Measuring microelastic properties of stratum corneum

Colloids and Surfaces B: Biointerfaces 48 (2006) 6–12

Measuring microelastic properties of stratum corneum Yonghui Yuan ∗ , Ritu Verma Unilever Research and Development, US, Edgewater, NJ 07020, USA Received 7 January 2004; accepted 14 December 2005 Available online 3 February 2006

Abstract We explore the compression moduli of a thin biological tissue through probe microscopy. The elastic modulus (E ) of isolated stratum corneum is measured at varying depths through the use of an atomic force microscope (AFM) as well as a nano-indentor (Hysitron Triboscope). In addition, a nano-DMA is used to measure visco-elastic properties. Measurements on dry and hydrated stratum corneum show an order of magnitude difference in E and the measured tan δ (E /E ) is seen to increase from ∼0.1 to 0.25. In addition, extensive validation of the experiments is conducted with different indentation probes at different force ranges to reveal the effects of indentor geometry and indentation depth on the measured elastic modulus. The sensitivity of the measurements is tested with applying known treatments to stratum corneum and exploring their effects on biomechanical parameters. © 2006 Elsevier B.V. All rights reserved. Keywords: Stratum corenum; Nano-indentation; AFM; Visco-elastic; Mechanical properties

1. Introduction Human skin is a layered composite biomaterial which protects our body from harsh environmental factors. The outermost layer of skin, the stratum corneum (SC), is only 20–40 ␮m thick and is composed of dead cells (corneocytes, which are 20–40 ␮m in diameter and about 0.5 ␮m thick) imbedded in an extracellular lipid matrix. This unique microstructure organization protects the aqueous environment in our bodies by preventing water loss. It protects body against mechanical, chemical and microbiological insults, and acts as the contact surface in tactile perception. The biomechanical properties of this outer layer are ultimately responsible in transmitting stresses and strains to the mechano-receptors that provide the neural coding for tactile perception. In addition, mechanical perturbations to the SC are also used to signal metabolic processes to preserve barrier function. Furthermore, a better understanding of SC tissue mechanics has wide ranging applications in artificial skin models for wound healing, medicine and cosmetics. In this paper we quantitatively measure the mechanical properties of stra-

∗

Corresponding author. Present address: Wyeth Research, 401 N. Middletown Rd., Pearl River, NY 10965, USA. Tel.: +1 845 602 3304; fax: +1 845 602 5585. E-mail address: [email protected] (Y. Yuan). 0927-7765/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfb.2005.12.013

tum corneum and explore the changes that occur under different material treatments. Previous investigations into skin mechanics are clustered around macroscopic measurements using clinical instruments such as the ballistometer and the cutometer, etc. [1]. These techniques are primarily macroscopic in nature and extract properties of skin in its entirety. They also rely on models to extract information about the SC layer. The studies that focus on SC layer mechanics can be broadly divided into two categories: investigations of mechanical properties in plane and out of plane. Most investigations have concentrated on in plane properties where the extensional properties of in vitro SC has been measured using techniques such as the linear extensometer [2] and dynamic mechanical spectroscopy [3–5]. However, recent investigations by Wu et al. [6] have measured the fracture properties of SC in the direction normal to the skin surface. All these studies still probe SC mechanical properties on a macroscopic length scale. There are no published studies of the compression modulus of SC possibly due to its limited thickness (20–40 ␮m). The compression force is easily transferred from the thin SC to the underlying substrate, which can cause the substrate to yield. This makes interpretation of the data more complicated. The focus of this investigation is to understand the mechanical properties of SC in the normal direction to the skin surface at the sub-micron length scales. Recent advances in probe microscopy techniques

Y. Yuan, R. Verma / Colloids and Surfaces B: Biointerfaces 48 (2006) 6–12

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have shown that thin film properties [7] can be successfully measured avoiding the complications of substrate coupling effects. Here, we present the first micro-scale study of SC mechanical properties utilizing these techniques. We investigate the changes in mechanical properties of the first few layers of SC under both quasi-static and dynamic conditions. The quasi-static tests were conducted by nano-indentation using an atomic force microscope (AFM) and a nano-indentor. Dynamic studies were carried out by a nano-dynamic mechanical analyzer (DMA). In addition, effects of hydration on SC mechanical properties are also considered in these experiments. We used porcine SC as our test material because of its similarity to human SC and its easy availability. Fig. 1. Typical AFM force curve on dry SC measured by Si3 N4 tip.

2. Materials and methods

√ 4E R 3/2 δ 3(1 − ν2 )

2.1. Stratum corneum preparation

F=

In our studies we use porcine SC isolated from the underlying tissue by trypsin treatment. The isolation technique is described in detail by Golder et al. [8]. Isolated stratum corneum (2 mm × 2 mm) was attached to a glass slide with double-sided tape and care was taken to maintain the SC orientation so that the outside surface of skin faces up. Measurements were also carried out on SC bonded to glass slide by epoxy and crazy glue to verify that the bonding layer did not affect the measured SC parameters. The measurements on isolated SC were performed under ambient conditions for the AFM and nano-indentor quasi-static measurements. Hydrated samples were prepared by soaking isolated stratum corneum in deionized water for 15 min, and then patting it dry with a Kimwipe tissue. The samples were measured immediately after preparation. Sample preparation for nanoDMA studies were similar, however, hydrated SC was presoaked in deionized water for ∼1 h and then loaded on an imaging chamber (Covelwell, Sigma, St. Louis, MR) for measurements.

In Eqs. (1) and (2), E is the elastic modulus, δ the indentation depth, R the radius of the indentation probe, α the half opening angle of the cone shaped indentor (we used 18 ◦ based on the specifications of the manufacture) and ν is the Poisson ratio (we assume ν = 0.5 in our calculations). Here, we use the conical probe equation Eq. (1) to approximate force between the AFM tip and the SC surface. It is easy to rewrite Eq. (1) as demonstrated by Domke and Radmacher [7]. Using the fact that F = kd = k(z − δ) and δ = z − d, where k is the cantilever spring constant, z the position of the AFM tip and d is the deflection of the cantilever, Eq. (1) can be rewritten as
k(d − d0 ) z − z0 = d − d0 + (3) 2/π(E/1 − ν2 ) tan(α)

2.2. AFM measurements A Dimension 3100 (Digital Instruments, Santa Barbara, CA) atomic force microscope was used to conduct force measurements. Several cantilever and tip geometries were used to mechanically stimulate the samples. In each case the tip approach and pull back frequency was maintained at 1 Hz. Each sample was probed at several different sites (5–10) and the data presented is an average of these measurements. Contact mode imaging and force maps were also generated on SC samples to probe the modulus variability over a region. The measured deflection curves are fit to a standard Hertzian model to extract the elastic modulus of the material. The geometric factors were modified to accommodate the different tip shapes. The Hertzian model for the force between a conical tip and a flat surface is given by F=

2 E δ2 tan α π 1 − ν2

(1)

whereas the force between a tip and a spherical indentor is given by

(2)

In this equation, E, z0 and d0 are three unknown parameters. AFM force measurements generally yield cantilever deflection as a function of the tip position, as shown in Fig. 1. When the measured retraction force curves region A and B in Fig. 1 are fitted to Eq. (3), the unknown parameters E, z0 and d0 can be obtained. The lower deflection limit B for fitting is determined by using the range of data that can give positive fitting parameters. The upper deflection limit A is chosen to be the highest deflection point. 2.3. Hysitron Triboscope measurements The Hysitron Triboscope (Hysitron Incorporated, Minneapolis, MN) was used in two modes: (a) the quasi-static mode and the (b) nano-DMA mode. In the quasi-static measurement we explored indentations with a Berkovich tip (pyramidal with a half angle of 65.35◦ and radius of curvature 100–200 nm) and in the nano-DMA mode we used a liquid tip with a spherical probe with a diameter of 100 ␮m. 2.3.1. Quasi-static measurements The operating mechanism of the quasi-static mode on the Hysitron Triboscope is similar to AFM indentation. The applied load, P, and the penetration depth, h, are constantly monitored by

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The solution to this differential equation is given by Fo

X0 = 

(6)

2

(k − mω2 ) + ((Ci + Cs )ω)2

where the subscripts i and s refer to indenter and substrate properties, respectively. The phase difference between the force and the displacement is given by φ = tan−1

Fig. 2. Typical force vs. penetration depth data obtained by Hysitron nanoindenter.

the transducer. A typical load–penetration depth curve is shown in Fig. 2. The sample hardness and elastic modulus are calculated using the technique described by Oliver and Pharr [9]. In this method, the reduced modulus, which is defined by √ ks π Er = √ (4) 2 Ac ks is the unloading stiffness, (dP/dh), and Ac is the projected contact area, is first obtained from the measured data. The modulus of the test material can be calculated from the following relationship, 1/Er = (1 − ν12 )/Ei + (1 − ν22 )/Es , where the subscript i corresponds to the indenter material, the subscript s refers to the indented material. The Poisson ratio ν1 and ν2 are assumed to be 0.5 for both the substrate and the indentor materials. Because in our system the indentor modulus is much higher than the material modulus (105 MPa versus 102 MPa), the reduced modulus Er is approximately equal to the material modulus Ei . This was validated by subsequent SC measurements presented later. In Eq. (4), the unloading stiffness ks is calculated by fitting the unloading curve to the power law relation, P = B(h − hf )m , where B, hf and m are arbitrary fitting parameters. The stiffness can be calculated from the derivative of the preceding equation at the highest indentation depth hmax : ks = dP/dh(hmax ) = mA(hmax − hf )m−1 . The contact area is determined from a tip calibration function A(hc ), where hc is the contact depth. 2.3.2. Nano-DMA Unlike the previous two models, the nano-DMA technique takes into account elastic as well as viscous contribution of the test material. This allows us to calculate the storage and loss moduli. The dynamic data is analyzed using the classical equation for single free harmonic oscillator as given by Fo sin(ωt) = mx + Cx + kx

(5)

where Fo is the magnitude of the sinusoidal force, ω the frequency of the applied force, m the mass, C the damping coefficient and k is the combined stiffness (k = ks + ki ) of the system.

(Ci + Cs )ω k − mω2

(7)

By assuming a linear visco-elastic response, Eqs. (6) and (7) can be used to calculate the stiffness and damping of a system from the displacement amplitude and phase lag. The stiffness and damping can in turn be used to calculate the storage modulus, loss modulus and tan δ of the material using Eqs. (8a)–(8c) √ ks π E = √ (8a) 2 Ac √ ωCs π (8b) E = √ 2 Ac tan δ =

ωCs ks

(8c)

where ks , Cs are the sample stiffness and damping coefficient and Ac is the projected contact area. Details of this model can be found in the paper by Syed Asif et al. [10]. The nano-DMA software we used is configured to operate in different modes. Varying frequency and varying load modes were used in the current study. In one set of measurements the applied load is specified (this sets the indentation depth) and the probe is oscillated across different frequencies. This provides the frequency dependent storage and loss moduli of SC. In the second set of measurements, the applied load is varied at a fixed frequency to measure SC moduli as a function of depth. 3. Results and discussion Dry stratum corneum is easily imaged using AFM in both contact and tapping mode. A typical contact mode height image of porcine SC is presented in Fig. 3, clearly showing the polygonal-shaped corneocytes. The measured size of each cell is about 30–40 ␮m which is consistent with literature reports [11]. The measured root-mean-square (RMS) roughness on a 100 ␮m scan size SC image is 375 nm with variations ranging between 230 and 750 nm. Investigations on imaging structural effects on SC under water and with other treatments are underway and will be reported later. AFM imaging was used to verify there was no obvious damage to SC while investigating the mechanical properties through indentation. In addition, comparison of AFM images and force maps can be used to correlate modulus variations with specific structural features of the SC.

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Fig. 3. AFM height image of pig stratum corneum imaged in contact mode with a 0.32 N/m silicon nitride tip. Scan size: 100 ␮m. Z range: 1000 nm.

3.1. AFM nano-indentation testing After obtaining the elastic modulus E by fitting the force curve in Fig. 1 to Eq. (3), the modulus can be used to calculate theoretical force versus indentation depth curve using Eq. (1) or (2) and compared with the experimental force–indentation curve. A typical force versus indentation curve for dry SC measured with a silicon tip is shown in Fig. 4. This graph shows a general trend of increase in force with relation to indentation depth. The deviation at low indentation depth is possibly attributed to the fact that the AFM tip is more sphere-like at low indentation depths and is inaccurately modeled by assuming a conical geometry. This has been observed elsewhere [7]. Using this fit we calculate the modulus for dry stratum corneum to be 200 MPa (with an error of 150 MPa for six measurements). The same measurement carried out under water on a presoaked SC yields a modulus of 50 MPa (with an error of 14 MPa for seven measurements).

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Because there are no published data for the compression modulus of SC, the results are compared with the elastic modulus of SC measured by other methods. Among these, in plane (parallel to skin surface) E measured by a DDV viscoelastometer (Vibron) is 102 –103 MPa [4], by a dynamic piezo-electrical analyzer [12] is of the order of 102 MPa, and out of plane (perpendicular to the skin surface) modulus E by stress separation tests is 0.7 MPa [6]. The nano-indentation modulus is closer to the in plane extension modulus. Both moduli are much higher than the out of plane modulus. This makes sense because although the fracture modulus is normal to the SC surface, it reflects the debonding strength between the layers of corneocytes, which is, to a significant extent, a property of the lipid multilayer region and expected to be much lower. The measured change in modulus of wet SC is as expected since wet skin is normally softer than dry skin. From literature reports SC swells about 8% in the area dimension and 26% in the thickness dimension under water [13]. We do not anticipate that the change in modulus arises due to disruptions in the lipid bilayer [14] since our hydration times are short. The indentation depth is determined from the AFM cantilever deflection and the sample deformation. A stiffer tip can indent deeper than a floppy tip on the same sample. We tested different cantilevers of various stiffnesses to explore moduli at different depths in our SC sample. In addition, we also looked at the effect of altering the tip shape and reducing probe pressures. The measured elastic moduli are summarized in Table 1. These results indicate that tips with different cantilever stiffness do not show a significant difference, even though the indentation depths are an order of magnitude different. This suggests that SC elastic properties are similar over a range of 100 nm indentation depth. However, changing the probe geometry yields considerably different moduli. Measurements using sphere probes yield much lower elastic moduli. We believe that these measurements might be affected by surface roughness. From our imaging data we know that the SC surface is not flat on the length scales of the sphere probe diameter. The radius of curvature of an AFM tip is 4–10 nm, so the contact area for the tip is very small (∼20 nm2 ) during indentation. Thus, locally the stratum corneum surface is relatively flat. However, when we use a spherical tip (R = 10.9 and 20 ␮m) to do the indentation, the contact area is much larger (∼␮m2 ). At this large contact area, the SC surface is not as smooth as was the case with the AFM tip (with a RMS about 500 nm at 100 ␮m scan size). For the same material, the rough surface is much more Table 1 Elastic moduli of dry and wet SC measured by AFM indentation using different tips

Fig. 4. Force vs. indentation depth curve for AFM indentation with a silicon tip on dry stratum corneum.

Cantilever (spring constant)

Indentation depth (nm) at 100 nm cantilever deflection

Dry stratum corneum moduli (Pa)

Si3 N4 tip (0.32 N/m) Silicon tip (50 N/m)

15.3 ± 0.4 170 ± 23 at 50 nm deflection 61 ± 8.3 17 ± 1.5

(2 ± 1.5) × 108 (2 ± 1.9) × 108

Sphere 20 ␮m (0.06 N/m) Sphere 10.9 ␮m (0.58 N/m)

(4 ± 0.9) × 104 (9.5 ± 1.8) × 105

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easily compressed due to higher effective pressure applied at the peaks, hence the effective moduli are lower. While AFM nano-indentation can give us useful elastic properties of material, the force measurements have to be conducted very carefully and there still might be some problems, such as thermo-drift, piezo hysteresis and creep [15]. To verify our findings, a commercial nano-indentor Hysitron Triboscope was used to do similar measurement on SC and the results were compared. 3.2. Quasi-static testing Utilizing the Hysitron Triboscope nano-indenter to probe SC allows us to circumvent some of the issues mentioned above. In addition, mechanical properties can be evaluated much deeper in the material due to the higher applied forces. The force versus displacement curves for the indentation on dry and wet SC obtained by the Hysitron Triboscope are shown in Fig. 5. There are a few things that are important to note from the graph. First, the trend of force versus indentation curve is similar to that shown in Fig. 4 (nano-indentation using the AFM). However, the Triboscope indentation depth is much deeper than the AFM (1–2 ␮m compared to 10–100 nm). Secondly, we noticed a creep when the tip is held in the sample at a certain load. This behavior is a signature of viscous relaxation in the SC and highlights the fact that SC is not purely elastic. As mentioned before, applying the Hertzian model may not be appropriate for characterizing SC mechanical properties. Once again we see that hydrated SC is much softer than dry SC, this is reflected in the slope of the two curves in Fig. 5. Elastic moduli were calculated from the retracting curve, again based on a purely elastic material model [15]. For dry and wet SC the elastic moduli are 120 and 26 MPa, respectively, for Fig. 5 at applied load of 500 ␮N. Several measurements were conducted at different spots of the sample and the error for the modulus is found to be fairly small when applied load is the same. Another interesting observation is that both wet and dry moduli are lower than those obtained by AFM using contact and tapping mode tips, but higher than that obtained by spherical tips. Once again, these differences could be due to the tip shape

Fig. 5. Force vs. displacement curves for dry and wet stratum corneum obtained by Hysitron Triboscope at 500 ␮N loading force.

Fig. 6. Reduced modulus (as in Eq. (4)) measured at different indentation depths.

and indentation depth differences. The Berkovich tip is about 100–200 nm in radius of curvature, which is much larger than an AFM tip, but still smaller than the glass sphere used for the sphere probes. We see that the AFM indentation and quasi-static Hysitron Triboscope nano-indentor give us qualitatively similar results. The quantitative comparison is harder to make given the different probe geometries. However, the larger issue with these techniques concerns applying purely elastic models to interpret the data when our material (SC) clearly exhibits visco-elastic characteristics. The indentation depth was changed by varying the applied load in the Hysitron Triboscope experiments. The reduced modulus measured at different indentation depths is shown in Fig. 6. At small indentation depth, the modulus values are close to that measured by AFM. However, as the indentation depth increases, there is a decline in the modulus value for both dry and wet SC. This phenomenon was also observed in the dynamic measurements, and will be discussed later. 3.3. Dynamic measurement with nano-DMA To better understand visco-elastic properties of SC, we used nano-DMA to characterize both the storage (E ) and loss modulus (E ). In these measurements the Hysitron nano-DMA oscillates the Berkovich tip in the sample, at different depths and frequencies. The oscillating amplitude and phase change are detected and used to calculate E and E . The results for a frequency sweep (1–100 Hz) experiment with a loading force of 500 ␮N are shown in Fig. 7. The change in E and E due to oscillating frequency change is very small, with a slight increase in E and a slight decrease in E with frequency for both dry and wet SC. Thus SC does not exhibit any cross-over behavior typically observed in visco-elastic materials. The E for dry and wet SC obtained by this method are 102 and 60 MPa, respectively. The measured ratio of E and E (tan δ) is ∼0.1 for dry SC and ∼0.25 for wet SC. These results also show that the loss moduli are fairly small compared to the storage moduli, especially for dry SC, which explains the realistic values obtained by the AFM and Hysitron quasi-static indentation

Y. Yuan, R. Verma / Colloids and Surfaces B: Biointerfaces 48 (2006) 6–12

Fig. 7. E and E for dry and wet SC obtained by nano-DMA in varying frequency mode at 500 ␮N force load.

measurements (200 and 120 MPa). The increase in loss modulus for wet SC is possibly attributed to the plasticization of the material by disrupting bonds during hydration. We also explored the change in moduli as a function of indentation depth by varying the applied load. The results of this experiment, conducted on dry and wet SC, are shown in Fig. 8. The measurements once again show that at similar indentation depths, dry SC has an order of magnitude higher values of E and E than wet SC. Similar to what was observed in the quasi-static indentation experiments, an interesting feature in these measurements is the apparent modulus variation with indentation depth. We do not fully understand the origin of this behavior. It is possible that a “skin”-like feature with higher modulus forms on SC. We observed a similar trend on a polydimethylsiloxane (PDMS) film (data not shown). Another possible reason for the change might be that the material yields as the applied load and pres-

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sure increases, and therefore the effective modulus drops. This phenomenon is still under investigation. The nano-DMA measurements were then used to explore mechanical properties of SC following treatment with glutaraldehyde, a well-known protein cross-linker [16], and sodium dodecyl sulfate (SDS), an anionic surfactant reported to change SC structure [17]. The initial measurements were made while the SC was wet and monitored until the SC dried. Preliminary experiments indicate that as expected glutaraldehyde ‘hardened’ SC. In the wet stage the elastic modulus is only slightly higher (1.2×) than SC that is hydrated in plain water. However, in the dry stage the modulus of SC increased significantly (a factor of 2) beyond the original value of the dry porcine SC. On the other hand, treatment with SDS yields moduli that ‘soften’ the SC to half its typical wet value, but drying leads to modulus recovery ending at 0.9 of typical dry SC numbers. Thus, we see that this technique is capable of distinguishing effects of material treatments. 4. Conclusion To summarize we have measured the elastic and viscoelastic properties of stratum corneum at the micron level. These mechanical properties were explored through the use of nanoindentation techniques using an AFM and a Triboscope nanoindentor. The elastic moduli values obtained with a purely elastic model are on the order of 100 and 10 MPa for dry and wet SC, respectively. Variations caused by differences in shape of the indentation probe and indentation depth are also reported. A more appropriate dynamic method using a nano-DMA was also used to highlight the visco-elastic nature of stratum corneum. The elastic component, i.e. the storage modulus, is consistent with the elastic modulus obtained by nano-indentation measurements. In addition, we measured the ratio of the loss modulus and storage modulus (tan δ), which is 0.1 for dry SC and 0.25 for wet SC. Furthermore, we also explored the effects of surfactants and known protein cross-linkers. These measurements open up the possibility of future probing of other material treatments of stratum corneum as well as extending the measurements to other biological tissues. Acknowledgements We thank Srividya Ramakrishnan, Yan Zhou for providing stratum corneum; Prem Chandar, David Moore, Alex Lips for useful discussions. References

Fig. 8. E and E for dry and wet SC obtained by nano-DMA in varying load mode at frequency 100 Hz.

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