| Abstract|| |
Background: Vitiligo surgery has come up a long way from punch skin grafts to epidermal cell suspension and latest to the extracted hair follicle outer root sheath cell suspension (EHFORSCS) transplantation. The progressive development from one technique to the other is always on a quest for the best. In the latest development, EHFORSCS, which is an enriched source of follicular inactive melanocyte (melanocyte stem cells), seems to be a good addition to the prevailing cell-based therapies for vitiligo. However, it needs to be explored further in larger, clinical trials. Methodology: A total of 11 patients with sixty stable vitiligo sites attending dermatology outpatient department were included for the open-labeled, prospective, comparative study. The sites were sequentially distributed into two groups of thirty each. Sites of one group were subjected to epidermal melanocyte transfer (EMT) and the others to hair follicular melanocyte transfer (HFMT). Response to treatment was evaluated on the basis of degree of repigmentation; final evaluation of area of involvement was done after completion of 6 months. Results: At the end of 6 months, repigmentation >90% was observed in 83.33% patches of EMT group and 43.33% in HFMT group. Repigmentation >75% was observed in 90% of patches in Group A and 43.34% of patches in Group B, respectively. There was statistically significant difference in the overall pigmentation between these two groups. Conclusion: Both noncultured autologous epidermal cell suspension transfer and noncultured EHFORSCS transfer are safe and effective surgical modalities in the management of stable vitiligo though EMT has shown a better response in the present study. Outer root sheath cell suspension transfer is a novel, minimally invasive technique in its nascent stage in the surgical management of vitiligo which requires further larger clinical trials for evaluation of its efficacy.
Keywords: Epidermal cell suspension, follicular unit extraction, noncultured autologous melanocyte transfer, outer root sheath cell suspension, stable vitiligo
|How to cite this article:|
Donaparthi N, Chopra A. Comparative study of efficacy of epidermal melanocyte transfer versus hair follicular melanocyte transfer in stable vitiligo. Indian J Dermatol 2016;61:640-4
|How to cite this URL:|
Donaparthi N, Chopra A. Comparative study of efficacy of epidermal melanocyte transfer versus hair follicular melanocyte transfer in stable vitiligo. Indian J Dermatol [serial online] 2016 [cited 2021 Jun 13];61:640-4. Available from: https://www.e-ijd.org/text.asp?2016/61/6/640/193671
What was known?
Epidermal Melanocyte transfer has become a known and popular modality for treatment of stable vitiligo.
| Introduction|| |
Vitiligo is an acquired disorder of pigmentation characterized by depigmented or hypopigmented macules caused by loss of epidermal melanocytes. It is a multifactorial disorder  related to both genetic and nongenetic factors. Vitiligo is separated into two main variants - nonsegmental vitiligo (NSV) and segmental vitiligo, each with a different presentation and clinical course. 
Various therapeutic options for repigmentation of vitiligo are available including corticosteroids, calcineurin derivatives, and phototherapy. Surgical treatment is indicated in stable disease not responding to medical treatment. There are various surgical modalities  available for vitiligo, which are based on the idea of restoring melanocytes on the recipient site. They are classified into tissue grafts and cellular grafts. Tissue grafts can treat only a small area; therefore, cellular grafts such as basal cell layer-enriched epidermal suspension are preferred over tissue grafts when a large area is to be treated.
Since the introduction of the noncultured cell transplantation technique by Gauthier and Surleve-Bazeille  in 1992, the field of vitiligo surgery has progressed a lot. However, most of the surgeries done till date focus mainly on taking the skin biopsy by one or the other ways and preparing the cell suspension and finally transplantation. Noncultured autologous extracted hair follicular melanocyte transfer (HFMT) is a new addition to the prevailing techniques and it utilizes melanocytes in hair follicle instead of epidermis.  Since it is a novel procedure in its nascent stage, clinical trials are required to prove its efficacy in the management of stable vitiligo though theoretically it is better than existing noncultured cellular grafting techniques.
| Methodology|| |
This was a prospective, single-center, open-labeled comparative study of the efficacy of epidermal melanocyte transfer (EMT) versus HFMT in the management of stable vitiligo. The patients in this study were drawn from the outpatient Department of Dermatology, Venereology and Leprosy at Command Hospital Air Force, Bengaluru, for 1 years from October 2013 to March 2015. Ethical clearance was obtained before starting the study.
A total of sixty stable sites, thirty in each group, were enrolled for the study. All patients above 12 years of age and both sexes with stable vitiligo, which is defined as no new lesions or increase in the size of existing lesions in the past 1 year, were included in the study. Patients with progressive vitiligo, patches on scalp, chronic diseases, history of koebnerization, and keloidal tendencies were excluded from the study.
Informed consents were taken from all the patients. Each patient in the study underwent a detailed clinical, general physical, systemic, and a thorough dermatological examination. A set of investigations consisting of hemoglobin, total leukocytic count, differential leukocytic count, and blood sugar (fasting and postprandial) was carried out before the procedure. Clinical photographs were taken before the surgical procedure and each month thereafter using standard aperture and lighting for the assessment. The sites were sequentially distributed into two groups of 30 each: Initial 30 into Group A and later 30 into Group B. Sites of Group A were subjected to EMT. Sites of Group B were subjected to HFMT. To calculate the size of vitiligo lesions, margins of rectangular lesions and radius of circular lesions were measured with a centimeter scale. Irregular big lesions were first divided into nearest geometrical shapes, and then the area was calculated.
Preparation of autologous noncultured basal cell layer-enriched epidermal cell suspension
After surgical preparation, a split-thickness skin graft (STSG) of size of about one-tenth of the recipient site was obtained from donor area, preferably, anterolateral aspect of thigh with the help of dermatome. The epidermal graft was washed in normal saline and then transferred to a petridish containing trypsin ethylenediaminetetraacetic acid (EDTA). The petridish along with the grafts was incubated in an incubator at 37°C for 50 min with epidermal side facing upward. The grafts were then transferred to a petridish containing trypsin inhibitor and left immersed in it for 5 min to neutralize the action of trypsin-EDTA with dermal side facing upward. Following this, the grafts were transferred to a petridish containing melanocyte nourishment medium, i.e., Dulbecco's Modified Eagle Medium/nutrient mixture F12 (DMEM/F12). The epidermis was gently separated from the dermis with forceps and the dermal pieces were discarded. The epidermal pieces were gently teased to separate the melanocytes which fell into the medium. The epidermis was teased till the point where there was no pigment left on their surface. The transparent epidermal pieces were discarded and the remaining DMEM solution containing melanocytes was transferred to a centrifuge tube. The tube was centrifuged for 10 min at 2000 rpm. The cell pellet settled down to the bottom. The floating epidermal pieces along with supernatant fluid were discarded leaving the cell pellet at the bottom. The pellet was resuspended in a small quantity of DMEM.
Preparation of noncultured extracted hair follicle outer root sheath cell suspension
After surgical preparation and local anesthesia, 15-25 hair follicular units (FUs) are harvested randomly from occipital scalp to cover approximately an area of 10-25 cm 2 . One millimeter punch was used, rotated in the direction of the hair follicle until it reached the mid-dermis. Then, the FU was pulled out gently using hair follicle holding forceps and collected in a petridish containing trypsin-EDTA. Hemostasis was achieved by pressure and the donor site was either kept open or dressed with antibiotic ointment with pressure dressing using roller bandage which was retained for 6-12 h. The petridish along with the grafts was incubated in an incubator at 37°C for a total duration of 90 min with serial incubations of 30 min each to separate outer root sheath cells. Trypsin inhibitor was added and left for 5 min to neutralize the action of trypsin-EDTA. Cell suspension was then filtered through 70 micron cell strainer. Cell suspension containing melanocytes was transferred to centrifuge tubes. The tubes were centrifuged for 5 min at 1000 rpm to obtain a cell pellet. The cell pellet was resuspended in a small quantity of DMEM.
Transfer of noncultured basal cell-enriched epidermal suspension or noncultured extracted hair follicle outer root sheath cell suspension to recipient area
Technique of melanocyte transfer is similar in both groups. Superficial dermabrasion of the vitiliginous areas was done with a manual dermabrader. Ideal level was when pinpoint bleeding spots appeared. The denuded areas were covered with gauze pieces moistened with normal saline/isotonic sodium chloride solution after achieving adequate hemostasis. The cell suspension obtained was applied evenly on the denuded area and spread uniformly with a spatula. The transplanted areas were covered with a dry collagen sheet and Tegaderm Transparent Dressing. This was covered with sterile gauze pieces moistened with DMEM/F12, dry gauze pieces and held in place with micropore and elastoplast dressings. The patient was immobilized for 30 min and then allowed to go with the instructions to do only restricted movements for next 7 days. Oral antibiotics, analgesics, and anti-inflammatory drugs were given for 5 days. Dressing of donor area was changed on alternate days and of the recipient area was removed after 7 days. Scrubbing was avoided for further 1 week although patient was allowed to have a bath. On alternate day, sun exposure of the transplanted site was started 15 days after removing the dressing.
The patients in both groups were followed up at monthly intervals for 6 months. The response to the therapy was assessed subjectively as well as objectively. Subjective assessment was done by the patient. Objective assessment was carried out using photographic evidence with standard aperture and lighting by two separate observers. Response to treatment was evaluated on the basis of degree of repigmentation, which was carried out at monthly intervals. The final evaluation of area of involvement was done after completion of 6 months. The response to the treatment based on the percentage of visible repigmentation was graded as follows: Poor: 0-25%; good: 26-50%; very good: 51-75%; and excellent: 76-100%.
The response in terms of onset of repigmentation, type of repigmentation, and degree of repigmentation in both groups was considered for the analysis. The results were statistically analyzed using "Chi-square test" and "statistical significance" (P value) methods.
[Figure 1] [Figure 2] [Figure 3] [Figure 4] [Figure 5]
| Results|| |
|Figure 1: Group A response to epidermal melanocyte transfer over foot. a) Before treatment, b) Patches showing > 90% pigmentation 6 months after treatment|
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|Figure 2: Group A response to epidermal melanocyte transfer in two patches over abdomen. a) Before treatment, b) Patches showing 90% and 50% pigmentation after treatment|
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|Figure 3: Group B response to hair follicular melanocyte transfer over leg. a) Before treatment, b) Patches on leg showing 100% and >75% repigmentation at the end of 6 months |
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|Figure 4: Group B response to Hair follicular melanocyte transfer around knee joint. a) Before treatment, b) Perifollicular repigmentation <25% 6 months after treatment|
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|Figure 5: Comparison of degree of repigmentation (objective) in both groups at the end of 6 months|
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All patients completed the study period of 24 weeks. Therefore, data of thirty sites each of both groups were considered for the final statistical analysis.
Demographic characteristics are represented in [Table 1]. Patients were not comparable with regard to age and sex but type of vitiligo. The majority (9/11) of the patients had NSV. Distribution of vitiligo lesions was not comparable with regard to duration of vitiligo and period of stability. Both Group A and Group B had a maximum number of vitiligo lesions over the extremities (70% and 93.3%, respectively) whereas only a few lesions were seen over trunk and acral parts with no lesions on flexures.
Group A exhibited earlier onset of repigmentation when compared to Group B. In Group A, 40% of the patches started to repigment by 1-14 days postprocedure and all the patches started to repigment by the end of the 1 st month. Onset of pigmentation was later in Group B (between 15 and 30 days postprocedure). This was statistically significant. All three types of repigmentation-diffuse, perifollicular, and marginal were observed in the study. Group A showed diffuse type of repigmentation in maximum patches (66.67%) whereas marginal type of repigmentation was seen more (50%) in Group B. At the end of 6 months, in Group A, a larger number of patches (80%) were assessed to have "excellent" repigmentation subjectively as compared to Group B (33.33%). Objectively, 90% of Group A patches and 43.34% of Group B patches exhibited > 75% pigmentation. Statistically significant difference in the degree of repigmentation was observed between the two groups. The degree of repigmentation was independent of gender, type or duration of vitiligo, period of stability, and anatomical site of the patch. However, smaller patches repigmented better than the larger ones in the present study. Minimal complications were observed over both the donor and recipient areas in both groups. There was hyperpigmentation in Group A and insignificant and invisible scarring in Group B over donor area. The main complication at the end of 6 months over recipient sites was perigraft halo in Group A and color mismatch in Group B, noted over a few patches. The repigmented sites retained their pigmentation till the end of follow-up period in both groups.
| Discussion|| |
Vitiligo surgery has come up a long way from punch skin grafts to epidermal cell suspension and latest to the extracted hair follicle outer root sheath cell suspension (EHFORSCS) transplantation.  Surgical treatment is indicated in stable disease not responding to medical treatment. Surgical modalities are divided into tissue grafts and cellular grafts. Tissue grafting procedures include transplantation of minigrafts, thin and ultrathin STSG, and suction blister epidermal grafts. Cellular grafting techniques include transplantation of noncultured basal cell-enriched epidermal suspension (NCES) and cultured melanocytes or cultured epidermis. 
Since the introduction of the noncultured cell transplantation technique by Gauthier and Surleve-Bazeille  in 1992, the field of vitiligo surgery has progressed a lot. In the latest development, EHFORSCS, which is an enriched source of follicular inactive melanocyte (melanocyte stem cells), seems to be a good addition to the prevailing cell-based therapies for vitiligo. 
The hair follicle is an important reservoir of melanocytes and their precursor cells. They are localized in the outer layer of the outer root sheath of the infundibulum and midfollicle and the matrix of the hair bulb.  Comparison of epidermal and follicular melanin units  reveals that there are certain important differences between both of them. The ratio between keratinocytes and melanocytes is 1:6 in hair follicle whereas it is 1:36 in epidermis. Hair follicular melanocytes are more dendritic and size of the melanosomes is larger. They are located at immunologically protected sites, and hence, less susceptible to immune destruction. Furthermore, they are influenced by aging (hair graying).
Vanscheidt and Hunziker,  in a small case series, used single-cell suspension of "plucked" hair follicles in the treatment of vitiligo. They found almost complete (>90%) repigmentation in 3 of 5 patients with vitiligo, around 50% repigmentation in 1 patient and <10% repigmentation in 1 patient. Their technique was simple, noninvasive, and allowed easy, immediate, and repeated application. However, the cell yield was less in case of plucked hair follicles. Mohanty et al.  have modified the method of obtaining the follicular tissue by the FU extraction (FUE) method. They used autologous EHFORSCS for transplantation in vitiligo patients. This method resulted in significant repigmentation in the majority of the patients. Three of their patients had <1 year of disease stability and none of them had >75% repigmentation while 9 of 11 patients with at least 1 year stability had >75% repigmentation.
As per the literature search, only one group, Singh et al.,  has done a comparative study of two methods. The extent of repigmentation was excellent (90-100% repigmentation) in 83% of lesions in the NCES group and 65% of lesions in the Non cultured extracted hair follicle outer root sheath cell suspension (NCEHFORSCS) group. Repigmentation >75% (good repigmentation) was observed in 92% of lesions in the NCES group and 78% of lesions in the NCEHFORSCS group. They concluded that both epidermal cell suspension and hair follicle outer root sheath cell suspension are safe and effective techniques with comparable efficacy.
We observed similar results in Group A (repigmentation >90% was observed in 83.33% and repigmentation >75% was observed in 90% of patches) but inferior results in Group B (repigmentation >90% was observed in 43.33% of patches). Probable reasons for inferior response obtained in Group B compared to Group A are inclusion of more number of elderly people in Group B and lack of proper surgical skills in FUE due to which insufficient (less number of FU) and ineffective (transected FU) donor area was utilized during the procedure.
Limitations of study
The study is an open-labeled study and not randomized. Furthermore, cell counting and viability testing have not been done which has been one limitation of this study.
| Conclusion|| |
Both autologous noncultured epidermal and HFMT methods are safe and effective surgical modalities in the management of stable vitiligo though EMT has shown a slightly better response in the present study. The degree of repigmentation in HFMT may be dependent on age as follicular melanocytes are influenced by aging. Hence, young patients are preferred for the HFMT technique. The degree of repigmentation is independent of gender, type or duration of vitiligo, period of stability, and anatomical site of the patch. However, it depends on the size of the lesion. Smaller patches repigmented better than the larger ones in the present study. Minimal complications were observed in both groups.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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What is new?
Epidermal melanocyte grafting and Hair follicular melanocyte transfer both are effective modalities of treating stable vitiligo though epidermal melanocyte grafting being more efficacious.
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]