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University Gugliemo Marconi, Rome, Italy
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New concept for treatment of vitiligo

Transplantation of noncultured epidermal cell suspension (ncecs) and laser-assisted repigmentation  in refractory nonsegmental vitiligo: a prospective, randomized comparative study.

Background: Vitiligo is a disfiguring depigmenting dermatosis that affects approximately 0.5% to 1% of the general population regardless of race and sex/1 /. Although it does not cause physical debilitation, the psychosocial effect it has on those who are affected can be crippling. Depigmenting patches, particularly on the face and hands, are a source of social embarrassment and stigmatization; this is especially true in dark skinned people./2 / Despite intensive research and varying theories, the pathomechanism underlying this disfiguring disorder is not completely understood. This gap in knowledge translates into a therapeutic shortfall; no treatment is uniformly successful in repigmenting vitiligo. The key principle of any vitiligo treatment (medical or surgical) is inducing repopulation of active melanocytes that are able to migrate and proliferate to repopulate the depigmented skin resulting in repigmentation. 

I. Surgical options for repigmentation in vitiligo  - tissue and cellular grafting.

Several surgical procedures have been reported to be effective in vitiligo./3-9/. Treatment of leucoderma by transplantation of autologous pigmented skin grafts or blister roofs has been carried out for about 45 years/10/. Surgical options for repigmenting vitiligo include tissue and cellular grafting. /11/. In patients with stable vitiligo who fail conventional therapy (topical and phototherapies), surgical transplant offers a viable alternative.

In tissue grafting, donor skin is transplanted into vitiliginous areas in the form of mini-punch, thin epidermal, or suction blister grafts. The size of the donor skin restricts the size of the area to be grafted. In contrast, cellular grafting allows transplantation of vitiliginous areas much larger than the size of the donor skin. This advantage can be further exploited by culturing and cryopreserving autologous melanocytes, although expansion of cells involves the use of well-equipped laboratories, highly skilled personnel, and high costs. Because culture media and supplements are used in the cultivation of melanocytes, long-term safety concerns remain when these cells are transplanted onto human skin. In contrast, noncultured cellular grafting offers the advantage of repigmenting vitiligo. 5 to 10 times the size of the donor skin at a lower cost, without the need of culture media, and can be completed on the same day on an outpatient basis./10/ In recent years, ways to simplify this procedure have been explored, including the use of commercially available kits./12/

The results have been encouraging in cases where the vitiligo has been stable, i.e. has not increased during the last few years. Punch grafts can give rise to cobble-stone-like scars, but this could be avoided by using blister roofs. The blisters have been produced on normal skin by suction and transplanted to white areas denuded by sandpaper scrubbing or by freezing. Falabella et al./13/ treated vitiliginous areas  with the use of cultured epidermal sheets. 

Surgical methods, mainly transplantation of noncultured epidermal cell suspension (NCECS), are emerging as the treatment of choice for stable vitiligo. The survival of transplanted melanocytes is more likely in SV than in NSV, as the grafted cells can be harvested from disease-free areas.  Major advantages of NCECS are a smaller amount of donor skin being needed to cover a large recipient area, little postoperative pain and discomfort, easier placement of the cellular graft and excellent colour match.

In 1992 Gauthier and Surleve-Bazeille/14/ introduced a simpler and more effective use of noncultured cellular grafts in patients with vitiligo. They developed a simplified grafting method that was carried out in the following two steps: (i) production of blisters on the depigmented lesions by freezing with liquid nitrogen; and (ii) injection into each blister of an NCECS. The cellular suspension was obtained from samples of skin of the hair scalp after trypsinization. This very simple technique could be used at the dermatologist’s clinic./15/ Several years later, in 1998, Olsson and Juhlin18 proposed comparable but improved and more sophisticated techniques for the surgical treatment of vitiligo. Van Geel et al./16/ also made some changes in comparison with the original technique. Hyaluronic acid was added as a carrier of the cellular suspension to obtain a higher viscosity. Goh et al./17/ also modified this technique as a ‘6-well plate’ in the extraction of epidermal cells from donor skin. In that study, they simplified the extraction of epidermal cells from donor skin using a 6-well plate, a microfilter and three reagents: trypsin, soybean trypsin inhibitor and phosphate-buffered saline (PBS). Kumar et al./18/simplified this procedure so that it can be performed anywhere in sterile conditions. The new technique was termed the „ four compartment (FC) method“. 

II. Laser-assisted repigmentation: new concepts

Laser-assisted surgery is a well-established method of skin resurfacing, particularly used nowadays in aesthetic dermatology issues. Particular “fractional” CO2 laser systems have been developed in order to achieve good clinical results in resurfacing without causing extensive tissue damage and subsequent long downtime periods for the patients. These devices produce “micro-ablation” of the epidermis and thermal effects of the dermis although the areas of ablated tissue are microscopic, thanks to their particular pulse shape. Normal skin is preserved in the areas surrounding each microcolumn of laser irradiation, and healing time as well as scarring risks are minimized compared with conventional ablative systems. Wound repair after ablative fractional resurfacing CO2 laser seems to possess some peculiarities. In particular, it is an effective wound healing modulator by acting on the cytokine pathways of tissue repairing mechanisms (19). Consequently, fractional CO2 laser may promote cell replication, but at the same time, it seems able to balance collagen organization against excessive fibrosis, thus avoiding aberrant wound healing.

Thus, fractional CO2 laser may be proposed in the management of vitiligo in selected patients who did not receive any evident benefit from previous treatments. In fact, the proliferation and repairing mechanisms following fractional laser treatment could also consistently lead to stimulation and reactivation of pigmentation. Various means of skin ablation besides fractional CO2 laser, either alone or in combination with other treatments, were reported to induce repigmentation of vitiliginous lesions. /20/.

Using lasers can be an effective drug permeation-enhancement approach for facilitating drug delivery into or across the skin. The controlled disruption and ablation of the stratum corneum (SC), the predominant barrier for drug delivery, is achieved by the use of lasers. The possible mechanisms of laser-assisted drug permeation are the direct ablation of the skin barrier, optical breakdown by a photomechanical wave and a photothermal effect. It has been demonstrated that ablative approaches for enhancing drug transport provide some advantages, including increased bioavailability, fast treatment time, quick recovery of SC integrity and the fact that skin surface contact is not needed.

Lasers with different wavelengths and types are employed to increase drug permeation. These include the ruby laser, the erbium:yttrium-gallium-garnet laser, the neodymium-doped yttrium-aluminumgarnet laser and the CO2 laser. Fractional modality is a novel concept for promoting topical/transdermal drug delivery. The laser is useful in enhancing the permeation of a wide variety of permeants, such as small-molecule drugs, macromolecules and nanoparticles.

III. Fractional Lasers and Drug Delivery

Response to topical drug therapy depends on uptake of the compound, which is limited by the skin’s permeability barrier. The uptake of most drugs is poor, typically only 1% to 5% of a topically applied dose, and many delivery strategies have been developed to aid drug uptake. Commonly used techniques have included tape-stripping to remove the stratum corneum (SC), electroporation, iontophoresis, microneedling, and sonophoresis. These techniques have various advantages and disadvantages, and in general, are cumbersome in practice. 
In 2010, ablative fractional laser (AFXL) was introduced as a new drug delivery-enhancement technique. /21/ The technique is based on fractional photothermolysis, which uses focused laser beams to create an array of very small thermal injuries in the skin./22/ Available AFXL systems include the carbon dioxide (CO2, l = 10,600 nm) and erbium-doped yttrium aluminum garnet (Er:YAG, l = 2,940 nm) lasers. These far-infrared lasers vaporize tissue efficiently, creating an array of very small channels into the skin known as microscopic ablation zones (MAZs). The MAZs cross the skin barrier, providing direct access to viable epidermis and dermis until the channels close by local wound repair, without scarring. Geometrically, the diameter, depth, and number of channels per unit skin area can be independently adjusted to regulate uptake and penetration of topical drugs./ 23/ Ablative fractional laser itself is an evolving treatment modality with numerous clinical applications. Ablative fractional laser-assisted delivery of drugs, fillers, and other substances is rapidly emerging; this review examines the authors’ present state of knowledge, efficacy, and safety concerns.

Clinical Applications: Multiple studies have demonstrated that AFXL-pretreatment can assist topical drug delivery into the skin./24-28/ Investigated compounds include topical 5-aminolevulinic acid, methyl 5-aminolevulinate (MAL), 5-fluorouracil (5-FU), ascorbic acid, corticosteroids, diclofenac, imiquimod, lidocaine, methotrexate (MTX), allogeneic stem cells, autologous platelet-rich plasma, and therapeutic antigens.

Ablative fractional laser-assisted delivery potentially alters everything—delivered amount, penetration depth, pharmacokinetics, metabolism, retention, and systemic uptake. The channels created by AFXL are typically several hundred micrometers in diameter, large enough to admit particles, cells, or tissue fragments applied to the skin. Each channel includes an empty volume space, lined with a thin layer of thermally denatured tissue that may or may not act as a secondary barrier. The dermal channels can be full-thickness or even deeper, and access to the vascular system raises the concern of potential systemic toxicity. Other concerns include introduction of pathogens from the skin surface or from nonsterile topical preparations.

The potential of AFXL-assisted delivery is to safely enhance and control intracutaneous delivery of topically applied drugs to different layers of the skin. The therapeutic window of an AFXL treatment, that is, how long the delivery of a topical agent is effectively enforced, is debated. After laser exposure, the skin barrier is presumably broken until reepithelialization occurs (2–4 days)/27 /; within hours after AFXL, tissue fluids, and granulocytes are suspected to crowd the MAZs, and to utilize the channel depth, a more rapid application is thought to be necessary.  Although the depth and density of laser-induced channels can be precisely controlled, the effects of laser settings on delivery of various substances are not well understood.

Although SC is the main barrier for classical topical drug delivery, the interstitial matrix consisting of collagen fibers, elastin, and hyaluronic acid can present a substantial diffusion barrier for drugs with higher molecular weights. Geometry of the channels and their coagulation zones might also affect the diffusion of drugs, in particular with higher molecular weight into the skin. Potentially, AFXL can deliver not only drugs and cells, but also particles and scaffold materials. Ablative fractional laser-assisted drug delivery is a promising modality, and the technology is potentially adaptable to other organs or tissues including mucosal surfaces. With responsible development, AFXL-assisted drug delivery may become a new important part of both medical and surgical dermatology.

Growing knowledge about the possible pathomechanisms of vitiligo has led to the development of novel and alternative treatments based on the use of therapies that may act on the balancing of the oxidative stress, the stimulation of the cutaneous blood flow, and/or having a positive impact on selective neuropeptide- or cytokine mediated pathomechanisms of vitiligo.

Prostaglandin E2 (PGE2) has been shown to have immunomodulatory effect and play an important role in melanogenesis, having growth-stimulatory effects on melanocytes and being capable of regulating their proliferation and maturation. In animal models, the topical application of PGE2 has been shown to increase melanocyte density (29 ), while lately, an observational study on humans showed significant results when a gel formulation containing PGE2 was topically applied twice daily for 6 months in subjects with stable vitiligo patches involving <5% body surface area (30). These positive results were also supported by evidence that UV light exerts a major part of its therapeutic efficacy in vitiligo by the increased production of PGE2 (31). In fact, UV light may induce the production of cyclooxygenase-2 (COX-2), an enzyme linked to mitogenic and inflammatory stimuli which leads to the production of PGE2 in keratinocytes./42/

Latanoprost, a prostaglandin F2alpha analog well established in the treatment of open-angle glaucoma and ocular hypertension, is well known for causing, among the possible side effects, increased pigmentation of the iris and periocular hyperpigmentation (32). Follow-up studies of patients withdrawn from latanoprost treatment show that the increased iridial pigmentation is irreversible, whereas changes in periocular skin pigmentation are reversible after cessation of therapy (33). These effects, however, do not seem to be attributed to a direct action of latanoprost itself on melanogenesis and/or melanocyte proliferation. In both bovine and human iridial melanocytes, latanoprost acid has been shown to cause a significant increase of the PGE2 production, indicating an involvement of COX-2 in the overall process (34). Endogenous prostaglandins (auto- and/or paracrine) could thus stimulate melanogenesis in skin depigmentation disorders such as vitiligo.

Various combination treatments have already been attempted in an effort to enhance efficacy in patients with vitiligo. It has been reported that CO2 fractional laser abrasio has an additive effect in enhancing the rate and degree of repigmentation with application of pimecrolimus by enhancing drug absorption and autoinoculation of melanocytes from the margin./35/ Also, laser dermabrasion followed by NB-UVB in combination with a potent topical steroid increased the repigmentation rate in NSV./36/

IV.  Inflammatory infiltrate at the lesion margin - marker  for clinical staging of individual lesions and therapeutic decisions

Vitiligo is a common skin disorder characterized by the progressive development of areas of skin devoid of melanocytes. Establishing the disease stability is important for making therapeutic decisions, but the notion of stable disease is subject to interpretation. Clinically, a three-stage scoring method, namely progressive/regressive/stable disease, is often used. However, the clinical definition of ‘stability’ varies greatly. Moellmann et al. (1982)/37/ defined active disease as ‘when lesions are enlarging in 6 weeks before examination’; Cui et al. (1993)/38/ as the ‘development of new lesions or extension of old lesions in 3 months before examination’; Uda et al. (1984)/39/ as the ‘spread without regression within the last half year’; and Falabella et al. (1993)/13/ defined stable vitiligo as ‘a condition that has not progressed for at least 2 yr’. Falabella et al. further proposed a list of clinical criteria to define the stability: (i) lack of progression of old lesions within the past 2 yr; (ii) no new lesions developing within the same period; (iii) absence of recent KP either from history or experimentally induced; (iv) spontaneous repigmentation or repigmentation of depigmented areas by medical treatment; and (v) positive minigrafting test and lack of koebnerization at donor site. However, these criteria may be challenged by clinical observations in which KP and minigraft testing are discordant. Data obtained from minigraft testing in case series suggest that the minigraft test provides a reflection of the stability of defined individual lesions, which does not necessarily reflect global stability of the disease. One of the striking features of vitiligo, compared to other chronic skin conditions, is the absence of clinical symptoms and overt signs of inflammation. However, histological studies indicate that an inflammatory response can be detected at the progressing edge of depigmented lesions /40/. Absence/presence of inflammatory infiltrate at a lesion margin might be helpful for clinical staging of individual lesions. Indeed, the observation of a microinflammatory border in active vitiligo, and/or of other local markers of disease activity, might become useful to make appropriate therapeutic decisions.

Histological analysis of the perilesional margin surrounding the depigmented skin reveals a lymphocytic infiltrate consisting of activated T cells. It was found that these T cells were skin-homing, polarized toward type-1 effector function, and evidently cytotoxic while clustering near disappearing melanocytes. In addition, vitiligo patients often have melanocyte-specific antibodies in their blood /38/ and circulating skin-homing melanocyte-specific cytotoxic T-lymphocytes (CTLs). Upon melanocyte antigen-specific stimulation, the perilesional CD8 + T cells became activated./40/

Stability is considered the most important parameter before performing any melanocyte transplantation procedure in vitiligo; however, current criteria rely on the history given by the patients.

The proper selection of cases for surgical therapy is of paramount importance
and the stability of the vitiligo is taken as the most important parameter before opting for any transplantation technique. The significance of stability of vitiligo in transplantation surgery has long been recognized but there has been little consensus regarding the optimal required period of stability.

The recommended period of stability in different studies varied widely from 4 months to 3 years,2–6 but none of these criteria were based on evidence obtained from systematic
research. They were conceived on a subjective basis and documented arbitrarily. Vitiligo has a complex pathogenesis in which biochemical and immunological (cellular and humoral) factors have been proposed to play a role. In vitiligo, catalase levels are known to be decreased in blood and lesional skin.

In addition, vitiligo is known to be associated with altered immunological profiles of CD4, CD8, CD45RO, CD45RA, and FoxP3 in the affected skin. Therefore, it is obvious that in any attempt to predict the outcome of any surgery for vitiligo, along with the clinical stability, the assessment of biochemical and cellular stability is important. Rio et al. 2012 highlights that activity of cytotoxic T lymphocytes in vitiligo lesions may be responsible for the unsuccessful results after transplantation. In the responder group, the upper limit of the 95% confidence interval for the mean of CD8 was 2.31%.The authors recommend that transplantation should be undertaken only if the CD8 count in the perilesional skin is < 2.31% of the total lymphocyte population./41/

Materials and Methods

Patient selection

Inclusion criteria: stable vitiligo (defined as no new lesion or expansion preexisting lesion in the previous 12 months), ages 16 and older, poor or no response to conventional therapy (e.g., topical treatment or phototherapy), no history or clinical signs of the Koebner phenomenon, and no tendency to develop keloids.

The patients are divided into three therapeutic groups:

  1. Group 1. Patients, who undergo a therapy with с melanocyte-enriched cell suspension for the treatment of on refractory non-segmental vitiligo (NSV) and to investigate a simplified and cost-effective method for NCECS –„ the four compartment (FC) method“ combined with 311-nm narrow-band microphototherapy BIOSKIN®  . 
  2. Group 2. Patients, who undergo therapy with fractional CO2 laser-assisted drug delivery of topically applied  Prostaglandin E2 (PGE2), followed by 311-nm narrow-band microphototherapy BIOSKIN®. 
  3. Group 3. Patients, who undergo conventional therapy with calcipotriol ointment 50 µg/g twice a day in combination with 311-nm narrow-band microphototherapy BIOSKIN®.

Group 1.
Graft collection

To prepare the cell suspension, a split-thickness graft, usually of one-tenth the size of the recipient area, is taken under local anaesthesia from the anterior or lateral thighs, using a shaving blade held firmly by long straight artery forceps. The wound is dressed using a sterile dressing. The skin graft is taken from the patient and transferred to an FC Petri dish, where the following steps are performed in compartments in the following sequence.

Compartment 1: The skin graft is taken from the patient and transferred to compartment 1 in the Petri dish with 0.25% trypsin and 0.02% ethylenediaminetetraacetic acid solution and incubated at 37 °C for 1 h. Compartments 2 and 3: After 1 h, the graft is transferred in turn to the next two compartments, which contained PBS, and washed gently twice with PBS using a sterile syringe, so as to remove the remaining trypsin solution. Compartment 4: After washing, the graft is transferred to the fourth compartment for separation of the epidermis and dermis, and PBS is poured into the compartment. Thereafter, the dermis is separated from the epidermis using sterile forceps, and cells are dislodged gently from the basement membrane. After dissociating all the cells into the PBS, the hard parts of the dermis and epidermis are discarded by manually picking them up with forceps and throwing them away. In order to form a homogeneous single-cell suspension, it is aspirated several times with the help of a sterile syringe. This suspension of epidermal cells, comprising a mixture of keratinocytes and melanocytes, is used for transplantation over the dermabraded area of the patient.

Recipient Site

Recipient depigmented areas are anesthetized with 5 mg/g of lidocaine and 25 mg/g of prilocaine cream (AstraZeneca, Lund, Sweden) with plastic occlusion.  To check the separation of cells from the skin graft after trypsinization at 37 °C for 1 h in the FC method, histological sections of the trypsinized skin graft are prepared. Epidermal cell suspension cell viability are checked by staining a drop of the suspension with Giemsa stain. Further cell proliferation and growth are checked by culturing these cells in a melanocyte and keratinocyte medium for 7 days.


The NCECS is carefully transferred to a tuberculin syringe with an 18-gauge needle attached. Using this, a few drops of the suspension are placed onto the denuded surface, and these are then spread evenly with the help of the needle, after which the dermabraded skin is covered with collagen dressing. The collagen sheet is  then covered by a final piece of saline-moistened gauze. A sterile transparent occlusive is placed on top of the gauze, followed by a surgical pad, and finally elastic plaster. The patients are asked to remain lying down for 30 min after the procedure and are then allowed to go home. The dressing is removed on day 7.


Patients are followed up on day 8 and weeks 4,8, 12 and 16 after the transplantation procedure. Repigmentation Is assessed subjectively by digital photography as follows: < 50%, poor repigmentation; 50–74%, fair repigmentation; 75–89%, good repigmentation; 90–100%, excellent repigmentation. Also, the repigmentation pattern is noted as ‘diffuse’, ‘perifollicular’ or ‘dotted’.  At each visit, patients are followed up for adverse events.

Group 2. Tree  sessions of fractional 10 600 nm CO2 laser therapy on the vitiliginous lesions are performed at a 2-month interval. Latanoprost / prostaglandin F2alpha analog/ is applied locally, immediately after the procedure and throughout the whole follow-up period – once a day on the treated patch without any dressing. 311-nm narrow-band microphototherapy BIOSKIN® is administered 5 days after each laser treatment for 2 months - 1 session per  week.
Group 3. Patients, who undergo conventional therapy with с calcipotriol ointment 50 µg/g twice a day in combination with 311-nm narrow-band microphototherapy BIOSKIN®. The subjects are treated according to the following scheme:

  • 5 sessions, once a day for 5 consecutive days.
  • 10 days break.
  • 1 session every 15 days for 5 months.

Follow-up for Group 2 and 3

Photographs of the subjects are taken at the beginning of the therapy and then once a month for six months using Wood’s lamp and one month after the treatment - by planimetry based on two comparable photographs.  At each visit, patients are followed up for adverse events.

For  Group 1. and Group 2.  A 3-mm punch biopsy for immunohistochemistry is taken from the margin of the patch on the day of transplantation in patients from the abovementioned groups, having signed an informed consent form. Immunohistochemical staining of the tissue is carried out for CD4, CD8, CD45RO, CD45RA and FoxP3.

Objective:  The aim of this study is:

  1. to test the usefulness of a melanocyte-enriched cell suspension for the treatment of on refractory non-segmental vitiligo (NSV) and to  investigate a simplified and cost-effective method for NCECS –„ the four compartment (FC) method“ combined with 311-nm narrow-band microphototherapy . 
  2. to investigate  the effects of fractional CO2 laser-assisted drug delivery of topically applied  Prostaglandin E2 (PGE2), followed by targeted  /NB UVB 311nm/ BIOSKIN® microphototherapy on refractory non-segmental vitiligo (NSV). 
  3. to compare these two new therapeutic methods, in terms of effectiveness and side effects, with the conventional therapy -  calcipotriol ointment 50 µg/g twice a day in combination with 311-nm narrow-band microphototherapy for the treatment of refractory non-segmental vitiligo (NSV).  
  4. to underscore the importance of Stability of the disease as the most important parameter before performing any melanocyte transplantation procedure in vitiligo.
  5. to determine immunological factors, as objective tools for the stability of the disease in patients with vitiligo so as to facilitate better patient selection for melanocyte transplantation, based on the percentage of CD8 and CD45RO cells in a lesional skin biopsy, following the assumption that higher percentage of CD8 and CD45RO cells in a lesional biopsy is associated with failure of repigmentation after transplantation.


  1. Taueb A, Picardo M., VETF Members. The definition and assessmentof vitiligo: a consensus report of the Vitiligo European Task Force. Pigment Cell Res 2007;20:27–35.
  2. Lotti T, D'Erme A, Vitiligo as a systemic disease; Clin Dermatol 2014;32(3):430-4. 
  3. Mutalik S, Ginzburg A. Surgical management of stable vitiligo: a view with personal experience. Dermatol Surg 2000; 26:248–54.
  4. Malakar S, Dhar S. Treatment of stable and recalcitrant vitiligo by autologous miniature punch grafting: a prospective study of 1000 patients. Dermatology 1999; 198:133–9.
  5. Olsson M, Juhlin L. Epidermal sheet grafts for repigmentation of vitiligo and piebaldism, with a review of surgical techniques. Acta Derm Venereol 1997; 77:463–6.
  6. Kahn AM, Cohen MJ. Repigmentation in vitiligo patients. Melanocyte transfer via ultra-thin grafts. Dermatol Surg 1998; 24:365–7.
  7. Kumagai N, Uchikoshi T. Treatment of extensive hypomelanosis with autologous cultured epithelium. Ann Plast Surg 1997; 39:68–73.
  8. Olsson M, Juhlin L. Transplantation of melanocytes in vitiligo. Br J Dermatol 1995; 132:587–91.
  9. Kahn AM, Ostad A, Moy RL. Grafting following short-pulse carbon-dioxide laser de-epithelialization. Dermatol Surg 1996; 22:965–8.
  10. Goh BK, van Geel NAC, Ongenae K, Naeyaert JM. Cellular grafting and repigmentation in vitiligo. Expert Rev Dermatol 2006; 1:121–9.
  11. Falabella R. Grafting and transplantation of melanocytes for repigmenting vitiligo and other types of leukoderma. Int J Dermatol 1989; 28:363–9.
  12. Mulekar SV, Ghwish B, Al Issa A, Al Eisa A. Treatment of vitiligo lesions by ReCell vs, conventional melanocyte-keratinocyte transplantation: a pilot study. Br J Dermatol 2008; 158:45–9.
  13. Falabella R, Escobar C, Borrero I. Treatment of refractory and stable vitiligo by transplantation of in vitro cultured epidermal autografts bearing melanocytes. J Am Acad Dermatol 1992; 26:230–6.
  14. Gauthier Y, Surleve-Bazeille J. Autologous grafting with noncultured melanocytes: a simplified method for treatment of depigmented lesions. J Am Acad Dermatol 1992; 26:191–4.
  15. Gauthier Y, Benzekri L. Non-cultured epidermal suspension in vitiligo: from laboratory to clinic. Indian J Dermatol Venereol Leprol 2012; 78:59–63.
  16. Van Geel N, Ongenae K, DeMil M, Naeyaert JM. Modified technique of autologous non cultured epidermal cell transplantation for repigmenting vitiligo: a pilot study. Dermatol Surg 2001; 27:873–6.
  17. Goh BK, Chua M, Chong K et al. Simplified cellular grafting for treatment of vitiligo and piebaldism: the ‘6-well plate’ technique. Dermatol Surg 2010;36:203–7.
  18. Kumar R , Parsad D, Singh C, Yadav S. Four compartment method: a simplified and cost-effective method of noncultured epidermal cell suspension for the treatment of vitiligo. British Journal of Dermatology 2014;170:581–585 
  19. Prignano F, Campolmi P, Bonan P, et al. Fractional CO2 laser: a novel therapeutic device upon photobiomodulation of tissue remodeling and cytokine pathway of tissue repair.Dermatol Ther 2009;22:8–15.
  20. Lin CH, Ibrahim A et al. Lasers as an approach for promoting drug delivery via skin. Expert Opin. Drug Deliv. (2014) 11(4);599-614
  21. Haedersdal M, Sakamoto FH, Farinelli WA, Doukas AG, et al. Fractional CO(2) laser-assisted drug delivery. Lasers Surg Med 2010;42:113–22.
  22. Manstein D, Herron GS, Sink RK, Tanner H, et al. Fractional photothermolysis: a new concept for cutaneous remodeling using microscopic patterns of thermal injury. Lasers Surg Med 2004; 34:426–38.
  23. Taudorf EH, Haak CS, Erlendsson AM, Philipsen PA, et al. Fractional ablative erbium YAG laser: histological characterization of relationships between laser settings and micropore dimensions. Lasers Surg Med 2014;46:281–9.
  24. Togsverd-Bo K, Haak CS, Thaysen-Petersen D, Wulf HC, et al. Intensified photodynamic therapy of actinic keratoses with fractional CO2 laser: a randomized clinical trial. Br J Dermatol 2012;166:1262–9.
  25. Lee WR, Shen SC, Al-Suwayeh SA, Yang HH, et al. Laser-assisted topical drug delivery by using a low-fluence fractional laser: imiquimod and macromolecules. J Control Release 2011;153:240–8.
  26. Waibel JS, Wulkan AJ, Shumaker PR. Treatment of hypertrophic scars using laser and laser assisted corticosteroid delivery. Lasers Surg Med 2013;45:135–40.
  27. Oni G, Lequeux C, Cho MJ, Zhang D, et al. Transdermal delivery of adipocyte-derived stem cells using a fractional ablative laser. Aesthet Surg J 2013;33:109–16.
  28. Bachhav YG, Heinrich A, Kalia YN. Using laser microporation to improve transdermal delivery of diclofenac: increasing bioavailability and the range of therapeutic applications. Eur J Pharm Biopharm 2011;78:408–14.
  29. Parsad D, Pandhi R, Dogra S, Kumar B. Topical prostaglandin analog (PGE2) in vitiligo – a preliminary study. Int J Dermatol 2002: 41: 942–945.
  30. Kapoor R, Phiske MM, Jerajani HR. Evaluation of safety and efficacy of topical prostaglandin E2 in treatment of vitiligo.Br J Dermatol 2009: 160: 861–863.
  31. Namazi MR. Ultraviolet light exerts a major part of its therapeutic efficacy against vitiligo by production of prostaglandin E2. Dermatology 2008: 217: 149.
  32. Hommer A. A review of preserved and preservative-free prostaglandin analogues for the treatment of open-angle glaucoma and ocular hypertension. Drugs Today (Barc) 2010: 46: 409–416.
  33. Grierson I, Jonsson M, Cracknell K. Latanoprost and pigmentation. Jpn J Ophthalmol 2004: 48: 602–612.
  34. Bergh K, Wentzel P, Stjernschantz J. Production of prostaglandin e(2) by iridial melanocytes exposed to latanoprost acid, a prostaglandin F(2 alpha) analogue. J Ocul Pharmacol Ther 2002: 18: 391–400.
  35. Farajzadeh S, Daraei Z, Esfandiarpour I, Hosseini SH. The efficacy of pimecrolimus 1% cream combined with microdermabrasion in the treatment of nonsegmental childhood vitiligo: a randomized placebo-controlled study. Pediatr Dermatol 2009; 26:286–91.
  36. Bayoumi W, Fontas E, Sillard L et al. Effect of a preceding laser dermabrasion on the outcome of combined therapy with narrowband ultraviolet B and potent topical steroids for treating nonsegmental vitiligo in resistant localizations. Br J Dermatol 2012; 166:208–11.
  37. Moellmann G, Klein-Angerer S, Scollay DA, Nordlund JJ, Lerner AB (1982) Extracellular granular material and degeneration of keratinocytes in the normally pigmented epidermis of patients with vitiligo. J Invest Dermatol 79:321–30
  38. Cui J, Bystryn JC. Melanoma and vitiligo are associated with antibody responses to similar antigens on pigment cells. Arch Dermato 1995; 131:314–8
  39. Uda H, Takei M, Mishima Y. Immunopathology of vitiligo vulgaris, Sutton's leukoderma and melanoma-associated vitiligo in relation to steroid effects. II. The IgG and C3 deposits in the skin. J Cutan Pathol. 1984;11(2):114-24.
  40. van den Boorn JG, et al. Autoimmune Destruction of Skin Melanocytes by Perilesional T Cells from Vitiligo Patients. Journal of Investigative Dermatology 2009; 129:2220-32.
  41. Rao A , Gupta S, Study of clinical, biochemical and immunological factors determining stability of disease in patients with generalized vitiligo undergoing melanocyte transplantation. Br Assoc of Derm 2012; 1661:230–1236. 
  42. Lotti TM, Hercogová J, Schwartz RA, et al. Treatments of vitiligo: what's new at the horizon. Dermatol Ther  2012;25 Suppl 1:32-40.