Vitiligo: general principles of therapy

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)

One of the fairly common acquired skin diseases from the group of dyschromia that a dermatologist has to deal with in his daily practice is vitiligo. 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.

Introduction

The true cause of melanocyte destruction has not yet been established [1]. However, a number of the most substantiated theories of the pathogenesis of vitiligo can be identified, among them :

• autoimmune [2];
• neurogenic [3];
• biochemical [4];
• imbalance of epidermal cytokines [5];
• genetic [6].

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 [7].

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 [8] (Fig. 1).

Rice. 1. NB UVB therapy. Patient, 36 years old, suffers from generalized bilateral vitiligo
within ten years. After 90 sessions of NB UVB over a period of one and a half years, repigment was achieved.
tion of about 85% of the affected surface (IPH “Virtus”)

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 [9]. 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 [10].

In 2001, Geel et al. modified these two techniques [11]. In this case, the cell suspension 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.

When melanocyte (M2) culture medium is added, the cell suspension is enriched, promoting the repair of larger defects (Olsson and Juhlin, 1998) [12]. 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) [11]. The minimum number of melanocytes required to achieve sufficient repigmentation has been found to vary between ^{210–250⁄mm2} (Tegta et al., 2006) [13].

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 under various cultivation conditions were published [16], including the use of hyaluronic matrices. Several studies [17, 18] have shown the success of using an 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 identified. The donor site can be obtained by biopsy or blistering [19]. The recipient site can be prepared by dermabrasion, Er:YAG or CO ₂ laser.

The study conducted by the authors of the article [19] 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) [19]; in another study, 18 of 21 patients achieved 90% repigmentation (Guerra et al., 2003) [20].

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 [21].

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, good results were not achieved, despite repeated use of surgical methods (three patients had satisfactory results, four had no changes) [22].

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 successful repigmentation of vitiligo lesions that occurred suggests the great potential of cryopreserved cells for the successful treatment of vitiligo (Olsson et al., 1994) [23].

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 cultivating a 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 [24];
• 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; the buttocks, inner upper thigh, or forearm may be used [25].

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 [26]; during exacerbation of herpes infection [27], 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 [28, 29, 30, 31], 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.

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Read the full version of the article in Les Nouvelles Esthetiques 2014/№5