LAUSR

Surgical modalities are important in the management of stable vitiligo, which is refractory to medical treatment. These include tissue grafting such as minigrafting, suction blister epidermal grafting, and ultrathin split-thickness skin grafting, and cellular transplantations such as cultured melanocytes, noncultured epidermal cell suspension (NCES), and hair follicle outer root sheath cell suspension prepared from either excised hair follicles (EHFs) or plucked hair shafts (PHSs). Hair plucking is a less invasive procedure, which potentially provides a renewable source of cells for cell-based therapies in vitiligo. This study was the part of a larger ongoing comparative trial comparing different cell suspensions prepared from EHFs, PHSs, and epidermal shave biopsies, where the PHS outer root sheath cell suspension transplantation was discontinued after seeing poor outcomes in this arm of the trial. A total of 10 patients of age 12 years or more with a clinical diagnosis of segmental or generalized vitiligo and disease stability duration of at least 1 year were recruited. Each patient underwent all 3 transplantations on different randomized patches at similar anatomic sites or in a large patch divided into 3 areas of similar sizes. For preparation of suspension in NCES group, about one-fourth to one-tenth the size of the recipient area was selected as the donor site (thighs or buttocks). Under aseptic precautions and topical anesthesia, a thin split-thickness skin graft was taken from donor site. The skin graft was transferred to trypsin–EDTA (ethylenediaminetetraacetic acid) solution (0.25% trypsin and 0.05% EDTA; Gibco BRL, Grand Island, NY) in a Petri dish and incubated at 37°C for 40 minutes to separate the epidermis from the dermis. After 2 hours, the trypsin–EDTA solution was removed and trypsin inhibitor added to terminate the activity of trypsin. Then, this was pipetted out and ringer’s lactate added and the tissue teased with sterile forceps to separate the cells from the tissue. The solid waste of tissue was removed, and the suspension centrifuged at 2,500 rpm for 5 minutes. The supernatant was then discarded, and the pellet, containing cells from the basal layers, taken. For hair harvesting in EHF group, an area of occipital scalp around 43 4 cm in size was selected, and the hair on that area trimmed to a length of approximately 2 mm. Following an infiltration anesthesia with 2% lignocaine and 1:1,000 adrenaline, around 50 pigmented follicles were extracted depending on the area to be treated. To obtain follicular units, a 0.7or 1-mm power punch was first inserted in the direction of the hair follicle until it reaches the mid-dermis. Then, the follicular unit was pulled out gently using hair follicle holding forceps by holding the skin surrounding the hair shaft(s). For hair plucking, a rubbertipped depilation forceps was used. In this technique, hair were held firmly and pulled briskly. Approximately 100 to 200 pigmented follicles were plucked per patient and collected in transport media. For preparation of suspension in EHF and PHS group, these follicles were washed 3 times in phosphate-buffered saline containing antibiotics and antimycotics (Gibco BRL). The follicles were then incubated with 0.25% trypsin–0.05% EDTA (Gibco BRL) at 37°C for 90 minutes to prepare the single-cell suspension. After every 30 minutes, the hair follicles were placed in a new tube of trypsin–EDTA, and the reaction in the previous tube terminated by adding trypsin inhibitor (Sigma-Aldrich, St. Louis, MO). After cell separation, only thin keratinous shafts of the hair were left, which were discarded. The cell suspensions of all 3 tubes were then combined in a single tube and then filtered through a 70-mm cell strainer (Becton Dickinson, Sunnyvale, CA) to prepare a single-cell suspension. Finally, the cell suspension was centrifuged for 10 minutes at 2,500 rpm to obtain a cell pellet. The following steps of the procedure were identical in all the 3 groups. The pellet was assessed for viability of melanocytes using trypan blue and counted in Neubauer’s chamber under light microscope. Following this, the pellet was resuspended in 250 to 500 mL of Dulbecco’s modified eagle medium depending on the size of the recipient patch and transplanted onto the recipient site. The recipient area was surgically cleansed, anaesthetized with topical anesthesia, and dermabraded with a motorized dermabrader fitted with diamond fraises until tiny pinpoint bleeding spots were seen. The prepared suspension was taken in a pipette and spread evenly onto the denuded surface and covered with small pieces of collagen dressing of fish origin (Neuskin-F; Eucare Pharmaceuticals, Chennai, India) and then a sterile gauze piece followed by Tegaderm. The dressing was removed on the eighth day. The patients were followed up at 4, 8, 12, 16, 24, and 36 weeks after the procedure, and repigmentation was assessed subjectively by photographic analysis and drawing patch size on transparent sheets to calculate percentage repigmentation. Cell counts in NCES were about 6 times higher as compared to EHF (p–0.015) and about 94 times higher as compared to PHS (p–0.00) (Table 1). Mean repigmentation at 8 and 36 weeks was significantly higher in NCES and EHF as compared to PHS; however, there was no significant difference between NCES and EHF (Table 1, Figures 1 and 2).