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ORIGINAL ARTICLE

ABSTRACT

INTRODUCTION

METHODS

Animal study
Twelve-week-old Sprague–Dawley rats were used to observe the healing process after the transplantation of dermal orienta- tion grafts with different punch sizes: 3, 2, 1, 0.8 and 0.5 mm. Each graft was taken from the rat dorsum, turned upside-down (i.e. dermal orientation) and re-inserted in the same location. Tissues were harvested at 3 days, as well as at 1, 2 and 3 weeks after transplantation in order to examine sequential changes in the grafts and surrounding tissues over time. All animal procedures were approved by the Institutional Animal Care and Use Committee of St Vincent’s Hospital (approval no. IACUC 18-6).

Clinical trial
We conducted a comparative split-body trial from April 2017 to April 2018 at our clinic. We sought to compare the efficacy and safety of the two opposite grafting orientations (epidermal and dermal) of motorized 0.5-mm micropunch grafting. The inclusion criteria were as follows: (i) segmental or non-segmen- tal vitiligo; (ii) stable diseases over at least 1 year; (iii) failure with non-surgical interventions over at least 6 months; and (iv) absence of confetti-like depigmentation, tri- or hypochromic areas, and type B Koebner phenomenon.12
In total, 50 patients were enrolled. Two patches were selected from each patient and were randomly allocated to the epidermal orientation and dermal orientation groups. The study protocol was reviewed and approved by the institutional review board of St Vincent’s Hospital (approval no. VC17RESI0108).

Interventions
We performed micropunch grafting using a stainless-steel punch of 0.5 mm diameter loaded into the handpiece of a micromotor (i-graft; Ilooda, Suwon, Korea) for both the recipi- ent and donor sites. Donor sites were the normal skin on the posterior side of the ear, which was chosen because it is the most hidden area of others’ eyes, and also allows the practi- tioners to manipulate both the donor and recipient areas at the same time without the patient’s postural changes during facial vitiligo surgery. The recipient and donor sites were cleansed with an antiseptic solution and local anesthesia was induced using an injection of 2% lidocaine with epinephrine at a con- centration of 1:200 000. At the recipient sites, chambers for planting the grafts were constructed at intervals of 5 mm using the motorized micropunch, approximately 80 grafts per 25 cm2 area. At donor sites, the grafts were harvested at intervals of 1 mm using the same instrument. Harvested grafts were trans- planted to the recipient sites in the correct orientation for the epidermal orientation group, and in the upside-down orienta- tion (epidermis of the graft placed in the dermis of recipient chamber and dermis of the graft placed in the epidermis of recipient chamber) for the dermal orientation group. Hydrocol- loid and Steri-Strip (3M, Maplewood, MN, USA) dressings were applied to the donor and recipient sites, respectively. All partic- ipants were prescribed 20 mg of prednisolone for 1 week post- operatively.

At 1 week postoperatively, all patients began 308-nm xenon chloride excimer laser treatment (EXL-440 ; Laser and Physics, Yong-in, Korea) on 2 non-consecutive days, weekly for 3 months. The dose was initiated at 100 mJ/cm2 and increased by 50 mJ/cm2 at subsequent sessions until pink ery- thema appeared and did not persist for 24 h or more. Topical tacrolimus (0.1% w/v) ointment (Leo Pharma, Ballerup, Den- mark) was applied twice daily to all surgical sites throughout the laser treatment period.

Outcomes
The treatment response was rated by two dermatologists based on clinical photographs obtained at baseline and 3 months postoperatively. Treatment success was defined as 75% or more repigmentation of the surgery site using VESTA.13 A partial response was defined as 50–74% repig- mentation, and insufficient repigmentation was defined as less than 50% repigmentation.
For safety evaluation, we assessed adverse events, includ- ing a cobblestone appearance, color mismatch or scar, at every visit. Each item was evaluated using 4-point grades according to severity (grade 0, none; grade 1, some; grade 2, obvious; and grade 3, pronounced). Of these, grades 2 and 3 were considered adverse events.

Histopathological study
Among the participants, six gave informed consent for skin biopsy, and skin specimens were collected once from the der- mal orientation group at various time points after surgery. Immunohistochemical staining with hematoxylin–eosin and with Melan-A was conducted.

Statistical analyses
Because this was a preliminary study, a non-inferiority test was not performed. Instead, the proportions of treatment success, insufficient repigmentation and each adverse event were com- pared using McNemar’s tests between the two groups. All analyses were performed using R 3.6.1 (R Foundation for Sta- tistical Computing, Vienna, Austria).

RESULTS

Animal study
After punch grafting in the dermal orientation, a little inflamma- tion near the graft was observed at 3 days postoperatively. The grafts appeared to be resolved into the surrounding tissue at 1 week postoperatively. Grafts of all sizes (3, 2, 1, 0.8 and 0.5 mm) were well integrated into the recipient sites without any complications at 3 weeks postoperatively (Fig. 1).

Study population
The median age of the patients was 24.0 years (range, 6–67). Of the 50 patients, 52.0% had non-segmental vitiligo and 48.0% had segmental vitiligo. Of the 100 patches, 68 (68.0%) were on the face and neck, 18 (18.0%) were on the hands and feet, 10 (10.0%) were on the extremities and 4 (4.0%) were on the trunk. The sizes of the recipient sites were 22 lesions



Figure 1. Histological examination of a 1-mm punch graft in the dermal orientation in an animal model. (a) Three days after the transplantation of a 1-mm punch graft in the dermal orientation, a little inflammation was observed surrounding the graft (hema- toxylin–eosin [HE], original magnification 9100). (b) The graft appeared to be resolved into the surrounding tissue with reduced inflammation at 1 week postoperatively (HE, 9100). (c) The epidermal component of the graft had moved up to the epidermis at 2 weeks postoperatively (HE, 9100). (d) The graft was well integrated into the recipient site at 3 weeks postoperatively (HE, 9100).

(22.0%) in the size range of 1 cm2 or less, 76 (76.0%) in the size range of 1–9 cm2 and two (2.0%) in the size range of 50– 100 cm2 (Table 1).

Table 1. Demographics and baseline clinical characteristics of patients undergoing motorized micropunch grafting for the treatment of vitiligo

Clinical characteristics	Value
Total patients, n	50
Sex, n (%) Male Female	25 (50.0) 25 (50.0)
Age, median (range), years	24 (6–67)
Disease duration, median (range), years	5.5 (0.5–39)
Subtype, n (%) Segmental vitiligo Non-segmental vitiligo	24 (48.0) 26 (52.0)
Total enrolled patches, n	100
Enrolled body sites, n (%) Face and neck Extremities Trunk Hands and feet	68 (68.0) 10 (10.0) 4 (4.0) 18 (18.0)
Size, cm2 <1 1–9 10–49 ≥50	22 (22.0) 76 (76.0) 0 (0) 2 (2.0)

Treatment response
Overall, treatment success (≥75% repigmentation) was achieved in 36 (72.0%) patches in the epidermal orientation group and 38 (76.0%) patches in the dermal orientation group; however, there was no statistically significant difference between the groups (Fig. 2). Initial repigmentation was observed at the graft sites at 3 weeks postoperatively. No sig nificant difference in treatment response was observed between the two groups (Table 2).

Histopathological examination
Minimal inflammation was observed around the dermal-ori- ented grafts at 1 week postoperatively. The graft was inte- grated to the surrounding tissue and inflammation was absent at 2 weeks postoperatively. Melan-A staining indicated the pro- liferation of melanocytes in the basal layer of the lesion where grafting was performed, while melanocytes were not observed in the basal layer of the surrounding vitiligo lesion at 3 weeks postoperatively (Fig. 3).

Adverse effects
A cobblestone appearance was observed in 4% of grafts in the epidermal orientation group and 2% of grafts in the dermal orientation group. A color mismatch was reported in 2% of grafts in the epidermal orientation group and 2% of grafts in the dermal orientation group. No other side effects were observed. None of the adverse effects showed a significantly different incidence between the two groups.



Figure 2. The skin seeding technique using 0.5-mm micropunch grafting for refractory vitiligo. (a) The handpiece of the micromotor used for 0.5-mm micropunch grafting. A comparative split-body clinical trial was performed in 50 patients. Two patches were selected from each patient and were randomly allocated to the epidermal orientation and dermal orientation groups, respectively. A 62-year-old woman with vitiligo of the forehead (b) before and (c) 3 months after the procedure (right, dermal orientation; left, epi- dermal orientation). A 21-year-old woman with vitiligo of the eyelid (d) before and (e) 2 months after the procedure (right, epidermal orientation group; left, dermal orientation). A 42-year-old man with vitiligo of the philtrum (f) before and (g) 3 months after the proce- dure (dermal orientation). A 74-year-old woman with vitiligo of the right cheek (h) before and (i) 3 months after the procedure (epi- dermal orientation).

Table 2. Treatment response and adverse effects between the epidermal and dermal orientation groups

	Epidermal orientation (n = 50)	Dermal orientation (n = 50)	P*
Treatment success	36 (72.0%)	38 (76.0%)	0.480
Insufficient repigmentation	11 (22.0%)	10 (20.0%)	1.000
Cobblestone appearance	2 (4.0%)	2 (4.0%)	1.000
Color mismatch	1 (2.0%)	1 (2.0%)	1.000

*McNemar’s tests were used to assess differences between the two groups.

DISCUSSION

Surgical treatment is essential to manage vitiligo that is refrac- tory to non-surgical interventions. Since its introduction in 1980,14 punch grafting has been proven simple, safe and inex- pensive, especially for the treatment of small localized lesions of vitiligo. Motorized 0.8-mm micropunch grafting has markedly reduced the surgery time and improved the outcomes. In this study, we demonstrated the efficacy and safety of the SST with motorized 0.5-mm micropunch grafting, which differs from traditional punch grafting in that it is irrespective of the epider￾mal–dermal orientation.
In our comparative split-body trial, we found no significant difference in final repigmentation between the epidermal and dermal orientation groups. The overall results were also com￾parable to those of our prior study using motorized 0.8-mm micropunch grafting.11 Furthermore, the occurrence of adverse events, including a cobblestone appearance, was less frequent with the SST than in the prior report, although our findings may be influenced by the small sample size in the present study and differences in the postoperative follow-up periods between studies. This is consistent with previous studies, in which smal￾ler punch grafting sizes yielded better results with a less fre￾quent cobblestone appearance.15
In our preliminary animal study, we confirmed that punch grafts of all sizes (3, 2, 1, 0.8 and 0.5 mm) inserted in the der￾mal orientation were eventually well integrated into the sur￾rounding tissue at 2 weeks postoperatively. In addition, human skin samples from the dermal orientation group showed that



Figure 3. Melanocytes implanted in the basal layer of the epidermis after 0.5-mm micropunch grafting in the dermal orientation. (a) The dermal-oriented micropunch graft was found in the dermis at 1 week postoperatively (hematoxylin–eosin [HE], original magnifi- cation 940). (b) The dermal-oriented micropunch graft was well integrated into the recipient site at 2 weeks postoperatively (HE, 940). (c,d) Melanocytes were observed along the basal layer of the epidermis at the recipient site (black arrows), but not on the sur￾rounding vitiligo lesion (red stars) (Melan-A, [c] 9100, [d] 9400).

punch grafts were successfully incorporated at 2 weeks post￾operatively; moreover, active melanocytes were present in the basal layer of the epidermis, whereas no melanocytes were present in the epidermis near the graft sites. It is unlikely that the melanocyte stem cells in the dermis could be the source of repigmentation16,17 because no repigmentation was observed after grafts involving only the dermal compartment without transplanted epidermis in five patients (data not shown). There￾fore, we concluded that the repigmentation resulted from mela￾nocyte insertion into the dermis. It remains unclear how melanocytes implanted in the dermis
reached the basal layer of the epidermis. Presumably, E-cad￾herin (the major adhesion molecule between basal keratinocytes and melanocytes) is involved in the correct implantation.18 Dur￾ing transepidermal elimination, basal keratinocytes may capture melanocytes so that they can settle at the correct layer. An alter￾native hypothesis is that the injury and healing process of the punch grafting procedure itself may induce some pro-me￾lanogenic factors or cytokines, which create a favorable environ￾ment for melanocyte stimulation. Additional studies are needed for detailed pathogenesis.
Although punch grafting is often underestimated, it remains a promising option for patients with refractory vitiligo. Com￾pared with autologous non-cultured epidermal cell suspension transplantation, punch grafting does not require a highly trained medical team and specialized equipment. With the development of motorized micropunch grafting, including the SST, the punch grafting procedure could be performed by a novice without assistants at a private clinic. This may be help￾ful to treat scattered remaining vitiligo lesions after prior surgical treatment, as well as after non-surgical treatment. No single method is ideal for all patients; it is meaningful to have various treatment options depending on the patient’s condi￾tion.
This study has some limitations. First, possible complica￾tions of the procedure include inflammation or immune-medi￾ated rejection. We did not encounter any of these adverse events, potentially due to the use of prednisolone (20 mg) for 1 week postoperatively. Second, the sample size was small and the follow-up period was short. Lastly, the mechanism involved was not investigated in our study.
In conclusion, even though micropunch grafting has brought a great improvement, the procedure still requires extremely tedious and time-consuming work under naked eyes. In this study, we demonstrated that the 0.5-mm micropunch grafting with dermal orientation could successfully induce pigmentation as those with epidermal orientation, and motorized 0.5-mm micropunch grafting irrespective of the epidermal–dermal ori￾entation, which we named the SST, could make the procedure much easier and faster. Additional controlled trials and studies are needed to assess the mechanism involved.

ACKNOWLEDGMENTS: This study was supported in part by the Catholic Medical Center Research Foundation in program year 2017.

CONFLICT OF INTEREST: None declared.

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