Bulge Hair Follicle Stem Cells Accelerate Cutaneous Wound Healing in Rats

Objective. Skin wound healing is a serious clinical problem especially after surgery and severe injury of the skin. Cell therapy is an innovative technique that can be applied to wound healing. One appropriate source of stem cells for therapeutic use is stem cells from the adult bulge of hair follicles. This study examined the effects of adult bulge hair follicle stem cells (HFSC) in wound healing. Materials and Methods. Hair follicle stem cells were obtained from rat vibrissa and labeled with DiI (Invitrogen, Carlsbad, CA), then special markers were detected using flow cytometry. A full-thickness excisional wound model was created and DiI-labeled HFSC were injected around the wound bed. Wound healing was recorded with digital photographs. Animals were sacrificed at 3, 7, or 14 days after surgery, and were used for the following histological analyses. Results. Flow cytometry analysis showed that HFSC were CD34 positive and nestin positive, but K15 negative. Morphological analysis of HFSC-treated wounds exhibited accelerated wound closure. Histological analysis of hematoxylin and eosin stained and Masson’s trichrome-stained photomicrographs showed significantly more re-epithelialization and dermal structural regeneration in HFSC-treated wounds than in the control group. Immunohistochemical analysis of CD31 protein-positive cells showed angiogenesis was also more significant in HFSC-treated wounds than in the control group. Conclusion. Hair follicle stem cells accelerate skin wound healing. Isolating HFSC from a small skin biopsy could repair less-extensive full-thickness skin wounds by autologous stem cells and overcome major challenges regarding the use of stem cells in clinical application, while avoiding immune rejection and ethical concerns.

Skin wound healing is a serious clinical problem particularly after surgery and severe injury because it increases the risk of injury site infections, postoperative hospitalizations^1,2 and raises medical expenses.³ Skin has many important functions including sweat production and thermoregulation, and acts as a barrier against many pathogens; therefore, it is essential to treat any wound of the skin as soon as possible.⁴

Current treatments include wound dressing, surgery, topical negative pressure, and skin substitutes; however, these methods are not effective in all circumstances and there is an urgent requirement to develop innovative techniques to ameliorate wound healing.^5-7 Cell therapy is a new field of medicine that uses embryonic⁸ or adult stem cells⁹ to regenerate tissue. Stem cells are self-renewing and able to proliferate and differentiate into other cell lines,¹⁰ but the appropriate source of stem cells for therapeutic use remains a major challenge. The differentiation potential of adult stem cells provides an opportunity for scientists to apply these cells in clinical applications¹¹; and it is possible to obtain adult stem cells from several organs of the body.¹²

One candidate cell group for regenerative medicine is adult stem cells from the hair follicle bulge.¹³ The hair follicle is a self-renewing structure that reconstitutes itself through the cycle — the growing phase (anagen), regression phase (catagen), and resting phase (telogen) — suggesting the presence of its own stem cells.¹⁴ 

Adult hair follicle stem cells (HFSC) are located in the bulge region, between the insertion of the arrector pili muscle and the duct of the sebaceous gland.¹⁵ Bulge stem cells can differentiate into glial cells, neurons, smooth muscle cells, and keratinocytes.^16,17 Stem cells from the bulge region may be an accessible source for clinical applications.¹⁸

Based on previous studies that mentioned the therapeutic value of hair follicle bulge stem cells in regenerative medicine, the authors hypothesized that transplantation of HFSC to lesion sites may enhance cutaneous wound healing in excisional skin wounds. To the best of the authors’ knowledge, there were no reports about the effects of transplanted HFSC on healing of excisional skin wounds. To determine the effect of HFSC-mediated wound healing, this study evaluated wound closure, re-epithelialization, angiogenesis, and dermal structural regeneration, such as collagen formation, using histological and morphological assays. Results demonstrated that cell therapy using endogenous stem cell populations located in the hair follicle bulge is an appropriate and innovative approach in cutaneous wound healing.

Animals. All animal procedures were carried out in accordance with guidelines of the Iranian Council for Use and Care of Animals and were approved by the Animal Research Ethical Committee of Iran University of Medical Sciences. The animals were permitted free access to food and water at all times, and were maintained under 12 hour light/dark cycles throughout the experiments.¹⁹

Hair follicle adult stem cells isolation and culture. Male Wistar rats (n = 30, body weight 150-180 g) were sacrificed with ether, and their whisker follicles were dissected as described by Amoh et al¹¹ with a slight modification^20,21—they used Transgenic C57/B6-GFP mice, which were anesthetized with tribromoethanol via intraperitoneal injection. The current study changed other parts of the isolation method. The upper lip of each rat was cleaned with 70% ethanol and betadine and shaved completely. The upper lip containing the vibrissa pad was cut and its inner surface exposed to isolate the intact follicle. Samples were incubated for 10 min (37°C, 5% CO₂) in a collagenase I/dispase II solution (Sigma-Aldrich, St. Louis, MO). Then, connective tissue around the follicles was removed and the intact follicles were plucked gently with fine forceps. The follicles were transferred into another sterile dish. Two transverse cuts were made above and below the bulge region and the collagen capsule was incised longitudinally. The bulges were rolled out of the capsule and immersed in amphotericin B for 4 minutes. Then amphotericin B was exchanged for trypsin-ethylenediaminetetraacetic acid (EDTA) (Sigma-Aldrich, St. Louis, MO), follicles were cut into smaller pieces, and, finally, plated on collagen type І-coated tissue culture flasks (TCF). The TCF contained medium that consisted of a 3:1 supplemented mixture of Dulbecco’s Modified Eagle’s Medium (DMEM) and Ham’s F12 medium (DMEM/F12), containing 10% fetal bovine serum (FBS), 10 ng/mL epidermal growth factor (Sigma-Aldrich, St. Louis, MO), antibiotics (100 U/mL penicillin and 100 µg/mL streptomycin), 0.5 mg/mL hydrocortisone, and 0.1 U/mL insulin. The bulges were allowed to attach to the collagen in tissue culture flasks. Four to 5 days after emigration, bulge fragments were removed and cells were incubated (37°C, 5% CO₂) in the same medium. All surgical procedures and the cultivation of cells were done in sterile conditions.

Flow cytometry. To determine the percentage of special markers expressed by the cultured cells of the bulge region, cells were detached from the culture flasks using trypsin-EDTA and incubated with primary antibodies, against the CD34 antigen, nestin, and K15 antigen (Sigma-Aldrich, St. Louis, MO) for 1 hour at room temperature. The cells were washed with 0.2 M phosphate buffered saline (PBS) and were incubated with a secondary antibody conjugated to fluorescein isothiocyanate (FITC) (1:1,400), (Sigma-Aldrich, St. Louis, MO) for 1 hour at room temperature in the dark. The labeled cells were then analyzed using flow cytometry.²⁰ Details of antibodies used are summarized in Table 1. 

Labeling cells with DiI. Hair follicle adult stem cells  harvested after 14 days were labeled with a fluorescent carbocyanine dye (CellTracker CM-DiI; Molecular Probes; Sigma-Aldrich, St. Louis, MO), suspended in PBS, according to the manufacturer’s standard protocol as previously described,²² and applied to full-thickness excisional wounds in the HFSC group. Fourteen days later, when the animals were sacrificed, wound samples were gathered and sectioned at 5 µm, then analyzed using a microscope (Olympus, Tokyo, Japan).

Wound model and hair follicle adult stem cell transplantation. Male Wistar rats (n = 54, body weight 250-300 g) were selected and randomly divided into 3 groups of 18 rats each: the HFSC group, control group, and the PBS group. Each group was studied at 3, 7 and 14 days. 

After anesthesia with the effective dose of ketamine (50-70 mg/kg) and xylazine (7.5-10.5 mg/kg) the dorsum skin of rats was shaved and cleaned with betadine. Then a doughnut-shaped silicone splint was fixed by interrupted sutures. Single 6 mm full thickness excisional wounds were created using a biopsy punch such that the wound was centered within the splint.²³ A total of 1 × 10⁶ HFSC were injected intradermally around the excisional wound at 4 injection sites at 12, 3, 6, and 9 o’clock. In the PBS group, an equal volume of PBS was injected intradermally at the same sites. Then a transparent occlusive dressing (Tegaderm Transparent Film Dressing, 3M, St. Paul, MN) was used to protect the wound.²⁴ The animals were housed individually. 

Wound analysis. Digital photographs of surgical wounds were taken the day of surgery, and 3, 7, and 14 days postsurgery and treatment, using Canon PowerShot SX100 camera (Canon USA, Lake Success, NY)²⁵ for future analysis with publicly available ImageJ software, Version 1.46 (National Institutes of Health, Bethesda, MD). Measurements were conducted by investigators blind to the intervention and group. The percentage of wound closure was calculated as follows:²⁶ 

Wound area on day 0 - Wound area on the indicated day

________________________________________________

Wound area on day 0 × 100 

Histological assay. Rats were sacrificed at 3, 7, or 14 days after surgery, and paraffin sections of skin wound tissue were gathered for histological analysis. Tissue samples were fixed in 10% formalin and embedded in paraffin. Five-micron-thick sections were stained with hematoxylin and eosin (H&E) for light microscopy. Masson’s trichrome staining was performed²⁶ to identify collagen formation, and the density of newly formed collagen was analyzed using ImageJ software. 

Immunohistochemical assay. The expression of CD31 in wounded skin samples was examined by immunohistochemical staining for new vessel detection.²³ Briefly, the samples were fixed with formalin and embedded in paraffin and sectioned at 5 µm. Antigen retrieval was performed using a sodium citrate buffer (pH = 6), (Sigma-Aldrich, St. Louis, MO) for 11 minutes in an autoclave (120°C). Then sections were incubated with a primary antibody against CD31 (Abcam, Cambridge, UK), diluted 1:50 in serum containing 5% bovine serum albumin (BSA) in PBS (4°C overnight). In further steps, the sections were washed with PBS, and incubated with a secondary antibody conjugated to FITC (Abcam, Cambridge, UK), then diluted 1:700 in PBS for 1.5 hours at room temperature in the dark. To visualize the nuclei, specimens were incubated in 4’,6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich, St. Louis, MO), diluted at 1:700 in PBS for 5 minutes.²⁷ The representative areas containing CD31-positive cells were detected using a microscope. New blood vessels in each sample were evaluated in six fields (×400) and reported as percentages. Table 2 details the antibodies used. 

Statistical analysis. All values are expressed as mean ± SD. Analysis of variance and a postanalysis Tukey test were used for multiple group comparisons. A P value of less than 0.05 was considered significant.

Hair follicle isolation and cell culture. Bulge cells from dissected adult rat whisker hair follicles were isolated and cultured successfully. One isolated follicle and its bulge are shown in Figure 1. 

The authors observed an outgrowth of stem cells from isolated bulges 4-5 days after cultivation (Figure 2A). Given the rapid proliferation after 6-8 days, the bulge cells started to accumulate around the bulge fragment and formed cell colonies (Figure 2B). Then, cells migrated from the cell colonies (Figure 2C). These results showed bulge cells have high proliferative activity in vitro (Figure 2C). 

As soon as the primary culture reached 70%-80% confluency on day 9 of cultivation, cells were subcultured to other collagen-coated flasks and incubated at 37°C and 5% CO₂ in the same medium. The cells were transplanted on day 14 of cultivation after 2 passages. 

Flow cytometry. The authors used flow cytometry to confirm the bulge cells of rat vibrissa follicles were primitive stem cells. The results showed bulge cells were CD34-positive, nestin-positive, but K15-negative (Figure 3). The expression of CD34, nestin, and K15 was 93%, 70.9%, and 6.8%, respectively. 

Wound analysis. Digital images of wounds were obtained. Morphological photographs of full thickness excisional wounds in rats are shown in Figure 4 and Figure 5A). The quantitative analysis of wound closure showed the wound area in the rats that received HFSC was significantly smaller than that of the control group. However, no significant differences were observed between the PBS and control groups. As shown in Figure 5B, wound closure was significantly improved in the HFSC group, especially in days 1-3 after implantation. Wound closure was inhibited by doughnut-shaped splints and was mostly a result of cell proliferation and granulation tissue formation. 

Histological and immunohistochemical analysis. Samples from wounded skin tissue were sectioned and stained with H&E. Hair follicle stem cell-treated wounds had thicker granulation tissue, extensive re-epithelialization, and many functional erythrocyte-containing vessels in the dermis in all days assayed, whereas the control group showed thin granulation tissue with fewer functional blood vessels (Figure 6A,B). 

Analysis of Masson’s trichrome-stained sections revealed that during the first few days after the wound the collagen fibers in HFSC-treated wounds contained a high cellular component (perhaps fibroblasts), and were sparse and heterogeneous, suggesting the fibers were newly formed (Figure 7A). Analysis of collagen fiber density revealed that collagen formation was faster in the HSFC group compared with the control group (Figure7B). Fourteen days after wounding and treatment, collagen fibers were observed throughout the granulation tissue in HFSC-treated wounds, but were limited to the edges of the wounds in the control group (Figure 7A). Moreover, there were more collagen fibers in the center of wounds treated with HFSC than in the control group. Collagen fibers were thicker and the remodeling process occurred earlier in the HFSC group, where the fiber arrangements were more regular. Collagen formation in the control group was delayed at all measured times. Findings in the PBS group were similar to those of the control group (Figure 7A, B). 

Neovascularization (angiogenesis) of wounded skin samples was evaluated by immunohistochemistry for CD31 at different days after surgery. The HFSC group showed the highest number of newly formed vessels (Figure 8A) located immediately below the epidermis, concordant with an earlier onset of the matrix remodeling phase. 

Analysis of wounds with high magnification showed a significantly higher number of CD31-positive cells in the HFSC group (Figure 8B). The CD31-positive cells were mainly located around the sites of new vessel formation. A control experiment in which the primary antibody was not included showed no staining.

Imaging of hair follicle stem cell migration in excisional skin wounds. In this study, excisional wounds treated with labeled HFSC were analyzed 14 days after surgery. Hair follicle stem cells migrated to the center of wounds and persisted throughout the experimental time (day 14) (Figure 9). 

Any disruption of the normal anatomical structure of the skin with consequent loss of its function can be described as a wound.²⁸ Despite many developments in the field of tissue regeneration, wound healing remains a serious problem; and innovative treatments are necessary. A new method in wound healing is cell therapy, which involves applying live stem cells to the wound to repair damaged tissue.^{29, 30} The application of stem cells have been explored in several areas of regenerative medicine.³¹ A candidate source of pluripotent adult stem cells for regenerative medicine are HFSC because these cells are readily available, can be cultured easily and are not associated with ethical issues, unlike embryonic and fetal stem cells.^32,33 Applying such autologous grafts to a patient prevents additional surgical complications.¹⁷ In this study, HFSC situated in the hair follicle bulge were isolated and cultured successfully as previously described.³⁴ Flow cytometry results showed these cells strongly expressed the stem cell markers CD34 and nestin, but not the keratinocyte marker K15 (Figure 3). These findings were in agreement with previous studies that showed nestin-expressing stem cells in the hair follicle bulge are pluripotent³⁵ and can differentiate into nonfollicular cell types such as glia, neurons, smooth muscle cells, keratinocytes, Schwann cells, and melanocytes in vitro.^11,33,36-38 Thus, these cells may be suitable candidates for regenerative medicine.

In the present study, a full-thickness excisional-splinting wound model was created in the dorsal skin of rats. Previous studies reported that excisional wound models were easily producible and tissue harvesting for histopathological and other evaluations was simple. Moreover, splinting prevents skin contraction and allows skin excisional wounds to heal mainly through granulation tissue formation and re-epithelialization.³⁹ Lastly, this model minimized variations caused by skin contraction and wound dressings, and resulted in uniform wound closure.²³

In this study, the authors found the application of HFSC to the wound area accelerated wound closure, and observed a wound closure rate that was significantly higher in the HFSC group than in the control group. These findings are in agreement with another study that showed stem cells, such as mesenchymal stem cells, accelerated wound closure.²³

The purpose of wound healing is to regenerate biological function to an injured tissue or organ. It is a dynamic process that involves several events, including inflammation, cell proliferation, re-epithelialization, collagen formation, angiogenesis, and remodeling.⁴⁰

The results of this study show a shorter period of inflammation in the HFSC group than in the control group. Moreover, the epithelial layer thickness in HFSC-treated wounds was significantly higher than in wounds in the control group. Thick granulation tissue, extensive re-epithelialization, and many functional erythrocyte-containing vessels in the dermis were seen in all assayed days in the HFSC group, whereas thin granulation tissue and fewer functional blood vessels were seen in the control group (Figure 6). 

It is known that wound healing is dependent on neovascularization at the site of injury to maintain the newly formed granulation tissue and supply the ischemic tissue with adequate oxygen and nutrients for cell proliferation and wound closure.^41,42 New vessel formation takes place via 2 processes: angiogenesis (proliferation of existing endothelial cells) and vasculogenesis (de novo assembly of new blood vessels).^43-45

Historically, vasculogenesis was thought to occur only during embryogenesis, and angiogenesis was thought to be involved in wound healing.⁴⁵ To evaluate the role of HFSC transplantation in angiogenesis, the authors performed immunohistochemistry for the CD31 antibody. Results showed HFSC-treated wounds have more capillaries in granulation tissue of the wound bed than that of the control group, especially at 7 days and 14 days post-treatment. Consistent with these findings, recent studies^23,42,46 confirmed that stem cells play important roles in promoting the formation of new vessels, specifically during the early wound healing processes, so that higher numbers of new vessels formed in the presence of stem cells. These newly formed vessels were not associated with an inflammatory process, but were related to the early stages of tissue remodeling and the maturation of the vascular network. These newly formed vessels probably reduced the inflammation phase and accelerated the healing process.^23,42,46 

Collagen formation has an essential role in cutaneous wound healing, and the authors performed Masson’s trichrome staining to identify collagen in sections of the granulation tissue. Fourteen days after treatments, collagen fibers were observed throughout the granulation tissue in the HFSC-treated group; in the control group collagen fibers were restricted to the edges of the section. The findings in the PBS group were similar to those of the control group, suggesting using PBS as the vehicle did not induce collagen formation and had no effect on promoting wound closure during healing processes (Figure 7A, B). The findings of the present study are consistent with those of Nishimura et al.²⁶

Cells labeled with fluorescent dye showed the existence of transplanted HFSC in the center of excisional wounds on day 14, consistent with previous results that showed dye-labeled fibroblasts were present in skin after 28 days.²²

Based on the findings of this study, the authors conclude that hair follicle stem cells accelerate skin wound healing. This is an extremely important result because it raises the possibility that skin wounds could be healed by isolating autologous HFSC from a patient’s skin biopsy, thereby avoiding the possibility of immune rejection and the ethical concerns associated with using stem cells in clinical application.

The authors wish to thank the Anatomy Department and Cellular and Molecular Research Center of Iran University of Medical Sciences.

From the Department of Anatomy, School of Medicine, Iran University of Medical Science, Tehran, Iran; Department of Anatomy, School of Medicine, Qom University of Medical Science, Qom, Iran; Department of Anatomy, School of Medicine, Alborz University of Medical Science, Karaj, Iran; Cellular and Molecular Research Center, Faculty of Medicine, Iran University of Medical Science, Tehran, Iran; Department of Pharmacology, School of Medicine, Tehran University of Medical Science, Tehran, Iran; Physiology Research Center, Iran University of Medical Science, Tehran, Iran; and Anti-Microbial Resistance Research Center, Iran University of Medical Science, Tehran, Iran

Address correspondence to:
Maliheh Nobakht, PhD 
Department of Anatomy, School of Medicine,
Iran University of Medical Sciences,
Hemmat Highway, PO Box 14155-6183, Tehran, Iran
nobakht.m@iums.ac.ir 

Disclosure: This study was financially supported by Grant Number 23337 from the Iran University of Medical Sciences.