Topic Article:
An Extended Epidermal Response Heals Cutaneous Wounds in the Absence of a Hair Follicle Stem Cell Contribution
Abigail K Langton, Sarah E Herrick and Denis J Headon
Journal of Investigative Dermatology (2008) 128, 1311–1318; doi:10.1038/sj.jid.5701178

Wound Healing without Hair

Yvonne Romagosa ¹, Shasa Hu ¹ and Robert S. Kirsner ¹

Journal of Investigative Dermatology (2008), 128, 1058. doi:10.1038/jid.2008.91

Nearly half of the re-epithelialization of partial-thickness wounds occurs from within adnexal structures in the wound bed, in distinct contrast to deeper wounds where hair and other adnexal structures no longer remain, resulting in slower healing and scarring (Li et al., 2007). In partial-thickness wounds, stem cells from the hair follicle bulge are thought to contribute significantly to wound coverage. However, the functional requirements for this hair follicle input are unknown.

To better understand the role of hair follicle stem cells in healing, Langton et al. (2008) developed a novel mutant mouse model (Edaradd mouse) that, as a result of defects in hair follicle development, lacks primary hair follicles. The tail region of these mice proved especially interesting, because it lacked all adnexal structures. In analyzing stem cell behavior in embryonic skin, the authors found clonogenic keratinocytes to be relatively plentiful in the ectoderm prior to hair follicle formation. However, their frequency in the interfollicular epidermis dropped sharply by birth, when the majority of stem cells were located within hair follicles. The investigators found that in the absence of hair follicles wounds would heal with an acute delay in re-epithelialization, followed by expansion of the region of activated epidermis beyond that seen in normal haired skin, but resulting in appropriate wound closure.

Through the following questions, we examine this paper in greater detail.

QUESTIONS

1. How and why did the investigators create a mouse model for healing that lacked adnexal structures? Are there clinical examples of wound healing in the absence of hair follicles/appendages?

2. What is the scientific and clinical significance of Eda, Edar, and Edaradd?

3. Where can stem cells be found in human epidermis, and what proxy measures of stem cells did the researchers use in their experiments?

4. How is healing different in the presence and absence of hair follicles (for example, on palmar/plantar skin)?

5. What may be the clinical significance of this article?

ANSWERS

1. The investigators developed a genetically altered mouse carrying a genetic mutation in Edaradd—a member of the ectodermodysplasin receptor pathway, which prevents development of all appendageal structures in tail skin. By doing so these investigators created a model by which to study how wound healing occurs in the absence of hair follicles (HFs). It is known that HFs not only produce hair but also contribute to epithelial wound healing (Bishop, 1945). Normally HFs do not contribute cells to the epidermis (Ito et al., 2005), but wounding perturbs homeostasis, resulting in activation of epithelial cells in the HF, which assists in re-epithelialization. For example, without HFs and other adnexal structures, wounds must re-epithelialize from the margins, resulting in much slower healing as compared with wounds in skin with intact HFs.

HF development on the trunks of mice takes place in distinct phases: primary HFs develop at day 14, secondary HFs at day 16, and tertiary HFs at the time of birth. Only primary HFs develop on the tails of mice (Fuchs, 2007). Using a mutation that impairs the Eda receptor (Edar) signaling pathway, which prevents development of primary HFs (Headon et al., 2001), the investigators were able to produce a mouse model that lacked HFs on the tail (Langton et al., 2008). HFs on the trunk were still present, as secondary HFs are not affected by this mutated pathway. Of import, the tail skin of these mutant mice lacks other adnexal structures (scales and/or eccrine sweat glands), which allowed the investigators to study wound healing in the absence of HFs exclusively, without possible confounding issues. However, the possibility exists that unrecognized cellular or molecular differences in mutant skin of these mice might affect healing in other ways that might delay the early steps or accelerate the later stages of wound healing.

One clinical example of wound healing in the absence of HFs and appendages is full-thickness (stage 3 or 4) burn injuries. Both the epidermal and dermal components of skin are lost with full-thickness burns. In this circumstance, wound healing occurs with initial contraction and re-epithelialization by epithelial cells at the wound margins, followed by late contraction by myofibroblasts (Baur et al., 1984).

2. Eda, Edar, and Edaradd are the components of the Edar signaling pathway. Eda (ectodysplasin) is a signaling molecule that belongs to the tumor necrosis factor (TNF) family. TNF receptor family members, including Edar (Eda receptor), contain an intracellular death domain (Edaradd), which recruits cytoplasmic death-domain adapter proteins. In humans and mice, Eda and Edar are crucial for ectodermal differentiation in several organs, including hair and teeth. Edar or Eda mutations cause hypohidrotic (anhidrotic) ectodermal dysplasias characterized by defective development of teeth, hair, and exocrine (sweat) glands (Headon et al., 2001). Several studies have shown that the Edar signaling pathway is essential in HF development, odontogenesis, and submandibular salivary gland development (Laurikkala et al., 2002; Laurikkala et al., 2001; Jaskoll et al., 2003).

In a study by Kumar et al. (2001), Edar was shown to play an important role in the NF-κB, c-Jun N-terminal kinase, and cell-death pathways. This was elucidated by showing that Edar mutants had an impaired ability to activate those pathways.

3. In the epidermis, stem cells are located in the basal layer and in the HF. Cells of the epidermis are produced through proliferation, which occurs only in the basal layer, suggesting that stem cells are found there. It has been shown that the main source of continual cell proliferation is the center of the “epidermal proliferative unit” (Ito et al., 2005).

Keratinocyte (Cotsarelis, 2006) and melanocyte (Nishimura et al., 2002) stem cells reside in a specialized niche in the lower HF, called the bulge. The interfollicular epidermis (IFE) is repopulated by keratinocyte stem cells from the bulge during wound healing (Ito et al., 2005). Approximately 25% of the cells in a re-epithelialized wound originate in the bulge. These cells do not persist in the newly formed epidermis; rather, they behave as transient amplifying cells. Cells from other parts of the hair structure, either in the upper isthmus or in the infundibulum, behave differently than bulge cells and can contribute permanent residents to the epidermis after wounding (Levy et al., 2007).

The investigators used the BrdU label-retaining-cells method to label relatively quiescent stem cells. A distinguishing feature of stem cells is their slow-cycling nature. After administration of a BrdU pulse followed by a chase period of several weeks, only the slow-cycling cells will retain the isotope and be identified as label-retaining cells, enabling detection of stem cells (Bickenbach, 1981).

4. Results from this study demonstrate that the acute wound healing response is delayed in the absence of HFs. Using a linear incision model, the wounds in wild-type skin closed steadily from 3 to 6 days after wounding, whereas the wounds in mutant skin (with no HFs) showed no closure up to day 4 but recovered to match wild-type skin by day 6 (Langton et al., 2008). This later event suggests that the epidermis can compensate for loss of follicles by calling on its vast proliferative potential. From an evolutionary standpoint, the need for efficient wound healing is great. Delays in healing leave a person at risk for increased morbidity and mortality, and the benefit of a compensatory reservoir of keratinocytes in the follicle is clear.

By analyzing K6 expression—a marker of IFE response to a wound (Coulombe, 2003)—early wound closure has been found to be enhanced by follicle-derived stem cells. In the absence of HFs, a wider area of IFE is recruited to achieve wound closure (Langton et al., 2008).

Extrapolating these results to non-hair-bearing areas of the skin, such as palmar/plantar skin, indicates that epithelialization probably occurs from the proliferative reservoir of IFE. This is consistent with clinical observations that hair-bearing areas tend to heal more quickly than areas lacking follicles (Bishop 1945). Another example of differential healing may be the scalp of patients with alopecia. Although some types of alopecia (alopecia areata and early androgenetic alopecia) do not cause permanent destruction of the bulge area, even in some of these situations inflammation may be targeted to this area, causing decreased numbers of bulge-cell progeny (Whiting, 2003; Jaworsky et al., 1992).

5. Given the results showing that wound healing in the absence of HFs is delayed acutely but eventually catches up to that of normal skin, it may be that if IFE stem cells could be signaled to activate sooner, wound healing in skin that lacks HFs might be accelerated. The discovery of a mediator that would cause this earlier activation could be formulated into a topical or intralesional therapy to be used to promote faster and earlier wound healing.

However, it is unclear whether the quality of healing differs between wounds that heal with the aid of follicular epithelium. This may have clinical import in terms of recurrence of wounds and, additionally, cancer development, since squamous cell carcinomas develop at a higher rate within the re-epithelialized epidermis of healed burn wounds long after injury.

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_ ^1^Department of Dermatology and Cutaneous Surgery, University of Miami Miller School of Medicine, Miami, Florida, USA_