The Secrets of Beautiful Hair: Why Is It Flexible and Elastic?

cosmetics

Article The Secrets of Beautiful Hair: Why is it Flexible and Elastic?

Mikako Ezure * , Noriyuki Tanji, Yukari Nishita, Takashi Mizooku, Shinobu Nagase and Takahiro Osumi

R&D-Hair Care Products Research, Kao Corporation, 2-1-3, Bunka, Sumida-ku, Tokyo 131-8501, Japan * Correspondence: [email protected]; Tel.: +81-3-5630-9714

Received: 4 June 2019; Accepted: 23 June 2019; Published: 6 July 2019

Abstract: Beautiful hair, so called “SHINAYAKA” hair in Japanese, has a good appearance not only when stationary but also when in motion, and it is a highly desirable hair condition for Japanese consumers. We investigated such SHINAYAKA hair, which was selected by sensory evaluation, for the relationship between physical properties, such as flexibility and elasticity, and hair structure. It has already been reported that human hair cortical cells have two types, similar to wool: the ortho-like cortex and the para-like cortex. Microscopic observation revealed that the ortho-like cortex is distributed in the outer layer of the hair (near the hair surface) and the para-like cortex exists in the inner layer (near the center of the fiber). This cell distribution, a concentric double-layered structure, was deemed to be a characteristic of SHINAYAKA hair. Furthermore, analysis of physical properties showed the difference between the elasticity of the outer layer and inner layer, and that this difference was bigger in SHINAYAKA hair compared to other hair. This phenomenon was observed not only in Japanese hair, but also in Caucasian hair. In addition, we have developed a new technology for creating “SHINAYAKA” hair by treatment with succinic acid. Inflexible and inelastic hair can be changed by this treatment, and its flexibility and elasticity improve by selective reduction of stiffness of the outer layer.

Keywords: hair; ﬂexible; elastic; dynamic; motion; ortho-like; para-like; cortex; bending; mechanical; succinic acid; concentric double-layered

1. Introduction Beautiful hair has been desired since ancient times. For this purpose, for several decades we have investigated beautiful hair in depth, developing hair care technologies and launching various unique products for consumers in the world. As important elements for beautiful hair, hair shine, surface friction and hair shape are generally known. Based on our macroscopic and microscopic observations of the hair fibers and their structures, relationships between hair appearance and its structure were clarified. Well-ordered alignment of the hair fibers enhances the reflections by sharpening shine, and a smooth surface and pore-less inside structure contributes to a good appearance. Our products apply technologies developed on such knowledge. Some organic acids (e.g., malic acid, lactic acid, succinic acid) are able to repair the surface of the hair fiber, create a pore-less internal hair structure or alter the physical properties of the hair fiber [1–8]. In this study, we have focused on the motion of hair strands, especially beautiful movements, as if polar auroras were fluttering or green grasses were waving on a prairie in the wind. This motion of hair fibers is flexible and elastic like a fluid, but these fibers smoothly return to their original shape. Such flexible and elastic behaviors are traditionally preferred in Japan and described as “SHINAYAKA” in Japanese. In this report, thus flexible and elastic property is described as “flexible/elastic”, while the opposite inflexible and inelastic property is described as “inflexible/inelastic”.

Cosmetics 2019, 6, 40; doi:10.3390/cosmetics6030040 www.mdpi.com/journal/cosmetics Cosmetics 2019, 6, 40 2 of 15

Several evaluation methods for hair motion have been reported [9–11]. These reports suggest that hair motion arises from damage and can be improved after treatment with a conditioner. They have not mentioned, however, the difference in the motion among individual hairs. The motion of a hair strand is a complex phenomenon, with the main contribution from hair–hair interactions, hair shape and physical properties of hair fibers. In the cases of virgin straight hair, the individual differences of the hair–hair interaction and hair shape are probably not so large, but a difference in the hair motion is, however, still observed among individuals. We considered bamboo and a composite (multi-layer) bow as the models for a flexible and elastic fiber, because these are composite materials that have multipart mechanical properties such as flexible and elastic behavior. Bamboo is composed of hard elastic fibers (vascular bundles) and soft material (parenchyma cell) to absorb compressive deformation. A composite bow is composed of softer material in the convex side and harder material in the concave side. The bamboo can be deformed resiliently and flexibly without structural collapse [12]. The bow is easy to bend and returns to its original shape [13]. These composite materials can realize complex mechanical properties such as flexible and elastic behavior; however, it is difficult to realize such complex properties with a homogeneous material. Hair fibers are also composite materials; they are composed of a scaly cuticle, a porous medulla and a fibrous cortex. The main component is the cortex, which makes up 85% of the hair fiber, and is composed of fibrous intermediate filaments (IFs) and keratin-associated proteins (KAPs). This means that the cortex is also a type of composite material. It is known that there are two types of cortices in wool. The ortho-cortex shows relatively small and dispersed macrofibrils in the fiber cross-section and a spiral alignment of IFs. On the other hand, the para-cortex shows relatively large and fused macrofibrils with a parallel alignment of the IFs. Similar structures were found in human hair [14–17]. These hair fiber physical properties are not simple. In this study, we have investigated the relationship between the flexible/elastic behaviors of the hair fiber, the hair composite structure and its physical properties. Previously, we have partially reported this result [18], but here, in this report, we describe in detail the physical property changes due to hair damage and a new treatment. We have developed a useful treatment method using succinic acid, for both virgin and damaged hair, and demonstrate the effects of succinic acid on not only Japanese but also Caucasian hair.

2. Materials and Methods

2.1. Hair Samples

2.1.1. Selection of Hair Samples Chemically untreated hair was obtained from 50 Japanese female volunteers. Ten typical hair samples that had variation in the flexible and elastic features were selected. These hair samples were classified into flexible/elastic and inflexible/inelastic hair, according to the following sensory tests. The sensory tests were performed by five special sensory evaluators in the laboratory. The viewpoints to evaluate were the softness feeling and the bounce when the hair was swinging. The softness and bounce correspond to the flexible and elastic behaviors, respectively. Each evaluator selected the softest and the bounciest sample, and the stiffest and the least bouncy sample out of ten samples. The former was regarded as ‘score 1’, and the latter was regarded as ‘score 10’. Based on this standard, they evaluated these ten samples on this 1–10 scale. Finally, the average score of each sample was taken from five evaluators’ scores (Table1). Cosmetics 2019, 6, 40 3 of 15

Table 1. Results of the sensory evaluation.

Sample No. Sensory Score (SS) Evaluation 1 1.6 flexible/elastic 2 1.8 flexible/elastic 3 3.1 flexible/elastic 4 4.2 somewhat flexible/elastic 5 4.3 somewhat flexible/elastic 6 4.5 somewhat flexible/elastic 7 7.1 inflexible/inelastic 8 8.5 inflexible/inelastic 9 9.0 inflexible/inelastic 10 9.3 inflexible/inelastic

2.1.2. Chemical Treated Hair For colored hair, a section 25 cm from the root end of a Japanese female’s hair that was treated once with hair color one year ago was used for experiments. As an example of heavily damaged hair, parts of the root and tip (30 cm from the root end) of a Japanese female’s hair which was treated with hair color and permed once a month were used for the experiments.

2.1.3. Caucasian Hair Eight Caucasian straight hair samples (chemically untreated, gender unknown) were purchased from Kerling International Haarrfabrik GmbH (Backnang, Baden-Württemberg, Germany), and the most ﬂexible/elastic Caucasian hair was selected by the sensory tests as described in Section 2.1.1.

2.2. Preparation of the Hair Sample

2.2.1. Preparation of the Model Damaged Hair

A bleaching agent containing 3.33% H2O2 and 0.68% NH3, pH = 10 was used. Commercial shampoo and conditioner were used in this treatment. The bleaching agent was applied to the hair sample at a ratio of 1:1 for 30 min at room temperature, and then rinsed with water. The hair sample was washed with shampoo, treated with conditioner and dried using a hair dryer. Model damaged hair was obtained by treating undamaged hair with bleach one time followed by washing twelve times as describe above. This treatment was repeated four times, totaling in 4 bleaches and 48 washes.

2.2.2. Preparation of the Surface-Removed Hair Surface-removed hair (Figure1) was prepared by grinding with alumina beads (2–2.5 mm in diameter). The thickness of the removed surface layer was determined as approximately 5 µm by microscopic observation. This thickness corresponds to the cuticle layer (approximately 2 µm, 5–6 sheets) plus the cortical cell layer (approximately 3 µm) near the hair surface, in the cases of the hair samples used in this study. The tip of the hair fiber was attached to a drum, and the drum was rotated in alumina beads. A strand of hair 10 cm in length was fixed at a position that was 7 cm from the rotation axis so that the tip of the hair is at the front in the rotation direction. It was stirred for 8–12 h at a rotational speed of 40 rpm. The hair samples were periodically removed and the diameter scanned with a light microscope. The surface-removed hair prepared in this way was used to measure the bending elastic modulus of the hair’s inner layer [19]. Cosmetics 2019, 6, 40 4 of 15 Cosmetics 2019, 6, x FOR PEER REVIEW 4 of 15

Figure 1. Surface layer was removed by using alumina beads. rtotal = radius of whole hair, rinner = Figure 1. Surface layer was removed by using alumina beads. rtotal = radius of whole hair, rinner = radius of surface removed hair. radius of surface removed hair. 2.2.3. Treatment with Succinic Acid 2.2.3. Treatment with Succinic Acid A hair sample was treated with 2.0% succinic acid (SA) solution, pH = 3.7, for 30 min at 40 ◦C. A hair sample was treated with 2.0% succinic acid (SA) solution, pH = 3.7, for 30 min at 40 °C. 2.2.4. The Three-Step Treatment System 2.2.4. The Three-Step Treatment System In order to provide realistic usage during in-bath conditions, a three-step treatment system was developed.In order Theto provide first step realistic was just usage shampooing during in-bat withh conditions, a composition a three-step containing treatment 11.5% system ammonium was developed.polyoxyethylene(1.0) The first step alkyl(C10–16) was just shampooing ether sulfates, with 0.35% a composition hydroxyethyl containing cellulose 11.5% hydroxypropyl ammonium polyoxyethylene(1.0)trimethylammonium alkyl(C10–16) chloride ether ether and 1.2%sulfat succinices, 0.35% acid, hydroxyethyl pH = 3.9. Thecellulose second hydroxypropyl step was the trimethylammoniumapplication of the first chloride conditioner ether containing and 1.2% 20%succinic dipropylene acid, pH glycol, = 3.9. 0.6% The sodiumsecond 2-naphthalenestep was the applicationsulfonate, 1.6%of the hydroxyethyl first conditioner cellulose containing and 1.5% 20% succinicdipropylene acid, glycol, pH = 0.6%6.0. Thesodium third 2-naphthalene step was the sulfonate,application 1.6% of the hydroxyethyl second conditioner cellulose containing and 1.5% 2.6%succinic stearoxypropyl acid, pH = dimethylamine,6.0. The third step 7.0% was stearyl the applicationalcohol, 3.0% of dimethicone the second conditioner and 0.5% amodimethicone, containing 2.6% pH stearoxypropyl= 3.4, without rinsingdimethylamine, off the first 7.0% conditioner. stearyl alcohol,This combination 3.0% dimethicone was left on and the hair0.5% for amodimethicone, 5 min and then finally pH = all 3.4, materials without were rinsing rinsed off off .the first conditioner. This combination was left on the hair for 5 min and then finally all materials were rinsed2.3. Microscopic off. Observation Of Hair Cross-Sections 2.3.1. Transmission Electron Microscopy (TEM) 2.3. Microscopic Observation Of Hair Cross-Sections Hair cross-sections were prepared using a microtome and observed using a transmission electron 2.3.1.microscope Transmission (TEM) (H-7100; Electron Hitachi Microscopy High-Technologies (TEM) Corporation, Minato, Tokyo, Japan) to determine the structuralHair cross-sections difference inwere cortical prepared cell distribution using a microtome between the and flexible observed/elastic andusing inflexible a transmission/inelastic electronhairs. Hair microscope samples (TEM) were rinsed (H-7100; in ethanolHitachi toHigh-Technologies clean their surface Corporation, and then dried Minato, before Tokyo, being Japan) put in toembedding determine molds. the structural Hair samples differe embeddednce in cortical in Spurr’s cell resindistribution (Spurr Low-Viscositybetween the flexible/elastic Embedding Media; and inflexible/inelasticPolysciences Inc., Warrington, hairs. Hair Pennsylvania,samples were USA)rinsed were in ethanol sliced withto clean a microtome their surface (Leichert and then Ultracut dried N; beforeLeica Microsystems, being put Wetzalar,in embedding Germany). molds. The thicknessHair samples of thehair embedded section was in 200 Spurr’s nm. Then, resin the (Spurr section Low-Viscositywas stained with Embedding 0.2% uranyl Media; acetate Polysciences solution, left Inc for., 10Warrington, min and rinsed Pennsylvani with deionizeda, USA) water.were sliced Next, withit was a stainedmicrotome with (Leichert lead citrate Ultracut solution N; (Sigma-Aldrich, Leica Microsystems, St. Louis, Wetzalar, Missouri, Germany). USA) containing The thickness lead nitrate of the(II) hair and section lead acetate was 200 (II) nm. trihydrate, Then, the left section for 10 min,was stained rinsed withwith deionized0.2% uranyl water acetate and solution, dried naturally. left for 10The min TEM and observation rinsed waswith performed deionized at anwater. acceleration Next, it voltage was ofstained 85 kV. with lead citrate solution (Sigma-Aldrich, St. Louis, Missouri, USA) containing lead nitrate (II) and lead acetate (II) trihydrate, 2.3.2. Fluorescent Light Microscope (FLM) left for 10 min, rinsed with deionized water and dried naturally. The TEM observation was performedThe cortical at an cellacceleration distribution voltage was also of confirmed85 kV. with a differential staining method. Hair cross-sections of 5 µm thicknesses were prepared and sequentially stained with sodium fluorescein and sulforhodamine 2.3.2.101 [14 Fluorescent]. The hair Light samples Microscope were placed (FLM) on slide glass and a few drops of 0.002% fluorescein sodium phosphate buffer solution was applied. After being left for 18 h under conditions of darkness and high The cortical cell distribution was also confirmed with a differential staining method. Hair humidity so as not to dry out, the samples were rinsed with deionized water 6 times and dried naturally cross-sections of 5 µm thicknesses were prepared and sequentially stained with sodium fluorescein for 20 min. Then, drops of 0.005% sulforhodamine 101 solution phosphate buffer were applied and left and sulforhodamine 101 [14]. The hair samples were placed on slide glass and a few drops of 0.002% for 1.5 h under the same conditions above, rinsed with deionized water 6 times, and dried. Para- and fluorescein sodium phosphate buffer solution was applied. After being left for 18 h under conditions of darkness and high humidity so as not to dry out, the samples were rinsed with deionized water 6

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Cosmeticstimes and2019 ,dried6, 40 naturally for 20 min. Then, drops of 0.005% sulforhodamine 101 solution phosphate5 of 15 buffer were applied and left for 1.5 h under the same conditions above, rinsed with deionized water 6 times, and dried. Para- and ortho-like cortical cells in the hair sections were differentially stained ortho-like cortical cells in the hair sections were differentially stained with the green fluorescein and red with the green fluorescein and red sulforhodamine, respectively. The stained hair samples were sulforhodamine, respectively. The stained hair samples were observed using a fluorescent light microscope observed using a fluorescent light microscope (FLM) (Carl Zeiss AG, MPM 800, fluorescent filter: (FLM) (Carl Zeiss AG, MPM 800, fluorescent filter: BP450-490/FT 510/LP520; Carl Zeiss AG, Oberkochen, BP450-490/FT 510/LP520; Carl Zeiss AG, Oberkochen, Baden-Württemberg, Germany). Baden-Württemberg, Germany). 2.4. Measurement of Bending Elasticity in the Outer and Inner Layers 2.4. Measurement of Bending Elasticity in the Outer and Inner Layers Hair fibers that were equilibrated at 20 °C / 65% relative humidity (RH) for 24 h were used. The Hair fibers that were equilibrated at 20 ◦C/65% relative humidity (RH) for 24 h were used. bending elasticity of each hair fiber was measured under the same conditions and 20 hair fibers were The bending elasticity of each hair fiber was measured under the same conditions and 20 hair fibers used for each measurement. The bending elasticity in both the minor and major axes of the hair were used for each measurement. The bending elasticity in both the minor and major axes of the samples were measured using a fiber bending measurement system (FBS900/FDAS765; Dia-Stron hair samples were measured using a fiber bending measurement system (FBS900/FDAS765; Dia-Stron Limited, Andover, Hampshire, UK). Limited, Andover, Hampshire, UK). Bending elasticity (bending stress normalized with curvature and moment) was obtained as Bending elasticity (bending stress normalized with curvature and moment) was obtained as follows according to the general material dynamics relationship formula [20]. follows according to the general material dynamics relationship formula [20]. The following formula describes the relationship between bending stress (M), bending The following formula describes the relationship between bending stress (M), bending elasticity (E), elasticity (E), the second moment of cross-section (I) and the radius of curvature (ρ) (Equation (1)). the second moment of cross-section (I) and the radius of curvature (ρ) (Equation (1)). M = E × I / ρ, (1) M = E I/ρ, (1) The hair cross section is regarded as an ellipse;× the radius on the major axis side is R, and the radiusThe on hair the cross minor section axis side is regarded is r. Assuming as an ellipse; that th thee hair radius is bent on to the the major minor axis axis side side, is R, the and second the radiusmoment on theof cross minor section axis side of the is r. elliptic Assuming cylinder that thewith hair one is end bent fixed to the is minorI = πRr axis3/4. side,When the the second hair is momentdeformed of crossas shown section in Figure of the elliptic2 as an cylinderend-loaded with cantilever, one end fixedthe bending is I = π Rrelasticity3/4. When (E) theis given hair isby deformedEquation as(2) shown[21]. in Figure2 as an end-loaded cantilever, the bending elasticity (E) is given by Equation (2) [21]. E = 4ML3 / 3πRr3d, (2) E = 4ML3/3πRr3d, (2) wherewhere L L is is sample sample length length and and d d is is deflection deflection due due to to the the bending bending stress. stress.

Figure 2. Schematic diagram showing measurement image of bending elastic modulus of hair. LFigure= sample 2. Schematic length; d = diagramdeﬂection showing due to bendingmeasurement stress. image of bending elastic modulus of hair. L = sample length; d = deflection due to bending stress. First, when a load of 2 mg was detected, d was set to 0 mm, and deformation was performed from d = 0 toFirst, 0.1 mmwhen at a a load speed of of 2 0.01mg mmwas/ s.detected, d was set to 0 mm, and deformation was performed fromIn d order = 0 to to 0.1 determine mm at a speed the mechanical of 0.01 mm/s. properties in the outer and inner layers, a double-layered cylinderIn modelorder to was determine adopted. the The mechanical bending elasticity properties of the in wholethe outer hair and ﬁber inner (Ewhole layers,) can a be double-layered described by Equation (3) (Figure3)[22]: cylinder model was adopted. The bending elasticity of the whole hair fiber (Ewhole) can be described by Equation (3) (Figure 3) [22]:   R r3 R r3  inner inner  inner inner Ewhole = Eouter1  + Einner , (3)  𝑅3 𝑟 𝑅3 𝑟 − Rtotalr Rtotalr 𝐸 𝐸 1 total 𝐸 total , (3) 𝑅𝑟 𝑅𝑟 where Eouter and Einner are the bending elasticities of the outer and inner layers (surface-removed hair), where Eouter and Einner are the bending elasticities of the outer and inner layers (surface-removed hair), Rtotal and Rinner are the hair radii in the major axis of the intact whole hair and surface-removed hair, Rtotal and Rinner are the hair radii in the major axis of the intact whole hair and surface-removed hair, rtotal and rinner are the hair radii in the minor axis of the intact whole hair and surface-removed hair, rtotal and rinner are the hair radii in the minor axis of the intact whole hair and surface-removed hair, respectively, and bending direction is assumed to be in the direction of the minor axis. Ewhole,Einner,

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Cosmetics 2019, 6, 40 6 of 15 respectively, and bending direction is assumed to be in the direction of the minor axis. Ewhole, Einner, Rtotal, Rinner, rtotal and rinner are measurable, therefore Eouter can be calculated according to the above

EquationRtotal,Rinner (3)., rtotal Twentyand r innerfibersare measurable,were randomly therefore selectedEouter fromcan beeach calculated individual according and the to thebending above propertiesEquation (3). of whole Twenty hair fibers and were surface-removed randomly selected hair were from measured. each individual Then, and thethe average bending values properties of the bendingof whole elasticities hair and surface-removedin the outer and inner hair werelayers measured. of flexible/elastic Then, theand average inflexible/inelastic values of the hairs bending were obtained.elasticities in the outer and inner layers of flexible/elastic and inflexible/inelastic hairs were obtained.

FigureFigure 3. Geometry ofof double-layereddouble-layered model. model.E outerEouter= =the the bending bending elasticity elasticity of theof the outer outer layer, layer,Einner E=innerthe = thebending bending elasticity elasticity of the of inner the inne layerr (surface-removedlayer (surface-removed hair), R totalhair),= the Rtotal hair = the radius hair in radius the major in the axis major of the axiswhole of hair,the wholeRinner =hair,the R hairinner radius= the hair in the radius major in axis the of majo surface-removedr axis of surface-removed hair, rtotal = the hair,hair rtotal radius = the in hair the radiusminor axisin the of the minor whole axis hair, ofrinner the= wholethe hair hair, radius rinner in the= the minor hair axis radius of the surface-removedin the minor axis hair. of the surface-removed hair 2.5. Atomic Force Microscopy (AFM) 2.5. AtomicIn order Force to Microscopy investigate (AFM) the elasticity of each structure, hair cross-sections were measured usingIn atomic order to force investigate microscopy the elasticity (AFM) (MFP-3D-SA-J; of each structure, Oxford hair Instruments,cross-sections Abingdon-on-Thames,were measured using atomicOxfordshire, force U.K.). microscopy Hair samples (AFM) embedded (MFP-3D-SA-J; in light-curable Oxford resins Instruments, (ARONIX LCRAbingdon-on-Thames, D-800; TOAGOSEI Oxfordshire,Co., Ltd., Minato, U.K.). Tokyo, Hair Japan) samples were slicedembedded with ain microtome, light-curable as outlined resins previously. (ARONIX The LCR elasticity D-800; of TOAGOSEIeach structure Co., was Ltd., measured Minato, by Tokyo, pushing Japan) a cantilever were sliced (NCHV; with Bruker,a microtome, Billerica, as Massachusetts,outlined previously. USA) Theinto elasticity the samples of usingeach structure AFM. The was force measured curve was by analyzed pushing according a cantilever to the (NCHV; Hertz modelBruker, [23 Billerica,]. Massachusetts,2.6. Diﬀerential ScanningUSA) into Calorimetry the samples (DSC) using AFM. The force curve was analyzed according to the Hertz model [23]. In order to investigate the interaction between organic acid and hair, differential scanning calorimetry 2.6.(DSC) Differential (DSC7000X; Scanning Hitachi Calorimetry High-Tech (DSC) Science Corporation, Minato, Tokyo, Japan) measurement was performed. Hair samples were stored under 20 C, 65% RH for 4 days. An empty pan was used as In order to investigate the interaction between◦ organic acid and hair, differential scanning a reference. Samples were cooled to 70 C, followed by a temperature increase to 250 C at 10 C/min. calorimetry (DSC) (DSC7000X; Hitachi− ◦ High-Tech Science Corporation, Minato, ◦Tokyo, ◦Japan) measurement3. Results was performed. Hair samples were stored under 20 °C, 65% RH for 4 days. An empty pan was used as a reference. Samples were cooled to −70 °C, followed by a temperature increase to 2503.1. °C Distribution at 10 °C/min. of Cortical Cells

3.3.1.1. Results TEM Observation of Hair Cross-Sections Typical Japanese flexible/elastic and inflexible/inelastic hairs were selected by the sensory tests as 3.1.described Distribution in Table of Cortical1 above Cells and the microstructures of those hairs were observed using a TEM. There were no significant differences in the cuticle or medulla. On the other hand, there were 3.1.1. TEM Observation of Hair Cross-Sections some differences in the cortices. There are two types of cortices in human hair. One is called the “ortho-likeTypical cortex”, Japanese where flexible/elastic the intermediate and inflexible/inelastic filament (IF) alignment hairs were is spiral. selected It shows by the relatively sensory smalltests asand described dispersed in Table macrofibrils 1 above in and the the hair microstructure cross-section.s of The those other hairs is the were “para-like observed cortex” using a where TEM. the IF alignmentThere were is parallel.no significant It shows differences relatively in largethe cuti andcle fused or medulla. macrofibrils On the [14 other–17]. hand, The TEMthere image were someof the differences flexible/elastic in the hair cortices. (sensory There score are 3) istwo shown types in of Figure cortices4. The in human flexible hair./elastic One hair is showscalled the “ortho-likecharacteristic cortex”, double-layered where the distribution intermediate of thefilament two types (IF) alignment of cortical cellsis spiral. (ortho-like It shows and relatively para-like smallcells). and The dispersed ortho-like macrofibrils cells (Figure 4ina) the tend hair to becross- locatedsection. near The the other hair surface, is the “para-like while the cortex” para-like where cells the IF alignment is parallel. It shows relatively large and fused macrofibrils [14–17]. The TEM image

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Cosmeticsof the flexible/elastic 2019, 6, x FOR PEER hair REVIEW (sensory score 3) is shown in Figure 4. The flexible/elastic hair shows7 of the15 characteristic double-layered distribution of the two types of cortical cells (ortho-like and para-like of the flexible/elastic hair (sensory score 3) is shown in Figure 4. The flexible/elastic hair shows the Cosmeticscells). The2019 ortho-like, 6, 40 cells (Figure 4a) tend to be located near the hair surface, while the para-like7 cells of 15 characteristic double-layered distribution of the two types of cortical cells (ortho-like and para-like (Figure 4b) tend to be located near the center of the hair fiber. The thickness of the ortho-like cell cells). The ortho-like cells (Figure 4a) tend to be located near the hair surface, while the para-like cells layer and cuticle layer are nearly 3 µm and 2 µm, respectively. (Figure(Figure 44b)b) tendtend to to be be located located near near the the center center of theof th haire hair ﬁber. fiber. The The thickness thickness of the of ortho-like the ortho-like cell layer cell layerand cuticleand cuticle layer layer are nearly are nearly 3 µm 3 and µm 2 andµm, 2 respectively. µm, respectively.

Figure 4. The distribution of two types of cortices in SHINAYAKA (flexible/elastic) hair. (a) Ortho-like cortex, (b) para-like cortex [18]. FigureFigure 4. TheThe distributiondistribution of of two two types types of cortices of cortices in SHINAYAKA in SHINAYAKA (flexible /(flexible/elastic)elastic) hair. (a) Ortho-likehair. (a) cortex, (b) para-like cortex [18]. 3.1.2.Ortho-like FLM Observation cortex, (b) para-likeof Hair Cross-Sectionscortex [18]. 3.1.2. FLM Observation of Hair Cross-Sections 3.1.2. TheFLM distributions Observation of theHair ortho- Cross-Sections and para- like cortices in flexible/elastic and inflexible/inelastic hair Thewere distributions confirmed ofwith the a ortho- differential and para- staining like cortices method. in flexible One hundred/elastic and hair inflexible samples/inelastic from each hair The distributions of the ortho- and para- like cortices in flexible/elastic and inflexible/inelastic wereindividual confirmed (in Table with a1) di werefferential stained staining with method.two types One of hundredfluorescent hair dyes samples and observed from each using individual FLM. hair were confirmed with a differential staining method. One hundred hair samples from each (inShown Table in1) wereFigure stained 5 is an with image two that types clearly of fluorescent represents dyes this and feature. observed The using flexible/elastic FLM. Shown hairs in Figureshowed5 individual (in Table 1) were stained with two types of fluorescent dyes and observed using FLM. isthe an ortho-like image that cortex clearly distributed represents thisnear feature. the hair The surface flexible /elastic(outer hairslayer) showed and the the ortho-likepara-like cortex Shown in Figure 5 is an image that clearly represents this feature. The flexible/elastic hairs showed distributeddistributed nearnear thethe hair center surface of the (outer hair layer)fiber (inner and the layer), para-like as shown cortex in distributed Figure 5a. near On thethe centerother ofhand, the the ortho-like cortex distributed near the hair surface (outer layer) and the para-like cortex hairthe inflexible/inelastic fiber (inner layer), hairs as shown show in a Figuredispersed5a. Onpattern the other of the hand, two types the inflexible of cortical/inelastic cells, as hairs shown show in distributed near the center of the hair fiber (inner layer), as shown in Figure 5a. On the other hand, aFigure dispersed 5b, patternindicating of the a twostructural types of corticaldifferen cells,ce between as shown the in Figureflexible/elastic5b, indicating hairs a structuraland the the inflexible/inelastic hairs show a dispersed pattern of the two types of cortical cells, as shown in diinflexible/inelasticfference between hairs. the flexible /elastic hairs and the inflexible/inelastic hairs. Figure 5b, indicating a structural difference between the flexible/elastic hairs and the inflexible/inelastic hairs.

FigureFigure 5. DistributionDistribution of of two two types types of of cortical cortical cells: cells: (a) ( aflexible/elastic) ﬂexible/elastic hair hair (Table (Table 1;1 No. ; No. 1) [18], 1) [ 18(b],) (inflexible/inelasticb) inﬂexible/inelastic hair hair (Table (Table 1;1 No.; No. 10). 10). The The pa para-likera-like cortical cortical cells cells look green andand thethe ortho-likeortho-like Figure 5. Distribution of two types of cortical cells: (a) flexible/elastic hair (Table 1; No. 1) [18], (b) corticalcortical cellscells looklook redred inin thesethese pictures.pictures. inflexible/inelastic hair (Table 1; No. 10). The para-like cortical cells look green and the ortho-like 3.2.3.2. Distributioncortical cells look of Hair red Mechanicalin these pictures. Properties

3.2. DistributionNext,Next, the of mechanicalmechanical Hair Mechanical didifferencesfferences Properties betweenbetween flexibleflexible/elastic/elastic andand inflexibleinflexible/inelastic/inelastic hairhair werewere investigatedinvestigated byby variousvarious means.means. Next, the mechanical differences between flexible/elastic and inflexible/inelastic hair were investigated3.2.1. Bending by Elasticityvarious means. of Outer and Inner Layers 3.2.1. Bending Elasticity of Outer and Inner Layers The results of the bending elasticity of the outer and inner layers obtained with typical flexible/elastic 3.2.1. Bending Elasticity of Outer and Inner Layers hair (Table1: No. 1 /Sensory Score = 1.6) and inflexible/inelastic hair (Table1: No. 10 / Sensory

Score = 9.3) are shown in Figure6. These measured values were E outer = 3.4 GPa, Einner = 5.6 GPa and

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The results of the bending elasticity of the outer and inner layers obtained with typical Cosmetics 2019, 6, x FOR PEER REVIEW 8 of 15 flexible/elastic hair (Table 1: No. 1/Sensory Score = 1.6) and inflexible/inelastic hair (Table 1: No. 10/ Sensory ScoreThe results = 9.3) ofare the shown bending in Figure elasticity 6. Theseof the measured outer and values inner layerswere Eobtainedouter = 3.4 withGPa, typical Einner = 5.6 GPaCosmetics flexible/elasticand Eouter2019 ,=6 ,5.1 40 hair GPa, (Table Einner 1: No.= 5.3 1/Sensory GPa of each.Score In= 1.6) this and case, inflexible/inelastic the ratios of bending hair (Table elasticity 1: No.8 of 10/ 15(Eouter / Einnr)Sensory were 0.60 Score and = 9.3)0.96, are respectively. shown in Figure 6. These measured values were Eouter = 3.4 GPa, Einner = 5.6 GPaThe andbending Eouter =elasticity 5.1 GPa, inEinner the = outer5.3 GPa layer of each. is lower In this than case, that the in ratios the innerof bending layer elasticity and this (E differenceouter / Eouter = 5.1 GPa,Einner = 5.3 GPa of each. In this case, the ratios of bending elasticity (Eouter/Einnr) were Einnr) were 0.60 and 0.96, respectively. in the0.60 flexible/elastic and 0.96, respectively. hair is bigger than that in the inflexible/inelastic hair. The bending elasticity in the outer layer is lower than that in the inner layer and this difference in the flexible/elastic hair is bigger than that in the inflexible/inelastic hair.

Figure 6. Bendingelasticityoftheouterandinnerlayersofflexible/elastic(Table1; No. 1)andinflexible/inelastic FigureFigure 6. Bending 6. Bending elasticity elasticity of of the the outer outer andand innerinner layerslayers ofof flexible/elastic flexible/elastic (Table (Table 1; No.1; No.1) and 1) and (Table1; No. 10) hairs. Measurements at 20 C and 65% relative humidity (RH), n = 20, SS = sensory score. inflexible/inelasticinflexible/inelastic (Table (Table 1; 1;No. No. 10) 10) hairs. hairs.◦ Meas Measurements at at 20 20 °C °C and and 65% 65% relative relative humidity humidity (RH), (RH), n = 20,Then = SS 20, bending = SS sensory = sensory elasticity score score in the outer layer is lower than that in the inner layer and this difference in the flexible/elastic hair is bigger than that in the inflexible/inelastic hair. 3.2.2.3.2.2. Influence Influence by Hairby Hair Damage Damage 3.2.2.Damaged Influence byhair Hair is Damageusually classified as inflexible/inelastic because of its inflexible/inelastic Damaged hair is usually classified as inflexible/inelastic because of its inflexible/inelastic behavior [9–11]. Changes in the mechanical properties between untreated hair and model damaged behaviorDamaged [9–11]. Changes hair is usuallyin the mechanical classified as properties inflexible/ inelasticbetween becauseuntreated of hair its inflexible and model/inelastic damaged behaviorhair, in [the9–11 way]. Changes described in theabove, mechanical as well properties as the root between and tip untreated of heavily hair anddamaged model hair, damaged were hair, in the way described above, as well as the root and tip of heavily damaged hair, were hair,compared. in the way The describedresults are above, shown as in well Figure as the 7a,b. root When and tip untreated of heavily hair damaged (No. 1; hair,described were compared.in Table 1) compared.Thewas results model The aredamaged results shown are as in described Figureshown7 a,b.in above, Figure When the untreated7a,b. bending When hairelasticity untreated (No. of 1; describedthe hair outer (No. layer in Table1; increaseddescribed1) was modelfrom in Table 3.4 1) wasdamaged GPamodel to 4.2damaged as GPa. described The as ratios above,described of the bend bending above,ing elasticity elasticitythe bending were of the changed elasticity outer layer from of increased 0.60the outerto 0.74, from layer which 3.4 increased GPa is significant. to 4.2 GPa.from 3.4 GPaThe Whento 4.2 ratios comparingGPa. of bendingThe ratioschanges elasticity of in bend the were mechanicaling changed elasticity fromproperti were 0.60es changed to between 0.74, which from the root is 0.60 significant. and to tip0.74, ofWhen heavilywhich comparing isdamaged significant. Whenchangeshair, comparing the in outer the mechanical changeslayer of thein properties the tip mechanicalwas betweenharder thanproperti the rootthe androotes between tip(tip: of 4.8 heavily theGPa, root damaged root: and 4.2 tip hair,GPa). of the heavily The outer ratios layerdamaged of hair,ofbending the the outer tip elasticity was layer harder ofwere thanthe 0.89 tip the (tip) rootwas and (tip:harder 0.80 4.8 (root). GPa,than root: theThe root 4.2ratio GPa). (tip:of the The4.8 tip ratiosGPa,was bigger ofroot: bending than4.2 GPa). that elasticity of The the were root.ratios of bending0.89These (tip) elasticity results and 0.80 suggest were (root). 0.89that The the(tip) ratio mechanical and of the0.80 tip (root).property was bigger The is stiffenedratio than thatof theby of thetip root.damage, was Thesebigger especially results than suggest thatin the of outer thatthe root. Thesethelayer. results mechanical suggest property that the is sti mechanicalffened by the property damage, is especially stiffened in by the the outer damage, layer. especially in the outer layer.

FigureFigure 7. 7.Bending Bending elasticity elasticity of the ofouter the outer and inner and layers inner of layers healthy of hair healthy and damaged hair and hair: damaged (a) flexible hair:/elastic (a) hairflexible/elastic (Table1; No. hair 1) and (Table model 1; damagedNo. 1) and hair model (Table damaged1; No.1). (hairb) Root (Table and 1; tip No.1). of heavily (b) Root damaged and tip hair. of

Measurements at 20◦C and 65% relative humidity (RH), n = 20, SS = sensory score. Figure 7. Bending elasticity of the outer and inner layers of healthy hair and damaged hair: (a) flexible/elastic hair (Table 1; No. 1) and model damaged hair (Table 1; No.1). (b) Root and tip of

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heavily damaged hair. Measurements at 20°C and 65% relative humidity (RH), n = 20, SS = sensory Cosmetics 2019, 6, 40 9 of 15 score

3.3.3.3. ModificationModification of the Mechanical PropertiesProperties TheThe aboveabove resultsresults suggestsuggest thatthat thethe concentricconcentric double-layereddouble-layered distributiondistribution ofof thethe mechanicalmechanical propertiesproperties (softer(softer outer outer layer layer and and sti ffstifferer inner inner layer) layer) is important is important for flexible for flexible/elastic/elastic hair. These hair. resultsThese alsoresults suggest also suggest that the that softening the softening of the outerof the layerouter islayer possibly is possibly effective effective for the for improvement the improvement of hair of propertyhair property from inflexiblefrom inflexible/inelastic/inelastic to flexible to /elastic.flexible/elastic. We have We studied have how studied to modify how theto mechanicalmodify the propertiesmechanical of properties the hair fiber of bythe investigating hair fiber by the investigating effect of organic the effect acids. of Some organic organic acids. acids Some have organic strong – interactionsacids have strong with keratinousinteractions proteins with keratinous and can modifyproteins mechanical and can modify properties mechanical [3–8]. properties Several organic [3 8]. acids,Several i.e. organic maleic acid,acids, aspartic i.e. maleic acid, acid, or succinic aspartic acid, acid, which or havesuccinic hair acid, softening which abilities, have hair were softening tested to investigateabilities, were whether tested these to investigate acids could whether selectively these soften acids the outercould layer.selectively Succinic soften acid the (SA) outer was foundlayer. toSuccinic be the acid most (SA) effective was found of these to materials. be the most effective of these materials.

3.3.1.3.3.1. Treatment withwith SuccinicSuccinic AcidAcid TheThe coloredcolored hairhair (shown(shown inin SectionSection 2.1.22.1.2)) withwith modelmodel damagedamage treatmenttreatment (ratio(ratio ofof bendingbending elasticityelasticity == 0.99;0.99; sensory score score == 7.0)7.0) were were used. used. Th Thee hair hair was was treated treated with with the the solution solution containing containing SA SAby the by themethod method described described in Section in Section 2.2.3. 2.2.3 With. With this treatment, this treatment, only onlythe outer the outer layer layer was softened was softened from from5.4 GPa 5.4 GPato 3.7 to GPa, 3.7 GPa, and and the themechanical mechanical difference diﬀerence between between the the outer outer layer layer and and the the inner layerlayer becamebecame clear.clear. At thisthis timetime,, thethe sensorysensory scorescore changedchanged fromfrom 77 toto 3.3. Furthermore,Furthermore, thethe samesame tendencytendency waswas conﬁrmedconfirmed byby thethe methodmethod shownshown inin SectionsSections2 –2–55 (Figure (Figure8a,b) 8a,b) [18 [18].].

FigureFigure 8.8. TheThe difference diﬀerence in in mechanical mechanical properties properties of of model model damaged damaged hair hair and and hair hair treated treated with with a

asolution solution containing containing succinic succinic acid acid (SA) (SA) [18]. [18]. ( (aa)) Bending Bending elasticity, elasticity, MeasurementsMeasurements atat 2020°C◦C and 65% relativerelative humidityhumidity (RH), (RH), n =n 20,= 20, SS SS= sensory = sensory score score (b) elasticity(b) elasticity of each of structureeach structure measured measured using atomic using forceatomic microscopy force microscopy (AFM). (AFM).

3.3.2.3.3.2. Treatment withwith thethe Three-StepThree-Step TreatmentTreatmentSystem System TheThe coloredcolored hair hair with modelwith damagemodel treatmentdamage (sametreatment as above) (same and as untreated above) inflexible and untreated/inelastic hairinflexible/inelastic (No. 10 shown hair in Table (No.1, sensory10 shown score in =Tabl9.3)e were1, sensory treated score with the= 9.3) three-step were treated system usingwith SA.the Asthree-step a result, system only the using outer SA. layer As a was result, softened only th frome outer 5.4 GPalayer to was 3.7 GPasoftened (above) from and 5.4 fromGPa 5.1to 3.7 GPa GPa to 3.9(above) GPa (Tableand from1; No. 5.1 10).GPa The to 3.9 di GPafference (Table between 1; No. the 10). outer The layerdifference and thebetween inner layerthe outer became layer more and clearlythe inner defined layer (Figure became9), evenmore underclearly realistic defined usage (Figure in in-bath 9), even conditions. under realistic usage in in-bath conditions.

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Figure 9. Change in mechanical properties when treated with three-step system using SA. (a) Model Figure 9. Change in mechanical properties when treated with three-step system using SA. (a) Model damagedFigure 9. Changehair (b) ininflexible/inelastic mechanical properties chemically when truntreatedeated with hair. three-step Measurements system using at 20°C SA. (anda) Model 65% damaged hair (b) inﬂexible/inelastic chemically untreated hair. Measurements at 20◦C and 65% relative relativedamaged humidity hair (b (RH),) inflexible/inelastic n = 20, SS = sensory chemically score. untreated hair. Measurements at 20°C and 65% humidityrelative humidity (RH), n = (RH),20, SS n = 20,sensory SS = sensory score. score. 3.3.3. Application Application to to Caucasian Caucasian hair 3.3.3. Application to Caucasian hair Similar results were obtained on Caucasian hair hair.. The outer layer became harder after damage Similar results were obtained on Caucasian hair. The outer layer became harder after damage (the elasticity of the outer layer increased from 2. 2.99 GPa to 4.8 GPa), and the outer layer was softened (the elasticity of the outer layer increased from 2.9 GPa to 4.8 GPa), and the outer layer was softened with treatment by by the the three-step three-step system system using using SA SA (the (the elasticity elasticity of ofthe the outer outer layer layer decreased decreased from from 4.8 with treatment by the three-step system using SA (the elasticity of the outer layer decreased from 4.8 GPa4.8 GPa to 2.8 to GPa) 2.8 GPa) (Figure (Figure 10). 10 ). GPa to 2.8 GPa) (Figure 10).

Figure 10. BendingBending elasticity of of the outer and inner layer of CaucasianCaucasian hair.hair. MeasurementsMeasurements atat 2020°C◦C andFigure 65% 10. relative Bending humidity elasticity (RH), of the n = outer20;20; SS SS and== sensorysensory inner score.layer score. of Caucasian hair. Measurements at 20°C and 65% relative humidity (RH), n = 20; SS = sensory score. 4. Discussion 4. Discussion 4. Discussion 4.1. Characteristic Structural Pattern 4.1. Characteristic Structural Pattern 4.1. CharacteristicMicroscopic Structural observations Pattern revealed a structural difference between flexible/elastic hair and inflexibleMicroscopic/inelastic observations hair. For flexible revealed/elastic a hair, structural ortho-like difference cells tend between to concentrate flexible/elastic in the outer hair layers and Microscopic observations revealed a structural difference between flexible/elastic hair and inflexible/inelasticof the hair fiber (near hair. the For hair flexible/elastic surface), while hair, para-like ortho-like cells concentratecells tend to in theconcentrate inner layers in the (near outer the inflexible/inelastic hair. For flexible/elastic hair, ortho-like cells tend to concentrate in the outer layerscenter). of These the hair results fiber indicated (near the that hair flexible surface),/elastic while hair shows para-like a concentric cells concentrate double-layered in the distribution inner layers of layers of the hair fiber (near the hair surface), while para-like cells concentrate in the inner layers (nearcortical the cells, center). as observed These results by TEM indicated and FLM. that In contrast, flexible/elastic inflexible hair/inelastic shows hair a concentric demonstrated double-layered a dispersed (near the center). These results indicated that flexible/elastic hair shows a concentric double-layered distribution ofof thecortical two cortex cells, types.as observed by TEM and FLM. In contrast, inflexible/inelastic hair demonstrateddistribution of a dispersedcortical cells, distribution as observed of the by two TEM cortex and types. FLM. In contrast, inflexible/inelastic hair 4.2.demonstrated Characteristics a dispersed of Bending distribution Elasticities of the two cortex types. 4.2. Characteristics of Bending Elasticities 4.2. CharacteristicsWe considered of that Bending flexible Elasticities/elastic hair not only had structural differences but also had mechanical differencesWe considered when compared that flexible/elastic to inflexible /inelastichair not hair.only Generally,had structural a material’s differences structure but isalso closely had We considered that flexible/elastic hair not only had structural differences but also had mechanicalrelated to a material’sdifferences property. when compared The mechanical to inflexible property/inelastic of hair hair. should Generally, be important a material’s for hair structure motion. ismechanical closely related differences to a material’s when compared property. to The inflexible mechanical/inelastic property hair. Generally,of hair should a material’s be important structure for is closely related to a material’s property. The mechanical property of hair should be important for

Cosmetics 2019, 6, 40 11 of 15 Cosmetics 2019, 6, x FOR PEER REVIEW 11 of 15 hairIf we motion. could understandIf we could theunderstand relationship the relationsh between mechanicalip between propertiesmechanical and properties beautiful and hair beautiful motion, hairit would motion, be helpfulit would to developbe helpful new to technologiesdevelop new to technologies control hair motion.to control So, hair we motion. investigated So, we the investigatedmechanical dithefference mechanical between difference flexible/elastic between and inflexibleflexible/elastic/inelastic and hair. inflexible/inelastic The elasticity of hair. ortho-like The elasticitycortical cells of ortho-like is relatively cortical lower thancells thatis relatively of para-like lower cortical than cells that [24 of,25 para-like]. Therefore, cortical it is expectedcells [24,25]. that Therefore,for flexible it/elastic is expected hair, the that outer for flexible/elastic layer of the hair hair, fiber the is outer softer layer than theof the inner hair layer. fiber In is general,softer than it is theunderstood inner layer. that In the general, cuticle is it the is hardestunderstood tissue that in thethe hair. cuticle Most is outerthe hardest layers oftissue each in cuticle the hair. cell (calledMost outerthe A-layer) layers of are each the cuticle hardest cell tissue (called in the the hair. A-layer) On the are other the hardest hand, mosttissue inner in the layers hair. of On each the cuticleother hand,(called most the endocuticle)inner layers areof each softer cuticle than the (called cortex. the When endocuticle) bending are the softer hair, thethan softer the cortex. tissue likeWhen the bendingendocuticle the canhair, be the preferentially softer tissue deformed, like the endocuti as publishedcle can previously be preferentially [19]. Therefore, deformed, the as contribution published previouslyof the cuticle [19]. to Therefore, bending elasticity the contribution is small. of the cuticle to bending elasticity is small. TheThe bending elasticityelasticity waswas measured measured by by deformation deformation of aof straight a straight hair hair strand strand into ainto curled a curled shape shapewith awith 25 mm a 25 curl mm radius curl forradius 10 s, for creating 10 seconds, a deformation creating velocity a deformation of 0.004 pervelocity millimeter of 0.004 second. per millimeterThis velocity second. corresponds This velocity to the change corresponds in curvature to the from change straight in curvature to 220 mm from curl radiusstraight in to one 220 second. mm curlThe radius tip end in of one the 300second. mm The long tip hair end strand of the moved 300 mm approximately long hair strand 170 mm moved away approximately within one second. 170 mmTherefore, away within the deformation one second. velocity Therefore, of this systemthe deformation is comparable velocity to the of velocitythis system of actual is comparable hair swinging to thewhile velocity walking of actual and/or hair turning swinging around while (Figure walking 11). and/or turning around (Figure 11).

FigureFigure 11. 11. TheThe deformation deformation velocity velocity at at the the measuremen measurementt point point and and the the deformation deformation of of the the actual actual hairhair swing. swing.

TheThe results results suggest suggest that that the the bending bending elasticity elasticity in in the the outer outer layer layer is is lower lower than than that that in in the the inner inner layerlayer and and this this difference difference between between the the outer outer and and inner inner layers layers in in flexible/elastic flexible/elastic hair hair is is bigger bigger than than in in inflexible/inelasticinflexible/inelastic hair. hair. We We confirmed confirmed that that the the difference difference is is smaller smaller in in damaged damaged hair. hair. In In other other words, words, damageddamaged hair hair did did not not show show the the characteristics characteristics of of the the concentric concentric double-layer. double-layer. PlottingPlotting the the ratio ratio of of bending bending elasticity elasticity versus versus se sensorynsory score score of of all all samples samples shown shown in in this this report report showedshowed a relationshiprelationship between between them. them. A A lower lower ratio ratio of bending of bending elasticity elasticity correlated correlated with a with lower a sensorylower sensoryscore (Figure score 12 (Figure). A lower 12). sensory A lower score sensory indicates score a better indicates hair feela better and a hair lower feel ratio and of bendinga lower elasticity,ratio of bendingindicating elasticity, a strong indicating separation a between strong theseparati inneron and between outer layers. the inner The and above outer results layers. suggest The thatabove the resultsconcentric suggest double-layered that the concentric distribution double-layered of the mechanical distribution properties of the (softer mechanical outer layer properties and stiffer (softer inner outerlayer) layer is important and stiffer for flexibleinner layer)/elastic is hair. important These resultsfor flexible/elastic also suggest thathair. softening These results of the also outer suggest layer is thatpossibly softening effective of the for theouter improvement layer is possibly of these effe hairctive properties for the fromimprovement inflexible/ inelasticof these tohair flexible properties/elastic. from inflexible/inelastic to flexible/elastic.

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Figure 12. Relationship between the ratio of bending elasticity (outer layer/inner layer) and the Figure 12. Relationship between the ratio of bending elasticity (outer layer/inner layer) and the sensory Figuresensory 12. score.Relationship R2 = coefficient between of thedetermination ratio of bending derived el fromasticity regression (outer layer/ line byinner least layer) squares and method. the score. R2 = coefficient of determination derived from regression line by least squares method. sensory score. R2 = coefficient of determination derived from regression line by least squares method. 4.3. Modification4.3. Modification of the of Mechanical the Mechanical Properties Properties 4.3. Modification of the Mechanical Properties 4.3.1.4.3.1. Effect Effect of Succinic of Succinic Acid Acid 4.3.1. Effect of Succinic Acid We haveWe have investigated investigated the etheffect effect of organic of organic acids acids on the on mechanicalthe mechanical properties properties of hair of hair fibers. fibers. SuccinicSuccinicWe acid have selectivelyacid investigated selectively softened softenedthe effect the outer the of organicouter layer. layer. Weacids predictedWe on predicted the mechanical that SAthat only SA properties only penetrated penetrated of thehair outer thefibers. outer layer.Succiniclayer. On the acidOn other the selectively other hand, hand, malic softened acidmalic has theacid beenouter has found beenlayer. tofoun We penetrate dpredicted to penetrate deeper that intodeeper SA theonly into hair’s penetrated the inner hair’s layer the inner outer [6]. layer It waslayer.[6]. hypothesized ItOn was the hypothesized other thathand, succinic malic that succinic acid acid has interacts acid been interacts foun mored stronglytomore penetrate strongly with deeper hairwith proteins hairinto proteinsthe andhair’s thus and inner thus likely layer likely penetrates[6].penetrates It was less. hypothesized less. The interactionThe interaction that succinic between between acid the organicinteracts the organic acid more acid and strongly hairand proteinshair with proteins hair was proteins analyzedwas analyzed and using thus using DSC. likely DSC. Thepenetrates glassThe glass transition less. transition The temperature interaction temperature between (Tg) (Tg) is shown the is shownorganic by the by acid derivative the and derivative hair di proteinsfferential differential was scanning analyzed scanning calorimetry using calorimetry DSC. (DDSC)The(DDSC) glass spectrum transition spectrum at the temperatureat time the time of temperature of (Tg) temperature is shown rise (Figureriseby the (Figure derivative13). DDSC13). DDSC differential showed showed the scanning dithefferential differential calorimetry of the of the DSC(DDSC)DSC spectrum spectrumspectrum and it andat could the it indicatetimecould of indicate temperature the inflection the inflection rise point (Figure more point 13). clearly. more DDSC Bothclearly. showed malic Both acid-treated the malic differential acid-treated hair andof the hair succinicDSCand spectrum acid-treated succinic andacid-treated hair it could decreased hairindicate decreased in Tg the compared inflection in Tg tocomp point untreatedared more to hair. clearly.untreated In this Both case,hair. malic theIn thisacid-treated Tg ofcase, succinic the hair Tg of acidand treatmentsuccinic succinic acid decreased acid-treated treatment more. decreasedhair Assuming decreased more. that in Assuming theTg largercomp thearedthat Tg the to decrease, untreatedlarger the the Tghair. more decrease, In strongly this thecase, it more interacts the stronglyTg of withsuccinic hairit interacts proteins, acid treatment with it can hair be decreased saidproteins, that succinicmore.it can Assuming be acid said interacts that that succinic the more larger strongly acid the interacts Tg with decrease, hair more proteins the strongly more compared strongly with hair toit malic interactsproteins acid. Itcomparedwith follows hair that toproteins, malic succinic acid. it acidcan It follows tendsbe said to thatthat stay succinic insuccinic the surface acid acid tends layerinteracts to of stay the more hair.in the strongly In surface addition, with layer it hair is of the thoughtproteinshair. that In compared succinicaddition, acid to it malicis e ffithoughtciently acid. that softensIt follows succinic the that proteins, acid succinic efficiently as theacid decrease softenstends to inthe stay Tg proteins, indicatesin the surface as that the water decreaselayer andof thein Tg organichair.indicates In acid addition, plasticized that water it is thethought and protein organic that [26 succinic acid–28]. plasticized acid efficiently the protein softens [26–28]. the proteins, as the decrease in Tg indicates that water and organic acid plasticized the protein [26–28].

FigureFigure 13. (A 13.) DDSC (A) DDSC curves curves and ( Band) glass (B) glass transition transition temperature temperature of each of hair.each (a)hair. Without (a) Without treatment, treatment, (b)Figure treated(b) treated13. with (A) DDSC succinicwith succiniccurves acid, and (c)acid, ( treatedB )(c) glass treated withtransition malicwith temperature malic acid [18acid]. of DDSC[18]. each DDSC =hair.derivative (a)= derivativeWithout diﬀ erentialtreatment, differential scanning(b) scanningtreated calorimetry withcalorimetry succinic acid, (c) treated with malic acid [18]. DDSC = derivative differential scanning calorimetry 4.3.2. The Three-Step Treatment System 4.3.2. The Three-Step Treatment System We tried to add succinic acid (SA) to shampoo and/or conditioner. When SA is added to shampoo,We tried no to sufficient add succinic effect acid was (SA) obtained, to shamp becaoouse and/or shampoo conditioner. is usually When rinsed SA off is immediately. added to shampoo, no sufficient effect was obtained, because shampoo is usually rinsed off immediately.

Cosmetics 2019, 6, 40 13 of 15

4.3.2. The Three-Step Treatment System Cosmetics 2019, 6, x FOR PEER REVIEW 13 of 15 We tried to add succinic acid (SA) to shampoo and/or conditioner. When SA is added to shampoo, noWhen suffi SAcient is added effect to was a conditioner, obtained, because no sufficient shampoo effect is was usually obtained, rinsed becaus off immediately.e anionic SA When is possibly SA is addedtrapped to by a conditioner,cationic materials. no suffi SAcient has etwoffect pKa was values, obtained, at 4.2 because and atanionic 5.6. The SA pKa is means possibly acid trapped constant by cationicexpressed materials. in common SA has logarithm. two pKa values,The dissociation at 4.2 and atstate 5.6. Thediffers pKa depending means acid on constant pH. Therefore, expressed inpenetration common and logarithm. solubility The can dissociation be controlled state by di pH.ffers depending on pH. Therefore, penetration and solubilityA three-step can be controlled treatment bysystem pH. was developed. It is composed of shampoo, the first conditioner with ASA three-step but without treatment cationic system materials, was developed. and the It second is composed conditioner of shampoo, with thecationic first conditioner materials. withWe SAconsidered but without that cationicin the second materials, step, andSA can the secondeasily penetrate conditioner into with the cationichair duematerials. to its higher We solubility considered at thatpH 6 in without the second inhibition step, SA by can cationic easily materials. penetrate intoIn th thee third hair step, due toSA its can higher be fixed solubility into the at pH hair 6 withoutfiber by inhibitionthe cationic by materials cationic materials. at pH 3.4. In Based the third on step, this SAsystem, can be we fixed have into succeeded the hair fiber in controlling by the cationic the materialsmechanical at pHproperties 3.4. Based even on under this system, realistic we usage have succeededin in-bath inconditions, controlling even the mechanicalwhen only propertiesused one eventime. underThe effect realistic of the usage three-step in in-bath system conditions, was conf evenirmed when with only Japanese used one time.consumers. The effect Perceived of the three-step changes systemin hair properties was confirmed (softness, with Japanese bounce, consumers.flexible and Perceived elastic) after changes use of in the hair three-step properties treatment (softness, bounce,system flexibleare shown and in elastic) Figure after 14. use of the three-step treatment system are shown in Figure 14.

Figure 14. EffectEﬀect of the three stepstep systemsystem conﬁrmedconfirmed withwith JapaneseJapanese consumers.consumers. Perceived changes in hair properties when the test formula is used.

5. Conclusions Microscopic observation revealed the concentric double-layereddouble-layered distribution of the two types of cortical cellscells inin flexible flexible/elastic/elastic (SHINAYAKA) (SHINAYAKA) hair. hair. The The ortho-like ortho-like cortex cortex tends tends to distribute to distribute in the in outer the layerouter oflayer the hairof the (near hair the (near hair the surface), hair surface), while the while para-like the para-like cortex tends cortex to distribute tends to indistribute the inner in layer. the Basedinner layer. on this Based observation, on this observation, it was found it that was the foun concentricd that the double-layered concentric double-layered distribution of distribution mechanical propertiesof mechanical (softer properties outer layer (softer and sti ffouterer inner layer layer) and is important stiffer inner for the layer) flexible is/elastic important behavior for of the hair.flexible/elastic The characteristic behavior mechanical of the hair. properties The characteri werestic changed mechanical due to hairproperties damage were and changed our developing due to treatmenthair damage containing and our SA.developing It was demonstrated treatment containi that theng mechanical SA. It was propertydemonstrated change that occurred the mechanical not only forproperty Japanese change but alsooccurred Caucasian not only hair. for Japanese but also Caucasian hair. Author Contributions: Conceptualization, M.E., T.M., S.N. and T.O.; methodology, M.E., N.T., T.M. and S.N.; investigation,Author Contributions: M.E., N.T. Conceptualization, and Y.N.; writing, M.E.;M.E., supervision,T.M., S.N. and T.O. T.O.; methodology, M.E., N.T., T.M. and S.N.; investigation, M.E., N.T. and Y.N.; writing, M.E.; supervision, T.O. Funding: This research received no external funding. Acknowledgments:Funding: This researchWe received would like no toexternal thank E.funding. Terada and K. Koike of KAO Co., Japan, for the useful discussion. We would also like to thank S. Breaksear of KAO Germany GmbH and E. Pyrozoku of KAO Co., Japan, for the Acknowledgments: We would like to thank E. Terada and K. Koike of KAO Co., Japan, for the useful useful discussion and his kind English editing for this publication. Special thanks to R. Furukawa for gathering muchdiscussion. of the We data. would also like to thank S. Breaksear of KAO Germany GmbH and E. Pyrozoku of KAO Co., Japan, for the useful discussion and his kind English editing for this publication. Special thanks to R. Furukawa Conflicts of Interest: The authors declare no conflict of interest. for gathering much of the data.

Conflicts of Interest: The authors declare no conflict of interest.

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