John Libbey Eurotext

There have been reports of various surgical proposals involving melanocyte autografts for the treatment of vitiligo [1-5] that include grafting, suction blister grafting, ultra thin Thiersch grafts or minigrafts. Recent advances in melanocyte culture have made autologous transplantation of these cells possible. Olsson et al. [6] cultivated melanocytes from the donor site removed by shaving and successfully implanted on the dermabraded recipient surface. There are still concerns regarding hypo and hyperpigmentation at the donor site provoked by the shaving. Another problem encountered is reduced cell adhesion, with cell death on the recipient site due to excessive fluid exudation resulting from dermabrasion of sites larger than 300 cm².

Lerner et al. [7] injected a suspension of cultivated melanocytes inside a blister provoked by suction on recipient areas of patients suffering from piebaldism. Gauthier [1] presented a simplified method for transplanting non-cultured autologous melanocytes into blisters provoked by liquid nitrogen on the recipient sites. The treatment with non-cultured melanocytes would make the technique simpler.

In our study, we compared four different surgical techniques on the same individuals in order to evaluate the efficacy of each method. This involved a three months follow-up of the appearance of repigmentation and its evolution so that a comparative study could be conducted.

The study was performed to evaluate the effectiveness of the melanocyte transplantation for the repigmentation of the acromic area, to observe if there was significant difference between non-cultured melanocyte and cultured melanocyte transplantation, and to define if Only Cryotherapy (OC) and Cryoterapy plus Melanocytes Culture Medium (CM) react as placebo.

The study consisted of a pilot clinical controlled trial including 11 patients — 10 females and 1 male, with symmetrical, generalized, stable vitiligo (Stable vitiligo was defined as the condition that had not been progressing for at least 1 year [20]), whose ages ranged from 20 years to 47 years. Children, the elderly, pregnant women and individuals with associated diseases were excluded from the study. Four distinct achromic patches were randomly chosen for each of the 11 selected patients. Each of the patches received one specific treatment, which meant that the same individual had four concomitant treatment sites (recipient) (Table I).

The donor site was standardized as a 2 cm long and 1 cm wide healthy skin fragment removed by simple fusiform excision and suture from the inguinal region. This region was chosen because it is concealed and also for aesthetic reasons.

Each of the patients received four different simultaneous treatments in four distinct randomly chosen areas:

site treated with only cryotherapy (OC): a blister was produced with the application of liquid nitrogen and the
standard freezing time was from 15 to 30 seconds, while the thawing period was from 30 to 60 seconds. An occlusive dressing of gauze and melanocyte culture medium MMK2 (Sigma-USA) was maintained until natural desiccation of the blister;

site treated with cryotherapy plus melanocyte culture medium (CM): 0.2ml of culture for melanocytes medium MMK2 (Sigma-USA) were injected into a blister induced by liquid nitrogen (only medium). An occlusive dressing of gauze and melanocyte culture medium MMK2 (Sigma-USA) was maintained until natural desiccation of the blister;

site treated with cryotherapy plus autologous transplantation of non-cultured melanocytes and keratinocytes (KM): the autologous fragment from the donor site was processed in the laboratory. After trypsinization of the skin fragment (in a solution of 2.5 % trypsin + EDTA) in an incubator with CO₂(at 37 °C for 3 hours), filtration and centrifugation (at 4 °C, for 10 min, 1200 rpm), a concentrated cell solution of approximately 2 x 10⁶ keratinocytes and melanocytes was obtained, resuspended in 0.2 ml of melanocyte culture medium MMK2 (Sigma) and injected into the previously formed blister, as already described. The patient was placed on complete bed rest for 5 hours so that the cells could adequately settle down on the blister floor. An occlusive dressing of gauze and culture medium was maintained until the blister desiccated naturally;

site treated with cryotherapy plus transplantation of cultured autologous melanocytes (CC): the skin fragment from the donor site was trypsinized, maintained in a CO₂ incubator and centrifuged as previously described. The cell concentrate obtained was dissolved in a melanocyte culture medium MMK2 (Sigma), supplemented with 10 % bovine fetal serum (Sigma), 1 IU/ml of penicillin, streptomycin 0.001 mg/ml and L-glutamine 0.02 mM/ml (Gibco). The culture was stored in 75 cc flasks and the medium changed three times per week. After 2 — 3 weeks in the culture, the cells were trypsinized (0.25 % trypsin solution + EDTA), centrifuged, resuspended in 0.2 ml melanocyte culture and immediately transplanted by injection inside a blister that was previously provoked. The patient was placed on complete rest for 5 hours so that the cells could adequately settle down on the blister floor. An occlusive dressing of gauze and melanocyte culture medium, which was changed on alternate days, was maintained until the blister naturally desiccated.

MMK2 (SIGMA-USA), Melanocyte Basal Medium is a modified version of MCDB 151 and MCDB 104, which contains essential and non-essential amino acids, vitamins, trace minerals, organic compounds and inorganic salts. It is supplemented with bovine pituitary extract (BPE), fetal bovine serum, bovine insulin, bovine transferrin, hydrocortisone and heparin.

All the patients received oral prophylactic antibiotic therapy (Roxitromycin 300 mg/ day for 5 days).

The patients were oriented to a 10-minute daily solar exposure of the treated areas, after totally drying up the blisters, without any associated medicine, according to research already published [20].

Measurements were taken in square centimeters of the treated achromic regions at the following times: T0 = onset of treatment; T30 = 30 days after treatment; T60 = 60 days after treatment; T90 = 90 days after treatment. The receptive areas were drawn on transparent plastic film at times T0, T30, T60, T90; and submitted to an image analysis computer program, with the measurement of the achromic area in square centimeters (Table II).

These measurements were statistically compared among different treatments and over the follow-up period. Observations were made regarding the effect of the treatment in relation to reduction of the initial achromic region and to see if there were significant differences among the various treatments.

The software used for statistical analysis was the SAS System for Windows 8.0. The Student t test was applied to assess each treatment from T0 to T90. A comparison among treatments was obtained using a generalized linear model for repeated measures together with the mean Tukey’s test. The dependent variable used in the model was the percentage of reduction in the achromic area over a period of time with regard to T0.

A careful follow-up of the 11 selected patients was performed for 90 days. Table I shows their profile as well as the treated areas.

All the 44 treated areas were followed up in relation to the initial achromic area (at time T0, in cm²) and then at times T30, T60 and T90. The following results were observed:

Comparison of each treatment’s results with time:

Treatment OC did not present any significant differences with regard to the affected areas over time regarding the initial measurements (Student t test, 10DF, p = 0.70, 0.55 and 0.87 for 30, 60, and 90 days, respectively).

Treatment CM also did not present any significant difference for the measurements of the affected areas over time in relation to the initial measurements at 60 and 90 days (Student t test, 10DF, p = 0.54 and 0.23 respectively). However, significant differences were found after 30 days in relation to the initial measurements (Student t test, p = 0.018) with a discrete reduction in the mean affected area.

Treatment KM presented significant differences for the affected areas at 30 days, 60 days and 90 days in relation to the initial area (Student t test, 10DF, p = 0.021, 0.064 and 0.055, respectively. The mean size of the affected areas decreased with time.

Treatment CC presented significant differences size of the affected areas at 30 days, 60 days, 90 days in relation to the initial size (Student t test, 10DF, p = 0.049, 0.014 and 0.032 respectively). The mean affected areas reduced with time.

The above results can be seen in Figure 1 and Figure 2.

Comparison among different treatments over the time:

The mean reduction percentage of the affected areas did not differ significantly in the CC, KM and CM treatments.

Treatments KM and CM did not significantly differ from treatment OC regarding the mean reduction percentage of the affected area.

Treatment CC was in average superior to treatment OC (the mean reduction percentage was greater).

Treatments CC and KM did not differ significantly from each other regarding the mean reduction percentage of the affected areas.

Treatments OC, KM and CM did not differ significantly from each other regarding the mean reduction percentage of the affected areas.

Treatment CC was superior to the treatments OC and CM (the mean reduction percentage of the affected areas was greater).

Treatments CC and KM did not significantly differ from each other with regard the mean reduction percentage of the affected areas.

Treatments CM and OC did not differ significantly from each other with regard to the mean reduction percentage of the affected areas.

Treatments CC and KM were superior to Treatments CM and OC regarding the mean reduction percentage of the affected areas.

A comparison among treatments was obtained using a generalized linear model for repeated measures together with the mean Tukey’s test.

The Figures 3, 4, 5 and 6 show the evolution of the treatments KM and CC, in case 1.

Over the last decades, conventional clinical treatment with topical or systemic corticosteroids, topical or systemic psoralen therapy, PUVA, melagenin and others has presented limitations regarding pigmentation results [8-13] and therefore surgical techniques have been developed [1, 5, 6, 14, 15] to try to meet the requirements of clinical treatment programs. In most patients with stable vitiligo, transplantation with cultured autologous melanocytes or non-cultured melanocytes/keratinocytes is an effective method of repigmenting non-pigmented skin [14, 16].

The patient should be informed that only the destroyed melanocytes are replaced but that the underlying cause of vitiligo is not cured.

In our study, a comparative analysis of various surgical techniques was conducted. It was observed that treatment
with only cryotherapy (OC) did not pigment any of the 11 cases during a follow up of 90 days. Treatment with cryotherapy plus melanocyte culture medium (CM) also did not pigment any of the lesions during a follow up of 90 days. These two groups were therefore standardized as controls and could be considered as placebos that presented unsatisfactory treatment results.

Both the KM and CC groups that were respectively treated with a pool of non-cultured cell and cultured melanocytes presented a similar response.

The repigmentation started in the center of the blister as discreet striations and follicular pigmentation that intensified with time, progressively reducing the achromic area. Discreet pigmentation occurred after 30 days and attained significant levels in 90 days in all the cases (Figures 3-6). Although Figures 1 and 2 demonstrate a better CC curve than the KM curve, which makes it appear that treatment CC is more effective, this was not statistically confirmed. Neither treatment presented statistically significant differences up to a period of 90 days.

Although the non-cultured melanocytes/keratinocytes transplantation (KM) was as efficient as the transplantation of cultured autologous melanocytes (CC), utilization of the second technique is justified because besides being a more sophisticated technique, it will permit cell storage (cryostorage), in future studies, to be used later in the remaining achromic patches without a need for new donor sites [17].

An interesting fact that occurred in our study was the repigmentation of adjoining achromic regions of the treated areas. This had not been found in the literature. The repigmentation of adjoining achromic regions of the treated areas was an observed fact in several cases, but not foreseen. The patients were oriented to a 10-minute daily solar exposure of the treated areas, without any associated medicine. They were not oriented regarding the untreated area, except for the traditional daily wearing of sun block products because of our climate. So the repigmentation of untreated areas was just observed in areas close to those areas treated with CM and KM. The other untreated achromic areas and the acromic areas treated with OC and CM remained achromic.

Finally, we consider that the correct selection of patients for melanocyte transplantation is an extremely important factor. The patients should present stable vitiligo that does not exceed 30 % of the body and be highly motivated as well as psychologically receptive to the treatment. Many of our patients report that the prospect of new treatment has brought new hope into their lives, socially restricted by this disease.

The authors would like to thank FAPESP/Brazil for their support to the research and Professor Dr. Alain Taieb for his thoughtful suggestions.

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Melanocyte transplantation for the treatment of vitiligo: effects of different surgical techniques