Vitiligo is an acquired skin disease that significantly impacts the quality of life of patients. Medical treatment of vitiligo includes the use of melanocyte transplant, but the results are variable. We have treated 4 patients with either focal or generalized stable vitiligo using a graft of autologous melanocytes' culture on a denuded amniotic membrane (AM). A culture biopsy was obtained in every patient and grown in melanocytes' media for 10–14 days after which cells were transferred to a denuded AM and transplanted into the achromic lesions. Patients were followed for up to 6 months using clinical assessment of achromic lesions. Treated areas ranged between 4 cm2 and 210.6 cm2. Response to treatment was excellent in all patients with 90–95% repigmentation success rate. Our results demonstrate that transplantation of autologous melanocytes cultured on AM is a new, simple, and effective treatment for stable vitiligo.
Vitiligo is an acquired skin disease that affects 0.1–3% of the world’s population, characterized by loss of melanocytes from the epidermis, and leads to the development of achromic lesions. The basic pathogenesis of vitiligo remains unknown, although several studies suggest genetic predisposition, relationship to other autoimmune disorders, biochemical and neurohormonal imbalance, and environmental toxin/stressors [
The amniotic membrane (AM), the inner part of the placenta, consists of a thick basement membrane of collagen type IV and laminin and an avascular stroma. Davis [
Experimental and clinical studies using AM as a graft or patch have demonstrated that AM promotes re-epithelialization, decreases inflammation and fibrosis, and inhibits angiogenesis. AM acts like a basement membrane and facilitates the migration of epithelial cells [
In the current study, we report the clinical results obtained with the application of melanocytes cultivated on AM onto distinct achromic lesions.
The study was approved by the institutional ethics committee, and written informed consent was obtained from the patients or parents. From January 2005 to May 2006, 4 patients (1 male and 3 female) ranging in age from 13 to 29 years, 2 with stable generalized vitiligo and 2 with stable focal vitiligo were treated with autologous transplantation of pure melanocytes using AM as a carrier (Table
Patients | Age, years/gender | Type of vitiligo | Grafted area | Size, | % repigmentation at 6 months |
---|---|---|---|---|---|
1 | 29/F | Focal | Areola and nipple | 4 | 95 |
2 | 13/M | Focal | Chin | 9 | 90 |
3 | 26/F | Generalized | Legs | 185 | 90 |
4 | 17/F | Generalized | Legs | 210 | 95 |
Placentas were obtained during elective cesarean delivery from mothers who had been screened for HIV, hepatitis B, hepatitis C, HTLV-1, CMV, and syphilis at the time of birth, and 3 months after harvesting the AMs. Under a laminar flow hood, the placenta was washed free of all blood clots using balanced saline solution containing 50 ug/mL streptomycin, 50 ug/mL penicillin, and 2.5 ug/mL amphotericin. The amnion was separated from the rest of the chorion by blunt dissection. The membrane was then flattened onto a nitrocellulose paper, with the epithelium/basement membrane surface up. The membrane and the paper were then cut up into
Piece of amniotic membrane (a); immunohistochemistry for HMB-45 applied to cells cultured on slide. A large number of melanocytes can be observed (b); micrograph of cross-sectioned AM stained with HMB-45 after cell culture (c). Original magnification: (b, c) X100.
Melanocyte basal medium (MBM, PromoCell, Heidelberg, Germany) without PMA (phorbol 12-myristate 13-acetate) nor bovine pituitary extract, supplemented with basic fibroblast growth factor (1 ng/mL), human recombinant stem cell factor (50 ng/mL), and human recombinant granulocyte/macrophage colony-stimulating factor (10 ng/mL) for 10–14 days, was used for the melanocyte culture.
A superficial shave biopsy (1 cm2) was taken from pigmented buttock skin under local anesthesia. Skin samples were washed in phosphate-buffered saline (PBS), and the connective tissue was trimmed. The epidermis was separated from the dermis with 1% Dispase II (Boehringer Mannheim, Barcelona, Spain) in PBS with gentamicin (20 mg/mL) at 37°C overnight. Epidermal sheets were peeled from the dermis and stirred in 0.05% trypsin and 0.53 mM EDTA solution (trypsin-EDTA solution) for 10 min at 37°C. The trypsin-EDTA solution was inactivated with a trypsin inhibitor (Sigma, Madrid, Spain) and centrifuged at 1500 rpm for 5 min. Cell pellets were resuspended in 10 mL of supplemented MBM and cultured at 37°C in a humidified atmosphere containing 5% CO2 in 25 cm2 tissue culture flasks (Nunclon, Roskilde, Denmark). When 80–90% confluence was reached (Figure
To ensure the presence of melanocytes, immunohistochemical staining for HMB-45 (1 : 100; Biogenex, San Ramon, Calif, USA) was performed using the EnVision+ System (Dako, Glostrup, Denmark) according to the manufacture’s recommendations. Both AMs and cells cultured on chamber slides were fixed in 4% neutral buffered formalin for 10 min. After washing in PBS, free-floating amniotic membranes and slides were permeabilized with 5% Triton X-100 for 30 min and endogenous peroxidase activity was quenched with 3% H2O2 for 10 min. Samples were incubated with primary antibody overnight at 4°C. After rinsing, membranes and slides were incubated with goat antimouse (K4001, Dako) labeled polymer for 30 min at room temperature. Cells were visualized by incubating the sections with DAB+ (K3468, Dako). Washes between each step were carried out with Tris-HCl 0.05 M buffer, 0.5 M saline, pH 7.6 (TBS) containing 0.05% Tween 20 (TBS-T). Slides were counterstained lightly with hematoxylin, dehydrated in graded series of ethanol, cleared in xylene, and mounted in DPX. Amniotic membranes were mounted onto glass slides, dried overnight, cleared in xylene, and coverslipped with DPX. Samples were examined on a Nikon Eclipse E800M microscope, and 10 random images per slide were captured with Analysis Soft Imaging System Gmbh software. The number of melanocytes per mm2 was counted using the ImageJ software.
After disinfection with povidone-iodine, under local anesthesia with lidocaine hydrochloride (20 mg/mL), epidermal ablation was performed using the Silk Touch Flashscanner attached to a Sharplan 1030 CO2 laser at the setting of 5.5–7 W with a 0.2-second pulse duration (Laser Industries Ltd, Tel Aviv, Israel). To obtain an adequate depth of the wound bed in the deepithelialization of achromic areas, we ablated slightly less tissue than was necessary to achieve pinpoint bleeding, which indicates penetration into the papillary dermis. One laser pass was sufficient for all patients. After the papillary dermis was reached, the denuded lesions were treated with the AMs containing cultured melanocytes. AMs with the melanocytes were mounted on Vaseline gauze (LINITUL, Bama-Geve, Barcelona, Spain). Each amniotic membrane (surface 12.56 cm2) was secured, with the basement membrane with cultured melanocytes down, onto the denuded surface with one layer of Vaseline gauze, steri-strips, and several layers of dry gauze and traditional bandages.
Patients were asked to limit movement of the treated region for 3-4 days. At that time the bandage was removed. Two weeks after the procedure, all patients began to receive sun exposure or UVA irradiation twice per week for approximately 6 weeks to stimulate melanocyte proliferation.
The presence of melanocytes in the culture was confirmed on the basis of the morphology of the cells and immunocytochemical analysis with HMB-45. The number of melanocytes transplanted varied between 0.1 and
Achromic epidermis was removed by means of the CO2 laser. The largest area treated in one session was 210 cm2, and the smallest treated lesion was 4 cm2. Assessment of repigmentation was performed monthly for up to 6 months in every patient. While responses were observed after 2 months in some patients, the complete evaluation of the percentage of repigmentation in all patients was performed after 6 months (Figure
Patient 1. Preoperatively (a); the achromic epidermis was removed using CO2 laser, and AM carrying cultured melanocytes on the basement membrane surface was applied on the denuded area with one layer of Vaseline gauze. The amniotic membranes were fixed with steri-strips and covered with dry gauze and adhesive tape (b); postoperative photography (>90% repigmentation at 16 weeks) (c).
Response to treatment was excellent in all patients (Table
Patient 3. Preoperatively (a), desepidermized lesion after treatment with CO2 laser (b), and postoperative photographs (>90% repigmentation at 16 weeks) (c).
Despite the availability of various types of medical and surgical treatments for vitiligo, the therapeutic response of either focal or chronic stable generalized vitiligo to conventional treatment is poor, with a significant number of patients failing to respond with a satisfactory degree of repigmentation [
It has been hypothesized that epidermal trauma (dermabrasion or laser) could be a melanocyte-stimulating trigger to a reservoir of melanocytes, that is, hair follicles and the surrounding skin. On the other hand, the induced repigmentation might be postinflammatory, caused by the release of inflammatory mediators after epidermal trauma. We believe these are unlikely explanations for the observed effects in our patients in accordance to studies in which Van Geel et al. [
We also show that the use of CO2 laser allows precise removal of the epidermis, preservation of the papillary dermis, and absence of bleeding and inflammation being a user-friendly procedure, fast and uniform unlike more classical dermabrasion or diathermosurgery [
The results in this limited number of patients warrant further studies to demonstrate the usefulness of the AM as a scaffold for melanocytes. Only an intraindividual clinical trial evaluating the usefulness of AM as a scaffold versus the application of melanocytes in suspension can confirm the greater efficiency of using a carrier for transporting and transplanting the cells.
This study was partially supported by the Clinical Trial EudraCT no. 2009-017757-36 from the Fondo de Investigación Sanitaria (FIS), Spanish Ministry of Health.