J. Cosmet. Sci., 66, 15–29 (January/February 2015) 15 New aspects of the structure of human hair on the basis of optical microscopic observations of disassembled hair parts ASAO YAMAUCHI and KIYOSHI YAMAUCHI, Department of Biochemistry, Osaka Municipal Technical Research Institute, Joto-ku, Osaka 536-8553 (A.Y.), and Keratin Materials Research Laboratory, Nishikyo-ku, Kyoto 610-1101(K.Y.), Japan. Accepted for publication November 23, 2014. Synopsis Infant’ and adult’ scalp hair fi bers were disassembled to various cellular components and blocks by chemical and enzymatic treatments, followed by random scission with rapidly rotating cutters. The hair fi bers were also fractured by the use of a vise. The optical microscopic inspection of these specimens led to the discovery of many previously unknown structures in the hair shaft. In particular, a cuticular cell (Cu) was found to take a trowel-like shape consisting of a part with a blade-like shape (CuB) and a part with a handle-like shape (CuH), where CuB overlapped one another and fused partially to build the honeycomb-like structure on a large cuticular thin plate (CuP). Whereas CuH was closely similar to the cortical cell in dimensions and rich- ness of macrofi brils (Mf). It was considered that human hair is stabilized structurally and physicochemically by the presence of the honeycomb-like structure, the CuP and the Mf. INTRODUCTION Several studies on the science of human hair were carried out over the last several decades, using the traditional structural model that was chiefl y composed of spindle-like cortical cells (Co), fl at cuticular cells (Cu), and serially aligned medulla cells (1–3). It would be very important, especially in a cosmetic fi eld, to analyze how chemical and biochemical agents interact with these cellular components. As a matter of fact, mammals’ hair fi bers have been frequently investigated by electron microscopes (4–15). The technique, however, even ignoring a laborious process for the preparation of a specimen, works in vacuo and provides only a black-and-white photograph of the dried substance. On the other hand, optical micro- scopes have been conveniently employed for examination of various hair samples despite the fact that the resolution is limited to semimicro and micrometer levels. Indeed, the micros- copy is well suited to dealing with wet substances, but also is very useful to inspect small objects for the structural characteristics, providing a see-through image with a high depth of fi eld and permitting chromatic distinction between two similar matters in the specimen. Address all Correspondence to Asao Yamauchi at yamauchi@omtri.or.jp

JOURNAL OF COSMETIC SCIENCE 16 The purpose of this study was to discover the unknown structures hidden in the inner domain of a human hair shaft. For achieving this aim, scalp hairs were disassembled or fractured to cellular components and blocks by combining chemical or enzymatic treatment with special cutting and compression processes. Taking advantages of the aforementioned optical microscopy, many specimens were analyzed for the inner structure of the hair shaft. EXPERIMENTAL HAIR SAMPLES AND REAGENTS Two- and six-year-old Japanese girls kindly gave black hairs I and II, respectively. Their hair was cut from more than 1 cm from the scalp surface. Virgin black hair III was simi- larly obtained from a healthy 16-year-old girl of the Miao ethnic group living in the mountains of China’s Yunnan province, and white hair IV from a 65-year-old Japanese male. All the hair fi bers were straight, neither being stained with dyes nor subjected to any permanent setting processes. The hairs were successively washed with aqueous 1.5 wt.% aqueous sodium dodecyl sulfate (SDS), deionised water, and aqueous 70 vol.% ethanol then the hairs were stored at 4°C in a sealed plastic container. All reagents, in- cluding 2-mercaptoethanol (ME), staining dyes, and papain, were commercially available. PREPARATION OF THE SPECIMENS FOR OPTICAL MICROSCOPY† The following four methods were adapted to prepare the specimens for microscopy. Meth- ods 1–3 consisted of the chemical and enzymatic pretreatments of the hair fi bers and the special cutting processes. Method 4 utilized a mini-vise to fracture the hair shaft. Method 1: Simple swelling treatment of hair fi bers by heating in the presence of urea and SDS (a general procedure). Hair (I–IV, 20 mg, about 1 cm length) was kept in the aqueous solu- tion (5 ml) of 8 M urea and 4 wt.% SDS at 55°C for 5 h without stirring. Since the treat- ment solution did not contain ME, the hair shaft becomes soft without disturbance of the structure see the swelling degree.‡ The swollen shaft was washed briefl y with pure water, dispersed in water (1 ml), and subjected to the cutting process I (vide infra). The resulting †The benefi ts of staining of the dyes employed in the present study are as follows: CBB—The Mf of the Co and the Cu were nonspecifi cally stained blue. Congo red—In contrast with the Co, the Cu, particularly the blade-like shaped parts (CuB), were stained preferentially pale red. Gentian violet—This dye was milder than CBB, staining the cellular components in various depth of blue-violet. Giemsa—The Co, the handle-like shaped parts (CuH) of the Cu and the medulla’s inner substance were more intensely stained than CuB and the medulla’s wall. Silver—Almost of all proteinous substances were stained black. Silver staining was useful to recognize the presence of CuB that was hardly stainable with any of the organic dyes. SM—This dye has an Hg2+ ion in the molecule. Therefore, sulfur-rich cellular components or sites are preferentially stained in red (the color of the ligand) presumably due to a formation of a strong Hg–S linkage. ‡When hair (I–IV about 20 mm length in plain water at 25°C) was heated in an aqueous mixture of 7 M urea, 3.5 wt.% SDS and 0 or 15 wt.% ME at 80°C for 20 min–1 h, the shafts were swollen in the follow- ing degree which was estimated using the equation, 100 × (L2 – L1)/L1, where L1 and L2 are the breadth and length of the hair shaft before and after the treatment, respectively. On heating for 1 h in the absence of ME: (breadth) I 34 ± 6%, II 28 ± 4%, III 25 ± 7%, and IV 21 ± 8% (length) I~IV about 0%. On heating for 20 min in the presence of ME: (breadth) I 143 ± 7%, II 150 ± 15%, III 150 ± 13%, and IV 200 ± 20% (length) I 13 ± 4%, II 21 ± 2%, III 35 ± 2%, and IV 43 ± 3%. All the hair samples swelled maximally after heating for about 1 h: (breadth) I~IV 200 ± 15% (length) I~II 40 ± 3%, and III~IV 50 ± 10%.