Cellular technologies in the treatment of vitiligo: a review of techniques

Vladimir Tsepkolenko, Doctor of Medical Sciences , Honored Doctor of Ukraine, Professor of the Department of Dermatovenereology and Cosmetology of Donetsk National Medical University. M. Gorky, General Director of the Ukrainian Institute of Plastic Surgery and Cosmetology “Virtus” (Ukraine)

Ekaterina Karpenko, dermatologist at Virtus Clinic (Ukraine)

Dmitry Pykhteev, head of the cell transplantation department at the Virtus clinic, head of the biotechnological laboratory at Smart Cell (Ukraine)

The prevalence of this disease in the world varies depending on the region and ranges from 0.5 to 8.2%. Its characteristic feature is sharply limited depigmented areas of the skin, which have a significant negative impact on the psycho-emotional state of the patient, and consequently on his quality of life. That is why today it is extremely important to choose the right treatment tactics to achieve the maximum therapeutic and aesthetic result.

Introduction

The true cause of melanocyte destruction has not yet been established. However, a number of the most substantiated theories of the pathogenesis of vitiligo can be identified, among them :

• autoimmune;
• neurogenic;
• biochemical;
• imbalance of epidermal cytokines;
• genetic.

According to recent data, keratinocytes play a significant role in the development of vitiligo: they are more susceptible to apoptosis, and changes in the melanocyte-keratinocyte composition can lead to a decrease in the production of keratinocyte-mediated melanocyte growth factor and, accordingly, the death of melanocytes.

Traditional therapy typically involves the use of topical steroids, Psoralen, and UV(A). As practice shows, narrow-band UVB does not always lead to a satisfactory result: after long-term and consistent treatment, only half of the patients achieve skin repigmentation of 75% or more.

In relation to forms of vitiligo that are resistant to traditional methods and do not progress, until recently the most relevant was the use of tissue grafting techniques. Today, with the development of cellular technologies, it has become possible to use cell grafting, for which cultured melanocytes and uncultured cell suspensions of melanocytes and keratinocytes are used. I would like to dwell on these methods in more detail.

Treatment of vitiligo using uncultured melanocytes

The method is used to treat patients with segmental and focal vitiligo. It was first used by Gauthier and Surleve-Bazeille in 1992 by injecting a suspension of cells obtained from the scalp into a blister formed by freezing an area of vitiligo skin. However, the applicability of this technique was limited due to the possibility of local cooling only in some areas of the body, the difficulty of dosing cooling, and the danger of hypopigmentation due to cryodamage of peripheral melanocytes. In 1997, Olsen and Juhlin used a basal cell layer enriched with an epidermal cell suspension and achieved 85% repigmentation.

In 2001, Geel et al. modified these two techniques. In this case, a suspension of cells is distributed using a pipette onto the dermabraded surface of the vitiligo spot and covered with a sterile occlusive dressing for one week. After three weeks, PUVA or narrowband UVB may be used. This method requires special equipment, although compared to culturing melanocytes it is considered relatively inexpensive and less time consuming. In addition, 70% of patients achieved repigmentation of about 55% after three months.

By adding melanocyte (M2) culture medium, the cell suspension is enriched, promoting the repair of larger defects (Olsson and Juhlin, 1998). When hyaluronic acid is added to a cell suspension, its viscosity increases, which allows for improved cell attachment to recipient sites (van Geel et al, 2001). The minimum number of melanocytes required to achieve sufficient repigmentation has been found to vary between 210–250⁄mm2 (Tegta et al., 2006).

The study by S. Mulekar (2004) included transplantation of an uncultured melanocyte-keratinocyte cell suspension into 50 patients with segmental vitiligo and 17 with focal vitiligo, followed by a 5-year follow-up period. A skin sample was obtained by shave biopsy measuring 1⁄10th of the recipient area. The prepared cell suspension was applied to a dermabraded depigmented area of skin and covered with a collagen bandage.

In the segmental vitiligo group, 41 patients (84%) showed excellent repigmentation, 3 (6%) had good repigmentation, and 5 patients (10%) had poor repigmentation, which was not maintained until the end of the corresponding observation period.

In the focal vitiligo group, 11 patients (73%) showed excellent repigmentation, 1 (7%) had satisfactory repigmentation, and 3 (20%) had poor repigmentation, which did not persist until the end of the observation period.

In a study by E. Gan et al. (2011), transplantation of uncultured melanocyte-keratinocyte cell suspension (cell grafting) was carried out in 84 patients, in 13 of them the transplantation was carried out in areas of leukotrichia. An ultrathin skin graft measuring 1/5 of the vitiligo area was taken from the gluteal region under local anesthesia. Donor tissue was crushed and incubated in a 0.25% trypsin solution for 30 minutes at 37 °C. Subsequently, the donor epidermal tissue was mechanically separated from the dermis, the cell suspension was neutralized with soybean trypsin inhibitor and centrifuged. The resulting cell pellet was resuspended in 0.5–1 ml of phosphate-buffered saline. After applying an anesthetic cream under occlusion to the vitiligo areas (exposure - 1 hour), ablation was performed with a CO2 laser. After this, the ablated surface was covered with a cell suspension, a sterile collagen dressing was applied and secured with a plaster for up to one week.

Twelve patients (92%) had poor leukotrichia repigmentation three months after transplantation. After six months, satisfactory repigmentation was achieved in eight patients (67%). After 9–12 months of follow-up, 91% of patients (10 of the remaining 11) achieved repigmentation, the quality of which was rated from “good” to “excellent”.

Thus, good to excellent repigmetation in leukotrichia can be achieved using non-culture cell grafting, eliminating the need for hair transplantation. This work is of particular interest due to the difficulty in achieving repigmentation in vitiligo patients in areas of leukotrichia, which is associated with the scarce number of melanocytes outside the orifices of the hair follicles.

The mechanism of leukotrichia repigmentation in non-cultured cell grafting has not yet been sufficiently studied, but it may presumably be associated with the retrograde migration of transplanted melanocytes or epidermal stem cells into hair follicles and/or their production of cytokines that stimulate melanogenesis of the hair follicle. This mechanism may explain the longer time required for repigmentation of a leukotrichia site compared to epidermal repigmentation.

Treatment of vitiligo using cultured melanocytes

This technique was described by Lerner et al (1987), who achieved successful repigmentation by injecting cultured melanocytes into patients with Piebaldism. Subsequently, other approaches to melanocyte transplantation were published under various cultivation conditions, including the use of hyaluronic matrices. Several studies have shown the success of using artificial hyaluronic matrix for the growth of keratinocytes and melanocytes.

Cultured melanocytes can be introduced as a pure cell suspension or co-cultured with keratinocytes. The latter option may be more appropriate due to the fact that keratinocytes regulate the growth and differentiation of melanocytes by releasing a large number of cytokines. Melanocytes retain their physiological characteristics when co-cultured with keratinocytes, which can also be produced in large numbers.

Mixed cell culture transplantation has been used for 20 years. However, no significant correlation of the therapy with an increase in the risk of skin carcinoma or melanoma was found. The donor site can be obtained by biopsy or blister formation. The recipient site can be prepared by dermabrasion, Er:YAG or CO2 laser.

The study conducted by the authors of the article involved 32 patients with vitiligo who were prescribed therapy with cultured melanocytes. As a result of the treatment, repigmentation of the limbs and perioral areas was 8%, and in other areas - 88–96% (Guerra et al., 2000); in another study, 18 of 21 patients achieved 90% repigmentation (Guerra et al., 2003).

In a study by Chen et al. (2004), when transplanting cultured melanocytes, 84% of 120 patients with persistent localized vitiligo received from 90 to 100% repigmentation, among patients with persistent generalized vitiligo, the percentage of complete repigmentation was 54%, and only 14% of patients with active generalized vitiligo, significant repigmentation was observed.

In a study by Kyoichi Matsuzaki and Norio Kumagai (2013), cultured melanocytes were transplanted into 27 patients with stable vitiligo resistant to conventional therapy. The study included 20 patients with segmental vitiligo and seven patients with generalized vitiligo, and was monitored for a year after transplantation.

Keratinocytes were obtained from a primary culture or from the first passage. Before transplantation, the vitiligo area was de-epithelialized using an abrasive method, after which cultured cells were applied and covered with a wet bandage. After complete epithelialization, the recipient site was subjected to UV irradiation.

In the group with segmental vitiligo, 12 out of 20 patients had a good therapeutic effect (90% or more repigmentation) after the first transplant, and after the second transplant their number increased to 14. The remaining six patients had satisfactory results (50–90% repigmentation) . In patients with generalized vitiligo, no good results were achieved, despite repeated use of surgical methods (three patients had satisfactory results, four had no changes).

The use of various biotechnological techniques allows for very flexible therapy for the disease in question. Of particular interest are methods of cryopreservation of the resulting cell suspensions.

Melanocytic suspensions, kept frozen for 6–12 months, were recultured after thawing and transplanted into four patients; The subsequent successful repigmentation of vitiligo lesions suggests the great potential of cryopreserved cells for the successful treatment of vitiligo (Olsson et al., 1994).

The method of culturing melanocytes requires specialized equipment, the creation of autologous expensive cultures, the presence of highly qualified specialists, and also takes considerable time.

The biological process of culturing the keratinocyte-melanocytic mixture consists of sampling a skin area (if necessary, pre-induced pigmentation). The biopsy samples are then sent to a biotechnology laboratory. There they undergo enzymatic processing and separation of the dermis from the epidermis. The epidermal layer is used in work after isolating a keratinocyte-melanocytic suspension, which is usually cultured in DMEM medium, sometimes in a mixture of DMEM-F 12 media, in different ratios.

A significant advantage of this method is the ability to treat large areas of vitiligo using a small donor site. Also, transplantation of cultured keratinocytes was more effective in treating segmental vitiligo rather than generalized vitiligo.

Currently, the method of culturing melanocytes is successfully used in the treatment of resistant forms of vitiligo at the Virtus Institute of Physics (Odessa). This was largely possible thanks to close cooperation with the only biotechnological laboratory in the south of Ukraine, equipped in accordance with GMP standards. Highly qualified biotechnology laboratory specialists carefully monitor each stage of the preparation of cellular material and guarantee its quality and safety.

General principles of vitiligo therapy using cell technologies

Choice of method. Any correctly performed technique from the above leads to positive results. An important principle is to use the least invasive method in order to obtain the best results, and it is also important to maintain the integrity of the donor sites.

Patient selection. When performing skin repigmentation using cellular technologies, a number of factors should be taken into account, such as:

• resistant form of vitiligo: the degree of resistance of vitiligo has no special definition, but the longer the observation period, the more resistant the form of the disease is considered. Segmental (unilateral) vitiligo is the most resistant form that responds well to cell therapy;
• results correspond to the expectations of patients: the appearance of the skin after repigmentation does not always coincide with the appearance of healthy skin, in addition, the results differ from patient to patient. Slight hyperpigmentation is possible, but its degree is insignificant compared to the positive changes obtained;
• degree of depigmentation: the best results are observed in patients of Fitzpatrick phototypes III–VI in completely depigmented areas, hypopigmented areas repigment much worse;
• localization of vitiligo in open areas: most patients are interested in the treatment of vitiligo with such localization. Although the dorsum of the hands and fingers are the most difficult areas to treat, however, with a clearly defined stable form, it is possible to achieve successful repigmentation in these areas;
• stages of treatment: in some cases, to achieve the best result, the cell suspension can be re-introduced, but not earlier than three months after the first administration;
• psychological aspects: some patients have increased requirements for minimal or moderate depigmentation, patients should be carefully examined to determine the real need for surgical treatment;
• Photo documentation: Photo documentation is always recommended to assess the quality and percentage of repigmentation, as well as possible side effects;
• choice of method and donor sites: it is recommended to use minimally non-invasive methods. Donor sites should be covered as much as possible; it is possible to use the buttocks, inner upper thigh or forearm.

Contraindications. Patients with hypertrophic and keloid scars, hyperpigmentation at sites of burns or injuries should be carefully examined to avoid such complications when undergoing treatment using cellular technologies.

Side effects:

• infection: rare, but it is necessary to remember to follow the rules of asepsis and antisepsis to prevent bacterial infections; in case of exacerbation of herpes infection, antiviral drugs are used systemically;
• Post-inflammatory hyperpigmentation: Mostly temporary in most patients, especially in people with skin phototypes IV to VI. Anti-inflammatory cytokines activated during surgical procedures may play an important role in the pathogenesis of hyperpigmentation. Persistent hyperpigmentation in injured areas of the skin may be a contraindication for cell therapy.

conclusions

The use of cell technologies in dermatology has opened up new opportunities in the search for effective methods for treating vitiligo. Today, many patients receive traditional therapy for vitiligo, however, partial or incomplete repigmentation occurs in a fairly large number of patients. Various types of therapy have been successfully used to treat vitiligo in the face and neck, on the trunk and proximal parts of the extremities - with satisfactory results, on the acral parts of the extremities there is little or no effect.

In case of extensive generalized bilateral vitiligo, the best results are achieved with NB UVB and PUVA therapy, while in the case of localized vitiligo, topical corticosteroids and calcineurin inhibitors show the greatest effectiveness.

The modern comprehensive approach to the treatment of resistant forms of vitiligo, used in the Virtus Institute of Chemical Physics, includes a combination of narrow-band phototherapy with an emission maximum at 311 nm, the introduction of a cultured melanocyte-keratinocyte suspension, as well as external therapy (calcioneurin inhibitors, emollients, etc.) and treatment of associated diseases.

When treating segmental vitiligo using cellular methods, in 60–100% of patients the level of repigmentation is 70–100%, and when treating persistent generalized vitiligo, a similar percentage of repigmentation is observed in only 50% of patients. The progress of further research into new and effective therapies is based on the study of the pathogenesis of vitiligo. Standardization of assessment methods for both effectiveness and quality of life of the patient is important to determine the most effective therapy for vitiligo.

First published: Les Nouvelles Esthetiques Ukraine, No. 5 (87), 2014