Hair is an essential element of physical appearance, well-being and attractiveness. The hair follicle (HF) is the hair shaft-producing unit that has been described as a complex and complete organ; indeed, it is characterized by continuous cycling with successive phases of growth (anagen), illustrated by the intense activity of hair matrix keratinocytes. The growth stage is followed by a complete regression of the lower segment of follicle and bulb (catagen) and relative quiescence (telogen), resulting in hair shedding (exogen).1 At the end of telogen, the HF is reactivated by intrafollicular and extrafollicular signals in order to re-initiate a new cycle.2
This intense cellular activity is the result of complex interactions taking place in different compartments of the HF. In particular, the concentric organization of the HF implicates regulated interactions between the epithelial and connective tissue sheath. These epithelial-mesenchymal interactions have been shown to play a central role in HF activity and involve components of the HF basement membrane,3 which is located between the connective tissue sheath that surrounds the HF and the basal layer of the outer root sheath (ORS) as seen in Figure 1. This basement membrane also continues at the level of the dermal papilla, which is composed of mesenchymal tissue and considered a conductor of hair regeneration and growth.4
In the skin, the basement membrane has been well-described as far as its molecular organization and role in maintaining epidermal-dermal cohesion.5 However, its role in relation to hair growth remains incompletely understood. The aim of the present study was to characterize the main components of the HF basement membrane by immunohistology to evaluate the modulation of their synthesis by a compound designed to boost cell energy levels.
Integrins are transmembrane adhesion proteins that are involved in cell-cell and cell-matrix interactions and exert signalling functions. In the HF, they play key roles in interactions between the outermost cell layer of the ORS and the basement membrane. In particular, b1 integrin mediated signaling has been implicated in hair growth control.6 Moreover, b1 and a6 integrin subunits have been identified as epidermal stem cell markers.7, 8
With respect to their role in cellmatrix adhesion, a6b4 and a3b1 integrins have been described as main receptors for laminin-332 and laminin- 511, respectively, which regulate hair growth. In particular, laminin-511, through interaction with its b1 integrin receptor, has been shown to promote dermal papilla development.9 Moreover, blocking laminin-511 has been shown to provoke alopecia on human scalp grafts.10
In the connective tissue, integrins are responsible for structural integrity by mediating the interactions between fibroblasts and the extracellular matrix. In particular, a5b1 is the main receptor for fibronectin, which is strongly involved in cell-matrix adhesion and plays a role in dermal papilla formation and HF development.11 Considering these mechanisms, the authors aimed to develop an active to positively Figure 2. Immunostaining of a3, a6 and b1 integrins on human scalp biopsies (nonspecific staining of hair shaft) Figure 3. Immunostaining of laminin-332 and laminin-511 on human scalp biopsies affect these markers and improve hair elongation.
The active corn extract designed to this end (INCI name: Water (aqua) (and) glycerin (and) hydrolyzed corn protein) is obtained after several enzymatic hydrolyses. The peptidic fraction, having a molecular weight lower than 5 kDa, is purified and concentrated; the final corn extract is rich in amino acids and proteins.
The expression of proteins associated with hair growth were then assessed after treatment with the active and compared with a placebo. In addition, hair shaft elongation measurements on scalp skin biopsies were taken after treatment with the active, as described here.
Materials and Methods
The study was carried out on 6-mm punch biopsies taken from human scalp skin grafts collected from face-lift surgeries. The biopsies were maintained in emersion on a porous membrane and fed by a serum-free medium composed of: William’s E mediuma, 10 μg/mL of insulinb, 10 ng/mL of hydrocortisonec, 2 mM of L-Glutamine and 100 μg/mL of a reagent to prevent contaminationd.12
Biopsies were treated by topical application of 20 μl of the extract at 1%, while the control biopsies received 20 μL of the placebo (phosphate buffered saline or PBS). The active was previously characterized as a cell energy-boosting compound that enhanced ATP level in human fibroblasts at this selected dose. The application was renewed every 24 hr and the biopsies were maintained in culture at 37°C in a 5% carbon dioxide-humidified atmosphere for a total incubation period of 48 hr.
At the end of the incubation period, scalp skin biopsies were formol-fixed and embedded in paraffin or optimal cutting temperature (OCT) embedding matrixe and frozen in liquid nitrogen, depending on the primary antibody selected to reveal a specific marker. For paraffin-embedded biopsies, 3 μm-thick sections were cut, deparaffinized in xylene, and rehydrated in successive alcohol baths and water. The sections were then washed with PBS and submitted to antigen retrieval.
For frozen biopsies, 6 μm-thick cryostat sections were air-dried at 37°C and fixed in cold acetone for 10 min. After blocking nonspecific sites with 5% bovine serum albumin (BSA) for 30 min, the sections were processed for immunofluorescence detection.
The expression of different integrin subunits and components of the basement membrane in addition to fibronectin, a mediator of cell adhesion, were evaluated. Improvement in the expression of these markers suggests a reinforcement of the basement membrane zone of the HF. This effect would further correspond with the improvement of cellular activity markers nuclear proteins Ki67 and p63, which are implicated in cell renewal. The following primary antibodies were used on paraffin sections: gamma-2 chain of laminin-332 (diluted 1:100) and beta1-integrin (diluted 1:50). On frozen sections, the following primary antibodies were used: alpha-5 chain of laminin-551 (diluted 1:200), alpha-3 and alpha-6 integrins (diluted 1:100), collagen IV (diluted 1:500), fibronectin (diluted 1:100), Ki67 (diluted 1:100) and p63 (diluted 1:100). Secondary anti-bodies conjugated to fluorescent dyef were then incubated for 1 hr (diluted 1:1000) and after washing, slides were mountedg.
Stained sections of the skin biopsies including hair follicles were observed by fluorescence microscopy using a microscope with a 20X objectiveh.
Photos were captured using a digital camera and softwarej. Hair follicle morphology and stage were controlled by hematoxylin-eosin staining.
Hair Shaft Elongation
Hair shaft elongation was determined according to a protocol modified from Lu et al.12 Biopsies of scalp skin were initially shaved to just above the level of the epidermis, maintained in culture, and treated as previously described for up to 17 days. To determine the hair growth, photographs of the biopsy surface were taken at days 0, 8, 14 and 17 with a camerak. The hair shaft length protrusion from the epidermis was measured on the photographs using softwarem, and hair shaft elongation was determined by calculating the difference between hair length at day 0 and on the days measurements were taken. Placebo and treated samples were compared by an unpaired student’s t-test, with a minimum of three measurements taken for control and treated biopsies; p < 0.05 was considered the level of significance, as indicated by an asterisk.
Effect on basement membrane zone proteins, connective sheath: The staining of the different integrin subunits (a3, a6 and b1) was localized in the outermost cell layer of outer root sheath (see Figure 2); a6 integrin expression was restricted to the basal pole of the basal ORS keratinocytes. Since these integrins play a crucial role for cell adhesion and signalling, the fact that they are located in the basal layer of ORS indicates this cell layer is an important zone for cell communication. Staining of these integrin subunits appeared to be positively modulated following treatment with the active ingredient. Moreover, the staining of laminin-332 and laminin-511 in the basement membrane was more intense with the active than in the control (see Figure 3).
The effect of the active’s application to the basement membrane zone was also observed on collagen IV expression, which was boosted by the treatment (see Figure 4). Furthermore, the staining of fibronectin, visualized in the dermal sheath, was enhanced by application of the active (see Figure 5). Therefore, the effect observed in the presence of the active strongly suggests optimized cellular adhesion and better cell communication, necessary to ensure growth of the hair shaft during the anagen phase.
Effect on cell renewal proteins: The second study evaluated the effects of the active on Ki67 and p63 by double immunofluorescence staining. Ki67 and p63 have been found to be deficient in areas of the scalp affected by alopecia, which emphasizes the role they play in hair growth and regeneration.13, 14 Additionally, Ki67 has been found to decrease in situ when stress on the HF is induced by UV irradiation.15 Results indicated the active positively modulated the expression of these two markers, favoring the activity of hair growth and regeneration (see Figure 6).
In vitro hair shaft elongation: To study hair shaft elongation, scalp skin biopsies were maintained in culture for up to 17 days. It is interesting to note that this ex vivo model maintained the vitality of scalp biopsies in vitro and provided the required conditions to actually enable the measurement of hair shaft elongation (see Figure 7). Application of both the active and placebo resulted in enhancement of the hair shaft length on biopsies treated daily; although the active produced 71% greater growth than the placebo at day 8 and 110% greater growth than the placebo at day 14 (see Figure 8).
Other work showed significant hair shaft elongation in a full thickness, organ-cultured human scalp skin model during the first 16 days of culture.12 Like the present ex vivo model, longer times in culture resulted in slower hair growth, mainly due to cell apoptosis in the hair follicle and surrounding tissue.
In addition to the described full thickness scalp skin models, hair shaft elongation measurements were performed on hair follicles isolated from the human scalp skin via microdissection and maintained in culture.16 This model has been used to characterize compounds with activity on hair growth and has shown good predictivity regarding in vivo effects.17, 18
The basement membrane plays an essential role in maintaining the health and cohesion of the HF. Further, in vitro tests indicate that boosting the expression of proteins implicated in cell adhesion and cell-matrix interactions results in a positive effect on hair growth. The present ex vivo model of scalp skin biopsies takes into account interactions within the HF as well as between the HF and its cutaneous environment. Moreover, it allows for the assessment of topically applied active compounds.
However, further studies are required to better characterize the delivery of actives through the hair follicle. This study emphasizes the role played by the components of basement membrane and basal layer of ORS in maintaining integrity of the hair follicle, which may affect hair vitality. The hair follicle is a complex and complete organ with much left to reveal. Studying the expression of specific proteins in the hair follicle sheds more light on the multiple interactions taking place in the hair follicle and allows for the development of specific actives to target particular functions in the hair follicle. This could be of use to improve the current understanding of hair follicle functioning and thus develop new generations of hair care biofunctional ingredients related to hair renewal, antiaging and vitality.
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