Efficacy of Transplantation of Combination of Noncultured Dermal and Epidermal Cell Suspension vs Epidermal Cell Suspension Alone in Vitiligo: A Randomized Clinical Trial

Key Points

Question  Is surgical management of vitiligo with duration of clinical stability (DS) of 3 to 6 months feasible using a novel combination of noncultured dermal and epidermal cell suspension (NCES and NDCS)?

Findings  In this randomized clinical trial of 40 patients, more than 75% repigmentation at 24 weeks was observed in all 10 participants with vitiligo (100%) with DS of 3 to 6 months treated with NCES and NDCS compared with 3 of 10 (30%) patients with DS treated with NCES alone.

Meaning  Combined NCES and NDCS is a novel technique that can be used successfully in patients with vitiligo with shorter duration of disease stability (3-6 months).

Importance  Surgical interventions, notably noncultured epidermal suspension (NCES), are the next line of treatment in patients with vitiligo who fail to respond to medical therapy. Noncultured epidermal suspension is usually performed in patients with vitiligo with duration of clinical stability (DS) of 12 months or longer because DS is a vital parameter in determining outcome of NCES. In this pilot study, we planned to assess the efficacy of a novel combination of noncultured epidermal cell suspension and noncultured dermal cell suspension (NCES and NDCS) in patients with vitiligo with shorter DS (3-6 months).

Objective  To compare the efficacy of transplantation of NCES and NDCS vs NCES alone in patients with vitiligo with DS of 3 to 6 months.

Design, Setting, and Participants  A single-center randomized clinical trial including 40 patients with focal, segmental, or generalized vitiligo with DS of 3 to 6 months or more than 12 months was carried out. Based on DS, 2 groups including 20 patients each were recruited (DS in group 1, 3 to 6 months; DS in group 2, more than 12 months). Each group was further randomized into 2 subgroups, A and B.

Intervention  Patients in subgroups 1A and 2A underwent NCES alone, whereas patients in subgroups 1B and 2B underwent NCES and NDCS.

Main Outcomes and Measures  Extent of repigmentation, color match, and pattern of repigmentation at 24 weeks.

Results  Of the 40 study participants, mean (SD) age was 24.9 (4.0) years and 24 (60%) were women; in group 1 with DS for 3 to 6 months, more than 75% repigmentation at 24 weeks was observed in all 10 patients in subgroup 1B (NCES and NDCS) compared with 3 of 10 patients in subgroup 1A (NCES) (100% vs 30%, P = .003). In group 2 (DS > 12 months), the same was observed in 6 of 10 patients in subgroup 2A and 7 of 10 patients in subgroup 2B (NCES) (60% vs 70%, P > .99). The 2 groups and subgroups did not show any significant differences with respect to color matching and pattern of repigmentation.

Conclusions and Relevance  Combination of NCES and NDCS resulted in excellent response in patients with vitiligo with shorter duration of clinical stability compared with NCES alone. This combination may be used early in the course of stable vitiligo without waiting for a period of 12 months or more since last clinical activity.

Trial Registration  ClinicalTrials.gov identifier: NCT03013049

Vitiligo is a complex pigmentary disorder, associated with selective and progressive melanocyte loss resulting in development of depigmented patches on the skin. It is often associated with considerable psychosocial repercussions on patients. The prevalence of vitiligo worldwide is approximately 1%. It is more common in India, with a prevalence of approximately 4% (range, 0.46%-8.8%).^1,2

Although medical therapy is the mainstay of treatment, it is not uniformly effective in all patients and residual lesions often require surgical intervention to achieve complete repigmentation. Surgical methods, notably transplantation of noncultured epidermal cell suspension (NCES) have proven to be effective in stable vitiligo. A proper selection of cases for surgical therapy is of paramount importance and vitiligo stability is considered a vital parameter before considering any melanocyte transplantation technique. Although consensus regarding the ideal period of stability is lacking, usually duration of clinical stability (DS) longer than 1 year is considered optimal.^3,4

Various studies suggest that perilesional cytotoxic CD8⁺ T cells in vitiligo may adversely affect the outcome of melanocyte transplantation, particularly in patients with shorter DS, and strategies against CD8⁺ T cells may be beneficial for patients undergoing transplantation.^5-7 Dermal mesenchymal stem cells (DMCs) are known to inhibit T-cell (both CD4⁺ and CD8⁺) proliferation and induce T-cell apoptosis,^8-10 thus, the possible immunoregulatory properties of DMCs from noncultured dermal cell suspension (NDCS) might be used as an auxiliary agent to improve the efficacy of NCES in vitiligo patients particularly with DS of less than 12 months.¹⁰

We intended to carry out this prospective pilot study to compare a novel method of autologous transplantation of NCES and NDCS vs NCES alone in vitiligo patients.

This randomized clinical trial was carried out in the department of Dermatology, Venereology, and Leprology, Postgraduate Institute of Medical Education and Research (PGIMER), Chandigarh, India. Institutional ethical clearance was obtained and the trial was registered on ClinicalTrials.gov (NCT03013049). The trial protocol is available in Supplement 1.

Patients 18 years or older visiting outpatient department from January 2016 to November 2016 with focal, segmental, or generalized vitiligo with DS of either 3 to 6 months or more than 12 months, with lesions nonresponsive or partially responsive to medical therapy were included in the study after obtaining written informed consent. Pregnant women, and patients with history of koebnerization, keloidal tendencies, or bleeding disorders were excluded. Baseline clinical and demographic data of recruited patients was recorded. Size of vitiligo patch selected for surgical interventions in the study was less than 100 cm².

Based on DS, 2 groups comprising 20 patients with vitiligo each were recruited (Figure 1). Patient DS was defined as the time elapsed since last noticed activity in vitiligo lesions in the form of appearance of new lesions or the increase in the size of existing lesions. Group 1 comprised patients with DS of 3 to 6 months whereas group 2 comprised patients with DS longer than 12 months. Patients in both the groups were further randomized (simple randomization) into 2 subgroups of 10 patients each using a computer-generated random number table. Patients in the first subgroups of both the groups (subgroups 1A and 2A) underwent NCES alone, whereas those in the second subgroup of both the groups (subgroups 1B and 2B) underwent combination of NCES and NDCS. Centralized allocation concealment was performed. Random number table was generated by one of the authors (S.K.), participant enrollment was done by another one of us (M.S.K.), surgery was performed by 2 authors (V.T. and S.K.), and the outcomes were assessed by another author (D.P.). Cell suspension was prepared by 2 authors (H.K. and N.S.). The physician assessing the outcomes was blinded to the information regarding the patient’s group and subgroup. The design of the study is depicted in Figure 1.

A donor site with an area of about one-tenth the area of the recipient site was selected and a split-thickness skin graft (STSG) was harvested. The STSG was transferred to a trypsin-EDTA solution (0.25% trypsin and 0.02% EDTA) maintaining aseptic precautions. The specimen was incubated overnight at 4°C. A cell suspension was prepared after washing 3 times in phosphate buffer solution (PBS). The suspension was centrifuged at 1000 rpm for 5 minutes. The pellet-containing cells from stratum basale (rich in melanocytes) were taken. Using trypan blue staining, viable epidermal cells (keratinocytes and melanocytes) were counted in Neubauer's chamber under light microscopy and the blue stained nonviable cells were excluded from the count. Phosphate buffer solution was added to the pellet to prepare a suspension of noncultured epidermal cells, which was used for autologous transplantation.

A 4-mm skin punch biopsy specimen from the donor site was collected under aseptic precautions in PBS with added antibiotics. The epidermis was carefully separated from the dermis and the dermis was incubated overnight in collagenase (1 mg/mL) at room temperature. After incubation, the contents were diluted in PBS and centrifuged at 1000 rpm for 5 minutes. Pellet was washed 3 times with PBS to remove any remnant collagenase. With trypan blue staining, viable cells were counted in Neubauer's chamber as described above. Phosphate buffer solution was added to make a suspension of noncultured dermal cells for use in autologous transplantation. For patients undergoing transplantation with NCES and NDCS, a suspension containing both components in a 1:1 ratio was used.

Dermal mesenchymal stem cells in NDCS were identified by flow cytometric analysis using CD90 (559869, BD Pharmingen). This was done in the initial 3 samples to confirm the presence of DMCs. The proportion of CD90⁺ cells was found to be 22.2%, 24.7%, and 20.6% in these 3 samples. A representative plot of one of the patients is depicted in the eFigure in Supplement 2.

The recipient site was prepared and dermabrasion was done until appearance of tiny pinpoint bleeding spots. The denuded area was washed with PBS. The suspension of interest (NCES alone or NCES/NCDS) was carefully transferred to a tuberculin syringe. Using an 18-gauge needle, the suspension was placed over the denuded surface and evenly spread. This was covered with a meshed collagen sheet (Kollagen M, Eucare Pharmaceuticals). After this, Bactigras dressing (Smith & Nephew Pty, Ltd) was placed followed by an additional layer of small gauze piece moistened with PBS. Tegaderm (3M Medical) was then placed and finally an elastic plaster (Dynaplast, Johnson & Johnson) was applied. The patients were kept under observation for 1 hour and were subsequently discharged. The patients were followed up on day 8 and weeks 4, 8, 16, and 24 after the transplantation procedure. No additional treatment was given during the follow-up period.

The outcome parameters were extent of repigmentation, color matching of repigmentation, and pattern of repigmentation. The extent of repigmentation was assessed subjectively using serial photographs taken in the same settings and objectively by serial paper markings and was categorized using the following criteria⁴: 25% or less repigmentation (minimal repigmentation), 25% to 50% repigmentation (mild repigmentation), 51% to 75% repigmentation (moderate repigmentation), 76% to 90% repigmentation (marked repigmentation), and more than 90% repigmentation (excellent repigmentation). The color matching of repigmented skin to surrounding skin was done and categorized as lighter color, same color, or darker color compared with the surrounding skin. Repigmentation pattern was assessed and classified as diffuse, perifollicular, or marginal.¹¹

Statistical analysis was carried out using Statistical Package for Social Sciences (SPSS; version, 22.0 for Windows; IBM Corp). Normalcy of continuous data was checked using Kolmogorov-Simrnov Test. Mean and standard deviation were calculated for all normally distributed continuous variables (eg, age, duration of disease, size of graft, and percentage body surface area [BSA] treated). Unpaired t test was used to compare means between study groups. χ² or Fisher exact tests were used to compare categorical variables.

Baseline clinical characteristics, operative sites, and mean surface area operated as described in Table 1 were comparable among the groups and subgroups. There were no statistically significant differences in the demographic characteristics (age, sex, duration of disease, and duration of clinical stability) between the 2 subgroups of each group.

All patients completed 24 weeks of follow up. Comparisons of extent of repigmentation, color matching, and repigmentation patterns among the subgroups are summarized in Table 2. With increasing follow-up interval, a gradual increase in the extent of repigmentation was noted in both the groups. In group 1, the extent of repigmentation at each follow-up was significantly higher in patients of subgroup 1B compared with those in subgroup 1A (Figure 2). At the end of 24 weeks, more than 75% repigmentation was observed in all the 10 patients of subgroup 1B compared with 3 patients in subgroup 1A (100% vs 30%, P = .003). In group 2, the extent of repigmentation was comparable in both the subgroups at all the follow-up visits (Figure 3).

No significant difference in color matching was noted among any of the subgroups. Most of the patients demonstrated a diffuse pattern of repigmentation. None of the patients showed marginal pattern of repigmentation. Overall, there was no statistical significant difference in the pigmentation pattern among the subgroups.

Overall, the intervention was well tolerated in all the patients. Superficial donor site infection was seen in 1 patient (subgroup 2A), which was treated successfully with topical antibiotics. Mild hyperpigmentation or hypopigmentation at donor site was seen in 2 patients (1 each in subgroup 1A and 2B). There were no complications at the recipient site. Postsurgery perilesional halo developed in a total of 8 patients.

Since its introduction, the technique of NCES in treating vitiligo has underwent multiple modifications and simplifications.^12,13 The outcome of NCES in terms of repigmentation rate has varied considerably in different studies probably owing to differences in duration of follow-up, disease characteristics, method of assessment of repigmentation, technique of preparation of the cell suspension, and postoperative treatment.¹² A recent study¹⁴ that evaluated the novel combination of follicular and epidermal cell suspension in vitiligo found it to be superior compared with epidermal cell suspension alone. In this study, we compared another novel combination of NCES and NDCS with NCES alone in patients with vitiligo. In patients with vitiligo with shorter DS (3-6 months), the outcome of NCES and NDCS was excellent in terms of repigmentation rate and was also significantly superior to NCES. Whereas in vitiligo patients with longer DS (>12 months), the outcome of NCES and NDCS and NCES alone were comparable in terms of repigmentation rate. The findings of our study are encouraging and suggest that surgical interventions using the novel combination of NCES and NDCS can be used in vitiligo patients with shorter DS (3-6 months) without waiting for 12 months or more since last clinical activity.

No significant difference in terms of color matching and patterns of repigmentation was observed with both the techniques in either of the groups. Most patients showed a diffuse pattern of repigmentation, few showed a perifollicular pattern, and none showed a marginal pattern of repigmentation. It is noteworthy that perifollicular or marginal pattern of repigmentation is observed when the repigmentation is owing to stimulation of melanocyte reserves in hair follicles, which occurs after dermabrasion and phototherapy.⁴ Predominantly diffuse pattern of repigmentation observed in our study suggests that the cause of repigmentation was transplantation of melanocytes by NCES and not the induction of the melanocyte reserve by dermabrasion. In previous studies on vitiligo surgery as well, diffuse pattern was the most commonly observed pattern.⁴

The results of our study can probably be explained by the findings of Rao et al⁵ and Zhou et al.¹⁰ Rao et al⁵ found a significantly higher lesional CD8⁺ T-cell count in patients with vitiligo with shorter DS (3-12 months) compared with those with longer DS (>12 months). They also observed higher number of lesional CD8⁺ T cells in patients having poor repigmentation after melanocyte transplantation compared with those with favorable repigmentation, concluding that activity of cytotoxic CD8 cells in vitiligo lesions may be responsible for inferior posttransplant outcome. Zhou et al¹⁰ studied the factors influencing efficiency of autologous melanocyte transplantation in patients with vitiligo while focusing on perilesional skin CD8⁺ T lymphocytes, and investigated the potential effects of dermal mesenchymal cells (DMCs) on CD8⁺ T-cell activity in vitro. Their patients with a high number of perilesional CD8⁺ T cells showed poor rate of repigmentation. When cocultured, DMCs induced apoptosis and inhibited proliferation of CD8⁺ T cells, which, they concluded, may enhance the efficacy of melanocyte transplantation.

In terms of extent of repigmentation (primary outcome), the response in subgroup 1B was significantly better than subgroup 1A at every follow-up visit. At 24 weeks’ follow-up, 100% patients (10/10) achieved more than 75% repigmentation in subgroup 1B, which was significantly higher compared with subgroup 1A (3/10, 30%). However, in group 2 (duration of disease stability more than 1 year), degree of repigmentation at 24 weeks was comparable in subgroups 2A and 2B. These findings suggest that there is significant benefit of addition of dermal cells to the epidermal cell suspension in vitiligo patients with 3 to 6 months DS but not in patients with DS more than 1 year.

Higher number of lesional CD8⁺ T-cells population in patients of group 1 (shorter duration of clinical stability), may have resulted in the difference in extent of repigmentation in patients who underwent NCES and NDCS compared with those who underwent NCES alone. Dermal mesenchymal cells or other dermal components from NDCS probably have inhibited the activity of cytotoxic T cells in subgroup 1B leading to better acceptance of transplantation while unabated activity of CD8⁺ T cells in subgroup 1A may have destroyed melanocytes in transplanted NCES thus resulting in poorer repigmentation. In group 2 (patients with longer DS), lower number of CD8⁺ T cells could have resulted in relatively better repigmentation result in both the subgroups with no significant difference.

Singh et al⁴ in their study reported that 92% of their patients exhibited more than 75% repigmentation after NCES; however, mean area treated in their study was much less than in our study. In another study by Bao et al,¹⁵ 81% of the patients showed more than 50% repigmentation following NCES method. Thus, the degree of repigmentation achieved in our subgroup 2A (NCES in patients with duration of stability >1 year) is comparable to other previous reports.

It may be logical to compare the results of our study, particularly subgroups 1B and 2B, with previous studies that have evaluated the technique of minipunch grafting in vitiligo patients because punch grafts contain both the dermal and epidermal component from the nonlesional area. Malakar et al¹⁶ reported more than 70% repigmentation in 83% of patients with vitiligo with DS longer than 6 months after minipunch grafting, which is comparable to more than 75% repigmentation in 70% of the subgroup 2B patients and 100% of the patients in subgroup 1B. Almost every patient in the study by Malakar et al¹⁶ was subjected to additional PUVASOL therapy after surgery, which may have improved the outcome. Boersma et al,¹⁷ who performed minipunch grafting in vitiligo patients with DS longer than 6 months reported more than 80% repigmentation in only 39% of lesions.

Because our trial was a pilot study with a follow up of 24 weeks, a small sample size and relatively short follow up were the major limitations. Prior sample size estimation was not performed because this was the first study evaluating NCES and NDCS. The process of postsurgery repigmentation may proceed well beyond 24 weeks, implying that longer follow up in participants may lead to different results. However, most of the previous studies on vitiligo surgery had similar sample sizes and follow-up period.^4,14,18

Our study suggests that NCES and NDCS is an effective approach and superior to NCES alone in patients with vitiligo with DS ranging from 3 to 6 months. This novel approach (NCES and NDCS), may be used in patients with vitiligo early in the course of stable disease without waiting for 12 months or more to elapse since last clinical activity. Future studies with longer follow up and larger sample size are warranted to monitor the long-term stability of achieved repigmentation, to establish the efficacy of this novel approach and further investigate the implication of DMCs in patients with active vitiligo.

Corresponding Author: Davinder Parsad, MD, Professor, Department of Dermatology, Venereology and Leprology, Postgraduate Institute of Medical Education and Research, Sector 12, Chandigarh 160012, India (parsad@mac.com).

Accepted for Publication: November 5, 2018.

Published Online: January 2, 2019. doi:10.1001/jamadermatol.2018.4919

Author Contributions: Drs Thakur and Parsad had full access to all of the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis.

Study concept and design: Thakur, Kumar, Sendhil Kumaran, Srivastava, Parsad.

Acquisition, analysis, or interpretation of data: Thakur, Kumar, Kaushik, Srivastava, Parsad.

Drafting of the manuscript: Thakur, Kumar, Kaushik, Srivastava, Parsad.

Critical revision of the manuscript for important intellectual content: Thakur, Kumar, Sendhil Kumaran, Srivastava, Parsad.

Statistical analysis: Thakur, Kumar, Parsad.

Administrative, technical, or material support: Thakur, Srivastava, Parsad.

Study supervision: Thakur, Sendhil Kumaran, Parsad.

Conflict of Interest Disclosures: None reported.

Data Sharing Statement: See Supplement 3.

Additional Contributions: We thank the patients for granting permission to publish this information.