Morphogenetic Types of Cuticles and Melanin Granules in the Anagen Phase of Hair Follicles

Full Length Research Paper

Morphogenetic types of cuticles and melanin granules in the anagen phase of hair follicles

Ju-Young Lee 1,2 * and Byung Soo Chang 3

1Mirae Biotech. Co.,Ltd., 227-217 Daeheung Bld. Jayang 3 Dong, Gwangjin-GU, Seoul, Republic of Korea. 2Sammyook Health College, Seoul 143 -854, Seoul, Republic of Korea. 3Department of Cosmetology HanSeo University, SeaSan, Republic of Korea.

Accepted 16 February, 2012

Our studies on the scalp and hair follicles have a particular interest in the formation of white hair and melanin; however, representative images of white hair and melanin formation have been difficult to find. The morphogenetic types of melanosomes and cuticles in hair follicles are diverse. The generated form of melanin granules include soft melanosomes that penetrate through the outer root sheath (ORS) of the hair follicle where they remain between the ORS and the inner root sheath (IRS) as irregular large masses of soft melanosomes. Subsequently, they break into small fragments, pass though the IRS and reach the hair cuticle layers. The melanosomes then adhere to the cuticle layers; however, and a portion of them continue penetrating the hair, finally entering the hair cortex where they become regular melanin granules. However, white hair roots are not formed in this manner as contain little melanosomes and melanin granules. The types of cuticle layers formed in both white and black hair roots include the curved board, mass and cogwheel types.

Key words: Morphogenetic type, white and black hairs, hair root, melanin granules, cuticles.

INTRODUCTION

The prenatal morphogenesis of hair follicles depends Following such a synthesis, they are transferred from upon a precisely regulated series of molecular genetic melanocytes to keratinocytes. Melanocytes are melanin- processes. Human follicles start forming at the eyebrows producing cells located in the bottom layer (the stratum and mustache from approximately 3 months after basale) of the skin's epidermis (Plonka et al., 1995; pregnancy, following which hair follicles are formed all Rogers and Gelfand, 1998). Melanin is a substance that over the body by 5 to 6 months after the pregnancy. The gives color to skin, eyes and hair, and may also be factors involved in this process are the cytokine and referred to as pigment. Hair color is a result of growth factors, which consist of more than 50 types pigmentation of hair follicles due to two types of melanin, including β-catenin, transforming growth factor β2 eumelanin and pheomelanin. (TGF β2), and sonic hedgehog homolog(SHH) (Akiyama A hair follicle undergoes 3 dynamic changes from an and Shimiz, 2000; Botchkarev and Kisshimoto, 2003; active growing phase (anagen) to a remodeling of Stephan et al., 2007). degeneration phase (catagen) and finally, to a quiescent It has been reported that melanosomes may template phase (telogen). Botchkarev et al. (2003) divided hair melanin polymerization by way of amyloidogenesis of the follicle changes into 4 phase by adding the exogen protein Pmel17, which is present in abundant quantities phase, which is the phase before the hair is falling out as in melanosomes. Some portion of the granules within a result of such things as hair combing or shampooing. It melanocytes contains tyrosinase and synthesizes melanin. is known that one of the longest phase in the hair growth

*Corresponding author. E-mail: [email protected]. Tel: +82 2 525 5875. 808 Sci. Res. Essays

observed by TEM on the hair shaft ranged from 4 to 7, with the number of layers varying with age and whether the person was a smoker or nonsmoker. Smokers had the smallest number of layers (4); in addition, their melanin granules were found to have very low electron density in the regions where collapse on the outermost site into the hair shaft. After hair coloring treatment, most of the cuticle layers membranes disappear due to damage caused by the hair dyeing solutions. Recently, there has been increased interest in the cuticle and melanin structural patterns of the hair root rather than those of the hair shaft. The present study was conducted to provide baseline data on the morphogenetic types of cuticle and melanin from the white and black hair roots during their anagen phase. Those differences were investigated using electron microscope.

MATERIALS AND METHODS

Hair root sample collection

Black and white hair roots (4 to 5 each) were donated by a healthy male in his 30s. All samples were pulled out slowly using tweezers, then dipped in phosphate buffered saline (PBS), stored in a micro tube. When hair root samples are slowly removed by tweezers, the hair root shape of the hair follicle provides high-quality reference for study. The donor was a nonsmoker and agreed with the use to the samples in this study.

Methods

The samples were cut into 5 sections using an ultra-microtome. The cross sections were form the lower section of the dermal papilla (DP) (crosscut A), the lower section of the bulge area located below the stem cell of the hair follicle (crosscut B) and the upper section of the bulge area above the stem cell (crosscut C). In addition, vertical sections were obtained from the lower section of the bulge area near the stem cell of the hair follicle (vertical cut A) and from the upper section of the bulge area(vertical cut B) (Figure 1). Figure 1. The distribution of cut sections. Sections CA, CB, For TEM analysis, each hair root sample was fixed for 2 h at 4°C CC, VSA and VSB correspond to crosscut A, crosscut B, in 0.1 M PBS containing 2.5% glutaraldehyde (pH 7.3). The tissues crosscut C, vertical cut A and vertical cut B, respectively . were then post-fixed in 1% osmium tetroxide in 0.1 M PBS on ice HFSC, hair follicle stem cell. for 1 h, dehydrated in an ethanol series and embedded in Epon 812 resin. Ultra-thin sections were cut on an ultramicrotome (Leica ultracut UCT, Wetzlar Germany) with a diamond knife (Diatome, Nidau Switzerland), and stained with uranyl acetate for 60 min followed by lead citrate staining for 5 min. The prepared samples cycle and the one in which most scalp hairs may be were examined by using a Zeiss EM912 electron microscope found, is the anagen phase. (CarlZeiss, Oberkochen, Germany) at 120-kV accelerating voltage Recently, it has been reported that the stem cell of a (Using the method of the Korea Basic Science Institute). hair follicle (Stephen et al ., 1998; Oshima et al., 2001; Taylor et al., 2000) is located at the follicle’s bulge area, near the follicle’s arrector pili muscle. Research on stem RESULTS cell separation, via the cultivation method (Yi Zhang et al ., 2006) and on formation of cell lines is very active, but TEM observations of melanosomes and cuticles from morphological study of the forming types and melanin anagen phase hair roots granules of hair follicle using transmission electron microscope (TEM) has been rarely conducted. Observations on the lower section of the dermal According to our previously published report (domestic papilla journal; J. of Korea Society of Cosmetology. (Ju-Young et al., 2007. 13(3). 873-884)}, the number of cuticle layers The images in Figure 2 were taken from a crosscut A Lee and Chang 809

Figure 2. A black hair root from crosscut A. Several dermal sheaths in oval shapes (arrows) are present (arrows , A). At the border area of B, connective tissue sheaths are appear (arrow), and while in C and D (arrows), several black holes which appear to be melanocytes are indicated. (Scale bars; A, 2 µm; B, 5 µm; C, 500 nm; D, 200 nm).

section from the lower section of the DP. Well arranged arrows) may be observed. The melanin penetrates dermal sheaths with an oval shape may be observed through the ORS of the hair follicle and forms an irregular (Figure 2A arrows). At the border area, there appear large mass type (Figure 3B arrow). Beyond the ORS, the many connective tissue sheaths (CTS) which are melanosomes are broken into small fragments (Figures arranged irregularly (Figure 2B). Black spots in the middle 3C and 3D) reach the cuticle layers after passing through of the CTS are clearly shown (Figures 2B, 2C and 2D), the IRS (Figure 3C arrow heads). Some melanosomes and seem to be traces of melanocytes which produced adhere to the cuticle layers while some keep penetrating melanin. into the hair cortex (Figure 3C). Numerous melanosomes passing through the soft cuticle layers were observed (Figure 3C arrow heads), appearing as black flakes; Observation on the lower section of the bulge area however, in these images, the melanin granules in the cortex are not present (Figures 3B and 3C). Nonetheless, The images in Figure 3 were taken from crosscut B numerous melanosomes appear to have moved to the section from the lower section from the bulge area. The inside of the follicle by penetrating through the cuticle cortex, the cuticle layers, the inner root sheath (IRS) and layers (Figures 3C and 3D). the outer root sheath (ORS) may be seen, in that order, beginning from the inside of the image in (Figure 3B). The balloon types of the ORS are arranged side by side Observations on the cortex area (Figure 3A), and while outside of them, CTS and the melanocytes with irregular oval shapes (Figure 3A The images in Figure 4 were from a crosscut B section. 810 Sci. Res. Essays

Figure 3. A black hair root from crosscut B. Melanocytes (arrows, A) are present and the melanin has formed an irregular big mass types (arrow, B). Numerous melanosomes of (arrow heads and arrow, C and D) are shown passing through the cuticle layers. CO, cortex; CU, cuticle layers; IRS, inner root sheath; ORS, outer root sheath (Scale bars: A and B, 1 µm; C,, 200 nm; D, 100 nm).

Numerous intermediate filaments (Figure 4D) appear to bulge area, respectively. The images clearly show that be gathered and together to form bundles formed (Figure the cuticle layers are arising from the IRS. Soft cuticle 4A), appearing like fully contained pipe type (Figure 4B). layers are produced and adhere to the IRS; subsequently Although three melanosomes appear (Figure 4A, arrows), another cuticle layers is produced and adhered to the in most bundles, melanosome are not present. In Figure IRS. Therefore, the signatures of cuticle layers will be left 4C, empty spaces among bundles and bundles uniting on them (Figure 5A). The types of cuticle layers formed with other bundles were observed. It appears that such the lower section of the bulge area and are a curved bundles may keep uniting together to become a macro board type and a mass type (Figure 5A); however, in the bundles; ultimately becoming a piece of the cortical upper section from the bulge area, the type of cuticle tissue. layers formed are of the cogwheel type (Figure 5B, arrows).

Observation on the cuticle layer in vertical cuts A and B Observations on the upper section of the bulge area

The images in Figures 5A and 5B were taken from the The images in Figure 6 were taken from the crosscut C vertically section of the lower and upper sections of the section in the upper section of the bulge area. It shows Lee and Chang 811

Figure 4. Hair cortex from crosscut B. Numerous IF (D) are gathered and formed into the bundles (A). Melanosomes are present (arrows, A). Bundles that have united are also present (B and C). (Scale bars: A, 200 nm; B, 70 nm; C, 50 nm; D, 20 nm).

that 7 or 8 cuticle layers together with regular melanin Observation on white hair roots in crosscut C granules into the hair cortex (Figure 6A). Such layers and granules were not observed in the lower section of the The images in Figure 8 were taken from the crosscut C bulge area. The 7 or 8 cuticle layers may be divided into section of the white hair root. In Figures 8A and 8B, the epicuticle, exocuticle, endocuticle and the strong melanosomes and melanin granules are not observed, layer (S-layer). In the epicuticle layer, numerous small which indicates that the function of melanocytes in white melanosomes particles have adhered (Figure 6A). hair is weak. In addition, the number of cuticle layers is shown to be 5 to 6 layers, 1 to 2 layers less than that in the black hair samples. Observation on white hair roots in the vertical cut A and B DISCUSSION The images in Figure 7 were taken from a vertical cut A section from the lower section of the bulge area of a When hair root samples are slowly removed by tweezers white hair root. It shows that the cuticle layers are being rather than by the follicular unit extraction (HUE) method, produced from the IRS, as was the case in the black hair the hair root shape of the hair follicle is a high-quality samples. Also, it shows that a few melanosomes (Figure reference standard. In the white hair samples examined, 7A, arrows) have penetrated into the cortex area. We few melanosomes were found and none in the cortex, suggest that the hair color is white because the number which we considered as an indication that the function of of melanosomes is significantly lower than that in black melanocytes has deteriorated. The cause of such hair. In the white hair, the type of cuticle layers formed hypofunction is considered to be hereditary or related to a are the curved board and mass types, as was observed nutritive condition or some other unknown reasons. When in the black hair samples (Figures 6A and 6B, an arrows). observing hairs, some hairs have both black and white 812 Sci. Res. Essays

Figure 5. A black hair root from vertical cuts A and B. Cuticles are shown as a curved board type and a mass type in the lower section of photo A. In the upper section from the bulge area, the cuticle layers have formed a cogwheel types (arrows, B). (Scale bars: A, 1 µm; B, 5 µm).

Figure 6. A black hair root from crosscut C. It shows 7 or 8 cuticle layers together with the regular melanin granules in the hair cortex (A). In the epicuticle layer, numerous small melanosomes particles have adhered (arrows, A). In the exocuticule layer (B), melanosomes have adhered (arrows, B) and the lengthy white stick forms are exocuticule membranes (B). (Scale bars: A, 500 nm; B, 55 nm).

Figure 7. A white hair root from crosscut C. The type of cuticle layers formed are a curved board and mass types similar to that black hair (arrow, B). Melanosome (arrows, A) have penetrated into the cortex area passing through the ORS and the IRS. (Scale bars: A, 5 µm; B, 1 µm). Lee and Chang 813

Figure 8. A white hair root from crosscut C. The melanosome and melanin granules are not present in the cuticle or the cortex (A and B), Five to six are shown , 1 to 2 layers less than that in a black hair. (Scale bars: A, 500 nm; B, 200 nm).

colors in a single hair. Such hairs might be produced in a cuticle layers and melanin granules that are formed. The process, in which the function of melanocytes is hairs should be pushed up to outside of skin like mummy fluctuating. hair by the finished formation of the hairs. In the microstruture of the hair cortex in lower bulge Each follicle is an independent organ and depending on area of the hair follicle, it is known that IF might be their functions, various forms of hair can appear. To present, and that low sulfur proteins might be synthesized prevent the occurrence of white hair and balding of the into the cortical cells. Such proteins may become head requires studies into restraining white hair formation incorporated into IF, and, with subsequent growth form a and controlling the transition of anagen hair into catagen macro fibril. In this study, it seemed that IF had gathered, hair. became macro bundles and grow more to become a mass bundle, subsequently, the mass bundles gathered to form a cortical tissue. Conclusion In hair cuticles, the layers in the upper section of the hair follicle bulge area are generally divided into The initial morphogenetic type of melanin granules is a epicuticle, exocuticle and endocuticle, and a so-called soft melanin which penetrates through the ORS of the strong layer (S-layer), which prevents the interior of the hair follicle and then remains between the ORS and the hair from damage related to hair tinting or hair permanent IRS as an irregular, large mass and soft melanosome beauty treatment. However, it has not been determined type. Subsequently, they break into small fragments types which layer(s) among the typical 7 cuticle layers and those melanosome types adhere to the cuticle layers specifically corresponds to the epicuticle, exocuticle, before becoming regular melanin granules in the hair endocuticle and S-layer. We previously repot that, after cortex through the cuticle layers; however, white hair hairs underwent three successive coloring treatments, the roots form hair with little or no melanin, and melanin most of the cuticle layer membranes had disappeared, granules are not found in the hair cortex of white hair due to damage caused by the hair dyeing solutions. But roots. the S-layer is appeared in that treatment. Thus, the S- Each 7 cuticle layers of a hair can be classified as: first layer appears to be able to prevent damage to the hair’s outer layer as the epicuticle, middle layers (from the interior. second layer to the fifth layer) as the exocuticle and the Accordingly, on the basis of this study and our previous layer adjacent to the cortex as the endocuticle. The repot, we suggest that it is reasonable to classify the first middle most layers of the cuticles is classified as the S- outer cuticle layer as the epicuticle, the middle layers layer. (from the second layer to the fifth layer) as the exocuticle, In white hair, there are 5 to 6 cuticle layers also shown the layer adjacent to the cortex as the endocuticle and as 5 to 6 layers, 1 to 2 layers less than that of black hair. the layer that is most in the middle (fourth to fifth) as the The types of hair cuticle layers formed are the curved S-layer. In this study, we observed the various types of board type, a mass type and a cogwheels type. 814 Sci. Res. Essays

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