Multimodal Evidence of Mesostructured Calcium Fatty Acid Deposits in

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Article Cite This: ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX

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Multimodal Evidence of Mesostructured Calcium Fatty Acid Deposits in Human Hair and Their Role on Hair Properties Jennifer M. Marsh,*,† Marc Mamak,† Fred Wireko,† Ariel Lebron,† Tom Cambron,† Daniel Huber,§ Isabel Boona,§ Robert E. A. Williams,§ and David W. McComb§ †

The Procter & Gamble Company, Mason Business Center, 8700 Mason-Montgomery Road, Mason, Ohio 45040, United States Center for Electron Microscopy and Analysis, The Ohio State University, 1305 Kinnear Road, Columbus, Ohio 43212, United States

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ABSTRACT: We provide the first conclusive evidence for the presence of exogenous calcium fatty acid deposits, which not only form in-between the cuticle layers in the lipid-rich cell membrane complex, but also grow to dimensions large enough to cause the structure to bulge, thereby impacting the optical and mechanical properties of the hair fiber. The composition and phase of these deposits were probed using a multimodal analytical approach with spatially resolved techniques including synchrotron micro X-ray fluorescence coupled with X-ray scattering, focused ion beam (FIB)scanning electron microscopy (SEM), scanning transmission electron microscopy, X-ray energy dispersive spectroscopy, and Fourier transform infrared and Raman imaging where the collective analysis is consistent with a meso-phase composed of calcium C16/C18 saturated fatty acids from natural sources such as sebum. X-ray microtomography and serial “slice and view” FIB/SEM both reveal the location and volumetric shape of the deposits. KEYWORDS: hair, calcium fatty acid, lipid crystals, self-assembly, mesostructures packed spindle shaped cells that are rich in keratin filaments, which are composed of 400 to 500 amino acid residues paired together to form proto-filaments, which make up a keratin chain. These are oriented parallel to the long axis of the hair shaft and are embedded in an amorphous matrix of high sulfur proteins. The cortex contains melanin granules, which provide the basis for coloring the fiber based on the number, distribution, and type of melanin granules. The medulla is present generally in thicker hair and is a less dense proteinaceous core that is also rich in lipids. Also, critically important to hair integrity are structural lipids, which are located at cortical and cuticle cell interfaces and contribute between 4 and 7% weight of hair. These lipids can be chemically bound to the protein structure as is the case with the F-Layer but are also present unbound but in ordered structures.3 About 85% of these lipids are saturated and unsaturated fatty acids, which range in chain length from C14 to C24 but are predominantly C16 and C18. In addition to fatty acids, there are wax esters, ceramides, cholesterols, and low levels of sphingoid bases.4 Metals are also present, particularly

1. INTRODUCTION Human hair is a keratinized fiber composed of protein, lipids, melanins, and metals with a diameter is 50−100 μm. The state of one’s hair is extremely important to our culture’s perception of beauty, which is reflected by the fact that caring for hair is a 11 billion dollar business within the US alone.1 Its growth rate from the hair follicle is approximately one centimeter per month so that the very tips of shoulder length hair are more than 18 months old. Thus, protection of the hair’s structural integrity from daily insults including, but not limited to shampooing, brushing, coloring, and heat treatments such as blow dryers or flat irons is paramount. The hair fiber is an interesting and complex biologically derived hierarchical composite structure consisting of three major protein-rich parts: the cuticle, cortex, and medulla.2 The cuticle is composed of specialized keratins and consists of six to eight layers of flattened overlapping cells with their free edges directed upward to the tip of the hair shaft. On the outer surface of each cuticle is a hydrophobic lipid layer of 18methyleicosanoic acid attached by a covalent chemical bond to the surface of the fiber, commonly known as the F-Layer. The cortex forms the main bulk of a fully formed (keratinized) hair shaft and contributes almost all the mechanical properties of the hair including strength and elasticity. It consists of closely © XXXX American Chemical Society

Received: July 31, 2018 Accepted: September 13, 2018 Published: September 13, 2018 A

DOI: 10.1021/acsabm.8b00386 ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX

Article

ACS Applied Bio Materials

For cross-sectional viewing of single fibers by SEM, the hair fiber was peeled in half by first notching the fiber with a sharp single edge Teflon coated razor blade and then using a pair of #4 or #5 Dumont tweezers to pull the two halves away from each other using a stereomicroscope as a viewing aid. The newly exposed half was mounted facing up by carefully affixing with double sided carbon tape. The very ends of the peeled strand were painted with a small amount of carbon or silver paint to improve charging issues. Several peeled strands were mounted on a single copper SEM stub, after which the specimen was coated with Au/Pd for about 100 s, then transferred to the SEM using a Gatan Alto Cryo-prep station. SEM imaging was performed with a Hitachi S-4700 FE-SEM. 2.2.2. Dual Beam FIB/SEM, STEM/EDS. An FEI Helios 600 Dualbeam FIB/SEM was used to section serially through the “bubble” as well as to produce thin, cross-sectional STEM specimens for analysis. The hair specimens were mounted to a SEM stub, affixed with double sided carbon tape, and sputter coated with gold using a Leica ACE600 sputter coater to reduce electron beam charging and protect the sample during repeated milling. The serial sectioning image stack for 3D reconstruction was produced using a 30 kV, 2.8 nA Ga+ ion beam to remove 30 nm slices of material; subsequently, electron images were acquired of the freshly revealed, cross-sectional surface. Image processing and segmentation were performed using MIPAR9 3D reconstruction, and data set visualization was performed with Avizo. Thin, cross-sectional STEM specimens were produced by common dual-beam FIB/SEM lift-out techniques. Unnecessary ion beam exposure was avoided by using relatively high beam currents for short durations. Accurate milling minimized any unnecessary milling and ion imaging steps, trenches, and undercuts were milled using a 30 kV, 6.5 nA Ga+ ion beam. An OmniprobeAutoProbe 200 in situ micromanipulator was used to extract the cross-sectioned lamellae and the specimen was attached to a Mo grid. Final thinning to electron transparency was performed rapidly with a 30 kV, 300 pA Ga+ ion beam. This procedure produced specimens free from obvious damage which were sufficiently thin for STEM imaging and EDS analysis. STEM imaging and X-ray energy dispersive spectroscopy (EDS) spectrum imaging (SI) were performed with a FEI Titan G2 60−300 STEM equipped with a Super-X quad-silicon drift detectors (SDD). High angle annular dark field (HAADF) and bright field (BF) STEM imaging was performed at 60 kV accelerating voltage, and EDS SI mapping was performed at 300 kV with a 20 μs dwell time and acquired over 30 min using drift correction. 2.2.3. FT-IR and Raman Imaging. FT-IR images were collected using a Bruker Hyperion 3000 microscope equipped with a 128 × 128 focal plane array coupled to a Vertex 80 spectrometer. Sample preparation involved compressing individual hair fibers including peeled hair fibers (see SEM/EDS section) in a diamond anvil cell and collecting hyperspectral images of the thinned fiber in transmission mode. The sample preparation procedure is performed under an optical microscope prior to transferring the sample to the FTIR microscope for collecting the hyperspectral image. A key observation made during sample preparation in the optical microscope is material associated with bubble formation flattens and smears displaying rheology similar to a wax-like material during fiber compression. Bubbles at the fiber edges are less deformed than bubbles at the top and bottom of the hair fiber. Spectra were collected from 3895−848 cm−1 using a spectral resolution of 8 cm−1. The spectral images were analyzed using Isys Chemical imaging analysis software (version 5.0.0.14). Contrast shown in the FTIR image illustrated in Figure 7 is based on a partial least squares discriminate analysis (PLS-DA) model developed from spectroscopic libraries of keratin and calcium stearate. Confocal Raman images of whole hair fibers (blond with low melanin levels to prevent burning of the fiber) were obtained using a Witec alpha300 confocal Raman microscope with 785 nm laser excitation, Zeiss 100× 0.9NA objective, NIR optimized UHTS400 spectrometer set to 300 g/mm grating blazed at 750 nm and spectral center at 2100 cm−1, and back-illuminated deep-depletion CCD camera. Spectral data at each pixel were processed using the Witec

calcium and magnesium but also trace levels of other metals such as iron, copper, and zinc. As hair continues to grow, modifications occur to the protein and lipid components of the structure due to damage from external insults, which include hair treatments with oxidative colorants or perming solutions, exposure to UV radiation, and exposure to heated implements such as flat irons and blow dryers. These events mainly degrade the protein and lipid structure and lead to observable hair changes such as increased breakage and lack of shine. In addition to these alterations, there is the possibility that exogenous materials will build up within the hair fiber itself. For example, after hair coloring, the disulfide bonds in cystine are oxidized to cysteic acid. These charged groups provide sites for copper binding, and, in many cases, relative copper levels have been observed to increase from root to tip.5 Calcium has also been shown to accumulate in hair fibers. In one example, time-of-flight secondary ion mass spectroscopy (ToF SIMS) has been used as a technique to map calcium in hair and to identify the source as either exogenous or endogenous.6,7 In this work, the authors identified calcium deposits at the cuticle scale edges and within the cuticle layer itself. Separately, Merigoux et al.8 used X-ray microfluorescence imaging to detect two different types of calcium, distinguished by whether they could be removed by hydrochloric acid, but could not correlate these with the source of the metals. In this work, we provide the first conclusive evidence for the presence of exogenous calcium fatty acid deposits, which not only form in-between the cuticle layers in the lipid-rich cell membrane complex, but also grow to dimensions large enough to cause the structure to bulge, thereby impacting the optical and mechanical properties of the hair fiber. The composition and phase of these deposits were probed using a multimodal analytical approach with spatially resolved techniques including synchrotron micro X-ray fluorescence coupled with X-ray scattering where the maximum d-spacing from the scattering pattern was consistent with C16/C18 saturated fatty acids. By utilizing high resolution scanning electron microscopy (SEM), scanning transmission electron microscopy (STEM), and focused ion beam (FIB) techniques the volumetric shape and internal mesophase microstructure of the deposits have been identified for the first time. The spatial distribution of the deposits in the cuticle cell membrane complex has been visualized with high resolution X-ray microtomography (XMT) highlighting how extensive these deposits are distributed throughout the hair structure. Fourier transform infrared (FT-IR) and Raman imaging have confirmed similar size and shape deposits while identifying spectral similarities to calcium stearate. The multimodal data sets obtained have been used to propose a mechanism of how these deposits can form and how their presence impacts the hair’s optical properties as well as hair strength.

2. METHODS 2.1. Samples. Single source hair was harvested from over 100 individuals across all hair colors and ethnicities and screened for high levels of deposits using either an optical microscope or SEM. Approximately 60% were found to contain bubbles. 2.2. Experimental Setups. 2.2.1. SEM/EDS. To view the surface of hair fibers, many hair fibers were cut to about 1 cm in length and mounted to a SEM stub by gently affixing with double sided carbon tape. Low-magnification screening of hair fibers was performed with a Hitachi S-3000N SEM using variable pressure mode (no conductive coating required). B

DOI: 10.1021/acsabm.8b00386 ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX

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ACS Applied Bio Materials

Figure 1. SEM images of subsurface deposits in hair. (a, b) Surface views for different fibers (scale bars = 50 μm); (c−f) cross-sectional views (scale bar = 5 μm in panels c−e, and 1 μm in panel f). Arrows in panels d and e identify the deposit material. All samples are from hair collected from Caucasian women. Suite 4 software for cosmic ray removal, baseline correction, and basis spectra analysis for color coded image generation.10 2.2.4. X-ray Fluorescence/X-ray Mapping. Experiments were conducted at the European Synchrotron Research Facility (ESRF) on ID 13 Microfocus Beamline. Data were recorded using a FReLoN CCD camera for recording the scattering signal and a Vortex micro Xray fluorescence detector. Two hair fibers (female Caucasian hair, one blond and one brown) were preselected for having a high quantity of subsurface calcium deposits by SEM. Each hair fiber was analyzed as a whole fiber as well as a peeled hair. An area of 120 μm × 24 μm or smaller was scanned with step size of 0.8 μm for each sample. The following parameters were utilized: wavelength, 0.832e-10 m; beam size, 200 nm × 200 nm; pixel size 1, 5.167133e-5 m; pixel size 2, 5.095279e-5 m; sample−detector distance, 186.79 mm; beam center 1, 1003; beam center 2, 1029.8; detector rotation, rot1 0.00046144, rot2 0.00016105, rot3 0. 2.2.5. X-ray Microtomography. XMT was performed using an Xradia 810 Ultra 3D X-ray Microscope using a photon energy of 5.4 keV. The sample was cut and mounted to a pin using epoxy. The imaging ROI was selected to be as near to the epoxy as possible without interfering with the X-ray beam. Phase contrast data sets were recorded using (a) a large field of view (65 um) using a voxel size of 64 nm and (b) a high resolution FOV (16 um) with a 32 nm voxel size. 2.2.6. Goniophotometer Measurements. To measure single fiber shine, a modified Brice-Phoenix goniophotometer was used to record the intensity of scattered light as a function of angle. Measurements were carried out on 50 randomly chosen single hair fibers. A He−Ne laser with a wavelength of 632 nm and a quartz tungsten halogen lamp emitting white light were used as illumination sources. A single hair fiber was placed in the sample holder horizontally at an angle of incidence of 45° and the reflected light was detected by the photomultiplier as a function of angle. Measurements were carried out with the fiber in the root-to-tip position at approximately the same distance from the root end. The luster was calculated by L (%) = S/[S + D] × 100

was calculated from three diameter measurement points along each 30 mm crimped fiber. The average cross-sectional values for each of the fibers were then used to set the Dia-Stron Cyclic Tester (CYC801) in controlled stress mode of 0.014 g/μm2 and rate of 40 mm/s. The relative humidity was set 50% RH and temperature at 23 °C. Data were analyzed by Weibull and Kaplan−Meier statistical tools (JMP Pro 12.1.0, SAS Cary, NC). Fibers with break cycles less than ten were omitted from the analysis due to premature breakage. Fifty fibers per leg were measured.

3. RESULTS AND DISCUSSION The SEM images in Figure 1 show hair both from the surface and in cross-section. From the surface view, many cuticles along the fiber surface are deformed by subsurface “bubbles”, which are approximately 5−10 μm in diameter. The size of these “bubbles” varies between individuals and between different fibers within the same hair sample. In addition, on a single fiber, a variety of “bubble” sizes are observed as seen in Figure 1a and b. The occurrence of very similar “bubbles” on the hair surface has been noted in the literature11 and was claimed to be caused by a combination of thermal and extension cycles but no further details were given. Larger cuticle “bubble” deformation features have also been noted to be caused by high temperatures from repeated flat ironing12 but these are very different in morphology to the “bubbles” of this study. After peeling “bubble” containing fibers in half so that the hair is split to bisect through the cuticle deformation features, deposits can be clearly seen just under the fiber surface, whereby they reside in-between the individual cuticle cells in the cell membrane complex region. The deposit shape as shown via cross-section projection is half of an oblate sphere that often results in a bulging deformation, which we have described as “bubbles” when viewed normal to the fiber surface. It is interesting to note that the deposits have been observed across the full depth of cuticle layers depending on how many layers are present on an individual hair fiber, and in some situations the deposit is not large enough to appreciably deform the surface (Figure 1e). Figure 1f shows a higher magnification image of much smaller deposits seen in between the cuticle cells, and it is proposed that these images represent the progression of growth for these deposits. We believe that submicron deposits are initially formed that over time grow to

(1)

Where S is defined as the specular peak area obtained from the goniophotometer curve using a Gaussian distribution and (S + D) is the total area under the curve. 2.2.7. Fatigue Measurements. Fibers were cut for fatigue strength measurements from the middle of the tress and ends crimped at 30 mm using a Dia-Stron Auto-Assembly System (AAS 1600) (Andover, Hampshire, UK). The average cross-sectional area along each fiber was analyzed using a Dia-Stron Fiber Dimensional Analysis System (FDAS 770), which incorporated a Mitutoyo laser micrometer (LSM6200) (Marlborough, MA, USA). The average cross-sectional area C

DOI: 10.1021/acsabm.8b00386 ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX

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ACS Applied Bio Materials

Figure 2. STEM imaging and EDS mapping of hair cross-section through a “bubble” deposit: (a) STEM-HAADF image, (b) calcium, (c) nitrogen, (d) carbon, (e) oxygen, (f) sulfur.

Figure 3. STEM images ((a) HAADF mode, (b, c) BF mode) of calcium fatty acid deposits in hair. Panel a provides a lower magnification overview of the deposit material as it sits between the cuticle layers where panels b and c provide views at increased magnification within the calcium rich deposit region.

a large enough size to deform the flexible cuticle and to become visible from the surface view as shown by SEM in Figure 1a and b. Sampling of hair from root to tip of a single fiber agrees with this hypothesis whereby smaller deposits are observed at the hair root end, which progressively become larger toward the tip. The hair splitting method described herein has a unique advantage of exposing the “bubble” deposits without impact of mechanical or chemical means. However, the cross-sectional plane achieved with this approach is fairly rough and the deposit is still sandwiched on three sides by cuticle cells; therefore, mapping across this plane by EDS presents difficulties. Traditional biological ultrasectioning techniques for TEM have also resulted in difficulties in obtaining crosssectional views of the “bubble” material due to dissolution during the fixation/embedding process or mechanical removal during sectioning and transfer to the TEM grid. Thus, the cuticle regions that contained “bubbles” were observed to be empty when viewed by TEM. Dual beam FIB/SEM was explored as an alternative approach to overcome these adversities whereby select regions of the hair fiber were identified and milled via focused ion beam of Gallium to expose the “bubble” material for analysis.

FIB/SEM sample preparation methods are employed commonly for studying the internal structure of hard materials such as semiconductors, ceramics, and metals but have only recently been applied to the study of soft materials such as polymers and biological substrates.13,14 By using the dual beam FIB/ SEM, “bubble” areas on the fiber surface were identified by SEM, and then the Ga+ FIB was used to mill away the surrounding cuticle cells to reveal the cross-section of the “bubble” deposits. Three key aspects of the “bubble” material were investigated using the FIB/SEM approach: elemental composition, microstructure, and 3D morphology. FIB lamellae cross-sections of the “bubble” material and surrounding hair structure were extracted and thinned to image and for STEM-EDS SI. The cross-sections were imaged under STEMHAADF conditions and EDS mapping for calcium, carbon, oxygen, nitrogen and sulfur are shown in Figure 2a−f. The HAADF image shows that the “bubble” deposit has brighter contrast versus the surrounding cuticle cells, as well as most of the cortex region of the hair, suggesting the average atomic number is higher for the deposit material versus the native hair. EDS mapping confirms that these deposits are enriched in calcium, oxygen, and carbon versus the native hair but contains less than 0.5 atom % sulfur or nitrogen. At higher D

DOI: 10.1021/acsabm.8b00386 ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX

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ACS Applied Bio Materials magnification, HAADF imaging of the deposit material reveals an interesting microstructure, which consists of a channel motif with periodic repeat distance; however, the channels are organized in nonuniform patterns having curvature (Figure 3). These curved channel patterns span across the full crosssection of the “bubble”. Measurements across the periodic repeat distance in both bright field and HAADF STEM mode reveal a ∼7 nm spacing. Materials with basic repeat motifs between 2−50 nm combined with curved nongeometric longrange order are typical for self-assembled materials based on liquid crystals with inorganic constituents as well as natural lipid extracts.15−19 The broad classification of these materials is mesophases or mesostructured materials. The field of synthetic inorganic mesostructures began with silicate liquid crystals MCM-41 in 1992 by Kresge and has since expanded to many different inorganic and amphiphilic combinations not least of which includes fatty acids and hydroxyapatite20 and surfactant templated aluminophosphates;21 however, literature reports for these structures being formed in nature from a process that is caused by exogenous factors are very few. Cross-sections of hair using both SEM and TEM show the flattened oblate shape of the deposits but this is only one projection of the deposit. To reveal the deposit’s full 3D volume and shape, a serial “slice and view” approach was accomplished utilizing the dual beam FIB/SEM where alternating FIB slices of about 30 nm followed by SEM images of the exposed plane were taken through a complete deposit structure comprising a total volume of approximately 10 μm in length/width by 3 μm in depth. The SEM image stack was processed using MIPAR software to create a 3D rendering of the deposit (Figure 4). Neither the cross-sectional view nor the surface SEM view adequately provides a true representation of the deposit’s full volume, as the FIB/SEM approach reveals that the deposit is much larger and flatter than previously indicated; it is approximately 10 μm long × 8 μm wide × 2 μm deep and thus extends extensively through the hair’s lipid cell membrane structure. XMT was completed for a single source hair sample that had been prescreened using an optical microscope as having calcium deposits. This microscope measures attenuation or phase shift of the incident X-ray beam in a series of 2D projections, which can then be reconstructed to create a 3D image with a spatial resolution of better than 150 nm. The calcium deposits were identifiable due to their relative contrast versus the surrounding cuticle cell, which allowed for segmentation and reconstruction of the data to achieve a 3D rendering of the fiber surface and internal structure. A length of hair ∼30 μm was imaged using this approach to identify cuticle-based calcium fatty acid deposits as well as to quantify several key dimensions (Figure 5). It is clear from the comparison of the surface projection versus the 3D rendering of the calcium fatty acid deposits that there are significantly more deposits present than are visible from surface. In addition, the shapes of the deposits are all flattened spheres with many having the thickest dimension at the center, which closely corresponds to the shape and dimensions achieved via dual beam FIB/SEM “slice and view” data for one selected deposit. From this data, quantitative metrics on the deposit size and shape can be calculated. The aspect ratio was mainly in the 0.2 to 0.3 range, but extended up to 0.45 which was indicative of the flattened shape of the deposits. The volume of the deposits ranged from 5−20 μm3 with a maximum at 48 μm3.

Figure 4. 3D reconstruction from a FIB/SEM serial sectioning image stack showing the secondary electron image (gray scale) and rendered image (green pseudocolored) at progressive depths across the volume of a single calcium deposit spanning from the beginning of the deposit (a) to the approximate midpoint of the deposit (c). The SEM image shows the cross-sectional plane of the fiber cuticle region containing a calcium deposit and the rendered image shows the reconstructed fiber surface. The size of the frame is 10 μm x 10 μm x 3 μm. E

DOI: 10.1021/acsabm.8b00386 ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX

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ACS Applied Bio Materials

Figure 5. X-ray microscopy of hair showing (a) surface rendering and (b) subsurface calcium fatty acid deposits color coded for local thickness

Figure 6. (a) FT-IR hyperspectral image collected from peeled hair with a high deposit count. The approximate cortex region is marked with arrows and the cuticle region is located only along the edges of the fiber. (b) PLS-DA loading that dominates contrast in the hyperspectral image superimposed on a spectrum of calcium stearate.

Figure 7. (a) Optical microscopy image of the hair surface and “bubble” deposit. The red line represents the line scan region of 30 um in length; (b) 30 × 10 μm2 line scan depth profile (x−z direction, 60 points per line) of the “bubble” region where the blue map depicts the distribution of the hydrocarbon rich region represented by the (d) basis spectrum versus the hair matrix (red map) and represented by the (c) basis spectrum; (e) library spectrum of calcium stearate obtained with a 1064 nm FT-Raman.

on a length scale consistent with the size of deposits on the hair. Hair samples were prepared for hyperspectral imaging by compressing individual hair fibers in a diamond anvil cell and

FT-IR hyperspectral images were collected from these samples to improve spectral selectivity and specificity for detecting deposits and to spatially resolve spectral information F

DOI: 10.1021/acsabm.8b00386 ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX

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ACS Applied Bio Materials

Figure 8. Micro XRF Log Intensity maps across the same region of peeled hair fiber plotting (a) Ca K peak, (b) S K peak.

Figure 9. (a) Regions of interest (Spot 1 and 2) chosen at high and low calcium regions and corresponding X-ray scattering patterns. (b) Overlay of SAXS patterns corresponding from Spot 1 and 2.

collecting hyperspectral images of the thinned fiber in transmission mode. A representative hyperspectral image for a peeled hair fiber is shown in Figure 6 where bubble shaped features are readily identified by high intensity at regions along the edges of the fiber where the deposits within the cuticle layers are commonly exposed. A molecular factor analysis was performed on the hyperspectral image. This numerical algorithm deconstructs the spectral data set into spectra associated with individual components. This approach is also referred to as self-modeling mixture analysis or multivariate curve resolution. The molecular factor (PC spectrum) responsible for the contrast in Figure 6a is shown in Figure 6b superimposed on a spectrum of calcium stearate for comparison. The hyperspectral image in Figure 6a shows a similar spatial distribution of deposits in the hair fiber as observed in the SEM and X-ray microtomography and indicates the deposits consist of an aliphatic hydrocarbon that has a very similar spectrum to calcium stearate. There is no evidence for calcium carbonate deposits or calcium salts of unsaturated fatty acids. Imaging of the deposits using intact hair was accomplished using confocal Raman imaging. Collecting Raman spectra from hair is challenging due to localized heating in the sample associated with exposing hair to the intense power density at the focal point of a laser beam. This challenge can be overcome by using hair that contains a minimal amount of melanin responsible for pigmentation in hair. In these experiments, the sample was blond with low melanin levels but contained a high number of deposits. The depth profile confocal Raman image depicted in Figure 7 reveals that the chemical nature of the hair inclusion forming the “bubble” (Figure 7d) is different from hair keratin (Figure 7c) and consistent with that of a long chain aliphatic hydrocarbon fatty acid (Figure 7e), in agreement with the results from hyperspectral FT-IR imaging. The dimension of the hydrocarbon inclusion is within the size

range of the “bubbles” revealed by X-ray microtomography and SEM. Micro-X-ray fluorescence and X-ray scattering were mapped across regions of hair fibers using the ESRF ID13 beamline, which allows for acquisition of fluorescence and scattering in parallel using a 200 × 200 nm2 X-ray beam scanned at 800 nm step size. Each pixel of the map across the fiber contains data sets for the X-ray fluorescence intensity for a range of elements including calcium and sulfur as well as the corresponding X-ray scattering pattern. Two single source hair samples (one blond and one brown color, female Caucasian hair) were analyzed as both whole fibers as well as peeled fibers where the calcium fatty acid deposits were exposed in the cross-section. An alignment microscope was used to focus on a visible “bubble” as observed on the hair surface, and then the X-ray fluorescence and X-ray scattering patterns were collected across the surrounding region. Figure 8 shows the calcium and sulfur X-ray fluorescence maps for one hair sample, and the calcium deposits are clearly visible as higher than background calcium levels. The corresponding sulfur maps showed lower sulfur levels in these same areas supporting the microscopy data that indicated the deposits did not contain sulfur. To note the X-ray beam travels through the sample so deposits can either be on the top or bottom surface of the sample. Regions of interest were chosen in the fluorescence map at high and low levels of calcium to compare the X-ray scattering patterns in these two regions as shown in Figure 9. For regions with high calcium intensity, a scattering pattern was consistently observed with a high intensity peak at q = 1.45 nm−1 with two smaller peaks at around 2.9 and 4.2 nm−1 indicative of a lamellar structure with the first order reflection having a d-spacing of ∼4.5 nm. Two smaller peaks were observed at 14.3 nm−1 and 28.6 nm−1, which correspond to a d-spacing of 0.44 nm. These d-spacing values were compared to the ICDD (International Center for Scattering Data) and G

DOI: 10.1021/acsabm.8b00386 ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX

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ACS Applied Bio Materials

of these deposits being naturally derived calcium fatty acid soaps, mainly calcium palmitate and stearate, rather than fatty alcohol, unsaturated fatty soaps, or sulfate surfactant compounds found in hair products.31 It is hypothesized that these deposits are formed from exogenous fatty acids from sebum penetrating into hair along with exogenous calcium from water hardness ions during shampooing. The deposits start small and slowly grow over time forming the observed meso-phase structures that are very stable due to interchain hydrophobic interactions.32 It is hypothesized that the majority of these deposits form in the cuticle lipid-rich cell membrane complex due to its unique lipid structure that is rich in free fatty acids (saturated and unsaturated) versus the cortex cell membrane complex that contains a higher level of cholesterols and ceramides. Formation of saturated fatty acid calcium salts is favored over unsaturated fatty acid calcium salts due to the favorable hydrophobic packing of the straight chain saturated fatty acids. The consequences of these exogenous deposits on hair properties were measured with two methods. The first was single fiber goniophotometer measurements where single source hair with and without deposits was measured. A He− Ne laser beam was impinged on a single fiber and the reflected light mapped using a photomultiplier detector.33 The reflection profile was analyzed for its specular and diffuse components and luster calculated using the formula

compared well to X-ray scattering patterns for calcium palmitate (C16:0) and calcium stearate (C18:0), which have a main d-spacing of 4.5 and 5.0 nm, respectively, and which correspond to the distance between the calcium ions with the repeating unit being Ca(FA)2. There is also an additional peak at 0.45 nm for calcium palmitate and 0.20 nm for calcium stearate, which corresponds to the distance between the fatty acid chains and it similar to the 0.44 nm spacing found in the hair deposits. There is some variation in d-spacing found in the hair deposits implying that they may be a mixture of chain lengths of fatty acid but no evidence was found for the presence of unsaturated fatty acids such as oleic acid, which is an important hair lipid or shorter chain fatty acids such as calcium myristate (C14:0), which has a d-spacing of 4.0 nm. In addition, no evidence was found for the mineral form of calcium carbonate, which has well established d-spacings beginning around 0.3−0.4 nm. The possible formation of calcium lipid species inside hair has been noted in the literature but the exact nature of these lipid structures in terms of content and spatial distribution has not been well determined. The existence of a unique “ring” or “band” at about 45−49 Å in X-ray scattering patterns for hair has been reported back to 1963.22 This d-spacing is unique from any scattering response of keratin itself and was attributed at that time to the existence of “lipid crystals”. In 1999, this seemingly forgotten topic was revisited due to the claim by Prof. Veronica James that the existence of a ring at about 44 Å could be used as a screening method for breast cancer.23 In her publications, James suggested this ring was due to randomly oriented lipid bilayers either in the plasma membrane or membranous inclusion in hair cells. While several groups questioned the reproducibility of her claims,24−26 one group responded by contributing a study aimed at better understanding the X-ray scattering response from different regions of the hair.27 In this study, Busson and co-workers used a microfocus beamline at ESRF with 3−6 μm X-ray beam diameter to show that the unique scattering due to lipids (lamellar periodic d-spacing of 45 Å) was most intense within the first 10 μm from the surface. The authors speculated that the lipids may exist as 50 to 100 nm diameter “granules” colocated with α-keratin zones. More than 10 years later, a connection was demonstrated between the periodic structure of lipid “granules” and high calcium content through the use of a combination of microscattering and XRF techniques.28 Within this study, the authors found that Pb2+ could be used to replace a large amount of Ca2+, which thereby enhances the Xray scattering intensity of the “lipid crystals” by a factor of 5, thereby providing an improvement in accurate peak detection and fitting of the scattering pattern. The authors of the Pb2+ study suggest that the Pb and Ca lipid structure is most likely a “soap” formed by C16/C18 fatty acids, which is likely like the thermotropic mesophase structure reported for synthetically produced lead alkanoates.29,30 The combination of microscopy, FT-IR hyperspectral imaging, and X-ray microtomography has confirmed the location of these deposits within the cuticle cell membrane complex along with their flattened oval shape concomitant with deformation to the cuticle surface and their wide distribution along the hair fiber. STEM−EDS has confirmed the presence of calcium, carbon, and oxygen in these deposits as well as an absence of sulfur and nitrogen, which suggests the absence of sulfur based surfactants. Raman and FT-IR spectroscopy and X-ray scattering all provide strong evidence

Luster L (%) = [S/(S + D)] × [W std1/2 /W sam1/2] × 100 (2)

where S is the amount of specular light in the reflection profile, D is the diffuse component, and W1/2 values are the peak widths at half-maximum for a black standard (std) and the sample (sam). The second is single fiber fatigue measurements where a repeated constant stress is applied until hair breaks where the applied stress for fatigue testing is in the Hookean region of the stress−strain curve.34 This method is proposed to more closely link to breakage induced by repeated combing and to be related to formation and propagation of cracks and flaws induced by repeated stress.35 A constant stress of 0.014 g/μm2 and a rate of 40 mm/s was used, and all fibers were checked to ensure this stress was within the Hookean extension region. As the data are not a normal distributed mean, the cycles to break is not an appropriate number to compare samples but instead alpha value is more commonly used. The alpha value is obtained from the Weibell distribution function and is the characteristic lifetime when 63.2% of fibers have broken. Weibell and Kaplan−Meier statistics using alpha values were used to determine significance between hair with and without “bubbles”. Table 1 shows the results from single fiber shine and fatigue testing for single source hair with and without calcium fatty acid deposits. Hair with high deposits had at least 80% of Table 1. Goniophotometer Shine and Fatigue Testing

low calcium fatty acid deposits high calcium fatty acid deposits H

single fiber luster (%)

single fiber fatigue strength alpha value (no. of cycles to 63.2% of fibers broken)

23.23 (2.77)

29 200

16.16 (2.01)

9675

DOI: 10.1021/acsabm.8b00386 ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX

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ACS Applied Bio Materials fibers with visible “bubbles” as measured by SEM and hair with low deposits had less than 20% of fibers with visible “bubbles”.

(5) Godfrey, S.; Staite, W.; Bowtell, P.; Marsh, J. M. Metals in Female Scalp Hair Globally and its Impact on Perceived Hair Health. Int. J. Cosmet. Sci. 2013, 35, 264−271. (6) Kempson, I. M.; Skinner, W. M.; Kirkbride, P. Calcium Distributions in Human Hair by ToF-SIMS. Biochim. Biophys. Acta, Gen. Subj. 2003, 1, 1624. (7) Kempson, I. M.; Skinner, W. M. ToF-SIMS Analysis of Elemental Distributions in Human Hair. Sci. Total Environ. 2005, 338, 213−227. (8) Merigoux, C.; Briki, F.; Sarrot-Reynauld, F.; Salome, M.; Fayard, B.; Susini, J.; Doucet, J. Evidence for Various Calcium Sites in Human Hair Shaft Revealed by Sub-Micrometer X-ray Fluorescence. Biochim. Biophys. Acta, Gen. Subj. 2003, 1619, 53−58. (9) Sosa, J. M.; Huber, D. E.; Welk, B.; Fraser, H. L. Development and Application of MIPAR: a Novel Software Package for Two- and Three-Dimensional Microstructural Characterization. Integrating Materials and Manufacturing Innovation 2014, 3, 10. (10) Schmidt, U.; Hild, S.; Ibach, W.; Hollricher, O. Characterization of Thin Polymer Films on the Nanometer Scale with Confocal Raman AFM. Macromol. Symp. 2005, 230, 133−143. (11) Gamez-Garcia, M. Morphological Changes in Human Hair Cuticles Upon the Simultaneous Action of Cyclical Mechanical and Thermal Stresses; Their Relevance to Grooming Practices. J. Cosmet. Sci. 1998, 49, 40−41. (12) Gummer, C. L. Bubble Hair: A Cosmetic Abnormality Caused by Brief, Focal Heating of Damp Hair Fibres. Br. J. Dermatol. 1994, 131, 901−903. (13) Deng, B.; Freria, C. M.; Williams, R.; Huber, D. E.; Sosa, J. M.; Popovich, P.; McComb, D. W. 3D Visualization of Motor-Neurons in Mice Spinal Cord Using FIB\SEM Tomography. Microsc. Microanal. 2014, 20 (Suppl. 3), 1400−1401. (14) Marsh, J. M.; Davis, M. G.; Flagler, M. J.; Sun, Y.; Chaudhary, T.; Mamak, M.; McComb, D. W.; Williams, R. E.; Greis, K. D.; Rubio, L.; Coderch, L. Advanced Hair Damage Model from Ultra-Violet Radiation in the Presence of Copper. Int. J. Cosmet. Sci. 2015, 37, 532−541. (15) Yang, H.; Coombs, N.; Dag, O.; Sokolov, I.; Ozin, G. A. Standing Mesoporous Silica Films; Morphogenesis of Channel and Surface Patterns. J. Mater. Chem. 1997, 7, 1755−1761. (16) Mann, S. The Chemistry of Form. Angew. Chem., Int. Ed. 2000, 39, 3392−3406. (17) Ozin, G. A. Panoscopic Materials: Synthesis Over ‘All’ Length Scales. Chem. Commun. 2000, 419−432. (18) Barriga, H. M. G.; Parsons, E. S.; McCarthy, N. L. C.; Ces, O.; Seddon, J. M.; Law, R. V.; Brooks, N. J. Pressure-Temperature Phase Behavior of Mixtures of Natural Sphingomyelin and Ceramide Extracts. Langmuir 2015, 31, 3678−3686. (19) Meyer, H. W.; Bunjes, H.; Ulrich, A. S. Morphological Transitions of Brain Sphingomyelin are Determined by the Hydration Protocol: Ripples Re-Arrange in Plane, and Sponge-Like Networks Disintegrate into Small Vesicles. Chem. Phys. Lipids 1999, 99, 111− 123. (20) Jiang, J.; Fan, Y.; Zhang, L.; Yang, H.; Chen, Y.; Zhao, D.; Zhang, P. Synthesis and Characterization of Multi-Lamellar Mesostructured Hydroxyapatites Using a Series of Fatty Acids. J. Mater. Sci. 2011, 46, 3828−3834. (21) Coombs, N.; Khushalani, D.; Oliver, S.; Ozin, G. A.; Shen, G. C.; Sokolov, I.; Yang, H. Blueprints for Inorganic Materials with Natural Form: Inorganic Liquid Crystals and a Language of Inorganic Shape. J. Chem. Soc., Dalton Trans. 1997, 3941−3952. (22) Fraser, R. D. B.; MacRae, T. P.; Rogers, G. E.; Filshie, B. K. Lipids in Keratinized Tissues. J. Mol. Biol. 1963, 7, 90−91. (23) James, V.; Kearsley, J.; Irving, T.; Amemiya, Y.; Cookson, D. Using Hair to Screen for Breast Cancer. Nature 1999, 398, 33−34. (24) Rogers, K. D.; Hall, C. J.; Hufton, A.; Wess, T. J.; Pinder, S. E.; Siu, K. Reproducibility of Cancer Diagnosis using Hair. Int. J. Cancer 2006, 118, 1060. (25) Briki, F.; Busson, B.; Salicru, B.; Estéve, F.; Doucet, J. Breast cancer diagnosis using hair. Nature 1999, 400, 226.

4. CONCLUSION The existence of “bubble” shaped calcium fatty acid deposits lodged between cuticle cells has been conclusively proven for the first time. A multimodal analytical approach using spatially resolved techniques was used to characterize the deposits as specifically as possible including size, shape, location, composition, and microstructure. Our results aim to resolve historical observations for peaks/rings in the SAXS region for hair fibers and especially for those with high in calcium content as well as provide insight into the formation of deposits via selfassembly mechanism. It has been demonstrated that the occurrence of these deposits has an impact on hair shine as well as mechanical properties.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsabm.8b00386.

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Movie file showing several viewpoints of “bubble” deposit, starting with secondary electron image stack from serial FIB/SEM serial sectioning, followed by view of “bubble” deposit after image analysis (reconstruction and segmentation), then to surface rendered image across single “bubble” deposit, and finally to internal view of “bubble” deposit (MPG)

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: 513-622-0445. ORCID

Marc Mamak: 0000-0003-0582-5071 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors would like to thank The Procter & Gamble Company for support of this work; Jeremie Gummel (P&G) and Michael Sztucki (ESRF) for X-ray fluorescence and X-ray scattering measurements; and Ying Boissy (P&G) for selected SEM images, Tim Felts (P&G) for fatigue measurements, the TRI in Princeton for single fiber goniophotometer measurements, and Jeff Gelb and Steve Kelly (Carl Zeiss X-ray Microscopy, Inc.) for XMT measurements.

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REFERENCES

(1) MarketResearch.com: The U.S. Beauty and Cosmetics Market Expected to Exceed $62 Billion in 2016. Cision; PR Newswire, 2016. https://www.prnewswire.com/news-releases/marketresearchcom-theus-beauty-and-cosmetics-market-expected-to-exceed-62-billion-in2016-300209081.html. Accessed Date: February 2, 2018. (2) Robbins, C. R. Chemical and Physical Behavior of Human Hair, 5th ed; Springer-Verlag, 2012; pp 42−63. (3) Robbins, C. R. The Cell Membrane Complex: Three Related but Different Cellular Cohesion Components of Mammalian Hair Fibers. Int. J. Cosmet. Sci. 2010, 32, 235. (4) Masukawa, Y.; Narita, H.; Imokawa, G. Characterization of the Lipid Composition at the Proximal Root Regions of Human Hair. Int. J. Cosmet. Sci. 2005, 56, 1−16. I

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ACS Applied Bio Materials (26) Aksirov, A. M.; Gerasimov, V. S.; Kondratyev, V. I.; Korneev, V. N.; Kulipanov, G. N.; Lanina, N. F.; Letyagin, V. P.; Mezentsev, N. A.; Sergienko, P. M.; Tolochko, B. P.; Trounova, V. A.; Vazina, A. A. Biological and Medical Application of the SR from the Storage Rings of VEPP-3 and “Siberia-2”. The Origin of Specific Changes of SmallAngle X-ray Diffraction Pattern of Hair and Their Correlation to Elemental Content. Nucl. Instrum. Methods Phys. Res., Sect. A 2001, 470, 380−387. (27) Busson, B.; Engström, P.; Doucet, J. Existence of Various Structural Zones in Keratinous Tissues Revealed by X-ray Microdiffraction. J. Synchrotron Radiat. 1999, 6, 1021−1030. (28) Bertrand, L.; Doucet, J.; Simionovici, A.; Tsoucaris, G.; Walter, P. Lead-Revealed Lipid Organization in Human Hair. Biochim. Biophys. Acta, Gen. Subj. 2003, 1620, 218−224. (29) Amorim Da Costa, A. M.; Burrows, H. D.; Geraldes, C. F. G. C.; Teixeira-Dias, J. J. C.; Bazuin, C. G.; Guillon, D.; Skoulios, A.; Blackmore, E.; Tiddy, G. J. T.; Turner, D. L. Mesophase Formation in Lead(II) Decanoate. Liq. Cryst. 1986, 1, 215−226. (30) Bazuin, C. G.; Guillon, D.; Skoulios, A.; Amorim Da Costa, A. M.; Burrows, H. D.; Geraldes, C. F. C. C.; Teixeira-Dias, J. J. C.; Blackmore, E.; Tiddy, G. J. T. Thermotropic Polymorphism in LiquidCrystalline Lead(II) Alkanoates. Liq. Cryst. 1988, 3, 1655−1670. (31) Jandacek, R. J. The Solubilization of Calcium Soaps by Fatty Acids. Lipids 1991, 26, 250−253. (32) Pereira, R. F. P.; Valente, A. J. M.; Fernandes, M.; Burrows, H. D. What Drives the Precipitation of Long-Chain Calcium Carboxylates (Soaps) in Aqueous Solution? Phys. Chem. Chem. Phys. 2012, 14, 7517−7527. (33) Keis, K.; Ramaprasad, K. R.; Kamath, Y. K. Studies of Light Scattering from Ethnic Hair Fibers. Int. J. Cosmet. Sci. 2004, 55, 49− 63. (34) Evans, T. R. Fatigue Testing of Hair − a Statistical Approach to Hair Breakage. Int. J. Cosmet. Sci. 2010, 60, 599−616. (35) Evans, T. R. Fatigue Testing of Hair − a Statistical Approach to Hair Breakage II. Repeated Grooming Experiments. Int. J. Cosmet. Sci. 2010, 61, 439−455.

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DOI: 10.1021/acsabm.8b00386 ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX