The Journal Of Dermatology
Japanese Dermatological Association
아래 내용은 JDA에 게재된 논문내용입니다.
[ 유레카피부과에서 치료받은 환자 케이스입니다. ]
Skin seeding technique with 0.5-mm micropunch grafting for
vitiligo irrespective of the epidermal–dermal orientation:
Animal and clinical studies
Dong Seok KIM,1,* Hyun Jeong JU,2,* Han Na LEE,2 In Hye CHOI,3 Sung Hye EUN,2
Jiehoon KIM,4 Jung Min BAE2
1 Eureka Skin & Laser Clinic, Seoul, 2 Department of Dermatology, St Vincent’s Hospital, College of Medicine, The Catholic University
of Korea, Suwon, 3 Department of Dermatology, Bucheon St Mary’s Hospital, College of Medicine, The Catholic University of Korea,
Bucheon, 4 Dr Kim’s Skin & Laser Clinic, Suwon, Korea
Micropunch grafting is the simplest surgical intervention for refractory vitiligo but is tedious and time-consuming.
Therefore, we aimed to verify the efﬁcacy and safety of dermal orientation grafting using motorized 0.5-mm
micropunch grafting for vitiligo. In a preliminary animal study, 12-week-old rats were used to observe the healing
process after the transplantation of dermal orientation grafts with various punch sizes. In a clinical trial, a total of
100 vitiligo patches in 50 patients with stable vitiligo were randomly allocated to motorized 0.5-mm micropunch
grafting in epidermal and dermal orientations, respectively. The grafts were implanted at intervals of 5 mm at the
recipient site. Treatment success was deﬁned as greater than 75% repigmentation. In the animal study, all grafts
were shown to be well integrated into the recipient site within 3 weeks. In the clinical trial, treatment success
was achieved in 72% and 76% of the epidermal and dermal orientation groups, respectively; a cobblestone
appearance was observed in 4% and 2%, respectively. In conclusion, we demonstrated that this new grafting
method irrespective of epidermal–dermal orientation using motorized 0.5-mm micropunch grafting was effective
and safe. We have named this the “skin seeding technique” and it differs from traditional punch grafting in that it
can be performed regardless of the graft orientation.
Key words: intervention, orientation, punch grafting, surgery, transplantation.
Vitiligo is a common depigmentation skin disorder with 1%
prevalence worldwide that profoundly inﬂuences the quality
of life of affected patients.1,2 Although phototherapy is the
mainstay for the treatment of vitiligo, not all patients can be
treated with such intervention.3,4 Thus, surgery that trans-
plants melanocytes from normal pigmented skin to depig-
mented areas has been used as an alternative for refractory
vitiligo that has failed to improve with non-surgical interven-
Surgical interventions for vitiligo include punch grafting, suc-
tion blister grafting and cellular grafting.7 While autologous
non-cultured epidermal cell suspension transplantation has
gained popularity in recent years,8,9 punch grafting remains the
simplest and most readily available technique for small vitiligo
lesions.10 It has often been noted that punch grafting is time
consuming and less efﬁcient, and that it involves a risk of
cobblestoning. However, the introduction of motorized 0.8-mm
micropunch grafting has remarkably reduced the procedure
time and improved the ﬁnal outcome while minimizing the inci-
dence of cobblestoning.11 Nevertheless, a smaller graft size
can cause difﬁculty in ensuring implantation in the correct ori-
entation with the unaided eyes. Therefore, we developed a
new surgical technique using motorized 0.5-mm micropunch
grafting that allows implantation regardless of the orientation,
thus saving time and effort. We have named this method the
skin seeding technique (SST), which differs from the classic
punch grafting method in which the grafts must be implanted
in the upright direction.
In the present study, we investigated whether the melano-
cytes in dermal orientation can settle in the basal layer as in
epidermal orientation. We also assessed the efﬁcacy and
safety of the SST in a clinical 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).
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).
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-
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.
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.
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.
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).
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).
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
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).
Demographics and baseline clinical characteristics of
patients undergoing motorized micropunch grafting for the
treatment of vitiligo
Total patients, n
Sex, n (%)
Age, median (range), years
Disease duration, median (range), years
Subtype, n (%)
Total enrolled patches, n
Enrolled body sites, n (%)
Face and neck
Hands and feet
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).
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).
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.
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-
Treatment response and adverse effects between the
epidermal and dermal orientation groups
*McNemar’s tests were used to assess differences between the two
(n = 50)
(n = 50)
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 epidermal–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 comparable 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 smaller punch grafting sizes yielded better results with a less frequent 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 dermal orientation were eventually well integrated into the surrounding tissue at 2 weeks postoperatively. In addition, human
skin samples from the dermal orientation group showed that
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 surrounding vitiligo lesion (red stars) (Melan-A, [c] 9100, [d] 9400).
punch grafts were successfully incorporated at 2 weeks postoperatively; 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). Therefore, we concluded that the repigmentation resulted from melanocyte insertion into the dermis.
It remains unclear how melanocytes implanted in the dermis
reached the basal layer of the epidermis. Presumably, E-cadherin (the major adhesion molecule between basal keratinocytes
and melanocytes) is involved in the correct implantation.18 During transepidermal elimination, basal keratinocytes may capture
melanocytes so that they can settle at the correct layer. An alternative hypothesis is that the injury and healing process of the
punch grafting procedure itself may induce some pro-melanogenic factors or cytokines, which create a favorable environment 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. Compared 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 helpful 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 condition.
This study has some limitations. First, possible complications of the procedure include inflammation or immune-mediated 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 orientation, 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.
This study was supported in part
by the Catholic Medical Center Research Foundation in program year
CONFLICT OF INTEREST:
1. Lee H, Lee MH, Lee DY et al. Prevalence of vitiligo and associated
comorbidities in Korea. Yonsei Med J 2015; 56: 719–725.
2. Richard MA, Corgibet F, Beylot-Barry M et al. Sex- and age-adjusted prevalence estimates of five chronic inflammatory skin diseases in France: results of the << OBJECTIFS PEAU >> study. J
Eur Acad Dermatol Venereol 2018; 32: 1967–1971.
3. Bae JM, Jung HM, Hong BY et al. Phototherapy for vitiligo: a systematic review and meta-analysis. JAMA Dermatol 2017; 153: 666–674.
4. Lee JH, Kwon HS, Jung HM et al. Treatment outcomes of topical
calcineurin inhibitor therapy for patients with vitiligo. A systematic
review and meta-analysis. JAMA Dermatol 2019; 155(8): 929.
5. Mulekar SV, Isedeh P. Surgical interventions for vitiligo: an evidence-based review. Br J Dermatol 2013; 169(Suppl 3): 57–66.
6. Taieb A, Alomar A, Bohm M et al. Guidelines for the management
of vitiligo: the European Dermatology Forum consensus. Br J Dermatol 2013; 168: 5–19.
7. Mohammad TF, Hamzavi IH. Surgical therapies for vitiligo. Dermatol
Clin 2017; 35: 193–203.
8. Altalhab S, AlJasser MI, Mulekar SV et al. Six-year follow-up of vitiligo patients successfully treated with autologous non-cultured melanocyte-keratinocyte transplantation. J Eur Acad Dermatol Venereol
2019; 33: 1172–1176.
9. Silpa-Archa N, Griffith JL, Huggins RH et al. Long-term follow-up of
patients undergoing autologous noncultured melanocyte-keratinocyte transplantation for vitiligo and other leukodermas. J Am
Acad Dermatol 2017; 77: 318–327.
10. Thakur V, Kumar S, Kumaran MS, Kaushik H, Srivastava N, Parsad
D. Efficacy of transplantation of combination of noncultured dermal
and epidermal cell suspension vs epidermal cell suspension alone
in vitiligo. A Randomized Clinical Trial. JAMA Dermatol 2019; 155(2):
11. Bae JM, Lee JH, Kwon HS, Kim J, Kim DS. Motorized 0.8-mm
micropunch grafting for refractory vitiligo: a retrospective study of
230 cases. J Am Acad Dermatol 2018; 79(4): 720–727.e721.
12. Van Geel N, Passeron T, Wolkerstorfer A, Speeckaert R, Ezzedine
K. Reliability and validity of the Vitiligo Signs of Activity Score
(VSAS). Br J Dermatol 2020; https://doi.org/10.1111/bjd.18950
13. Bae JM, Oh SH, Kang HY et al. Development and validation of the
Vitiligo Extent Score for a Target Area (VESTA) to assess the treatment response of a target lesion. Pigment Cell Melanoma Res 2019;
14. Kumar SA, Sarin RC, Puri VK. Evaluation of replacement grafts and
punch grafts in the treatment of vitiligo. Indian J Dermatol Venereol
Leprol 1980; 46: 140–145.
15. Komen L, Vrijman C, Prinsen CA, van der Veen JP, Luiten RM,
Wolkerstorfer A. Optimising size and depth of punch grafts in autologous transplantation of vitiligo and piebaldism: a randomised controlled trial. J Dermatolog Treat 2017; 28: 86–91.
16. Lee JH, Fisher DE. Melanocyte stem cells as potential therapeutics
in skin disorders. Expert Opin Biol Ther 2014; 14: 1569–1579.
17. Kovacs D, Abdel-Raouf H, Al-Khayyat M et al. Vitiligo: characterization of melanocytes in repigmented skin after punch grafting. J Eur
Acad Dermatol Venereol 2015; 29: 581–590.
18. Tang A, Eller MS, Hara M, Yaar M, Hirohashi S, Gilchrest BA. Ecadherin is the major mediator of human melanocyte adhesion to
keratinocytes in vitro. J Cell Sci 1994; 107(Pt 4): 983–992.