Hair Follicle Melanocyte Cells as a Renewable Source of Melanocytes for Culture and Transplantation

Porcine model

All animals were treated in an AAALAC-approved facility under an approved animal protocol. Three 2-month-old Yucatan mini pigs (Charles River, Wilmington, MA) were housed and treated in the animal care facility at Brigham and Women's Hospital (Boston, MA) for at least 3 days before the start of each experiment. After acclimation, the pigs were sedated (telazol 5 mg/kg IM and xylazine 2 mg/kg IM) and transported into our animal operating room. General anesthesia was administered via snout mask using halothane (1.5%–3.0%), oxygen (4–6 L/min), and nitrous oxide (2–3 L/min) and monitored using continuous pulse oximetry. The dorsum and scalp of the pig was scrubbed and painted 3 times using a betadine solution. Hair follicles were then plucked from these areas and immediately transferred into an enriched Gibco keratinocyte serum-free media (S-FM; Invitrogen, Carlsbad, CA).

Sixteen 3.0×3.0-cm full-thickness skin wounds were incised on the paraspinous region of the dorsum. Hemostasis was obtained with direct pressure and electrocautery. Collagen-glycosaminoglycan (GAG) matrices, either with or without hair follicles, were sutured in place and stabilized using a semiocclusive polyurethane dressing (Tegaderm, St. Paul, MN), gauze, and a protective cotton vest.

Harvesting hair follicles

Anagen phase hair follicles were plucked from the scalp and back of a Yucatan pig. All plucked hair follicles were immediately transferred into an enriched Gibco keratinocyte S-FM and subsequently examined under a microscope to ensure that hair root bulbs and sheath were intact. We also removed excess hair root debris from around each bulb. Hair follicles were then implanted into collagen-GAG matrices after being stained with a PKH26 fluorescent dye for cell tracking, and either immediately placed into newly ceated full-thickness skin wounds or grown in cell culture media for 2 weeks before implantation. After the matrices containing 3 hair follicles were applied to the dorsal wounds of each pig, they were covered with Tegaderm and followed daily until complete wound healing (Fig F1).

Collagen-CAG matrices

Porous matrices were prepared using a procedure initially described by Yannas et al.¹¹ A collagen dispersion was prepared using 0.5% bovine hide collagen (Sigma Chemical, St. Louis, MO) in 0.05 M acetic acid (pH 3.2) with chondroitin 6-sulfate (sodium salt, Type C, Sigma Chemical, St. Louis, MO) in a custom-built blender with an internal cooling apparatus. This was poured into a 3-mm thick stainless steel pan and transferred to a freeze dryer with a shelf temperature of −r°40°C (Virtis Unitop 800, Gardiner, NY) for 20 hours. After lyophilization, matrices were cross-linked using a dehydrothermal treatment at 105°C for 24 hours, followed by UV irradiation at 254 nm at 4510 μW/cm² for 45 minutes. Matrices were then cut into 3.0×3.0-cm squares under sterile conditions.

Cell culture

We used an enriched Gibco keratinocyte S-FM for storing harvested hair follicles, hydrating collagen-GAG matrices before follicle placement and coverage of wounds, and culturing follicular melanocytes in vitro. This media consists of using keratinocyte S-FM supplemented with pituitary extract (Invitrogen, Carlsbad, CA), epidermal growth factor (EGF) (R&D Systems, Minneapolis, MD), pen-strep-Glutamine (PSQ) (Shimazu, Kyoto, Japan), hFGF, and TPA (Sigma, St. Louis, MO).

PKH26 fluorescent red stain

Harvested hair follicles were rinsed using a phosphate buffered saline solution and then stained using PKH26 fluorescent cell linker (Sigma, St. Louis, MO) for cell tracking. One group (n = 8) was immediately placed on to collagen-GAG matrices for immediate transplantation, whereas a second group (n = 8) was grown in enriched keratinocyte S-FM for 2 weeks before transplantation.

Melanocyte transplantation

Follicular melanocytes using 3.0×3.0-cm collagen-GAG matrices as a delivery agent were transplanted into newly created 3-cm² full-thickness skin wounds on day 0 and day 14. Follicular melanocytes that were transplanted on day 0 were stained after being harvested and immediately reimplanted into dorsal wounds (n = 8). Melanocytes transplanted on day 14 were grown in vitro for 2 weeks before transplantation (n = 8). All wounds were covered with a semiocclusive polyurethane dressing and followed daily until complete wound healing.

Wound harvesting and histology

Wounds were harvested on day 14 and day 28 under general anesthesia with a 1-cm margin of normal tissue. After biopsies were taken on day 14, the resulting wounds were covered with isotonic sodium chloride solution–soaked collagen-GAG matrices and using the same polyurethane dressing. After biopsies were taken on day 28, each animal was euthanized. One set of biopsy specimens was stored in 10% formalin, transferred to cassettes, embedded, sectioned, and stained with hematoxylin and eosin. Two observers, both blinded to the specimen groups, independently counted the number of melanocytes present in each wound. The second set of biopsy specimens was frozen immediately using liquid nitrogen and stained with a DAPI solution in phosphate buffered saline for visualization under a fluorescent microscope (Axioplan 2 Imaging, Zeiss, TMS, Nikon). Images of histological slides were taken using an inverted microscope at 200× magnification. Cell counting was performed using the same microscope under 400× magnification.

Results

Hair follicle melanocytes begin to proliferate in the early Anagen II stage and continue through the Anagen III stage of the hair growth cycle. After harvesting, melanocytes were either grown in vitro using enriched keratinocyte S-FM culture for 2 weeks or transplanted immediately.

Among the cultured melanocytes, a pigmented area was identified around the implanted hair follicle in the matrix before transplantation (Fig F1). This area corresponds to an area of potential melanocyte migration from the hair follicle bulb. We could see the pigmented area around the base of cultured hair follicles on both day 7 and day 14. After the transplantation, the dark-brown-colored area (arrow) is still visible around the transplanted hair follicle (Fig F2).

When hair follicles were transplanted immediately after harvest and allowed to heal, we were also able to identify the pigmented area 2 weeks posttransplantation. This pigmented area is similar to that seen among cultured melanocytes. We could see a dark-brown area around the transplanted hair follicles, whereas no pigmentation was observed in the nontransplanted wound. Over time, as the surgical wounds healed, this dark-brown color became more pronounced (Fig F3).

After complete healing (4 weeks posttransplantation), central areas of pigmentation observed in hair follicle transplanted wounds appeared even darker and thicker than their counterparts at 2 weeks posttransplantation (Fig F4). Despite marked wound contraction, a rim of nonpigmented tissue visibly demarcated the central area of hair follicle melanocyte pigmentation from the rim of epidermal melanocyte pigmentation surrounding each wound.

Histological sections of the wound show that a large number of melanocytes per high-power field can be seen in transplanted wounds (Fig F5). The study wound contained 6.0 ± 1.4 pigmented melanocytes per high-power field, whereas control wounds had only 0.2 ± 0.4 pigmented melanocytes.

To distinguish follicular melanocytes from their epidermal counterparts, plucked hairs were stained with PKH26 fluorescent dye for cell tracking before transplantation (Fig F6). We detected PKH26-positive cells containing melanin pigments in the transplanted areas, indicating that melanocytes in the pigmented area are derived from the follicle implant (Fig F7).