Epidermal skin grafting in vitiligo: a pilot study

International Wound Journal ISSN 1742-4801 ORIGINAL ARTICLE Epidermal skin grafting in vitiligo: a pilot study Agata Janowska, Valentina Dini, Salvatore Panduri, Michela Macchia, Teresa Oranges & Marco Romanelli Wound Healing Research Unit, Department of Dermatology, University of Pisa, Pisa, Italy Key words Vitiligo; Skin grafting; Epidermal skin grafting Correspondence to Marco Romanelli, MD, PhD Wound Healing Research Unit, Department of Dermatology University of Pisa Via Roma, 67 Pisa 56126 Italy E-mail: m.romanelli@med.unipi.it doi: 10.1111/iwj.12632 Janowska A, Dini V, Panduri S, Macchia M, Oranges T, Romanelli M. Epidermal skin grafting in vitiligo. Int Wound J 2016; 13 (suppl. S3):47 51 Abstract Vitiligo is a multifactorial acquired dermatosis characterised by achromic or hypochromic macules and by the absence of functioning melanocytes. Treatment depends on the extent of the affected areas and on disease activity. Surgical techniques have proven to be effective in stable cases but can be time-consuming and, in some cases, aesthetically unsatisfying or painful for the patients. The aim of the study was to assess the clinical safety and effectiveness of a new automatic epidermal skin harvesting device in patients with stable localised vitiligo over a minimum 12-month period. This new system (CELLUTOME Epidermal Harvesting System, KCI, an ACELITY Company, San Antonio, TX) is a commercially available epidermal skin harvesting system that can be used without local anaesthesia or other pre-treatments and has been shown to have low rates of donor site morbidity. Epidermal skin grafts can used in patients with acute and hard to heal chronic wounds, burns and stable vitiligo. The use of advanced therapies may improve the quality of life, have cost benefits and accelerate re-pigmentation of patients with vitiligo. In our preliminary study, this system was seen to be a safe and efficacious means of harvesting epidermal micrografts containing melanocytes for use in patients with stable vitiligo unresponsive to standard therapies. Introduction Vitiligo is a multifactorial acquired dermatosis with a prevalence of 0 5 2 0% in the world population and a significant psychological impact. It is clinically characterised by achromic or hypochromic macules and, histologically, by the absence of functioning melanocytes (1). Treatment depends on the extent of the affected areas and on disease activity and the efficacy of the often limited treatments available. The standard therapies are systemic and topical corticosteroids, narrowband ultraviolet B (NB-UVB), vitamin D analogues, topical immunomodulators, surgical techniques or a combination of therapies (2). Surgical techniques have proven to be effective in stable cases, and melanocyte transfer may be performed by punch minigrafting, split-thickness skin grafting, hair follicle transplantation, suction blisters, epidermal curettage techniques and cultured and non-cultured autologous melanocytes. The previous surgical and cultured techniques can be time-consuming and, in some cases, aesthetically unsatisfying or painful for the patients (3,4). In recent years, new non-cultured autologous epidermal techniques have been developed with similar results to the cultured melanocytes techniques, but are simpler, less expensive and less time-consuming. These techniques do not require the use of a laboratory but only a split-thickness thin biopsy that is pre-treated with trypsin solution (5). Key Messages epidermal skin grafting can be used to transfer autologous melanocytes as a treatment for patients with stable vitiligo a commercially available epidermal skin harvesting system can be used in the health care provider s office and without anaesthesia five patients with stable vitiligo (stable with no disease activity for 12 months) unresponsive to standard treatments were treated with epidermal skin grafts complete repigmentation of the recipient site was seen in one patient, while the remaining four patients displayed 50 75% repigmentation the use of advanced therapies may improve the quality of life, have cost benefits and accelerate repigmentation in patients with vitiligo In our study, we used a commercially available epidermal skin harvesting system (CELLUTOME Epidermal Harvesting System, KCI, an ACELITY Company, San Antonio, TX) without local anaesthetic or other pre-treatments (6 8). The 2016 Medicalhelplines.com Inc and John Wiley & Sons Ltd 47

Epidermal micrografts in vitiligo aim of the study was to assess the clinical safety and effectiveness of this new automatic epidermal fractional skin harvesting system in patients with stable localised vitiligo over a minimum 12-month period. Materials and methods Study participants From January to June 2015, we recruited patients with stable vitiligo, defined as no progression over the preceding 12 months and with a Fitzpatrick Skin Type 3, to be treated with epidermal skin grafts. We evaluated the vitiligo disease activity (VIDA) score of these areas. VIDA is a score that assesses the stability of vitiligo over time, with a range between +4 (activity lasting 6 weeks or less) and 1 (stable vitiligo with spontaneous repigmentation) (9). All the vitiligo areas had been stable for at least 12 months (VIDA score 0) and were unresponsive to the standard therapies such as topical corticosteroids, NB-UVB and vitamin D analogues. Informed consent was obtained for all patients, and personal history was evaluated. Four patients reported no comorbidities and one patient reported celiac disease(table1). Table 1 Patient demographics A. Janowska et al. Patient Gender Age Body area Comorbidity 1 Woman 24 Shin Celiac disease 2 Man 26 Elbow None 3 Woman 23 Elbow None 4 Woman 61 Hand None 5 Man 67 Neck None Epidermal skin harvesting and grafting We obtained epidermal skin grafts using the epidermal harvesting system that combines negative pressure and heat to form epidermal micrografts (6 8). The donor area (inner thigh) was cleaned with saline solution, and body hair was removed where necessary. The harvester head was applied on the inner thigh. The heat and negative pressure facilitated the separation of dermis and epidermis through the lamina lucida in 30 40 minutes. The formation of micrografts was visible in the lighted viewing window. The time needed to form micrografts depended on the patients medical therapies and the thickness of their skin. Dermabrasion was used to prepare the recipient site as some studies have shown that dermabrasion of the achromic macules is a simple, economic and effective method with minimal risk of scarring (10). Curettage of the achromic site of vitiligo to be treated was performed using 2% lidocaine for local anaesthesia and with a sterile curette until the Auspitz s sign, without significant bleeding (Figure 1). Micrografts were transferred directly from the harvester to the recipient site using non-adherent dressing with a layer of silicone (ADAPTIC TOUCH Non-Adhering Silicone Dressing, Systagenix, an ACELITY Company, Gargrave, UK). The micrografts were covered with a hydrocolloid dressing to maintain adequate hydration. The donor area was covered by non-adherent dressing (ADAPTIC TOUCH Dressing, Systagenix, an ACELITY Company, Gargrave, UK) and patched with gauze. The duration of all procedures was approximately 50 minutes. Figure 1 Curettage of recipient site. Table 2 Patient-reported pain score during epidermal harvesting Patient Wong-Baker FACES score* 1 2 2 1 3 1 4 2 5 1 *Wong-Baker FACES score = patient-reported pain on a scale from 0 (no hurt) to 10 (hurts worse). was performed after 7 days, 14 days, 1 month, 2 months and 3 months. In order to assess engraftment, a digital camera was used for clinical evaluation, and dermoscopy (HEINE Delta 20 T Dermatoscope, HEINE Optotechnik, Herrsching, Deutschland) was performed through the non-adherent dressing. In the absence of spontaneous repigmentation, we performed an NB-UVB therapy twice per week over a 2-month period. A twice-weekly treatment schedule was used as in previous clinical studies, and the uninvolved skin remained protected (11). Patient-reported pain during epidermal harvesting Pain during and after harvesting was evaluated using the Wong-Baker FACES pain score. The pain score was recorded as:0 nopain;1 verylowpain;2 lowpain;3 moderate pain; 4 severe pain; and 5 very severe pain. Follow-up Assessment of repigmentation We used the vitiligo area scoring index (VASI) to evaluate the repigmentation. VASI is a standard method for calculating the percentage of repigmentation and de-pigmentation described by Hamzavi et al. in 2004 (12). The patients body is divided 48 2016 Medicalhelplines.com Inc and John Wiley & Sons Ltd

A. Janowska et al. Epidermal micrografts in vitiligo Figure 2 Representative photo depicting epidermal micrograft engraftments 7 14 days post epidermal grafting. The whole graft recipient site (A) and a 10 magnification of the micrograft (HEINE Delta 20T Dermatoscope, HEINE Optotechnik, Herrsching, Deutschland) (B) are shown. Figure 3 Photo showing 100% repigmentation following epidermal skin grafting. The patient s hand prior to epidermal grafting (A) and 1 month after epidermal grafting (B) are shown. into five areas: the hands, upper extremities, trunk, lower extremities and the feet. The face and neck are not included in this evaluation. The VASI is based on the extension of repigmentation of the treated area and ranges between 100% (total repigmentation) and 0% (no repigmentation). Possible middle values are 0 25%, 25 50%, 50 75% and 75 100%. The cosmetic results may be described as excellent, good, sufficient or bad compared with the adjacent normal skin (9). Results From January to June 2015, five patients (two men and three women) with a mean age of 40 2 years (range 23 67 years old) and with a Fitzpatrick Skin Type 3 were enrolled in our study and treated with epidermal skin grafts. The treated body sites were elbows (n = 2), hand (n = 1), neck (n = 1) and shin (n = 1). Micrograft engraftments were observed, both clinically and demoscopically, between 7 and 14 days post grafting in all patients (Figure 2A and B). The donor sites were fully healed without scarring within 14 days of harvesting and required no further treatment. No reactivation of the vitiligo or other complications, such as infection or Koebner phenomenon, were noted during the follow-up period. The patients reported only minimal discomfort caused by local anaesthetic at the recipient site during curettage of the vitiligo lesion and heat sensation during harvesting, with an average pain score of 1 4 (range 1 2) (Table 2). We saw the first repigmentation results after 1 month. A 61-year-old female with stable vitiligo of the left hand showed 100% repigmentation Table 3 Skin graft cosmetic and repigmentation outcomes Patient VASI Cosmetic outcome Time to repigmentation (months) 1 50 75% Good 3 2 50 75% Good 3 3 50 75% Good 3 4 100% Excellent 1 5 50 75% Good 3 VASI, vitiligo area scoring index. of the treated area, with an excellent cosmetic outcome after a month (Figure 3 and Table 3). The other four patients showed minimal repigmentation; as such, a standard dose of NB-UVB was performed twice per week for 2 months. After 2 months, a good cosmetic outcome was observed, with 50 75% repigmentation observed in the remaining four patients (Figure 4 and Table 3). Discussion Epidermal micrografts for repigmentation of vitiligo were originally described in 1971. The lack of a reliable harvesting methodology could cause improper orientation of the graft, resulting in possible graft failure. This early epidermal skin harvesting also had limited use in clinical practice because of the long duration (3 hours, approximately) and poor cosmetic outcomes (13). Other surgical standard therapies are available but can be time-consuming, in some cases, with unsatisfactory 2016 Medicalhelplines.com Inc and John Wiley & Sons Ltd 49

Epidermal micrografts in vitiligo A. Janowska et al. Figure 4 Representative photo of 50 75% repigmentation. Vitiligo area prior to epidermal grafting (A) and 2 months after epidermal grafting (B) are shown. outcomes and pain for the patients. The epidermal harvesting system is a commercially available, minimally invasive device that does not require anaesthesia, hospitalisation or an operating theatre. Additionally, epidermal skin grafting has successfully been utilised in patients with acute and hard to heal chronic wounds, burns and stable vitiligo (14,15). In our study, five patients with stable localised vitiligo were treated with epidermal skin grafts. The achromic areas were prepared by curettage with local anaesthetic, and the epidermal harvesting system was used to obtain epidermal micrografts from the inner thigh. In our preliminary experience, all patients showed micrograft engraftment between 7 and 14 days, with no adverse effects at the donor site. In comparison, a suction blister study (16) reported minimal scarring and minimal post-inflammatory hyperpigmentation at the donor sites. However, in another suction blister grafting study, hypopigmentation was observed at the donor sites of most study participants (17). It should be noted that these studies did not use the epidermal harvesting system and instead created epidermal blisters using standard suction blister epidermal grafting methods. Other published studies using the epidermal harvesting system in patients without vitiligo have also shown minimal to no adverse events at the donor or recipient site (18 20). These results suggest that epidermal skin harvesting using the commercially available epidermal harvesting system is safe for use in patients with stable vitiligo. Epidermal skin grafts were useful in our patients who did not respond to previous standard treatments, including phototherapy. One of our patients had a total repigmentation after one month; the remaining four patients showed a minimal repigmentation after one month and required adjuvant therapy with NB-UVB in order to obtain a better cosmetic outcome. These results are similar to other published studies. Patients who underwent suction blister epidermal grafting showed excellent repigmentation results and maintained persistent pigment retention at follow-up when grafts were applied to the face or lips (16). Additionally, 92% of patients in the Bao et al. study developed satisfactory repigmentation (>50% repigmentation) after 10 days (21). Li and colleagues used a modified suction blister method in 1100 patients (17). Complete repigmentation was observed in 20 6% of patients, and 51 6% of patients developed >50% repigmentation. However, 16 patients developed hypopigmentation at the recipient site within 6 12 months (17). In contrast, Budania et al. compared suction blisters with a cellular suspension grafting technique (22). Only 27% of patients that received suction blister grafting showed excellent repigmentation compared to 71% of the patients that received the cell suspension. In the suction blister group, 85% of the patients developed 75% repigmentation compared to 89% of the cell suspension group (22). These repigmentation results were lower than ours; however, they did not use the epidermal harvesting system nor did they apply NB-UVB treatment to encourage repigmentation. Available published literature does not describe the use of epidermal micrografts along with NB-UVB despite NB-UVB being the standard treatment for stable vitiligo. Our use of NB-UVB in patients that initially showed poor repigmentation may explain the improved results in our cohort. In our study, patients were asked to report pain during epidermal skin harvesting using the Wong-Baker FACES pain scale. Our patients reported minimal pain during epidermal harvesting (highest reported value was 2). While studies reporting pain following epidermal harvesting use the epidermal harvesting system are limited, these results are similar to the 50 2016 Medicalhelplines.com Inc and John Wiley & Sons Ltd

A. Janowska et al. Epidermal micrografts in vitiligo patient-reported pain from the Osborne et al. healthy human study (6,7). Here, the healthy study participants reported an averageof1 3 pain rating 20 minutes post epidermal harvesting system initiation. Immediately after harvesting, mean pain was reported at 1 0, which further decreased to 0 0 at 7 days post harvesting (6,7). Heat and negative pressure facilitate the separation of the dermis and epidermis through the lamina lucida, and the design of the disposable harvester permits the uniform creation of microdomes (epidermal skin micrografts). Studies have shown that new keratinocytes and melanocytes migrate and proliferate outward from the micrograft edge as well as secrete growth factors to further drive cell migration and proliferation until the entire area has been re-epithelialised (23 25). These findings were recently confirmed when Osborne et al. examined characteristics of epidermal skin grafts harvested from 15 healthy human study participants. In vitro experiments revealed that epidermal skin harvested from 12/15 patients displayed almost complete cell viability. Additionally, both keratinocytes and melanocytes were retained in the micrografts and were able to proliferate and secrete growth factors in culture (3/15 subjects) (6,7). In summary, the use of advanced therapies may help improve the quality of life, have cost benefits and accelerate repigmentation of patients with vitiligo. In our preliminary study, epidermal skin harvesting and grafting was seen to be a safe and efficacious treatment in patients with stable vitiligo, unresponsive to standard therapies. Follow-up studies and new clinical trials with a standard measure are needed to confirm the efficacy of this new treatment, but the current results have proved to be promising. 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