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Structural Aspects of Stratum Corneum
C H A P T E R
42 Structural Aspects of Stratum Corneum I. Hatta Nagoya Industrial Science Research Institute, Nagoya, Japan
42.1 INTRODUCTION In this chapter, I will focus my attention on microscopic structures formed in stratum corneum (SC). X-ray diffraction technique is a powerful tool to obtain structural evidence at the molecular level when we consider the function of stratum corneum. Generally, when an X-ray beam impinges on an object, part of the beam called a direct beam is transmitted through the object and part is scattered by the object. An object with periodic structure can give rise to diffraction at certain angles; at other scattered angles destructive interference causes the X-ray waves to cancel. In an object with a three-dimensional periodic crystal structure, the atoms or molecules are arranged in parallel layers or crystal planes that reflect X-ray beams as a mirror reflects light. Positive interference between X-rays reflected from the parallel layers or crystal planes occurs at angles, satisfying Bragg’s law: 2d sinð2qB =2Þ ¼ nl
(42.1)
where 2qB is the angle between the incident and the reflected X-ray beams called the Bragg angle, i.e., qB is the angle between the incident X-ray beam and the parallel layers or crystal planes, d is the perpendicular spacing between the parallel layers or crystal planes, l is the wavelength of X-ray, and n is an integer. The wavelength of X-rays is around 0.1 nm. Therefore, according to Bragg’s law the spacing d that is more than 0.2 nm can be detected by X-ray diffraction. We can observe X-ray diffraction when periodic structures take place in SC. Periodic structures formed in intercellular lipids of SC are characterized in terms of two orthogonal lattice spacings: one is the lamellar repeat distances and another is the lattice spacings of the hydrocarbon-chain packing structure (so-called “subcell structure”). From X-ray diffraction for the lamellar structure of hairless mouse SC that exhibits one of the typical X-ray diffraction in mammalian SC, the long lamellar structure with the repeat distance of 13.6 nm and the short lamellar structure with the repeat distance of about 6 nm have been obtained as illustrated schematically in Fig. 42.1A and B, respectively.1 The illustrations show only the presumable molecular arrangement of ceramides, free fatty acids, and cholesterol in SC. But there are a lot of arguments for the molecular arrangements. To solve them it is important to perform the detailed structural study at the molecular level not only on SC but also on SC lipid model system in which the constituent molecules are known. Based upon the structural study on an SC lipid model system, it has been pointed out that in the formation of the long lamellar structure a long ceramide molecule such as CER(EOS) is one of the key elements.2 For the short lamellar structure it is worthwhile pointing out that water molecules are incorporated into the short lamellar structure and therefore that a water layer between the successive lipid bilayers in the short lamellar structure takes place as shown in Fig. 42.1B.3e5 On the other hand, for the lateral packing of the lipids there are hexagonal and orthorhombic hydrocarbon-chain packing structures with the lattice constant of 0.42 nm and with the lattice constants of 0.42 and 0.37 nm, respectively, at room temperature as illustrated schematically in Fig. 42.2A and B.1 Here the periodic crystal planes are shown by straight lines, where centers of electron-density distribution in a hydrocarbon chain are connected with straight lines. It should be noted that the lattice constant is not the distance between the neighboring hydrocarbon chains but the spacing between the neighboring crystal planes. We have to pay attention to one more important result that based upon the wide-angle X-ray diffraction in the hydrocarbon-chain packing structures Doucet et al.6 have estimated the proportion of liquid-crystalline state that exhibits a diffraction pattern of a diffuse ring Cosmetic Science and Technology: Theoretical Principles and Applications http://dx.doi.org/10.1016/B978-0-12-802005-0.00042-2
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FIGURE 42.1 Schematic view for (A) long lamellar structure with lamellar repeat distance 13.6 nm and (B) short lamellar structure with about 6 nm. Both structures are composed of ceramides, free fatty acid, cholesterol, etc. In short lamellar structure water layers appear, where water molecules are indicated by blue dots (Gray in print versions).
FIGURE 42.2 Hydrocarbon-chain packing structures: (A) Hexagonal where lattice constant is 0.42 nm, (B) Orthorhombic where lattice constants are 0.37 and 0.42 nm.
near 4.6 nm and as a result the proportion of liquid-crystalline state reaches about 80%. This fact might be important in considering barrier function and penetration pathway in SC. It should be pointed out that the structure formed by soft keratin is one of the key factors in considering the behavior of water in SC.5 Soft keratin in corneocytes show a diffraction pattern of two very diffuse rings of 4.6 and 1 nm. However, the diffuse ring near 4.6 nm superposes on the diffuse ring due to the liquid-crystalline hydrocarbon-chain packing structure. Therefore it is generally hard to discriminate two broad peaks.6 From this viewpoint the X-ray diffraction measurement for 1 nm diffuse ring is promising in studying behavior of soft keratin. In addition, a variety of structural studies on SC, such as electron microscopy, electron spin resonance, infrared spectroscopy, Raman spectroscopy, atomic force microscopy, etc., have been performed. Here I will focus my attention on in vitro X-ray diffraction measurement in SC, since the basic structural knowledge obtained from X-ray diffraction is indispensable in structural study on SC at the molecular level.
42.2 X-RAY DIFFRACTION STUDY ON STRATUM CORNEUM The X-ray impinges on an SC sample and the X-ray diffraction pattern is recorded on a detector as shown in Fig. 42.3. The scattering vector is given by 2 2q S ¼ sin (42.2) l 2 where 2q is the scattering angle. Scattering angle is obtained from the distance (x) from the center of the detector and the sample-to-detector distance (z) as given by tan2q ¼ x/z. Here I will briefly remark that the definition of scattering vector S is a little different from the usual definition for scattering vector q or Q, which is equal to 2pS. Generally,
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FIGURE 42.3 X-ray diffraction measurement system. X-ray beam impinges on a sample, X-ray is diffracted by the sample, and the diffracted X-ray beams are detected by a detector; where z is camera length and 2q is scattering angle.
when there is a periodic electron-density distribution with periodicity of d, the X-ray diffraction peak takes place due to Bragg’s law. From Eqs. (42.1). and (42.2) the n-th order diffraction peak appears at the scattering vector: 2 2qB n Snth ¼ sin (42.3) ¼ l d 2 When the electron density in an SC sample is modulated periodically in the vertical direction as given in Fig. 42.1A and B, the diffraction peaks take place along the meridional axis. When the images shown in Fig. 42.1A and B are rotated by 90 , the diffraction peaks take place along the equatorial axis. Therefore when the crystal planes of a periodic structure distribute uniformly or randomly around 360 we can observe diffraction rings that are called DebyeeScherrer rings as shown in Fig. 42.3 schematically. The diffraction rings are circular averaged to obtain a radial intensity profile. In SC, in the small-angle diffraction region the diffraction peaks appear at S ¼ 1/ 13.6, 2/13.6, 3/13.6,. nm-1 for the long lamellar structure and at Sw1/6, 2/6,.1/6 for the short lamellar structure. On the other hand, in the wide-angle diffraction region the peaks appear at S ¼ 1/0.42 and 1/0.37 nm1 for the orthorhombic hydrocarbon-chain packing structure and at S ¼ 1/0.42 nm1 for the hexagonal hydrocarbon-chain packing structure.
42.3 HIGHLY SENSITIVE DETECTION OF MINUTE STRUCTURAL CHANGE ON APPLYING CHEMICAL AGENTS Frequently when solution with chemical agents, such as cosmetics and drugs, is applied on SC, we encounter a case to clarify the percutaneous route at the molecular level. For this purpose a sample cell that was used for X-ray diffraction measurement in SC was developed as schematically shown in Fig. 42.4.7 A stratum corneum sample was embedded in a central hollow surrounded by filter paper that was used to sustain the sample. The front and the rear surfaces of the cell were sealed by a pair of polymer thin films. Therefore when solution was applied to a stratum corneum sample, it was always exposed in sufficient solution. The incident X-ray beam impinged through the front surface. As mentioned previously, to obtain the total structural modification on applying solution with chemical agents in SC it is important to observe X-ray diffraction from small- to wide-angle region. It has been pointed out that there exist two potential penetration pathways: one is an intercellular route in which the penetration of chemical agents takes place via the intercellular lipid matrix lying between the corneocytes, and the other is a transcellular route in which the penetration takes place across both the corneocytes and the intercellular lipid matrix.8 However, whether the former is the dominant pathway or they both are equivalently important in the penetration is still controversial. Therefore, it is highly desirable to make clear the structural evidence at the molecular level when the chemical agents are applied to SC. In order to consider this problem further, we performed X-ray diffraction measurement in two kinds of the percutaneous penetration enhancers, hydrophilic and hydrophobic ones. Ethanol is one of the hydrophilic penetration enhancers.9e11 It has been pointed out that ethanol may extract some lipids from SC when it is used for prolonged times.12,13 On the other hand, terpene, for instance D-limonene, is wellknown as a hydrophobic penetration enhancer.11,14e16 So far the effects of penetration enhancers have been studied
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FIGURE 42.4 Sample cell for X-ray diffraction measurement to track structural change of a sample after applying solution. Using this method minute change of the diffraction profiles with time can be detected with high resolution.
by small-angle X-ray diffraction (SAXD) and wide-angle X-ray diffraction (WAXD). Cornwell et al.15 have carried out WAXD in SC to study the effects of terpene such as D-limonene, nerolidol, and 1,8-cineole and found that after 12-h treatment WAXD intensities for the hexagonal and the orthorhombic hydrocarbon-chain packing structures do not change significantly, and on the other hand a broad intensity hump caused by liquid terpenes incorporated into SC takes place. Cornwell et al.16 have performed SAXD in human SC to investigate the effects of D-limonene and 1,8-cineole and found that the intensity for the long lamellar structure decreases but the intensity for the short lamellar structure remains as a shoulder. The same behavior has also been pointed out by Cornwell et al.15 in human SC. By X-ray diffraction the effects of hydrophilic penetration enhancers on SC have been studied for acetone,16 ethanol,7 ethanol and water mixture,17 and also water.3,5 Generally when solution is applied to SC, the structure changes gradually with time. From the detection of the successive X-ray diffraction change we can obtain very minute modification of the structure. From the differences between the successive X-ray diffraction patterns we can distinguish only small changes of the structure. Furthermore, we can overcome problems of the individual differences among SCs since the change of the structure can be detected more or less by this method if any small structural change takes place. Based upon the result obtained from this method, we can get the effect of solutions at the molecular level. In the following discussion I will show typical results for hydrophilic ethanol and hydrophobic D-limonene on applying to SC.
42.4 PENETRATION ROUTE OF HYDROPHILIC MOLECULES IN STRATUM CORNEUM Ethanol is commonly used as one of the transdermal formulations. It is well known that ethanol with water permeates rapidly through skin with a steady-state flux.10 Here we have examined only the effects of pure ethanol. After application of ethanol to SC, as shown in Fig. 42.5A the X-ray diffraction intensity profiles change successively from red (0 s) to blue (7500 s) curves with time in the broad-angle region of S ¼ 0.05e3.0 nm1. In this figure a black curve indicates the X-ray scattering profile for ethanol in arbitral scale. For the SAXD of S ¼ 0.05e0.4 nm1 the intensity profiles are shown in Fig. 42.5B. In the middle angle X-ray diffraction of S ¼ 0.5e1.5 nm1 the intensity difference, which was obtained from the intensity profiles subtracted by the initial intensity profile successively, is shown in Fig. 42.5C. The analysis to use the intensity difference is a predominant point of the present method by which we are able to obtain very small structural modification on applying solution to a single SC sample. In SAXD, the peaks for the long lamellar structure take place at S ¼ 0.074, 0.148, 0.222, and 0.296 nm1 for first-, second-, third-, and fourth-order reflection, respectively, where the repeat distance of the lamellar structure is 13.6 nm. The repeat distance does not change with time. But the intensities decrease with time in superposition on a bigger scattering intensity slope in the smaller angle that increases with time. The growth of this smallerangle scattering intensity might be related to the incorporation of hydrophilic ethanol into corneocytes, since similar behavior has been observed when water is applied to SC.18,19 It is an important point that, although in Fig. 42.5B the peak for the short lamellar structure is weak, the swelling of the short lamellar structure must
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FIGURE 42.5 After applying ethanol on stratum corneum, (A) in the broad-angle region the X-ray diffraction profiles change with time from red (Light gray in print versions) to blue curves (Dark gray in print versions), (B) in the small-angle region the intensities change as illustrated in the same manner, (C) in the middle-angle region the intensity differences change as illustrated in the same manner, and (D) schematic view of transcellular route of hydrophilic molecules predicted based upon the above results.
take place. Because of the X-ray diffraction measurement for application of hydrophilic ethanol and water mixture,17 the peak shift of the short lamellar structure is clearly observable in contrast to unchanged behavior of the long lamellar structure. Furthermore, it is well known that uptake of water in SC yields swelling behavior of the short lamellar structure due to expansion of water layers.3e5 In medium angle X-ray diffraction, the diffraction peak around 1 nm1 (i.e., the lattice spacing: 1 nm) due to soft keratin decreases by applying ethanol as seen in Fig. 42.5A. The intensity difference is derived as in Fig. 42.5C. First of all, prior to consideration of soft keratin I will discuss the baseline, which has nothing to do with anomalous behavior of soft keratin. The baseline is composed of upward shift of an almost-flat curve and growth of the slope in the low-angle side. The former behavior seems to be due to taking up ethanol into SC and the latter due to a part of the slope observed in the SAXD.18,19 By taking into account the contribution of the baselines, we can deduce that the intensity difference for soft keratin exhibits a shallow dip, deepens with time, and slightly shifts low angle, that is, the peak intensity at about 1 nm1 decreases with time and the peak position shifts slightly low angle. This fact indicates that as a result of penetration of ethanol into corneocytes, ethanol partially disrupts the structure of soft keratin in corneocytes. In WAXD, the intensity peaks at 2.42 nm1 (lattice constant: 0.41 nm) and 2.67 nm1 (lattice constant: 0.37 nm), which appear in superposition on a broad diffraction peak around 2.4 nm1 (lattice constant: 0.42 nm), taking place as seen in Fig. 42.5A. The peak positions for the hydrocarbon-chain packing structures do not change with time, but the intensities decrease by a small amount. This might be due to either slight extraction or partial melting of lipids in intercellular matrix. Rise of the broad peak around 2.4 nm1 seems to be caused by uptake of ethanol in SC, because in pure ethanol we could observe a broad diffraction peak of ethanol around 2.4 nm1 as seen in Fig. 42.5A. This might be partly due to formation of pools composed of either ethanol or ethanol and water mixture. Based upon the previous results, it is proposed that when hydrophilic molecules are applied to SC they penetrate via a transcellular route, as shown schematically in Fig. 42.5D. Namely, hydrophilic molecules can penetrate into water layers of the short lamellar structure in intercellular lipid matrix, go partly through corneocytes, and make pools of either hydrophilic molecule or mixtures of water and hydrophilic molecule.
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42.5 PENETRATION ROUTE OF HYDROPHOBIC MOLECULES IN STRATUM CORNEUM Terpene, D-limonene, is a penetration enhancer.11,15,16 After application of D-limonene to the SC, the X-ray diffraction intensity profiles change successively from red (0 s) to blue (7500 s) curves with time as shown in Fig. 42.6A in the broad-angle range of S ¼ 0.05e3.0 nm1. In this figure a black curve indicates the X-ray scattering profile for D-limonene in arbitral scale. As seen in Fig. 42.6A, a broad hump near S ¼ 2.0 nm1 increases with time due to take-up of Dlimonene in SC. This behavior indicates formation of pools composed of D-limonene in SC. For the SAXD of S ¼ 0.05e0.4 nm1, the intensity profiles are shown in Fig. 42.6B. For the middle-angle X-ray diffraction of S ¼ 0.5e1.5 nm1, the difference intensities that were obtained from the intensity profiles subtracted by the initial intensity profile successively are shown with high sensitivity in Fig. 42.6C. On applying D-limonene to SC, the diffraction peak positions for the long lamellar structure in the SAXD shifts toward the lower angle as seen in Fig. 42.6B, that is, the swelling of the long lamellar structure takes place. In this figure, the repeat distance of the long lamellar structure expands from 13.5 nm and saturates near 14.5 nm with a relaxation time of 5000 s. This behavior is consistent with the fact that the hydrophobic molecules penetrate through the narrow band of the long lamellar structure with hydrophobic character during the percutaneous absorption as proposed by Bouwstra and Ponec.20 In the middle-angle X-ray diffraction the diffraction peak around 1 nm1 due to soft keratin does not change with application of D-limonene as seen in Fig. 42.6A. Namely in the intensity difference shown in Fig. 42.6C, except for growth of the slope above 1 nm1 is due to take-up of D-limonene in SC and the intensity difference for soft keratin is unchanged. This fact indicates that hydrophobic D-limonene does not penetrate into corneocytes. On applying D-limonene to SC, the peak positions of the hydrocarbon-chain packing structures do not change with time, but as seen in the WAXD region of Fig. 42.6A the peak intensities decrease and therefore the hydrocarbon-chain packing structures are slightly disrupted, that is, hydrophobic chemicals such as D-limonene are compatible with the hydrophobic and disordered region in intercellular lipid matrix. Based upon the previous results, it is proposed that when hydrophobic molecules such as D-limonene are applied to SC they penetrate via an intercellular route, as shown schematically in Fig. 42.6D. Namely, hydrophobic molecules
FIGURE 42.6 After applying D-limonene on stratum corneum, (A) in the broad-angle region the X-ray diffraction profiles change with time from red (Light gray in print versions) to blue curves (Dark gray in print versions), (B) in the small-angle region the intensities change as illustrated by a similar manner, (C) in the middle-angle region the intensity differences change as illustrated by a similar manner, and (D) schematic view of intercellular route of hydrophobic molecules predicted based upon the above results.
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can penetrate into disordered layers of the long lamellar structure in intercellular lipid matrix and make pools composed of hydrophobic molecules.
42.6 BEHAVIOR OF WATER IN STRATUM CORNEUM The role of water in SC is an important subject and a variety of studies have been performed. Transepidermal water loss and the water concentration gradient are useful indices to characterize the condition of SC. In principle to SC water is supplied from the body constantly and in the normal condition the same amount of water evaporates from the surface of skin. This fact means that the steady-state water permeation in SC occurs in nonequilibrium condition and then the water concentration results in gradient. By in vivo confocal Raman microscopy the water content in SC has been measured as a function of distance from skin surface.21 The water content near the skin surface is estimated to be about 25 wt% and continuously increases with the depth down to viable cell where the water content reaches to about 65 wt%.22 It is of interest to know the water behavior within SC when SC is instantaneously exposed to humid or dry conditions. Egawa and Kajikawa22 have performed in vivo confocal Raman microscopy as follows: When water is applied to skin surface the water distribution near the surface of the SC increases markedly; after a while it returns almost to the original distribution. This fact indicates that under the normal condition the water content of about 25 wt% is kept near the surface of SC, that is, despite varying circumstances, the water content near the skin surface is regulated to be kept in a normal condition. In connection with this fact, it is worthwhile to pay attention to the results obtained by in vitro differential scanning calorimetric measurement (DSC) for various hydrated SC. From DSC the nonfreezing water has been estimated to be about 25 wt%,23,24 where weight% is given by (weight of water incorporated into dried SC) 100/(sum of weights of water and dried SC). These water molecules exist as bound water within SC and might play an important role in keeping the normal water condition in SC. These facts indicate that, needless to say, although in vivo study is very important to know what goes on in a living state, performing in vitro X-ray diffraction study is indispensable since it is possible to make clear the fundamental hydration mechanism at the molecular level.
42.7 WATER REGULATION MECHANISM IN STRATUM CORNEUM AT THE MOLECULAR LEVEL We have carried out a detailed study on the SAXD in hairless mouse SC as a function of water content.7 The SAXD profiles are shown in Fig. 42.7 at water contents of 0, 12, 21, 35, 50, 70, and 80 wt%. The peaks denoted by an open arrow exhibit the first- to fifth-order diffraction peak for 13.6 nm lamellar spacing, and the peaks denoted by closed arrow exhibit the first- and the second-order diffraction peaks for about 6 nm lamellar spacing. As seen in Fig. 42.7,
FIGURE 42.7 Small-angle X-ray diffraction of hairless mouse stratum corneum as a function of water content. The water contents are 0 wt% (A), 12 wt% (B), 21 wt% (C), 35 wt% (D), 50 wt% (E), 70 wt% (F), and 80 wt% (G).
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with increasing water content the diffraction peak positions for the first- to fifth-order diffraction of 13.6 nm are almost unchanged, although the sharpness of these peaks depends on the water content. On the other hand, with increasing the water content the first- and second-order diffraction peaks for about 6 nm markedly shift toward lower angle, i.e., the short lamellar repeat distance becomes larger. This fact indicates the appearance of swelling due to the expansion of the water layers in the short lamellar structure. To elucidate the swelling effects further, we analyzed the shape of the diffraction profiles. For this purpose we focus our attention to the diffraction profile near Sw0.15 nm1, where the first-order diffraction peak of about 6 nm and the second-order diffraction peak of 13.6 nm lie. In Fig. 42.8, as a function of the water content the results on about 6 nm lamellar spacing are drawn together with the spacing of about 6.8 (¼13.6/2) nm that is obtained from the second-order diffraction of the long lamellar structure with the repeat distance of 13.6 nm. The long lamellar spacing is almost unchanged with the water content. On the other hand, the short lamellar spacing grows from 5.8 to 6.6 nm as the water content increases from 12 to 50 wt%, and above 50 wt% the short lamellar diffraction peak becomes small and seems to merge into the second-order diffraction of the long lamellar structure with the spacing of 6.8 nm. In Fig. 42.8B, full width at half maximum of the diffraction profiles for the short and the long lamellar structures are shown as a function of the water content. At about 25 wt%, full width at half maximum becomes narrow not only in the spacing of about 6 nm but also in the spacing of 6.8 (¼13.6/2) nm. To sum up, first, the behavior of the spacing of 6.8 nm for the second-order diffraction peak of the long lamellar structure is consistent with the results previously reported by Bouwstra et al.25 Second, the swelling of the short lamellar structure occurs undoubtedly in our measurement.3 Similar swelling behavior has been observed in the neutron diffraction on human SC.4 Third, at the low water content, both full widths at half maximum broaden markedly, near 25 wt% both lamellar diffractions for the spacings of about 6 nm, and furthermore 6.8 (¼13.6/2) nm become sharp, and at the higher water contents they become broad. As discussed before, the long and the short lamellar structures coexist and therefore form domains. I will consider the correlation between the long and the short lamellar structures. The result of Fig. 42.8B indicates that at a water content of about 25 wt% both lamellar structures are well arranged and below and above the water content become disordered simultaneously. Generally, there is mismatch of the hydrophobic parts at the boundary of the two domains. In the case when there is boundary between hydrocarbon chains of lipid membrane and a hydrophobic part of a membrane protein, mechanical strain due to the mismatch is relaxed by disorder of hydrocarbon chains at the domain boundary.26 If this is the case, I can propose that in intercellular lipids a domain composed of the long lamellar structure faces laterally a couple of the short lamellar structures where the domain boundary is constructed by a hydrophobic interface composed of hydrocarbon chains. At water contents lower than about 25 wt%, the hydrophobic part of the long lamellar structure is longer than twice the thickness of the short lamellar structure and therefore the distortion spreads over the both lamellar structures, i.e., the X-ray diffraction peaks for the long and the short lamellar spacings become broad simultaneously. At the water content of about 25 wt%, the distortion is relaxed and then both X-ray diffraction peaks become sharp. At the higher water content than about 25 wt%, distortion takes place again since the hydrophobic parts of the both lamellar structures cause mismatch, i.e., both X-ray diffraction peaks become broad again. The above aspect might be related to the existence of a disordered
FIGURE 42.8 In hairless mouse stratum corneum, we have obtained (A) spacings of the short lamellar structure at the first-order diffraction peak and the long lamellar structure at the second-order diffraction peak and (B) full width at half maximum for both peaks.
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intermediate region between the neighboring domains composed of the short and the long lamellar structures, although it is difficult to detect only by the X-ray diffraction measurement due to the irregular structures. Then as another role of the disordered region, Forslind27 has proposed that in the domain mosaic model molecule permeation takes place via the disordered region. The previous results indicate that the long and the short lamellar structures interact directly or indirectly with each other, the swelling of the short lamellar structure takes place, and as a result at a water content of about 25 wt% the lamellar structures are stabilized simultaneously. It should be pointed out that in SC almost all water is stored in corneocytes, so-called bricks, that become thick with increase of the water content in SC, but a small part of water comes out to the water layers of the short lamellar structure. Once the thickness of the water layer deviates from the steady-state water thickness, owing to the interaction between the neighboring domains a regulation mechanism to keep the water content of about 25 wt% in SC works so as to bring back to the steady-state thickness. Finally, the water content in the corneocytes is regulated to be kept at about 25 wt% in normal condition. In addition, in this regulation the bound water in corneocytes plays an important role subsidiarily. It is highly desirable to study the short lamellar structure on human SC as a function of the water content. We have obtained the X-ray diffraction in human SC as shown in Fig. 42.9.5 It has been confirmed that swelling of the short lamellar structure takes place and full width at the half maximum for this diffraction profile becomes narrow at the water content of about 25 wt% as shown in Fig. 42.10A and B, respectively, consistent with the results obtained from measurement on the hairless mouse SC.3 Therefore generally the short lamellar structure exhibits the swelling behavior and becomes stable at the water content of about 25 wt%. Furthermore, in human stratum corneum it has been found that soft keratin within corneocytes changes near the water content of about 25 wt% as shown in Fig. 42.11. This fact indicates uptake of water molecules into corneocytes, and the interaction between water and soft keratin changes near 25 wt% is consistent with the fact that until the water content of 25 wt% water molecules form bound water within corneocytes and above 25 wt%, water molecules become free. To sum up the behavior of water in SC is schematically shown in Fig. 42.12. Water molecules are supplied to SC always from viable cells and the same amount of water molecules is released continuously from skin surface.
INTENSITY (arb. units)
3500 3000 2500 2000 1500 1000 500 0 0.1
0.2
0.3
0.4
S (nm-1)
FIGURE 42.9 Small-angle diffraction profiles as a function of water content in human stratum corneum. The water content is 5, 10, 15, 20, 25, 30, 40, and 50 wt% from bottom to top in the profiles.
(B)
6.30
FWHM (nm-1)
SPACING (nm)
(A)
6.20
6.10
6.00
0
10
20
30
40
WATER CONTENT (wt%)
50
60
0.026 0.024
0.022 0.020 0.018
0
10
20
30
40
50
60
WATER CONTENT (wt%)
FIGURE 42.10 In human stratum corneum we have obtained (A) spacings of the short lamellar structure at the first-order diffraction peak and (B) full width at half maximum for this peak.
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SPACING (nm)
1.04
1.00
0.96
0.92
0
10
20
30
40
50
60
WATER CONTENT (wt%)
FIGURE 42.11
In human stratum corneum we have obtained spacing of soft keratin as a function of water content.
Atmosphere
Bound Water
Water Loss
About 25 wt% Water Supply
FIGURE 42.12
Viable Cells
Schematic view of water behavior in stratum corneum at a stationary state.
Therefore SC lies in a stationary state against water. Within SC water molecules exist up to about 25 wt% as bound water, and a large amount of water molecules are in corneocytes. Near viable cell sides there are a lot of unbound water molecules, and the concentration of unbound water molecules decreases with approach to the skin surface. Beyond a water content of about 25 wt% the unbound water molecules seem to lie in corneocytes and also to form water pools in intercellular lipid matrix of SC. In this process permeation of water molecules takes place via a transcellular route as shown in Fig. 42.5D.
References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19.
Hatta I, Ohta N. Photon factory activity report 2003 part A, highlights. 2004. p. 49. Groen D, Gooris GS, Bouwstra JA. Biophys J 2009;97:2242. Ohta N, Ban S, Tanaka H, Nakata S, Hatta I. Chem Phys Lipids 2003;123:1. Charalambopoulou GCh, Steriotis ThA, Hauss Th, Stubos AK, Kanellopoulos NK. Physica B 2004;350:e603. Nakazawa H, Ohta N, Hatta I. Chem Phys Lipids 2012;165:238. Doucet J, Potter A, Baltenneck C, Domanov Y. J lipid Res 2014;55:2380. Hatta I, Nakazawa H, Obata Y, Ohta N, Inoue K, Yagi N. Chem Phys Lipids 2010;163:381. Suhonen TM, Bouwstra JA, Urtti A. J Control Rel 1999;59:149. Kurihara-Bergstrom T, Knutson K, DeNoble LJ, Goates CY. Pharm Res 1990;7:762. Kai T, Mak VHW, Potts RO, Guy RH. J Control Rel 1990;12:103. Williams AC, Barry BW. Adv Drug Delivery Rev 2004;56:603. Golden GM, Guzek DB, Haris RR, McKie JE, Potts RO. J Invest Dermatol 1986;86:255. Garson J-C, Doucet J, Leveque J-L, Tsoucaris G. J Invest Dermatol 1991;96:43. Okabe H, Takayama K, Ogura A, Nagai T. Drug Design Deliv 1988;4:313. Cornwell PA, Barry BW, Stoddart CP, Bouwstra JA. J Pharm Pharmacol 1944;46:938. Cornwell PA, Barry BW, Bouwstra JA, Gooris GS. Intern J Pharm 1996;127:9. Bouwstra JA, de Graaff A, Gooris GS, Wiechers JW, van Aelst AC. J Invest Dermatol 2003;120:750. Charalambopoulou GCh, Steriotis TA, Mitropoulos ACh, Stefanopoulos KL, Ioffe A. J Invest Dermatol 1998;110:988. Horita D, Hatta I, Yoshimoto M, Kitao Y, Todo H, Sugibayashi K. Biochim Biophys Acta 2015;1848:1196.
IV. PHYSIOLOGICAL AND DERMATOLOGICAL ASPECTS
REFERENCES
20. 21. 22. 23. 24. 25. 26. 27.
Bouwstra JA, Ponec M. Biochim Biophys Acta 2006;1758:2080. Caspers PJ, Lucassen GW, A Carter E, Bruining HA, Puppels GJ. J Invet Dermatol 2001;116:434. Egawa M, Kajikawa T. Skin Res Tech 2009;15:242. Walkley K, Invet J. Dermatol 1972;59:225. Inoue T, Tsujii K, Okamoto K, Toda K. J Invet Dermatol 1986;86:689. Bouwstra JA, Gooris GS, van der Spek JA, Bras W. J Invest Dermatol 1991;97:1005. Mouritsen OG, Bloom M. Biophys J 1984;46:141. Forslind B. Acta Derm Venereol 1994;74:1.
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42 Structural Aspects of Stratum Corneum I. Hatta Nagoya Industrial Science Research Institute, Nagoya, Japan
42.1 INTRODUCTION In this chapter, I will focus my attention on microscopic structures formed in stratum corneum (SC). X-ray diffraction technique is a powerful tool to obtain structural evidence at the molecular level when we consider the function of stratum corneum. Generally, when an X-ray beam impinges on an object, part of the beam called a direct beam is transmitted through the object and part is scattered by the object. An object with periodic structure can give rise to diffraction at certain angles; at other scattered angles destructive interference causes the X-ray waves to cancel. In an object with a three-dimensional periodic crystal structure, the atoms or molecules are arranged in parallel layers or crystal planes that reflect X-ray beams as a mirror reflects light. Positive interference between X-rays reflected from the parallel layers or crystal planes occurs at angles, satisfying Bragg’s law: 2d sinð2qB =2Þ ¼ nl
(42.1)
where 2qB is the angle between the incident and the reflected X-ray beams called the Bragg angle, i.e., qB is the angle between the incident X-ray beam and the parallel layers or crystal planes, d is the perpendicular spacing between the parallel layers or crystal planes, l is the wavelength of X-ray, and n is an integer. The wavelength of X-rays is around 0.1 nm. Therefore, according to Bragg’s law the spacing d that is more than 0.2 nm can be detected by X-ray diffraction. We can observe X-ray diffraction when periodic structures take place in SC. Periodic structures formed in intercellular lipids of SC are characterized in terms of two orthogonal lattice spacings: one is the lamellar repeat distances and another is the lattice spacings of the hydrocarbon-chain packing structure (so-called “subcell structure”). From X-ray diffraction for the lamellar structure of hairless mouse SC that exhibits one of the typical X-ray diffraction in mammalian SC, the long lamellar structure with the repeat distance of 13.6 nm and the short lamellar structure with the repeat distance of about 6 nm have been obtained as illustrated schematically in Fig. 42.1A and B, respectively.1 The illustrations show only the presumable molecular arrangement of ceramides, free fatty acids, and cholesterol in SC. But there are a lot of arguments for the molecular arrangements. To solve them it is important to perform the detailed structural study at the molecular level not only on SC but also on SC lipid model system in which the constituent molecules are known. Based upon the structural study on an SC lipid model system, it has been pointed out that in the formation of the long lamellar structure a long ceramide molecule such as CER(EOS) is one of the key elements.2 For the short lamellar structure it is worthwhile pointing out that water molecules are incorporated into the short lamellar structure and therefore that a water layer between the successive lipid bilayers in the short lamellar structure takes place as shown in Fig. 42.1B.3e5 On the other hand, for the lateral packing of the lipids there are hexagonal and orthorhombic hydrocarbon-chain packing structures with the lattice constant of 0.42 nm and with the lattice constants of 0.42 and 0.37 nm, respectively, at room temperature as illustrated schematically in Fig. 42.2A and B.1 Here the periodic crystal planes are shown by straight lines, where centers of electron-density distribution in a hydrocarbon chain are connected with straight lines. It should be noted that the lattice constant is not the distance between the neighboring hydrocarbon chains but the spacing between the neighboring crystal planes. We have to pay attention to one more important result that based upon the wide-angle X-ray diffraction in the hydrocarbon-chain packing structures Doucet et al.6 have estimated the proportion of liquid-crystalline state that exhibits a diffraction pattern of a diffuse ring Cosmetic Science and Technology: Theoretical Principles and Applications http://dx.doi.org/10.1016/B978-0-12-802005-0.00042-2
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FIGURE 42.1 Schematic view for (A) long lamellar structure with lamellar repeat distance 13.6 nm and (B) short lamellar structure with about 6 nm. Both structures are composed of ceramides, free fatty acid, cholesterol, etc. In short lamellar structure water layers appear, where water molecules are indicated by blue dots (Gray in print versions).
FIGURE 42.2 Hydrocarbon-chain packing structures: (A) Hexagonal where lattice constant is 0.42 nm, (B) Orthorhombic where lattice constants are 0.37 and 0.42 nm.
near 4.6 nm and as a result the proportion of liquid-crystalline state reaches about 80%. This fact might be important in considering barrier function and penetration pathway in SC. It should be pointed out that the structure formed by soft keratin is one of the key factors in considering the behavior of water in SC.5 Soft keratin in corneocytes show a diffraction pattern of two very diffuse rings of 4.6 and 1 nm. However, the diffuse ring near 4.6 nm superposes on the diffuse ring due to the liquid-crystalline hydrocarbon-chain packing structure. Therefore it is generally hard to discriminate two broad peaks.6 From this viewpoint the X-ray diffraction measurement for 1 nm diffuse ring is promising in studying behavior of soft keratin. In addition, a variety of structural studies on SC, such as electron microscopy, electron spin resonance, infrared spectroscopy, Raman spectroscopy, atomic force microscopy, etc., have been performed. Here I will focus my attention on in vitro X-ray diffraction measurement in SC, since the basic structural knowledge obtained from X-ray diffraction is indispensable in structural study on SC at the molecular level.
42.2 X-RAY DIFFRACTION STUDY ON STRATUM CORNEUM The X-ray impinges on an SC sample and the X-ray diffraction pattern is recorded on a detector as shown in Fig. 42.3. The scattering vector is given by 2 2q S ¼ sin (42.2) l 2 where 2q is the scattering angle. Scattering angle is obtained from the distance (x) from the center of the detector and the sample-to-detector distance (z) as given by tan2q ¼ x/z. Here I will briefly remark that the definition of scattering vector S is a little different from the usual definition for scattering vector q or Q, which is equal to 2pS. Generally,
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FIGURE 42.3 X-ray diffraction measurement system. X-ray beam impinges on a sample, X-ray is diffracted by the sample, and the diffracted X-ray beams are detected by a detector; where z is camera length and 2q is scattering angle.
when there is a periodic electron-density distribution with periodicity of d, the X-ray diffraction peak takes place due to Bragg’s law. From Eqs. (42.1). and (42.2) the n-th order diffraction peak appears at the scattering vector: 2 2qB n Snth ¼ sin (42.3) ¼ l d 2 When the electron density in an SC sample is modulated periodically in the vertical direction as given in Fig. 42.1A and B, the diffraction peaks take place along the meridional axis. When the images shown in Fig. 42.1A and B are rotated by 90 , the diffraction peaks take place along the equatorial axis. Therefore when the crystal planes of a periodic structure distribute uniformly or randomly around 360 we can observe diffraction rings that are called DebyeeScherrer rings as shown in Fig. 42.3 schematically. The diffraction rings are circular averaged to obtain a radial intensity profile. In SC, in the small-angle diffraction region the diffraction peaks appear at S ¼ 1/ 13.6, 2/13.6, 3/13.6,. nm-1 for the long lamellar structure and at Sw1/6, 2/6,.1/6 for the short lamellar structure. On the other hand, in the wide-angle diffraction region the peaks appear at S ¼ 1/0.42 and 1/0.37 nm1 for the orthorhombic hydrocarbon-chain packing structure and at S ¼ 1/0.42 nm1 for the hexagonal hydrocarbon-chain packing structure.
42.3 HIGHLY SENSITIVE DETECTION OF MINUTE STRUCTURAL CHANGE ON APPLYING CHEMICAL AGENTS Frequently when solution with chemical agents, such as cosmetics and drugs, is applied on SC, we encounter a case to clarify the percutaneous route at the molecular level. For this purpose a sample cell that was used for X-ray diffraction measurement in SC was developed as schematically shown in Fig. 42.4.7 A stratum corneum sample was embedded in a central hollow surrounded by filter paper that was used to sustain the sample. The front and the rear surfaces of the cell were sealed by a pair of polymer thin films. Therefore when solution was applied to a stratum corneum sample, it was always exposed in sufficient solution. The incident X-ray beam impinged through the front surface. As mentioned previously, to obtain the total structural modification on applying solution with chemical agents in SC it is important to observe X-ray diffraction from small- to wide-angle region. It has been pointed out that there exist two potential penetration pathways: one is an intercellular route in which the penetration of chemical agents takes place via the intercellular lipid matrix lying between the corneocytes, and the other is a transcellular route in which the penetration takes place across both the corneocytes and the intercellular lipid matrix.8 However, whether the former is the dominant pathway or they both are equivalently important in the penetration is still controversial. Therefore, it is highly desirable to make clear the structural evidence at the molecular level when the chemical agents are applied to SC. In order to consider this problem further, we performed X-ray diffraction measurement in two kinds of the percutaneous penetration enhancers, hydrophilic and hydrophobic ones. Ethanol is one of the hydrophilic penetration enhancers.9e11 It has been pointed out that ethanol may extract some lipids from SC when it is used for prolonged times.12,13 On the other hand, terpene, for instance D-limonene, is wellknown as a hydrophobic penetration enhancer.11,14e16 So far the effects of penetration enhancers have been studied
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FIGURE 42.4 Sample cell for X-ray diffraction measurement to track structural change of a sample after applying solution. Using this method minute change of the diffraction profiles with time can be detected with high resolution.
by small-angle X-ray diffraction (SAXD) and wide-angle X-ray diffraction (WAXD). Cornwell et al.15 have carried out WAXD in SC to study the effects of terpene such as D-limonene, nerolidol, and 1,8-cineole and found that after 12-h treatment WAXD intensities for the hexagonal and the orthorhombic hydrocarbon-chain packing structures do not change significantly, and on the other hand a broad intensity hump caused by liquid terpenes incorporated into SC takes place. Cornwell et al.16 have performed SAXD in human SC to investigate the effects of D-limonene and 1,8-cineole and found that the intensity for the long lamellar structure decreases but the intensity for the short lamellar structure remains as a shoulder. The same behavior has also been pointed out by Cornwell et al.15 in human SC. By X-ray diffraction the effects of hydrophilic penetration enhancers on SC have been studied for acetone,16 ethanol,7 ethanol and water mixture,17 and also water.3,5 Generally when solution is applied to SC, the structure changes gradually with time. From the detection of the successive X-ray diffraction change we can obtain very minute modification of the structure. From the differences between the successive X-ray diffraction patterns we can distinguish only small changes of the structure. Furthermore, we can overcome problems of the individual differences among SCs since the change of the structure can be detected more or less by this method if any small structural change takes place. Based upon the result obtained from this method, we can get the effect of solutions at the molecular level. In the following discussion I will show typical results for hydrophilic ethanol and hydrophobic D-limonene on applying to SC.
42.4 PENETRATION ROUTE OF HYDROPHILIC MOLECULES IN STRATUM CORNEUM Ethanol is commonly used as one of the transdermal formulations. It is well known that ethanol with water permeates rapidly through skin with a steady-state flux.10 Here we have examined only the effects of pure ethanol. After application of ethanol to SC, as shown in Fig. 42.5A the X-ray diffraction intensity profiles change successively from red (0 s) to blue (7500 s) curves with time in the broad-angle region of S ¼ 0.05e3.0 nm1. In this figure a black curve indicates the X-ray scattering profile for ethanol in arbitral scale. For the SAXD of S ¼ 0.05e0.4 nm1 the intensity profiles are shown in Fig. 42.5B. In the middle angle X-ray diffraction of S ¼ 0.5e1.5 nm1 the intensity difference, which was obtained from the intensity profiles subtracted by the initial intensity profile successively, is shown in Fig. 42.5C. The analysis to use the intensity difference is a predominant point of the present method by which we are able to obtain very small structural modification on applying solution to a single SC sample. In SAXD, the peaks for the long lamellar structure take place at S ¼ 0.074, 0.148, 0.222, and 0.296 nm1 for first-, second-, third-, and fourth-order reflection, respectively, where the repeat distance of the lamellar structure is 13.6 nm. The repeat distance does not change with time. But the intensities decrease with time in superposition on a bigger scattering intensity slope in the smaller angle that increases with time. The growth of this smallerangle scattering intensity might be related to the incorporation of hydrophilic ethanol into corneocytes, since similar behavior has been observed when water is applied to SC.18,19 It is an important point that, although in Fig. 42.5B the peak for the short lamellar structure is weak, the swelling of the short lamellar structure must
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FIGURE 42.5 After applying ethanol on stratum corneum, (A) in the broad-angle region the X-ray diffraction profiles change with time from red (Light gray in print versions) to blue curves (Dark gray in print versions), (B) in the small-angle region the intensities change as illustrated in the same manner, (C) in the middle-angle region the intensity differences change as illustrated in the same manner, and (D) schematic view of transcellular route of hydrophilic molecules predicted based upon the above results.
take place. Because of the X-ray diffraction measurement for application of hydrophilic ethanol and water mixture,17 the peak shift of the short lamellar structure is clearly observable in contrast to unchanged behavior of the long lamellar structure. Furthermore, it is well known that uptake of water in SC yields swelling behavior of the short lamellar structure due to expansion of water layers.3e5 In medium angle X-ray diffraction, the diffraction peak around 1 nm1 (i.e., the lattice spacing: 1 nm) due to soft keratin decreases by applying ethanol as seen in Fig. 42.5A. The intensity difference is derived as in Fig. 42.5C. First of all, prior to consideration of soft keratin I will discuss the baseline, which has nothing to do with anomalous behavior of soft keratin. The baseline is composed of upward shift of an almost-flat curve and growth of the slope in the low-angle side. The former behavior seems to be due to taking up ethanol into SC and the latter due to a part of the slope observed in the SAXD.18,19 By taking into account the contribution of the baselines, we can deduce that the intensity difference for soft keratin exhibits a shallow dip, deepens with time, and slightly shifts low angle, that is, the peak intensity at about 1 nm1 decreases with time and the peak position shifts slightly low angle. This fact indicates that as a result of penetration of ethanol into corneocytes, ethanol partially disrupts the structure of soft keratin in corneocytes. In WAXD, the intensity peaks at 2.42 nm1 (lattice constant: 0.41 nm) and 2.67 nm1 (lattice constant: 0.37 nm), which appear in superposition on a broad diffraction peak around 2.4 nm1 (lattice constant: 0.42 nm), taking place as seen in Fig. 42.5A. The peak positions for the hydrocarbon-chain packing structures do not change with time, but the intensities decrease by a small amount. This might be due to either slight extraction or partial melting of lipids in intercellular matrix. Rise of the broad peak around 2.4 nm1 seems to be caused by uptake of ethanol in SC, because in pure ethanol we could observe a broad diffraction peak of ethanol around 2.4 nm1 as seen in Fig. 42.5A. This might be partly due to formation of pools composed of either ethanol or ethanol and water mixture. Based upon the previous results, it is proposed that when hydrophilic molecules are applied to SC they penetrate via a transcellular route, as shown schematically in Fig. 42.5D. Namely, hydrophilic molecules can penetrate into water layers of the short lamellar structure in intercellular lipid matrix, go partly through corneocytes, and make pools of either hydrophilic molecule or mixtures of water and hydrophilic molecule.
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42.5 PENETRATION ROUTE OF HYDROPHOBIC MOLECULES IN STRATUM CORNEUM Terpene, D-limonene, is a penetration enhancer.11,15,16 After application of D-limonene to the SC, the X-ray diffraction intensity profiles change successively from red (0 s) to blue (7500 s) curves with time as shown in Fig. 42.6A in the broad-angle range of S ¼ 0.05e3.0 nm1. In this figure a black curve indicates the X-ray scattering profile for D-limonene in arbitral scale. As seen in Fig. 42.6A, a broad hump near S ¼ 2.0 nm1 increases with time due to take-up of Dlimonene in SC. This behavior indicates formation of pools composed of D-limonene in SC. For the SAXD of S ¼ 0.05e0.4 nm1, the intensity profiles are shown in Fig. 42.6B. For the middle-angle X-ray diffraction of S ¼ 0.5e1.5 nm1, the difference intensities that were obtained from the intensity profiles subtracted by the initial intensity profile successively are shown with high sensitivity in Fig. 42.6C. On applying D-limonene to SC, the diffraction peak positions for the long lamellar structure in the SAXD shifts toward the lower angle as seen in Fig. 42.6B, that is, the swelling of the long lamellar structure takes place. In this figure, the repeat distance of the long lamellar structure expands from 13.5 nm and saturates near 14.5 nm with a relaxation time of 5000 s. This behavior is consistent with the fact that the hydrophobic molecules penetrate through the narrow band of the long lamellar structure with hydrophobic character during the percutaneous absorption as proposed by Bouwstra and Ponec.20 In the middle-angle X-ray diffraction the diffraction peak around 1 nm1 due to soft keratin does not change with application of D-limonene as seen in Fig. 42.6A. Namely in the intensity difference shown in Fig. 42.6C, except for growth of the slope above 1 nm1 is due to take-up of D-limonene in SC and the intensity difference for soft keratin is unchanged. This fact indicates that hydrophobic D-limonene does not penetrate into corneocytes. On applying D-limonene to SC, the peak positions of the hydrocarbon-chain packing structures do not change with time, but as seen in the WAXD region of Fig. 42.6A the peak intensities decrease and therefore the hydrocarbon-chain packing structures are slightly disrupted, that is, hydrophobic chemicals such as D-limonene are compatible with the hydrophobic and disordered region in intercellular lipid matrix. Based upon the previous results, it is proposed that when hydrophobic molecules such as D-limonene are applied to SC they penetrate via an intercellular route, as shown schematically in Fig. 42.6D. Namely, hydrophobic molecules
FIGURE 42.6 After applying D-limonene on stratum corneum, (A) in the broad-angle region the X-ray diffraction profiles change with time from red (Light gray in print versions) to blue curves (Dark gray in print versions), (B) in the small-angle region the intensities change as illustrated by a similar manner, (C) in the middle-angle region the intensity differences change as illustrated by a similar manner, and (D) schematic view of intercellular route of hydrophobic molecules predicted based upon the above results.
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can penetrate into disordered layers of the long lamellar structure in intercellular lipid matrix and make pools composed of hydrophobic molecules.
42.6 BEHAVIOR OF WATER IN STRATUM CORNEUM The role of water in SC is an important subject and a variety of studies have been performed. Transepidermal water loss and the water concentration gradient are useful indices to characterize the condition of SC. In principle to SC water is supplied from the body constantly and in the normal condition the same amount of water evaporates from the surface of skin. This fact means that the steady-state water permeation in SC occurs in nonequilibrium condition and then the water concentration results in gradient. By in vivo confocal Raman microscopy the water content in SC has been measured as a function of distance from skin surface.21 The water content near the skin surface is estimated to be about 25 wt% and continuously increases with the depth down to viable cell where the water content reaches to about 65 wt%.22 It is of interest to know the water behavior within SC when SC is instantaneously exposed to humid or dry conditions. Egawa and Kajikawa22 have performed in vivo confocal Raman microscopy as follows: When water is applied to skin surface the water distribution near the surface of the SC increases markedly; after a while it returns almost to the original distribution. This fact indicates that under the normal condition the water content of about 25 wt% is kept near the surface of SC, that is, despite varying circumstances, the water content near the skin surface is regulated to be kept in a normal condition. In connection with this fact, it is worthwhile to pay attention to the results obtained by in vitro differential scanning calorimetric measurement (DSC) for various hydrated SC. From DSC the nonfreezing water has been estimated to be about 25 wt%,23,24 where weight% is given by (weight of water incorporated into dried SC) 100/(sum of weights of water and dried SC). These water molecules exist as bound water within SC and might play an important role in keeping the normal water condition in SC. These facts indicate that, needless to say, although in vivo study is very important to know what goes on in a living state, performing in vitro X-ray diffraction study is indispensable since it is possible to make clear the fundamental hydration mechanism at the molecular level.
42.7 WATER REGULATION MECHANISM IN STRATUM CORNEUM AT THE MOLECULAR LEVEL We have carried out a detailed study on the SAXD in hairless mouse SC as a function of water content.7 The SAXD profiles are shown in Fig. 42.7 at water contents of 0, 12, 21, 35, 50, 70, and 80 wt%. The peaks denoted by an open arrow exhibit the first- to fifth-order diffraction peak for 13.6 nm lamellar spacing, and the peaks denoted by closed arrow exhibit the first- and the second-order diffraction peaks for about 6 nm lamellar spacing. As seen in Fig. 42.7,
FIGURE 42.7 Small-angle X-ray diffraction of hairless mouse stratum corneum as a function of water content. The water contents are 0 wt% (A), 12 wt% (B), 21 wt% (C), 35 wt% (D), 50 wt% (E), 70 wt% (F), and 80 wt% (G).
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with increasing water content the diffraction peak positions for the first- to fifth-order diffraction of 13.6 nm are almost unchanged, although the sharpness of these peaks depends on the water content. On the other hand, with increasing the water content the first- and second-order diffraction peaks for about 6 nm markedly shift toward lower angle, i.e., the short lamellar repeat distance becomes larger. This fact indicates the appearance of swelling due to the expansion of the water layers in the short lamellar structure. To elucidate the swelling effects further, we analyzed the shape of the diffraction profiles. For this purpose we focus our attention to the diffraction profile near Sw0.15 nm1, where the first-order diffraction peak of about 6 nm and the second-order diffraction peak of 13.6 nm lie. In Fig. 42.8, as a function of the water content the results on about 6 nm lamellar spacing are drawn together with the spacing of about 6.8 (¼13.6/2) nm that is obtained from the second-order diffraction of the long lamellar structure with the repeat distance of 13.6 nm. The long lamellar spacing is almost unchanged with the water content. On the other hand, the short lamellar spacing grows from 5.8 to 6.6 nm as the water content increases from 12 to 50 wt%, and above 50 wt% the short lamellar diffraction peak becomes small and seems to merge into the second-order diffraction of the long lamellar structure with the spacing of 6.8 nm. In Fig. 42.8B, full width at half maximum of the diffraction profiles for the short and the long lamellar structures are shown as a function of the water content. At about 25 wt%, full width at half maximum becomes narrow not only in the spacing of about 6 nm but also in the spacing of 6.8 (¼13.6/2) nm. To sum up, first, the behavior of the spacing of 6.8 nm for the second-order diffraction peak of the long lamellar structure is consistent with the results previously reported by Bouwstra et al.25 Second, the swelling of the short lamellar structure occurs undoubtedly in our measurement.3 Similar swelling behavior has been observed in the neutron diffraction on human SC.4 Third, at the low water content, both full widths at half maximum broaden markedly, near 25 wt% both lamellar diffractions for the spacings of about 6 nm, and furthermore 6.8 (¼13.6/2) nm become sharp, and at the higher water contents they become broad. As discussed before, the long and the short lamellar structures coexist and therefore form domains. I will consider the correlation between the long and the short lamellar structures. The result of Fig. 42.8B indicates that at a water content of about 25 wt% both lamellar structures are well arranged and below and above the water content become disordered simultaneously. Generally, there is mismatch of the hydrophobic parts at the boundary of the two domains. In the case when there is boundary between hydrocarbon chains of lipid membrane and a hydrophobic part of a membrane protein, mechanical strain due to the mismatch is relaxed by disorder of hydrocarbon chains at the domain boundary.26 If this is the case, I can propose that in intercellular lipids a domain composed of the long lamellar structure faces laterally a couple of the short lamellar structures where the domain boundary is constructed by a hydrophobic interface composed of hydrocarbon chains. At water contents lower than about 25 wt%, the hydrophobic part of the long lamellar structure is longer than twice the thickness of the short lamellar structure and therefore the distortion spreads over the both lamellar structures, i.e., the X-ray diffraction peaks for the long and the short lamellar spacings become broad simultaneously. At the water content of about 25 wt%, the distortion is relaxed and then both X-ray diffraction peaks become sharp. At the higher water content than about 25 wt%, distortion takes place again since the hydrophobic parts of the both lamellar structures cause mismatch, i.e., both X-ray diffraction peaks become broad again. The above aspect might be related to the existence of a disordered
FIGURE 42.8 In hairless mouse stratum corneum, we have obtained (A) spacings of the short lamellar structure at the first-order diffraction peak and the long lamellar structure at the second-order diffraction peak and (B) full width at half maximum for both peaks.
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intermediate region between the neighboring domains composed of the short and the long lamellar structures, although it is difficult to detect only by the X-ray diffraction measurement due to the irregular structures. Then as another role of the disordered region, Forslind27 has proposed that in the domain mosaic model molecule permeation takes place via the disordered region. The previous results indicate that the long and the short lamellar structures interact directly or indirectly with each other, the swelling of the short lamellar structure takes place, and as a result at a water content of about 25 wt% the lamellar structures are stabilized simultaneously. It should be pointed out that in SC almost all water is stored in corneocytes, so-called bricks, that become thick with increase of the water content in SC, but a small part of water comes out to the water layers of the short lamellar structure. Once the thickness of the water layer deviates from the steady-state water thickness, owing to the interaction between the neighboring domains a regulation mechanism to keep the water content of about 25 wt% in SC works so as to bring back to the steady-state thickness. Finally, the water content in the corneocytes is regulated to be kept at about 25 wt% in normal condition. In addition, in this regulation the bound water in corneocytes plays an important role subsidiarily. It is highly desirable to study the short lamellar structure on human SC as a function of the water content. We have obtained the X-ray diffraction in human SC as shown in Fig. 42.9.5 It has been confirmed that swelling of the short lamellar structure takes place and full width at the half maximum for this diffraction profile becomes narrow at the water content of about 25 wt% as shown in Fig. 42.10A and B, respectively, consistent with the results obtained from measurement on the hairless mouse SC.3 Therefore generally the short lamellar structure exhibits the swelling behavior and becomes stable at the water content of about 25 wt%. Furthermore, in human stratum corneum it has been found that soft keratin within corneocytes changes near the water content of about 25 wt% as shown in Fig. 42.11. This fact indicates uptake of water molecules into corneocytes, and the interaction between water and soft keratin changes near 25 wt% is consistent with the fact that until the water content of 25 wt% water molecules form bound water within corneocytes and above 25 wt%, water molecules become free. To sum up the behavior of water in SC is schematically shown in Fig. 42.12. Water molecules are supplied to SC always from viable cells and the same amount of water molecules is released continuously from skin surface.
INTENSITY (arb. units)
3500 3000 2500 2000 1500 1000 500 0 0.1
0.2
0.3
0.4
S (nm-1)
FIGURE 42.9 Small-angle diffraction profiles as a function of water content in human stratum corneum. The water content is 5, 10, 15, 20, 25, 30, 40, and 50 wt% from bottom to top in the profiles.
(B)
6.30
FWHM (nm-1)
SPACING (nm)
(A)
6.20
6.10
6.00
0
10
20
30
40
WATER CONTENT (wt%)
50
60
0.026 0.024
0.022 0.020 0.018
0
10
20
30
40
50
60
WATER CONTENT (wt%)
FIGURE 42.10 In human stratum corneum we have obtained (A) spacings of the short lamellar structure at the first-order diffraction peak and (B) full width at half maximum for this peak.
IV. PHYSIOLOGICAL AND DERMATOLOGICAL ASPECTS
708
42. STRUCTURAL ASPECTS OF STRATUM CORNEUM
SPACING (nm)
1.04
1.00
0.96
0.92
0
10
20
30
40
50
60
WATER CONTENT (wt%)
FIGURE 42.11
In human stratum corneum we have obtained spacing of soft keratin as a function of water content.
Atmosphere
Bound Water
Water Loss
About 25 wt% Water Supply
FIGURE 42.12
Viable Cells
Schematic view of water behavior in stratum corneum at a stationary state.
Therefore SC lies in a stationary state against water. Within SC water molecules exist up to about 25 wt% as bound water, and a large amount of water molecules are in corneocytes. Near viable cell sides there are a lot of unbound water molecules, and the concentration of unbound water molecules decreases with approach to the skin surface. Beyond a water content of about 25 wt% the unbound water molecules seem to lie in corneocytes and also to form water pools in intercellular lipid matrix of SC. In this process permeation of water molecules takes place via a transcellular route as shown in Fig. 42.5D.
References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19.
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