|Year : 2015 | Volume
| Issue : 1 | Page : 4-8
Follicular cell suspension: A new surgical modality for the treatment of vitiligo
Gitesh U Sawatkar, Keshavamurthy Vinay, Sunil Dogra
Department of Dermatology, Venereology and Leprology, Postgraduate Institute of Medical Education and Research, Chandigarh, India
|Date of Web Publication||26-Jun-2015|
Department of Dermatology, Venereology and Leprology, Postgraduate Institute of Medical Education and Research, Sector 12, Chandigarh - 160 012
Source of Support: None, Conflict of Interest: None
Follicular cell suspension is a recently described technique of cellular suspension for treatment of stable vitiligo. Its uniqueness lies in the fact that hair-follicle melanocytes from the outer root sheath are utilized as a source of melanocytes unlike traditional methods, which use epidermal melanocytes. Few distinctive properties of these hair-follicle melanocytes make them an attractive source of donor cells over epidermal melanocytes. In this article, we briefly discuss the theoretical basis, procedure and advantages of this novel technique.
Keywords: Follicular cell suspension, melanocytes, stem cells, surgery, vitiligo
|How to cite this article:|
Sawatkar GU, Vinay K, Dogra S. Follicular cell suspension: A new surgical modality for the treatment of vitiligo. Pigment Int 2015;2:4-8
| Introduction|| |
Vitiligo is chronic, acquired pigmentary disorder with a worldwide incidence. The prevalence of vitiligo is 1% and varies from 0.1 to more than 8.8% in different parts of the world with the highest incidence in the Indian subcontinent.  In vitiligo, there is a selective and progressive loss of functioning melanocytes from the basal layer of epidermis and hair-follicles, which clinically translates to depigmented patches over the skin and mucosa.  Apart from sunburns in amelanotic skin, the disease is asymptomatic, is not preceded by inflammation and does not have any adverse systemic symptoms. However, it causes severe cosmetic disfigurement with profound psychological implications. Social discrimination based on skin changes leads to a long lasting psychological morbidity causing a major impact on quality of life of the patients. 
The cause for the selective loss of melanocytes is to be clearly deciphered. But it seems to be a heterogeneous and multifactorial disease process, in which multiple factors, varying in different patients and with the stages of the disease, influence the biological behavior of the melanocytes.  Though a continuous research is still ongoing concerning its pathogenesis and treatment, a definitive solution is still awaited.
Various modalities of treatment (both medical and surgical) have been used in the treatment of vitiligo. Although medical modalities are the main stay of treatment, it is not effective in all and residual lesions require surgical treatment. A variety of surgical procedures like minigrafts, split thickness grafts, suction blister grafts, noncultured epidermal cell suspension (NCES) and cultured autologous melanocytes are available for the treatment of vitiligo. The key principle of surgical methods is to transfer melanocytes from uninvolved skin to the stable vitiligo patch so that they are able to migrate, and proliferate to repopulate the depigmented skin resulting in repigmentation. Autologous noncultured outer root sheath (ORS) hair-follicle cell suspension also known as follicular cell suspension (FCS) is a new novel surgical therapy for the treatment of vitiligo.
| Follicular Cell Suspension|| |
The core idea
Hair-follicle melanocytes play an important role in re-pigmentation of vitiliginous lesion. Repigmentation in vitiligo can occur in four clinical patterns: Follicular, marginal, diffuse and combined, follicular being the commonest. It has been suggested that the basis for such type of repigmentation is due to the reservoir of melanocyte stem cells (MelSCs) in the hair-follicle.  In vitiliginous lesions, there is a selective destruction of dihydroxyphenylalanine (DOPA)-positive melanocytes, whereas the DOPA-negative melanocytes and its precursors stem cells in the ORS of the hair-follicles are spared.  Further, hair-follicle melanocytes express antigens associated with alopecia areata, but not that associated with vitiligo.  MelSCs is located in the upper, permanent region of ORS and have the capacity to migrate and enter vacant niches in epidermis to cause perifollicular re-pigmentation, which later spread in a concentric pattern causing diffuse re-pigmentation. 
Advantages over epidermal cell suspension
Hair-follicle melanocytes have some unique properties. A follicular melanin unit is constituted by one melanocyte for every five keratinocytes in the hair bulb, which is much higher than epidermal melanin unit, which has one melanocyte for every 36 keratinocytes.  Furthermore, hair melanocytes have remarkable synthetic capacity, and a relatively small number of melanocytes can potentially produce sufficient melanin to pigment up to 1.5 m of the hair shaft. Hair-follicle is a rich source of three different types of stem cells, and it appears that all of them are important in hair growth. These stem cells include MelSCs, keratinocyte stem cells, and mesenchymal stem cells.  With all these properties, the hair appears to be a more attractive source of melanocytes than epidermis for cell based therapies in vitiligo.
In addition to the unique characteristics of hair-follicle melanocytes and stem cells, FCS has other added advantages. The procedure of hair extraction (described further) involves removal of much less volume of tissue in comparison with epidermal shave biopsy. Thus, the healing time of the donor site is much less compared with conventional shave biopsy from buttock or thigh. The minimal scars of follicular unit extraction (FUE) on the scalp are invisible and insignificant, unlike in NCES where there is a risk of keloid or scar formation over the donor site. In comparison with the conventional NCES, the technique of preparation of FCS is simpler, as it does not require separation of the epidermis from dermis and manual breaking of epidermal samples into small pieces.
| Procedure of Follicular Cell Suspension|| |
Follicular unit extraction
- Few hairs from the occipital area of the scalp are trimmed to a length of approximately 2 mm [Figure 1]a
|Figure 1: (a) Hairs from the occipital area trimmed to 2 mm length and field block being injected. (b) Hairs extracted from the occipital area using 1 mm disposable plastic punches|
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- Actively growing anagen hairs, which are usually more pigmented due to increased melanin content in this phase, are selected from the occipital area of the scalp
- 2% lignocaine is used to give a field block anesthesia encircling the trimmed hair area
- With the help of 1 mm punch, hair-follicles are obtained. A manual or motorized punch can be used; however manual punch is preferred to have precise control [Figure 1]b Utmost care is taken to avoid piercing the sub cutaneous space so as to avoid transection of hair-follicle
- Then the hair-follicular unit is gently pulled out using a hair-follicle holding forceps
- Depending on the area to be transplanted, approximately 30-40 pigmented follicles are extracted
- The transacted hair-follicles are discarded, and the suitable hair-follicles are collected in normal saline and are then transported to the laboratory for necessary processing
- The donor area is dressed using mupirocin ointment and sterile cotton pads.
Preparation of a single cell suspension
- In the laboratory under all sterile conditions, the extracted hair-follicles are washed with phosphate buffered saline (PBS) for about 3 times. The PBS contains suitable antibiotics and antimycotics such as penicillin, streptomycin, and amphotericin B
- The hair-follicles are then incubated with 0.25% trypsin − 0.05% ethylene diamine tetra acetic acid (EDTA) at 37°C for 90 min to prepare the single cell suspension. The trypsin used can be either porcine or bovine source derived or recombinant trypsin. The former has a risk of zoonotic disease transmission such as transmission of porcine endogenous retroviruses. Studies suggest the use of recombinant trypsin produce similar results as an animal derived trypsin 
- Within 15-20 min of incubation, the cells start loosening from each other. The hair-follicles are subsequently placed in another tube of trypsin and EDTA. Trypsin inhibitor is added in the former tube to cease the reaction and prevent the digestion of separated cells by trypsin
- At the end of three such cycles, a thin keratinous shaft is left behind [Figure 2]a and b
|Figure 2: (a) Extracted hair follicles in Petri dish More Details prior to trypsinization. (b) After treating with trypsin, thin keratinous hair shafts are left behind|
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- The cell suspensions of all the tubes are added in a single tube and then filtered through a 70 μm cell strainer to prepare a single cell suspension
- The final cell pellet is obtained by centrifuging the combined cell suspension at 1000 rpm for 5 min. The cell pellet thus obtained is again suspended in PBS. The total amount of time for the laboratory procedure takes about 2-3 hours [Figure 3].
|Figure 3: (a) Hair-follicles in normal saline prior to transportation to the laboratory. (b) Cell pellet formed by centrifuging for 5 min at 1000 rpm|
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- The recipient site is cleaned with betadine-surgical spirit and draped [Figure 4]a
|Figure 4: Clinical photography showing different stages of cellular grafting. (a) Lesional skin over retro auricular area. (b) Dermabrasion of the lesional skin till pinpoint bleeding spots are seen. (c) Placement of the cellular graft by a tuberculin syringe. (d and e) First layer dressing by collagen and chlorhexidine gauze. (f) Second layer dressing by sterile cotton pad. (g and h) Third layer dressing by a transparent dressing film (Tegaderm) and an elastic bandage (Dynaplast)|
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- Prior to dermabrasion, local anesthesia is given using 2% lignocaine
- Manual or motorized dermabrasion is done until tiny bleeding points appear over the recipient site. The appearance of bleeding points suggests the approach of dermo-epidermal junction. Dermabrasion should not be carried beyond this point or scaring will ensue [Figure 4]b
- The procedure of dermabrasion needs to be carried about 5 mm beyond the margin of vitiliginous skin so as to avoid the halo phenomenon
- Using an 18 g needle attached to a tuberculin syringe or a pipette, few small drops of suspension are placed over the denuded surface. This is then spread evenly to cover all the dermabraded area [Figure 4]c
- A meshed collagen sheet (Neuskin-F ® ; Eucare Pharmaceuticals, Chennai, India) is placed over the suspension, which is then covered with sterile vaseline/chlorhexidine gauze [Figure 4]d and e
- Over this surgical pad is put, and the dressing is stabilized by a transparent dressing film (Tegaderm) and an elastic bandage (Dynaplast) [Figure 4]f-h
- The patient should be observed for 1 h after procedure.
- The patient is advised to keep the dressing dry and minimize local manipulation
- Systemic antibiotics are prescribed for a minimum period of 7 days
- The dressing is then removed at 7 days follow-up
- At this stage, the vitiliginous site takes up graft.
| Response Following Follicular Cell Suspension Transplantation|| |
Vanscheidt and Hunziker  in a small case series, first used single cell suspension of "plucked" hair-follicles in the treatment of vitiligo. They found almost complete (>90%) repigmentation in three of five patients with vitiligo, around 50% repigmentation in one patient and <10% repigmentation in the other. Their technique was simple, noninvasive and allowed immediate and repeated application. However, the cell yield is known to be less in case of plucked hair-follicles and optimization of cell harvest form the hair-follicular unit had to be standardized for optimum yield. Later, in a pilot study on three subjects, it was also noticed that stem cell niche is partially lost during follicular plucking and mean percentage of CD200 positive cells (a marker of MelSCs) in FUE was significantly higher than those of plucked hairs. 
In an open label pilot study, Mohanty et al.  performed FCS in 14 patients of vitiligo and achieved >75% pigmentation in nine patients. The procedure involved removal of only 15-25 follicular units, which provided 25,000-50,000 cells sufficient to treat up to 25 cm.  Singh et al. in a randomized comparative study compared FCS with NCES in 30 patients with 47 Vitiligo lesions. Excellent repigmentation (90-100%) was observed in 83.3% of lesions in the NCES group and 65.2% of lesions in the FCS group. In NCES group, the dermatological life quality index reduced from a mean value of 10.73 before surgery to 2.13 after surgery; while in FCS Group, it reduced from a value of 10.47 before surgery to 3.27 after surgery. Though, the response in FCS was numerically less, the authors found no statistical difference in the repigmentation rate between both the groups.  This may be due to the low sample size in the study group. Alternatively, the inferior response may be due to lack of keratinocyte derived growth factors and cytokines since keratinocytes in the suspension supplies essential growth factors for melanocyte growth.  Melanocytic homeostasis is modulated via a complex network of autocrine and paracrine factors. Keratinocyte derived factors that help in melanogenesis include endothelin-1,  stem cell factor,  basic fibroblast growth factor,  nerve growth factor,  etc.
Further, Vinay et al.  studied the factors determining the repigmentation rate in patients of stable vitiligo undergoing FCS. The number of melanocytes, and hair-follicle stem cells transplanted, and absence of dermal inflammation were found to be important determinants of optimal repigmentation.
| Areas of Future Research|| |
It has been an observation that the results obtained by FCS are not to the par as expected theoretically. Even though ORS serves as a major source of melanocytes, their multiplication, and survival to achieve pigmentation might be governed by several other unidentified factors. One such factor may be a lack of keratinocyte-derived growth factors. Future studies should assess if enriching the FCS by adding these growth factors would enhance viability, proliferation and differentiation of hair-follicle melanocytes thus improving the repigmentation rate. Recent studies have assessed the immunological markers associated with poor surgical outcome in stable vitiligo patients.  Use of these immunological markers for patient selection can improve the repigmentation rate of FCS. Follicular melanocytes appear to be more sensitive than epidermal melanocytes to aging influence.  Therefore, long-term follow-up is required for assessing the stability of repigmentation.
Combining different treatment modalities are known to enhance repigmentation rate in patients undergoing NCES. Postsurgery ultraviolet light and excimer laser have been used successfully with other techniques such a punch grafting  and NCES.  This should be assessed further in combination with FCS.
| Conclusion|| |
Surgical interventions have revolutionized the whole management of vitiligo and brought down the overall burden of psychological stress among patients providing them a new ray of hope with pigmentation even on long standing patches. FCS provides a new direction to surgical interventions of vitiligo. The procedure of FCS is easy with requirement of basic lab amenities. The results with FCS are good, but better outcomes can be expected, as ORS is the main home for melanocytes. Further studies are needed that suggest techniques to improve melanocytes survival, function and multiplication to achieve higher pigmentation in the holistic management.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4]
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