example of type I treatment response. In (A) the bulb of a control HF is shown next to a HF bulb demonstrating a type I treatment response after 1 day in culture. A clear retraction of Fig. 2. A cultured control HF that showed normal hair growth the hair fiber can be seen from the DP and a characteristic up to at least 5 days. In (A) the HF is shown after 1, 2, 4, and shaped Melanin Condensate (MC) can be observed. In (B) the 5 days of culture. The hair fiber elongated each day. In (B) the follicle is shown after 1, 2, and 4 days of culture, illustrating bulb of the same follicle on day 2 is shown enlarged demon- continued fiber retraction. Note the small and rounded DP strating a clear bulb, a Dermal Papilla (DP) which was half on day 4 and the clear decrease in length of the hair fiber. covered with melanocytes (M), and a clear boundary between the (IRS) and (ORS). nocytes and melanin, has retracted further during the subsequent 3 days leaving an epithelial strand below. The advantage of this method over other previously The clear decrease of length of the hair fiber is visible. reported methods is that the behavior of the follicles can The DP itself is reduced in size and is relatively dark, be monitored in vitro and specific treatment effects can be possibly indicating loss of viability of the cells. This studied. Type I effect was seen in 27/44 follicles examined Control (sham treated) samples. Of the 45 control (61%). HFs, 37 (82%) showed normal hair growth for at least II. The characteristic feature observed in this group is 5 days. The remaining control HFs showed atypical or no a thickened epithelial column formed between the hair growth. A typical example of normal hair growth is retracted fiber base and the melanin zone, which can shown in Figure 2A. The HF is shown after 1, 2, 4, and be seen after approximately 4 days (Fig. 4). The 5 days of culture and the growth of the hair fiber is clearly visible with the follicle elongating each day, as described previously [30]. In Figure 2B an enlarged picture of the bulb of the same HF is shown during the second day of culture. The dermal papilla (DP) remains visible but is half covered with melanocytes. The bulb is intact and the border of the inner root sheath (IRS) and outer root sheath (ORS) of the follicle can be easily distinguished. These are all signs of healthy hair growth [31]. Treated samples. Cultured hair follicles from the skin samples exposed to IPL treatment in general showed atypical hair growth. The effects observed in the 44 HFs from treated skin can be classified into four types of responses, which will be referred to as type I to IV, described as follows:

I. Marked changes were observed in the lower part of the HF within 1 day of treatment (Fig. 3). As can Fig. 4. In (A) an example of a type II treatment response is be observed in Figure 3A, after 1 day in culture, the shown of a hair follicle on day 1, 2, 4, and 5 of culture. On days 4 base of the hair fiber is rounded and the melanocytes and 5 a keratinized epithelial column is observed under the have retracted upwards away from the DP and form hair fiber, which is the characteristic feature of type II a characteristic melanin condensate, in comparison response. In (B) another type II follicle is shown on day 5, the with the control. Figure 3B shows that the base of indicated area is shown enlarged and the keratinized epithelial the hair shaft, apparently containing trapped mela- column is indicated by the white arrow. 524 ROOSEN ET AL.

overall response is similar to type I response, that is, rounding of the base of the fiber, rounding of the DP and upward retraction of the hair fiber. The thicken- ing effect was seen in 6/44 HFs examined (14%). III. Type III HFs are characterized by visible melanin clumping in the area of the matrix and after 4–5 days these melanin clumps are visible in the hair shaft (Fig. 5). The fiber, however, remains close to the dermal papilla. This effect was seen in 5/44 follicles examined (11%). IV. In 3/44 HFs (7%), hardly any treatment effect was seen (type IV). There is a slight retraction of the melanin and appearance of isolated melanocytes around the DP. There is, however, no evidence of melanin clumping and the HFs continue to form pigmented hair (Fig. 6).

An overview of the treatment responses and their occurrence is given in Tables 1 and 2, respectively. In 2/44 Fig. 6. Example of a follicle that shows a type IV treatment HFs normal hair growth was observed and 1/44 HF response. In (A) the follicle is shown after 1, 2, 4, and 5 days of appeared to be not viable (referred to as ‘‘other’’ in Table 2). culture. The fiber elongates each day. No retraction of the hair Propidium iodide was used to assess follicle viability fiber is visible. In (B) the bulb of the same HF after 1 day of following treatment. Figure 7A,B shows a control and a culture is shown enlarged. treated HF, respectively, after 1 day in culture. In contrast to the control HF, the treated hair follicle shows an upward retracted hair fiber leaving the dermal papilla uncovered. A concentration of normal anagen hair follicle anatomy. clear concentration of bright matrix/DP cells can be seen at These cells probably have been subjected to a relatively the level of the white arrow in the treated hair follicle, high temperature increase during the IPL treatment. meaning these cells has lost their viability. The location of these cells coincides with the area of high melanin Histological Analysis of Biopsies Skin tissue. Immediately after treatment no signs of damage to the were observed, even in individuals having skin type IV. The biopsies taken 3 days after treatment did not reveal gross changes in morphology of the skin. One week after treatment, the epidermis also appeared normal with good differentiation and normal melanin distribution. The contained undamaged normal fibroblasts, collagen and micro-vessels at all times. Hair follicles. In one of the four biopsies taken 3 days after the treatment no HF was visible, the other three biopsies contained, respectively 1, 2, and 3 hair follicles. Analysis of these biopsies shows that the anagen HFs have been targeted in a very specific way. The upper bulb shows no signs of any damage; the ORS and IRS and fiber appear normal (Fig. 8A). Also no damage is observed in the . However, lower down the HF, signs of a trauma induced catagen-like state are evident. These signs include separation of the fiber base from the dermal papilla, clumping of the cells in the matrix area and trapping of melanin/melanocyte remnants (Fig. 8B). These features are uncommon for natural catagen HFs, indicating that this catagen-like state is induced by the light exposure. Melanin clumps are trapped in the keratinized clump of cells. As is normal for a catagen HF, the bulb size appears to Fig. 5. Example of a follicle that shows a type III treatment be reduced and the DP is rounded as compared to the response. The follicle is shown after 1, 2, and 4 days of culture. anatomy of anagen hair follicles (Fig. 8C). In the matrix The fiber elongates each day. The remains cells nuclear fragmentation is observed which is a sign of connected to the DP and a melanin clump is observed in the apoptosis. The connective tissue sheath shows signs of a hair shaft (arrow). thickening hyaline membrane and is highly cellular. In TEMPORARY HAIR REMOVAL BY LFP 525

TABLE 1. Morphology Descriptions of Treatment Effects as Observed in Hair Follicles of the In Vitro Model as a Result of the Low Fluence Photoepilation Treatment

Treatment effect Morphology description days 1 and 2 Morphology description days 4 and 5 Type I Rounding of the base of the fiber. Melanin trapped. Continued upward retraction of the hair fiber away Upward retraction of the hair fiber away from the from the DP DP. DP rounded Type II Rounding of the base of the fiber. Melanin trapped. Within the epithelial column, a ‘‘keratinised’’ Upward retraction of the hair fiber away from the structure appeared attached to the base of the hair DP. DP rounded shaft. Continued upward retraction of the hair fiber away from the DP Type III Some melanin clumping but no obvious retraction of The hair matrix remained connected to the DP with the shaft evidence of continued fiber formation. Might see melanin clump in the shaft Type IV Very little effect. Slight retraction of the melanin and HFs continued to form pigmented hair. No evidence appearance of isolated melanocytes around the DP of melanin clumping another HF taken after 3 days, melanin clumping in a hair follicle bulbs, which are loosely anchored in the disrupted lower hair matrix is observed (Fig. 9) subcutaneous fat. However, we assumed this to be a From the n ¼ 2 biopsies taken seven days post treatment relatively unimportant limitation, as only anagen hairs the first biopsy contained three HFs, the second contained are particularly sensitive to trauma from external one hair follicle. Again, the upper follicles showed no insults, including photoepilation [32]. Another assumption apparent damage and the hair fibers looked normal. we made was that the treatment response of anagen Catagen hair follicles were observed but it could be not HFs from human scalp skin can be extrapolated to determined whether these are treatment induced catagen anagen hairs of other body locations. This is more than transitions. Of all HFs from the biopsies taken 7 days after likely, as hair follicle structure and growth of anagen hairs treatment only one hair follicle bulb was visible. This bulb are largely comparable for HFs over different body again shows melanin clumping and trapping of melanin in locations and experience with photoepilation has shown the hair fiber (Fig. 10). treatment response on various anatomical areas, despite Due to the small size of the biopsies and the low density of differences in hair follicle geometry over these areas [5]. We leg hair, only two of the n ¼ 4 biopsies taken directly after used scalp skin because of its relatively good availability treatment contained HFs. The upper hair follicle regions from cosmetic procedures, and the proven suitability of looked normal and there was no damage to the hair shaft. HFs originating from this tissue for the culture procedure Only one hair bulb was identified and it looked normal. [31,33,34]. Models for hair follicle responses to photoepilation have been used by other groups but have certain drawbacks. DISCUSSION For instance, Poureshagh [35] and Liew et al. [25] have In this study, the mode of action of a single low-fluence investigated the acute impact of photoepilation treatment photoepilation (LFP) treatment has been investigated on hair follicles following illumination of ex vivo skin using an in vitro model and compared with histological samples without maintaining them in vitro. This approach, analysis of biopsies taken from skin following however, does not allow determination of whether an actual identical treatment regimes. The in vitro model enabled a catagen-transition would have taken place, as there was no detailed analysis of HF morphology and hair growth in vitro follow up. A published abstract of Shander et al. [36] dynamics during the first few days after exposure of the does report application of in vitro maintenance of HFs follicles to LFP. A limitation of this model is that only for investigating photoepilation. However, in this study anagen HFs can be investigated. Telogen hairs cannot HFs are exposed to light after isolation, such that optical be obtained by the procedure used due to their strong properties of the skin and their effect on the light anchorage in the upper dermis, in contrast to anagen propagation are not incorporated in the model.

TABLE 2. Occurrence of Treatment Effects in the Treated HFs and Normal Hair Growth in the Control Hair Follicles of the In Vitro Model

Type I Type II Type III Type IV Normal Other Control 45 HFs — — — — 37 (82%) 8 (18%) Treated 44 HFs 27 (61%) 6 (14%) 5 (11%) 3 (7%) 3 (7%) 526 ROOSEN ET AL. .

Fig. 9. Melanin trapping in a disrupted ‘‘clumped’’ lower hair matrix can be seen in a follicle from a second biopsy sample taken 3 days after LFP treatment (see arrow) Note that the ORS remains unaffected. Fig. 7. Two Propidium Iodide-stained hair follicles after 1 day in culture. A: A control hair follicle and (B) a hair follicle growth are not perturbed when applying the sham LFP following exposure to LFP treatment. The clear concentration procedure. Furthermore, as the light exposure procedure of bright cells in the area of the DP/matrix in (B) (arrow) closely resembled the in vivo situation, including the indicates a loss of viability of those cells. presence of whole skin during the light treatment, as well as correct initial tissue temperature, we expect the model to be an excellent means by which to explore the mechanisms To our knowledge this is the first report of a model in of LFP. which in vitro maintenance of hair follicles (HFs) follows a The majority of HFs in both ex vivo and in vivo treatment light treatment of ex vivo whole skin. The ability to study regimes showed a similar treatment effect, this being a hair growth dynamics during the first 5 days following catagen-like response in the subsequent days. Some HFs exposure to LFP is of particular importance. This is because from the in vitro model showed a less pronounced effect the actual course of the hair follicle changes induced by which may be due to variation in exposure, for example, the exposure is crucial for this specific photoepilation as a result of the position of the HF in the skin sample treatment, rather than the extent of initial damage only. (deep versus shallow). However, in this study we have not Westgate et al. [30] demonstrated in detail that in vitro investigated exact causes of the different responses. The hair growth resembles hair growth in vivo very well. The observed catagen-like response is in line with the proposed results we observed in the control HFs of the in vitro model mechanism for the temporary effect seen in the so-called also showed normal maintenance of hair growth and hair permanent photoepilation treatments [8,18,21]. follicle architecture. This indicates that the isolated HFs The localized nature of the initial thermal damage that are viable and confirms that the conditions for normal hair we observed is striking when compared to that which is

Fig. 8. Sections through a single HF in skin removed 3 days a condensation of pre-cortical matrix cells which contain after treatment. The section in (A) is at the level of the supra- melanin remnants. The connective tissue sheath is very bulbar region of the follicle showing the IRS and ORS; no cellular. In section (C) evidence of cellular degeneration can apparent changes in this region are seen. As the sections be observed in the DP and bulb matrix, which is much reduced progress towards the bulb, evidence of change in the follicle in size. These are all signs of a trauma induced catagen-like architecture can be observed; the arrows in section (B) point to state. TEMPORARY HAIR REMOVAL BY LFP 527

the melanin rich bulb matrix area causing local damage sufficient to precipitate .

ACKNOWLEDGMENTS We gratefully acknowledge the faculty of Biomedical Engineering of the Eindhoven University of Technology for making their facilities available for this research, the Catharina Hospital Eindhoven for the supply of samples, and the histology service ‘‘Stichting Laboratoria voor Pathologische Anatomie en Medische Microbiologie’’ (PAMM) for their assistance with sectioning.

REFERENCES 1. Anderson RR, Parrish JA. Selective photothermolysis: Pre- cise microsurgery by selective absorption of pulsed radiation. Science 1983;220:524–527. 2. Ross EV, Ladin Z, Kreindel M, Dierickx C. Theoretical Fig. 10. Clumping and trapping of melanin is seen in section of considerations in . Dermatol Clin 1999;17: a follicle taken 7 days after treatment. 333–355, viii. 3. Sadick NS, Weiss RA, Shea CR, Nagel H, Nicholson J, Prieto VG. Long-term photoepilation using a broad-spectrum intense pulsed light source. Arch Dermatol 2000;136:1336– 1340. generally reported for HF morphology after treatments 4. Ort RJ, Anderson RR. Optical hair removal. Semin Cutan aimed at permanent hair reduction, even those pertaining Med Surg 1999;18:149–158. to fluencies in the 10 J/cm2 range. We found that the fully 5. Price ML, Griffiths WA. Normal —A review. Clin Exp Dermatol 1985;10:87–97. keratinized pigmented hair shaft, the IRS and ORS were 6. Lin TY, Manuskiatti W, Dierickx CC, Farinelli WA, Fisher not damaged, as judged from morphology and histology. ME, Flotte T, Baden HP, Anderson RR. Hair growth cycle Within one to three days after the treatment, however, a affects hair follicle destruction by ruby laser pulses. J Invest Dermatol 1998;111:107–113. melanized epithelial cell condensate was observed just 7. Dierickx CC, Grossman MC, Farinelli WA, Anderson RR. above the dermal papilla and this feature was observed in Permanent hair removal by normal-mode ruby laser. Arch both in vivo and ex vivo treated skin samples. The Dermatol 1998;134:837–842. propidium iodide stained HFs confirmed that thermal cell 8. Grossman MC, Dierickx C, Farinelli W, Flotte T, Anderson RR. Damage to hair follicles by normal-mode ruby laser damage and death is induced by the LFP treatment and pulses. J Am Acad Dermatol 1996;35:889–894. that this is focused in the matrix area and region of the DP. 9. Chui CT, Grekin RC, Chui CT, Grekin RG, LeBoit PE, The observation that thermal damage is highly localized Zachary CB. Long-pulsed Nd:YAG for hair removal: Early histological changes. Laser News. net vol. 1, 1999, p 3–6. can be explained by the significantly higher density of 10. Sadick NS, Shea CR, Burchette JL Jr, Prieto VG. High- melanin in this area. Exposure of skin to IPL with our intensity flashlamp photoepilation: A clinical, histological, parameters (a fluence of 9 J/cm2 in a pulse of 15 milli- and mechanistic study in human skin. Arch Dermatol 1999; seconds) and subsequent generation of heat apparently 135:668–676. 11. Baugh WP, Trafeli JP, Barnette DJ, Jr., Ross. EV. Hair suffices to interrupt the hair growth cycle, without causing reduction using a scanning 800 nm diode laser. Dermatol damage to structures outside the matrix and DP area. Surg 2001;27:358–364. The clinical implications of our observations indicate that 12. Rohrer TE, Chatrath V, Yamauchi P, Lask G. Can patients treat themselves with a small novel light based hair removal even a single LFP treatment is sufficient to cause system? Lasers Surg Med 2003;33:25–29. temporary hair removal. Without significant hair follicle 13. Omi T, Honda M, Yamamoto K, Hata M, Akimoto M, damage, the follicle is likely to recover to produce a new hair Asano G, Kawana S. Histologic effects of ruby laser hair removal in Japanese patients. Lasers Surg Med 1999;25: some weeks after treatment. The clinical benefits of LFP 451–455. treatment have also been examined by our group and will 14. Bjerring P, Cramers M, Egekvist H, Christiansen K, Troilius be reported separately [23]. A. Hair reduction using a new intense pulsed light irradiator In conclusion, we have developed a unique model for and a normal mode ruby laser. J Cutan Laser Ther 2000; 2:63–71. studying the treatment effect of LFP on hair follicle 15. Sun TT, Cotsarelis G, Lavker RM. Hair follicular stem cells: behavior and our studies strongly suggest that LFP induces The bulge-activation hypothesis. J Invest Dermatol 1991;96: an immediate catagen-like transition in anagen hair 77S–78S. 16. McCoy S, Evans A, James C. Histological study of hair follicles, with minimal damage to the majority of the hair follicles treated with a 3-msec pulsed ruby laser. Lasers Surg follicle structures. This finding is further strengthened by Med 1999;24:142–150. the analysis of the clinical biopsies, where catagen-like hair 17. Ort RJ, Dierickx C. Laser hair removal. Semin Cutan Med follicles were found having similar characteristics. The Surg 2002;21:129–144. 18. Dierickx C, Alora MB, Dover JS. A clinical overview of hair absence of damage in structures of the hair follicle other removal using lasers and light sources. Dermatol Clin than the matrix area confirms the notion that LFP targets 1999;17:357–366. 528 ROOSEN ET AL. 19. Haedersdal M, Gotzsche PC. Laser and photoepilation for 28. Philpott MP, Green MR, Kealey T. unwanted hair growth. Cochrane Database Syst Rev 2006; in vitro. J Cell Sci 1990;97(Pt 3):463–471. 4:CD004684. 29. Philpott M. In vitro maintenance of isolated hair follicles: 20. Ort RJ, Dierickx C. Laser hair removal. Semin Cutan Med Current status and future development. Exp Dermatol 1999; Surg 2002;21:129–144. 8:317–319. 21. Anderson R. Laser hair removal, a lecture presented to the 30. Westgate GE, Gibson WT, Kealey T, Philpott MP. Prolonged 77th congress of the Japan society of aesthetic surgery, maintenance of human hair follicles in vitro in a serum-free November 14, 1999. 2005. medium. Br J Dermatol 1993;129:372–379. 22. Altshuler GB, Anderson RR, Manstein D, Zenzie HH, 31. Westgate GE, Gibson WT, Kealey T, Philpott MP. Prolonged Smirnov MZ. Extended theory of selective photothermolysis. maintenance of human hair follicles in vitro in a serum-free Lasers Surg Med 2001;29:416–432. medium. Br J Dermatol 1993;129:372–379. 23. Evers LJT, et al. Effective temporary hair removal by low 32. Braun-Falco O, Heilgemeir GP. The trichogram, structural fluence photoepilation. Lasers Surg Med in press. and functional basis, performance and interpretation. Semin 24. Drosner M, Stangl S, Hertenberger B, Klimek H, Pettke- Dermatol 1985;124:40–52. Rank. C. Low dose epilation by alexandrite laser: A dose 33. Philpott M. In vitro maintenance of isolated hair follicles: response study. Med Laser Appl 2001;16:293–298. Current status and future development. Exp Dermatol 1999; 25. Liew SH, Grobbelaar AO, Gault DT, Sanders R, Green CJ, 8:317–319. Linge C. The effect of ruby laser light on ex vivo hair follicles: 34. Philpott MP, Green MR, Kealey T. Human hair growth Clinical implications. Ann Plast Surg 1999;42:249–254. in vitro. J Cell Sci 1990;97(Pt 3):463–471. 26. Orringer JS, Hammerberg C, Lowe L, Kang S, Johnson TM, 35. Poureshagh M. Effects of fluence and pulse duration Hamilton T, Voorhees JJ, Fisher GJ. The effects of laser- for flashlamp exposure on hair follicles, 2006. http://www. mediated hair removal on immunohistochemical staining palomarmedical.com/Fileuploads/Altshuler-EffectsFluence- properties of hair follicles. J Am Acad Dermatol 2006;55: PulseLight.pdf (September 22, 2007). 402–407. 36. Shander D, Farinelli W, Anderson R. Modification of hair 27. Trelles MA, Allones I, Calderhead RG, Velez M. Hair removal cycle stage, growth rate an structure of hair shafts of cultured evaluated with a filterless flashlamp-based system: A pre- human hair follicles exposed to normal mode ruby laser liminary study in 10 patients. J Cosmet Laser Ther 2003; irradiation. J Invest Dermatol Proceedings, vol. 4, no. 3, 5:15–24. December 1999, p 351.