Journal of Biomechanics 47 (2014) 1186–1192
Contents lists available at ScienceDirect
Journal of Biomechanics journal homepage: www.elsevier.com/locate/jbiomech www.JBiomech.com
In vitro measurement of the mechanical properties of skin by nano/microindentation methods T. Jee, K. Komvopoulos n Department of Mechanical Engineering, University of California, Berkeley, CA 94720, USA
art ic l e i nf o
a b s t r a c t
Article history: Accepted 13 October 2013
The elastic behaviors of stratum corneum, viable epidermis, dermis, and whole skin were investigated by nano/microindentation techniques. Insignificant differences in reduced elastic modulus of skin samples obtained from three different porcine breeds revealed breed type independent measurements. The reduced elastic modulus of stratum corneum is shown to be about three orders of magnitude higher than that of dermis. As a result, for relatively shallow and deep indentations, skin elasticity is controlled by that of stratum corneum and dermis, respectively. Skin deformation is interpreted in the context of a layered structure model consisting of a stiff and hard surface layer on a compliant and soft substrate, supported by microscopy observations and indentation measurements. & 2014 Published by Elsevier Ltd.
Keywords: Epidermis Deformation Dermis Indentation Mechanical properties Skin Stratum corneum
1. Introduction Aside from teeth, cornea, hair, and nails, human organ surfaces consist of epithelial tissue. The outer epithelial layer (epidermis), commonly referred to as the skin, is the largest organ of the human body, providing vital protection to tissue and cells against external intruders, such as bacteria, virus, and fungi, and preventing the loss of water (McGrath and Uitto, 2010; Archer, 2010). Other important functional properties of skin include body temperature regulation, transmission of mechanical stresses, and absorption of light (radiation). Skin mechanical properties have been traditionally measured with macroscopic instruments. For example, different suction tests have been used to study in vivo skin elasticity (Grahame, 1969) and its dependence on ageing (Grahame and Holt, 1969), the role of natural tension on the mechanical behavior of skin (Alexander and Cook, 1977), the dependence of skin elasticity on age, sex, and anatomical region (Cua et al., 1990), the effect of hydration (Auriol et al., 1993) and ageing (Leveque et al., 1980) on skin extensibility, and the influence of fluid volume changes in hemodialysis on the biophysical properties of skin (Brazzelli et al., 1994). An in vivo mechanical model of the human skin (Diridollou et al., 2000) and an analysis of the relative contributions of different skin layers to the overall mechanical behavior of human skin in vivo (Hendriks et al., 2006) have also been used to study the mechanical response of skin subjected to suction. In addition to elastic stretching, the
n
Corresponding author. Tel.: þ 1 510 642 2563; fax: þ 1 510 642 5539. E-mail address: [email protected] (K. Komvopoulos).
0021-9290/$ - see front matter & 2014 Published by Elsevier Ltd. http://dx.doi.org/10.1016/j.jbiomech.2013.10.020
in vivo torsional elastic behavior of human skin has been examined in earlier studies (Finlay, 1971; Sanders, 1973). Several in vivo investigations have focused on the effects of ageing, stress, sex, and moisturizing treatment on human skin torsional elasticity (Agache et al., 1980; Kalis et al., 1990; Salter et al., 1993). Most measurements of skin mechanical properties have relied on mechanical instruments (Warren et al., 1991; Gunner et al., 1979; Berardesca et al., 1986; Ohura et al., 1980; Sugihara et al., 1991; Peck and Glick, 1956; Dikstein et al., 1984; Falanga and Bucalo, 1993; Diridollou et al., 1998; Cooper et al., 1985). Despite important insight into the mechanical properties of human skin provided by the aforementioned studies, very little is known about the mechanical behavior of individual skin layers. Obtaining such knowledge requires the use of microprobe-based methods, such as nano/microindentation, which can permit objectively probing the mechanical response of individual skin layers. Therefore, the objective of this study was to examine the mechanical behavior of individual skin layers using nano/microscale indentation techniques and identify the contribution of each skin layer to the overall mechanical behavior of skin. To accomplish this objective, indentation experiments were performed with porcine skin samples obtained from three different breeds to examine the effect of the breed type on the measurements. Porcine skin is appropriate for in vitro studies because its topology, texture, architecture, metabolic rate, and drug permeability are similar to those of human skin (Schmook et al., 2001). In addition, skin properties are not significantly affected by the lack of a physiological environment provided there is significant moisture (Agner and Serup, 1990). The morphology of porcine skin is similar to that of healthy human skin, even after 3 days from harvesting (Fig. 1).
T. Jee, K. Komvopoulos / Journal of Biomechanics 47 (2014) 1186–1192
1187
Stratum corneum samples were prepared by removing an outer layer of a few tens of micrometers (consisting of the stratum corneum and a portion of the viable epidermis) from skin samples using a surgical knife. The samples were then epoxyattached to a steel disk with the viable epidermis facing down or kept on a Petri dish until testing. A chemical method of removing the stratum corneum was not used to prevent any unknown effects on the skin properties. Although the present method does not ensure the full removal of the viable epidermis, this is not a problem because the maximum contact depth in nano/microindentation testing is only a few micrometers. The dermis samples were prepared by removing the hypodermis and a portion of the dermis with a surgical knife and then attaching the stratum corneum surface to a Petri dish covered with filter paper that was soaked in 0.9% NaCl or PBS solution. 2.2. Histology Skin samples were embedded in an optimal-cutting-temperature compound (TissueTek, Elkhart, IN) on dry ice and kept at a temperature of 62 1C until testing. Before performing histology, 10 mm-thick specimens were cut from the skin samples and stained with hematoxylin and eosin (H&E) following a standard protocol. 2.3. Nano/microindentation experiments Nanoindentation tests were performed with a custom-made apparatus consisting of an atomic force microscope (AFM) scanner (Nanoscope II, Digital Instruments, Santa Barbara, CA), a three-plate capacitor force–displacement transducer (Triboscope, Hysitron, Minneapolis, MN), and a detector assembly (head) that uses the AFM scanner and the software of a scanning tunneling microscope. Because of the large thickness of the dermis and skin samples, microindentation experiments were performed with a microindentation apparatus (Bruker, Campbell, CA) that has a very large depth range. Details about experimental set-up, associated testing procedures, and mechanics analysis for determining the reduced elastic modulus Er (Eq. (S5)) and hardness H (Eq. (S6)) as functions of maximum contact depth hc,max can be found in Supporting information. 2.4. Statistical analysis Significant differences among porcine breeds or samples were determined by a one-way analysis of variance (ANOVA). The F-test was used to validate the assumptions made in ANOVA. Statistically different data were discerned by the corresponding p-value, calculated for a significance level α ¼ 0.05. The null hypothesis was used to examine if the mean values were statistically different. The null hypothesis was rejected for p o 0.05. Sample sizes were determined for 80% power. Curve fits in figures indicate general trends.
3. Results
Fig. 1. Surface morphology of (a) human and (b) porcine (American Yorkshire) skin.
The effects of the mechanical properties of stratum corneum, viable epidermis, and dermis to the mechanical behavior of skin are discussed in the context of nano/microindentation, histology, and microscopy results.
2. Experimental methods 2.1. Sample preparation Skin samples were harvested from the belly parts of 4–12 months old Berkshire and Duroc–Berkshire Cross porcine breeds from a local abattoir within 3 days from sacrifice. In addition, skin samples of American Yorkshire porcine breed of a similar age range were obtained from the School of Medicine, University of California, San Francisco. To maintain a physiologically similar pH, the skin samples were placed on Petri dishes covered with filter paper, which was previously soaked in 0.9% NaCl or phosphate buffer saline (PBS) solution. Testing was performed within 1–2 days from sample acquisition without any chemical treatment. Before testing, the hairs were carefully removed with surgical blades and the samples were sectioned to the sizes needed for testing. To minimize sample dehydration, testing was performed within 30 min from sample preparation in a clean-air laboratory environment.
The results presented in this section were obtained from three types of layer/substrate samples: (1) stratum corneum/viable epidermis, (2) dermis/epidermis, and (3) whole skin with the hypodermis as the substrate. Samples were attached to a Petri dish via their substrates. Hereafter, these samples will be referred to as the stratum corneum, dermis, and skin samples, correspondingly. Also, hmax is measured from the undeformed surface of each sample. To avoid the effect of surface roughness (root-mean-square (rms) E200–300 nm) on the measurements, data were acquired for depths 4500 nm. For negligible substrate biasing of the measurements, hc,max must be r10% of the layer thickness (Bhattacharya and Nix, 1988). Thus, considering the thickness of the stratum corneum ( 10 μm) and viable epidermis left after sectioning, hc,max o1500 nm in all measurements. 3.1. Cross-sectional histology Optical micrographs of the cross-sectional histology of porcine skin obtained before testing (Fig. 2) showed overall histological features similar to those of human skin (McGrath and Uitto, 2010). As shown in Fig. 2b, the thickness of the darker layer (stratum corneum) is 10 μm, while that of the underlying tightly packed layer (viable epidermis) varies between 20 and 100 μm. Next is the dermis of several millimeters thickness, followed by the hypodermis.
1188
T. Jee, K. Komvopoulos / Journal of Biomechanics 47 (2014) 1186–1192
calculated by averaging the data obtained with the sharp indenter in the contact depth range 500–1000 nm to avoid the effects of the compliant viable epidermis and the high surface roughness of stratum corneum (rms¼200–300 nm), are equal to 0.8770.42 GPa and 15.6710 MPa, respectively. Much lower Er (Fig. 4c) and H (Fig. 4d) were obtained with the blunt indenter due to biasing of the measurements by the viscoelastic deformation of the viable epidermis. However, extrapolation of the fitted curve to a contact depth of 1 nm yields Er ¼0.80 GPa, which is close to that of stratum corneum obtained with the sharp indenter. Data corresponding to contact depths 4500 nm are considered to be indicative of the viable epidermis. Consequently, Er and H of the viable epidermis, obtained by averaging the data measured with the blunt indenter in the contact depth range 500–1300 nm, are equal to 0.2170.05 GPa and 1.370.5 MPa, respectively. As mentioned earlier, the mechanical properties of the dermis layer were measured with the microindentation apparatus, because the contact depth range was beyond the maximum z-displacement of the nanoindenter. Also, a blunt nanoindenter (R¼ 20 mm) was used to avoid excessive damage of the very soft dermis. Both Er (Fig. 4e) and H (Fig. 4f) of dermis decrease with the increase of hc,max, attaining steady-state values of 1.91 70.88 MPa and 0.857 0.45 MPa, respectively, for hc,max 4 300 mm. The mechanical properties of skin were also studied with the microindentation apparatus due to the large contact depths. Er (Fig. 4g) and H (Fig. 4h) of skin sharply decrease with increasing hc,max, reaching steady-state values of 3.777 1.7 MPa and 1.43 70.58 MPa, respectively, for hc,max 4100 μm. From curve fitting, Er at hc,max E5 μm is estimated to be 0.80 GPa, which is close to that of stratum corneum (0.87 GPa), whereas Er at hc,max E200 μm is equal to 2 MPa, which is close to that of the dermis layer (1.91 MPa). 3.4. Surface deformation Fig. 2. Cross-sectional optical microscopy images of porcine skin (American Yorkshire) at different magnifications. Image (b) shows (from top to bottom): stratum corneum ( 10 μm thick), viable epidermis (20–100 μm thick), and dermis (a few mm thick).
3.2. Effect of breed type on skin mechanical properties Er of stratum corneum does not show statistical differences among different breeds (Fig. 3a, p¼0.287). A similar observation can be made for data obtained from randomly selected samples of different breeds (Fig. 3b, p¼ 0.226). Similar to stratum corneum, Er of dermis does not show a dependence on breed type (Fig. 3c, p¼ 0.887) and sample selection or test order (Fig. 3d, p¼ 0.981). Insignificant differences in Er of skin are also shown for different breeds (Fig. 3e, p¼0.88) and sample selection or test order (Fig. 3f, p¼0.999); however, the corresponding standard deviation is very high due to the significant scatter of these measurements. Fig. 3 shows that stratum corneum, dermis, and skin of different breed types exhibit similar elastic properties. Therefore, skin mechanical properties presented and discussed next are independent of breed type and order of sample testing. In addition, considering the fairly large number of samples in the age range examined and statistically indifferent data shown in Fig. 3, it may be inferred that age and gender effects on the measured mechanical properties were insignificant. 3.3. Reduced elastic modulus and hardness Er and H of stratum corneum show a nonlinear decrease with increasing hmax for relatively sharp (R¼1 mm) and blunt (R¼20 mm) indenters (Fig. 4a–d). Er (Fig. 4a) and H (Fig. 4b) of stratum corneum,
Additional evidence of the indentation (contact) depth effect on skin deformation was obtained with optical microscopy performed after 1 h from testing. Shallow indentations caused only slight burnishing on the stratum corneum surface (Fig. 5a), presumably a result of localized sliding against the indenter surface, indicating mainly elastic deformation in the bulk of stratum corneum. Alternatively, deep indentations induced gross plastic deformation, resulting in localized fracture of the stratum corneum after unloading (Fig. 5b).
4. Discussion Insignificant differences in the mechanical properties of stratum corneum, dermis, and skin of different porcine breeds and randomly selected samples of each breed (Fig. 3) indicated that the measured mechanical properties were independent of breed type and order of sample testing. Table 1 summarizes the mechanical properties of individual skin layers and whole skin extracted from nano/microindentation experiments with relatively sharp (stratum corneum) and blunt (all other samples) indenters (to account for differences in material compliance and softness affecting the contact depth range) and indentation mechanics analysis (Eqs. (S1)–(S6) in Supporting information). To avoid biasing of the measurements by the high surface roughness (rms¼200–300 nm), nanoindentation measurements were obtained with a sharp (R¼ 1 mm) indenter for hc,max 4500 nm. Because of the less pronounced roughness effect on the measurements made with a blunt (R¼20 mm) indenter, reliable measurements were obtained for a smaller range of hc,max. However, these measurements were influenced by the high compliance of the viable epidermis, as evidenced by the nonlinear decrease of Er and H of
T. Jee, K. Komvopoulos / Journal of Biomechanics 47 (2014) 1186–1192
1189
Fig. 3. Effect of breed type and sample or test order on reduced elastic modulus of (a, b) stratum corneum, (c, d) dermis, and (e, f) skin of three different porcine breeds: American Yorkshire (AY), Berkshire (B), and Duroc–Berkshire Cross (DBC). The measurements were obtained with a sharp indenter (R ¼1 mm) and contact depth fixed at 1 mm for stratum corneum and a blunt indenter (R ¼20 mm) and contact depth fixed at 300 mm and 70 mm for dermis and skin, respectively. (Set size: (a) 53 (AY ¼ 10, B ¼ 21, DBC ¼ 22), (b) 51, (c) 35 (AY¼ 9, B ¼13, DBC ¼ 13), (d) 34, (e) 72 (AY ¼13, B ¼29, DBC ¼30), and (f) 71.)
stratum corneum with increasing hc,max (Fig. 4a–d). This trend is attributed to the deformation effect of the highly compliant and soft viable epidermis, which becomes more significant with increasing contact depth. This problem was overcome by extrapolating the fitted curve into the low range of hc,max. Using this approach, Er of stratum corneum measured with the blunt indenter (0.80 GPa) was found to be in good agreement with the directly measured value obtained with the sharp indenter (0.87 GPa). The Er of stratum corneum measured in this study is in good agreement with the results of previous studies (Park and Baddiel, 1972; Nicolopoulos et al., 1998). Using the same
curve-fit/extrapolation approach, H of skin is estimated to be 1.43 MPa, which is much lower than that of stratum corneum (15.6 MPa) and slightly higher than that of the viable epidermis (1.30 MPa). This is attributed to the dominant effect of the viable epidermis and dermis, which are much thicker and softer than the stratum corneum. The significantly lower Er (1.91 MPa) and H (0.85 MPa) of dermis compared to stratum corneum (by about three and one orders of magnitude, respectively) explains the sharp decrease of the mechanical properties of skin with increasing hc,max (Fig. 4g and h). For small contact depths, the mechanical properties of skin
1190
T. Jee, K. Komvopoulos / Journal of Biomechanics 47 (2014) 1186–1192
Fig. 4. Reduced elastic modulus and hardness of (a)–(d) stratum corneum, (e, f) dermis, and (g, h) skin versus maximum contact depth. The measurements were obtained with sharp (a, b) and blunt (c)–(h) indenters of radius equal to 1 and 20 mm, respectively. Curve fits indicate general trends.
are controlled by those of stratum corneum, whereas for relatively large contact depths, the mechanical behavior of skin is dominated by the properties of the viable epidermis and the dermis. Thus, the loading response of skin subjected to shallow indentation is governed by the mechanical behavior of stratum corneum, while the unloading response of skin recovering from deep indentation is mostly influenced by the elastic behaviors of the viable epidermis and the dermis.
The presented results indicate that irreversible skin deformation commences beyond a critical contact depth, on the order of the stratum corneum thickness ( 10 μm), due to the inadequate support of the highly compliant and soft viable epidermis and dermis. Because the rate of cell and tissue regeneration in injured viable epidermis is much larger than the replenishment rate of stratum corneum by the viable epidermis (Candi et al., 2005), understanding the role of the mechanical properties of individual
T. Jee, K. Komvopoulos / Journal of Biomechanics 47 (2014) 1186–1192
1191
blood or interstitial fluid sampling using microneedle-based procedures. Conflict of interest statement The authors confirm that there are no conflicts of interest.
Acknowledgments This work was partially funded by the Center for Information Technology Research in the Interest of Society (CITRIS), University of California, Berkeley. The authors thank Y. Seo and D.W. Gao from the School of Medicine, University of California, San Francisco, for the donation of the American Yorkshire porcine samples, Professor S. Li from the Bioengineering Department, University of California, Berkeley, for helpful discussions, and F. Yuan for assistance in histology staining.
Appendix A. Supplementary materials Supplementary information associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.jbio mech.2013.10.020. References
Fig. 5. Optical microscope images of skin samples (American Yorkshire) obtained after 1 h from testing with a blunt indenter of radius equal to 12.5 mm for a maximum displacement equal to (a) 60 and (b) 500 mm.
Table 1 Mechanical properties of individual skin layers and whole skin extracted from nano/microindentation experiments. Material a
Stratum corneum Viable epidermisb Dermisb Skinb,c a b c
Reduced elastic modulus Er (GPa)
Hardness H (MPa)
0.87 7 0.42 0.217 0.05 0.001917 0.00088 0.003777 0.0017
15.6 710.0 1.30 70.5 0.85 70.45 1.43 70.58
Sharp indenter (R¼ 1 mm). Blunt indenter (R ¼20 mm). hc,max 4100 mm.
layers on irreversible skin deformation is of paramount importance to skin repair. The results of this study provide insight into the mechanical behavior of skin due to indentation loading, which is of particular importance to minimally invasive procedures relying on effective penetration of the stratum corneum, such as transdermal drug delivery, local tissue and gene delivery, and
Agache, P.G., Monneur, C., Leveque, J.L., De Rigal, J., 1980. Mechanical properties and Young's modulus of human skin in vivo. Arch. Dermatol. Res. 269, 221–232. Agner, T., Serup, J., 1990. Sodium lauryl sulphate for irritant patch testing – a dose– response study using bioengineering methods for determination of skin irritation. J. Invest. Dermatol. 95, 543–547. Alexander, H., Cook, T.H., 1977. Accounting for natural tension in the mechanical testing of human skin. J. Invest. Dermatol. 69, 310–314. Archer, C.B., 2010. Functions of the skin. In: Burns, T., Breathnach, S., Cox, N., Griffiths, C. (Eds.), Rook's Textbook of Dermatology, eighth edition, Willey-Blackwell, Oxford, UK, pp. 4.1–4.11. Auriol, F., Vaillant, L., Machet, L., Diridollou, S., Lorette, G., 1993. Effects of shorttime hydration on skin extensibility. Acta Derm.-Venereol. 73, 344–347. Berardesca, E., Borroni, G., Gabba, P., Borlone, R., Rabbiosi, G., 1986. Evidence for elastic changes in aged skin revealed in an in vivo extensometric study at low loads. Bioeng. Skin 2, 261–270. Bhattacharya, A.K., Nix, W.D., 1988. Analysis of elastic and plastic deformation associated with indentation testing of thin films on substrates. Int. J. Solids Struct. 24, 1287–1298. Brazzelli, V., Borroni, G., Vignoli, G.P., Rabbiosi, G., Cavagnino, A., Berardesca, E., 1994. Effects of fluid volume changes during hemodialysis on the biophysical parameters of the skin. Dermatology 188, 113–116. Candi, E., Schmidt, R., Melino, G., 2005. The cornified envelope: a model of cell death in the skin. Nature Rev.: Mol. Cell Biol. 6, 328–340. Cooper, E.R., Missel, P.J., Hannon, D.P., Albright, G.B., 1985. Mechanical properties of dry, normal, and glycerol-treated skin as measured by the gas-bearing electrodynamometer. J. Soc. Cosmet. Chem. 36, 335–348. Cua, A.B., Wilhelm, K.-P., Maibach, I.H., 1990. Elastic properties of human skin: relation to age, sex, and anatomical region. Arch. Dermatol. Res. 282, 283–288. Dikstein, S., Hartzshtark, A., Bercovici, P., 1984. The dependence of low-pressure indentation, slackness, and surface pH on age in forehead skin of women. J. Soc. Cosmet. Chem. 35, 221–228. Diridollou, S., Berson, M., Vabre, V., Black, D., Karlsson, B., Auriol, F., Gregoire, J.M., Yvon, C., Vaillant, L., Gall, Y., Patat, F., 1998. An in vivo method for measuring the mechanical properties of the skin using ultrasound. Ultrasound Med. Biol. 24, 215–224. Diridollou, S., Patat, F., Gens, F., Vaillant, L., Black, D., Lagarde, J.M., Gall, Y., Berson, M., 2000. In vivo model of the mechanical properties of the human skin under suction. Skin Res. Technol. 6, 214–221. Falanga, V., Bucalo, B., 1993. Use of a durometer to assess skin hardness. J. Am. Acad. Dermatol. 29, 47–51. Finlay, B., 1971. The torsional characteristics of human skin in vivo. Biomed. Eng. 6, 567–573. Grahame, R., 1969. Elasticity of human skin in vivo. Ann. Phys. Med. 10, 130–136. Grahame, R., Holt, P.J.L., 1969. The influence of ageing on the in vivo elasticity of human skin. Gerontologia 15, 121–139. Gunner, C.W., Hutton, W.C., Burlin, T.E., 1979. The mechanical properties of skin in vivo – a portable hand-held extensometer. Br. J. Dermatol. 100, 161–163. Hendriks, F.M., Brokken, D., Oomens, C.W.J., Bader, D.L., Baaijens, F.P.T., 2006. The relative contributions of different skin layers to the mechanical behavior of human skin in vivo using suction experiments. Med. Eng. Phys. 28, 259–266.
1192
T. Jee, K. Komvopoulos / Journal of Biomechanics 47 (2014) 1186–1192
Kalis, B., De Rigal, J., Léonard, F., Lévêque, J.L., Riche, O., Le Corre, Y., De Lacharriere, O., 1990. In vivo study of scleroderma by non-invasive techniques. Br. J. Dermatol. 122, 785–791. Leveque, J.L., de Rigal, J., Agache, P.G., Monneur, C., 1980. Influence of ageing on the in vivo extensibility of human skin at a low stress. Arch. Dermatol. Res. 269, 127–135. McGrath, J.A., Uitto, J., 2010. Anatomy and organization of human skin. In: Burns, T., Breathnach, S., Cox, N., Griffiths, C. (Eds.), Rook's Textbook of Dermatology, eighth edition, Willey-Blackwell, Oxford, UK, pp. 3.1–3.53. Nicolopoulos, C.S., Giannoudis, P.V., Glaros, K.D., Barbenel, J.C., 1998. In vitro study of the failure of skin surface after influence of hydration and preconditioning. Arch. Dermatol. Res. 290, 638–640. Ohura, T., Sugihara, T., Honda, K., 1980. Postoperative evaluation in plastic surgery using the bio-skin tension meter. Ann. Plast. Surg. 5, 74–82. Park, A.C., Baddiel, C.B., 1972. Rheology of stratum corneum—I: a molecular interpretation of the stress–strain curve. J. Soc. Cosmet. Chem. 23, 3–12.
Peck, S.M., Glick, A.W., 1956. A new method for measuring the hardness of keratin. J. Soc. Cosmet. Chem. 7, 530–540. Salter, D.C., McArthur, H.C., Crosse, J.E., Dickens, A.D., 1993. Skin mechanics measured in vivo using torsion: a new and accurate model more sensitive to age, sex and moisturizing treatment. Int. J. Cosmet. Sci. 15, 200–218. Sanders, R., 1973. Torsional elasticity of human skin in vivo. Pflügers Arch. 342, 255–260. Schmook, F.P., Meingassner, J.G., Billich, A., 2001. Comparison of human skin or epidermis models with human and animal skin in in-vitro percutaneous absorption. Int. J. Pharm. 215, 51–56. Sugihara, T., Ohura, T., Homma, K., Igawa, H.H., 1991. The extensibility in human skin: variation according to age and site. Br. J. Plast. Surg. 44, 418–422. Warren, R., Gartstein, V., Kligman, A.M., Montagna, W., Allendorf, R.A., Ridder, G.M., 1991. Age, sunlight, and facial skin: a histologic and quantitative study. J. Am. Acad. Dermatol. 25, 751–760.
Contents lists available at ScienceDirect
Journal of Biomechanics journal homepage: www.elsevier.com/locate/jbiomech www.JBiomech.com
In vitro measurement of the mechanical properties of skin by nano/microindentation methods T. Jee, K. Komvopoulos n Department of Mechanical Engineering, University of California, Berkeley, CA 94720, USA
art ic l e i nf o
a b s t r a c t
Article history: Accepted 13 October 2013
The elastic behaviors of stratum corneum, viable epidermis, dermis, and whole skin were investigated by nano/microindentation techniques. Insignificant differences in reduced elastic modulus of skin samples obtained from three different porcine breeds revealed breed type independent measurements. The reduced elastic modulus of stratum corneum is shown to be about three orders of magnitude higher than that of dermis. As a result, for relatively shallow and deep indentations, skin elasticity is controlled by that of stratum corneum and dermis, respectively. Skin deformation is interpreted in the context of a layered structure model consisting of a stiff and hard surface layer on a compliant and soft substrate, supported by microscopy observations and indentation measurements. & 2014 Published by Elsevier Ltd.
Keywords: Epidermis Deformation Dermis Indentation Mechanical properties Skin Stratum corneum
1. Introduction Aside from teeth, cornea, hair, and nails, human organ surfaces consist of epithelial tissue. The outer epithelial layer (epidermis), commonly referred to as the skin, is the largest organ of the human body, providing vital protection to tissue and cells against external intruders, such as bacteria, virus, and fungi, and preventing the loss of water (McGrath and Uitto, 2010; Archer, 2010). Other important functional properties of skin include body temperature regulation, transmission of mechanical stresses, and absorption of light (radiation). Skin mechanical properties have been traditionally measured with macroscopic instruments. For example, different suction tests have been used to study in vivo skin elasticity (Grahame, 1969) and its dependence on ageing (Grahame and Holt, 1969), the role of natural tension on the mechanical behavior of skin (Alexander and Cook, 1977), the dependence of skin elasticity on age, sex, and anatomical region (Cua et al., 1990), the effect of hydration (Auriol et al., 1993) and ageing (Leveque et al., 1980) on skin extensibility, and the influence of fluid volume changes in hemodialysis on the biophysical properties of skin (Brazzelli et al., 1994). An in vivo mechanical model of the human skin (Diridollou et al., 2000) and an analysis of the relative contributions of different skin layers to the overall mechanical behavior of human skin in vivo (Hendriks et al., 2006) have also been used to study the mechanical response of skin subjected to suction. In addition to elastic stretching, the
n
Corresponding author. Tel.: þ 1 510 642 2563; fax: þ 1 510 642 5539. E-mail address: [email protected] (K. Komvopoulos).
0021-9290/$ - see front matter & 2014 Published by Elsevier Ltd. http://dx.doi.org/10.1016/j.jbiomech.2013.10.020
in vivo torsional elastic behavior of human skin has been examined in earlier studies (Finlay, 1971; Sanders, 1973). Several in vivo investigations have focused on the effects of ageing, stress, sex, and moisturizing treatment on human skin torsional elasticity (Agache et al., 1980; Kalis et al., 1990; Salter et al., 1993). Most measurements of skin mechanical properties have relied on mechanical instruments (Warren et al., 1991; Gunner et al., 1979; Berardesca et al., 1986; Ohura et al., 1980; Sugihara et al., 1991; Peck and Glick, 1956; Dikstein et al., 1984; Falanga and Bucalo, 1993; Diridollou et al., 1998; Cooper et al., 1985). Despite important insight into the mechanical properties of human skin provided by the aforementioned studies, very little is known about the mechanical behavior of individual skin layers. Obtaining such knowledge requires the use of microprobe-based methods, such as nano/microindentation, which can permit objectively probing the mechanical response of individual skin layers. Therefore, the objective of this study was to examine the mechanical behavior of individual skin layers using nano/microscale indentation techniques and identify the contribution of each skin layer to the overall mechanical behavior of skin. To accomplish this objective, indentation experiments were performed with porcine skin samples obtained from three different breeds to examine the effect of the breed type on the measurements. Porcine skin is appropriate for in vitro studies because its topology, texture, architecture, metabolic rate, and drug permeability are similar to those of human skin (Schmook et al., 2001). In addition, skin properties are not significantly affected by the lack of a physiological environment provided there is significant moisture (Agner and Serup, 1990). The morphology of porcine skin is similar to that of healthy human skin, even after 3 days from harvesting (Fig. 1).
T. Jee, K. Komvopoulos / Journal of Biomechanics 47 (2014) 1186–1192
1187
Stratum corneum samples were prepared by removing an outer layer of a few tens of micrometers (consisting of the stratum corneum and a portion of the viable epidermis) from skin samples using a surgical knife. The samples were then epoxyattached to a steel disk with the viable epidermis facing down or kept on a Petri dish until testing. A chemical method of removing the stratum corneum was not used to prevent any unknown effects on the skin properties. Although the present method does not ensure the full removal of the viable epidermis, this is not a problem because the maximum contact depth in nano/microindentation testing is only a few micrometers. The dermis samples were prepared by removing the hypodermis and a portion of the dermis with a surgical knife and then attaching the stratum corneum surface to a Petri dish covered with filter paper that was soaked in 0.9% NaCl or PBS solution. 2.2. Histology Skin samples were embedded in an optimal-cutting-temperature compound (TissueTek, Elkhart, IN) on dry ice and kept at a temperature of 62 1C until testing. Before performing histology, 10 mm-thick specimens were cut from the skin samples and stained with hematoxylin and eosin (H&E) following a standard protocol. 2.3. Nano/microindentation experiments Nanoindentation tests were performed with a custom-made apparatus consisting of an atomic force microscope (AFM) scanner (Nanoscope II, Digital Instruments, Santa Barbara, CA), a three-plate capacitor force–displacement transducer (Triboscope, Hysitron, Minneapolis, MN), and a detector assembly (head) that uses the AFM scanner and the software of a scanning tunneling microscope. Because of the large thickness of the dermis and skin samples, microindentation experiments were performed with a microindentation apparatus (Bruker, Campbell, CA) that has a very large depth range. Details about experimental set-up, associated testing procedures, and mechanics analysis for determining the reduced elastic modulus Er (Eq. (S5)) and hardness H (Eq. (S6)) as functions of maximum contact depth hc,max can be found in Supporting information. 2.4. Statistical analysis Significant differences among porcine breeds or samples were determined by a one-way analysis of variance (ANOVA). The F-test was used to validate the assumptions made in ANOVA. Statistically different data were discerned by the corresponding p-value, calculated for a significance level α ¼ 0.05. The null hypothesis was used to examine if the mean values were statistically different. The null hypothesis was rejected for p o 0.05. Sample sizes were determined for 80% power. Curve fits in figures indicate general trends.
3. Results
Fig. 1. Surface morphology of (a) human and (b) porcine (American Yorkshire) skin.
The effects of the mechanical properties of stratum corneum, viable epidermis, and dermis to the mechanical behavior of skin are discussed in the context of nano/microindentation, histology, and microscopy results.
2. Experimental methods 2.1. Sample preparation Skin samples were harvested from the belly parts of 4–12 months old Berkshire and Duroc–Berkshire Cross porcine breeds from a local abattoir within 3 days from sacrifice. In addition, skin samples of American Yorkshire porcine breed of a similar age range were obtained from the School of Medicine, University of California, San Francisco. To maintain a physiologically similar pH, the skin samples were placed on Petri dishes covered with filter paper, which was previously soaked in 0.9% NaCl or phosphate buffer saline (PBS) solution. Testing was performed within 1–2 days from sample acquisition without any chemical treatment. Before testing, the hairs were carefully removed with surgical blades and the samples were sectioned to the sizes needed for testing. To minimize sample dehydration, testing was performed within 30 min from sample preparation in a clean-air laboratory environment.
The results presented in this section were obtained from three types of layer/substrate samples: (1) stratum corneum/viable epidermis, (2) dermis/epidermis, and (3) whole skin with the hypodermis as the substrate. Samples were attached to a Petri dish via their substrates. Hereafter, these samples will be referred to as the stratum corneum, dermis, and skin samples, correspondingly. Also, hmax is measured from the undeformed surface of each sample. To avoid the effect of surface roughness (root-mean-square (rms) E200–300 nm) on the measurements, data were acquired for depths 4500 nm. For negligible substrate biasing of the measurements, hc,max must be r10% of the layer thickness (Bhattacharya and Nix, 1988). Thus, considering the thickness of the stratum corneum ( 10 μm) and viable epidermis left after sectioning, hc,max o1500 nm in all measurements. 3.1. Cross-sectional histology Optical micrographs of the cross-sectional histology of porcine skin obtained before testing (Fig. 2) showed overall histological features similar to those of human skin (McGrath and Uitto, 2010). As shown in Fig. 2b, the thickness of the darker layer (stratum corneum) is 10 μm, while that of the underlying tightly packed layer (viable epidermis) varies between 20 and 100 μm. Next is the dermis of several millimeters thickness, followed by the hypodermis.
1188
T. Jee, K. Komvopoulos / Journal of Biomechanics 47 (2014) 1186–1192
calculated by averaging the data obtained with the sharp indenter in the contact depth range 500–1000 nm to avoid the effects of the compliant viable epidermis and the high surface roughness of stratum corneum (rms¼200–300 nm), are equal to 0.8770.42 GPa and 15.6710 MPa, respectively. Much lower Er (Fig. 4c) and H (Fig. 4d) were obtained with the blunt indenter due to biasing of the measurements by the viscoelastic deformation of the viable epidermis. However, extrapolation of the fitted curve to a contact depth of 1 nm yields Er ¼0.80 GPa, which is close to that of stratum corneum obtained with the sharp indenter. Data corresponding to contact depths 4500 nm are considered to be indicative of the viable epidermis. Consequently, Er and H of the viable epidermis, obtained by averaging the data measured with the blunt indenter in the contact depth range 500–1300 nm, are equal to 0.2170.05 GPa and 1.370.5 MPa, respectively. As mentioned earlier, the mechanical properties of the dermis layer were measured with the microindentation apparatus, because the contact depth range was beyond the maximum z-displacement of the nanoindenter. Also, a blunt nanoindenter (R¼ 20 mm) was used to avoid excessive damage of the very soft dermis. Both Er (Fig. 4e) and H (Fig. 4f) of dermis decrease with the increase of hc,max, attaining steady-state values of 1.91 70.88 MPa and 0.857 0.45 MPa, respectively, for hc,max 4 300 mm. The mechanical properties of skin were also studied with the microindentation apparatus due to the large contact depths. Er (Fig. 4g) and H (Fig. 4h) of skin sharply decrease with increasing hc,max, reaching steady-state values of 3.777 1.7 MPa and 1.43 70.58 MPa, respectively, for hc,max 4100 μm. From curve fitting, Er at hc,max E5 μm is estimated to be 0.80 GPa, which is close to that of stratum corneum (0.87 GPa), whereas Er at hc,max E200 μm is equal to 2 MPa, which is close to that of the dermis layer (1.91 MPa). 3.4. Surface deformation Fig. 2. Cross-sectional optical microscopy images of porcine skin (American Yorkshire) at different magnifications. Image (b) shows (from top to bottom): stratum corneum ( 10 μm thick), viable epidermis (20–100 μm thick), and dermis (a few mm thick).
3.2. Effect of breed type on skin mechanical properties Er of stratum corneum does not show statistical differences among different breeds (Fig. 3a, p¼0.287). A similar observation can be made for data obtained from randomly selected samples of different breeds (Fig. 3b, p¼ 0.226). Similar to stratum corneum, Er of dermis does not show a dependence on breed type (Fig. 3c, p¼ 0.887) and sample selection or test order (Fig. 3d, p¼ 0.981). Insignificant differences in Er of skin are also shown for different breeds (Fig. 3e, p¼0.88) and sample selection or test order (Fig. 3f, p¼0.999); however, the corresponding standard deviation is very high due to the significant scatter of these measurements. Fig. 3 shows that stratum corneum, dermis, and skin of different breed types exhibit similar elastic properties. Therefore, skin mechanical properties presented and discussed next are independent of breed type and order of sample testing. In addition, considering the fairly large number of samples in the age range examined and statistically indifferent data shown in Fig. 3, it may be inferred that age and gender effects on the measured mechanical properties were insignificant. 3.3. Reduced elastic modulus and hardness Er and H of stratum corneum show a nonlinear decrease with increasing hmax for relatively sharp (R¼1 mm) and blunt (R¼20 mm) indenters (Fig. 4a–d). Er (Fig. 4a) and H (Fig. 4b) of stratum corneum,
Additional evidence of the indentation (contact) depth effect on skin deformation was obtained with optical microscopy performed after 1 h from testing. Shallow indentations caused only slight burnishing on the stratum corneum surface (Fig. 5a), presumably a result of localized sliding against the indenter surface, indicating mainly elastic deformation in the bulk of stratum corneum. Alternatively, deep indentations induced gross plastic deformation, resulting in localized fracture of the stratum corneum after unloading (Fig. 5b).
4. Discussion Insignificant differences in the mechanical properties of stratum corneum, dermis, and skin of different porcine breeds and randomly selected samples of each breed (Fig. 3) indicated that the measured mechanical properties were independent of breed type and order of sample testing. Table 1 summarizes the mechanical properties of individual skin layers and whole skin extracted from nano/microindentation experiments with relatively sharp (stratum corneum) and blunt (all other samples) indenters (to account for differences in material compliance and softness affecting the contact depth range) and indentation mechanics analysis (Eqs. (S1)–(S6) in Supporting information). To avoid biasing of the measurements by the high surface roughness (rms¼200–300 nm), nanoindentation measurements were obtained with a sharp (R¼ 1 mm) indenter for hc,max 4500 nm. Because of the less pronounced roughness effect on the measurements made with a blunt (R¼20 mm) indenter, reliable measurements were obtained for a smaller range of hc,max. However, these measurements were influenced by the high compliance of the viable epidermis, as evidenced by the nonlinear decrease of Er and H of
T. Jee, K. Komvopoulos / Journal of Biomechanics 47 (2014) 1186–1192
1189
Fig. 3. Effect of breed type and sample or test order on reduced elastic modulus of (a, b) stratum corneum, (c, d) dermis, and (e, f) skin of three different porcine breeds: American Yorkshire (AY), Berkshire (B), and Duroc–Berkshire Cross (DBC). The measurements were obtained with a sharp indenter (R ¼1 mm) and contact depth fixed at 1 mm for stratum corneum and a blunt indenter (R ¼20 mm) and contact depth fixed at 300 mm and 70 mm for dermis and skin, respectively. (Set size: (a) 53 (AY ¼ 10, B ¼ 21, DBC ¼ 22), (b) 51, (c) 35 (AY¼ 9, B ¼13, DBC ¼ 13), (d) 34, (e) 72 (AY ¼13, B ¼29, DBC ¼30), and (f) 71.)
stratum corneum with increasing hc,max (Fig. 4a–d). This trend is attributed to the deformation effect of the highly compliant and soft viable epidermis, which becomes more significant with increasing contact depth. This problem was overcome by extrapolating the fitted curve into the low range of hc,max. Using this approach, Er of stratum corneum measured with the blunt indenter (0.80 GPa) was found to be in good agreement with the directly measured value obtained with the sharp indenter (0.87 GPa). The Er of stratum corneum measured in this study is in good agreement with the results of previous studies (Park and Baddiel, 1972; Nicolopoulos et al., 1998). Using the same
curve-fit/extrapolation approach, H of skin is estimated to be 1.43 MPa, which is much lower than that of stratum corneum (15.6 MPa) and slightly higher than that of the viable epidermis (1.30 MPa). This is attributed to the dominant effect of the viable epidermis and dermis, which are much thicker and softer than the stratum corneum. The significantly lower Er (1.91 MPa) and H (0.85 MPa) of dermis compared to stratum corneum (by about three and one orders of magnitude, respectively) explains the sharp decrease of the mechanical properties of skin with increasing hc,max (Fig. 4g and h). For small contact depths, the mechanical properties of skin
1190
T. Jee, K. Komvopoulos / Journal of Biomechanics 47 (2014) 1186–1192
Fig. 4. Reduced elastic modulus and hardness of (a)–(d) stratum corneum, (e, f) dermis, and (g, h) skin versus maximum contact depth. The measurements were obtained with sharp (a, b) and blunt (c)–(h) indenters of radius equal to 1 and 20 mm, respectively. Curve fits indicate general trends.
are controlled by those of stratum corneum, whereas for relatively large contact depths, the mechanical behavior of skin is dominated by the properties of the viable epidermis and the dermis. Thus, the loading response of skin subjected to shallow indentation is governed by the mechanical behavior of stratum corneum, while the unloading response of skin recovering from deep indentation is mostly influenced by the elastic behaviors of the viable epidermis and the dermis.
The presented results indicate that irreversible skin deformation commences beyond a critical contact depth, on the order of the stratum corneum thickness ( 10 μm), due to the inadequate support of the highly compliant and soft viable epidermis and dermis. Because the rate of cell and tissue regeneration in injured viable epidermis is much larger than the replenishment rate of stratum corneum by the viable epidermis (Candi et al., 2005), understanding the role of the mechanical properties of individual
T. Jee, K. Komvopoulos / Journal of Biomechanics 47 (2014) 1186–1192
1191
blood or interstitial fluid sampling using microneedle-based procedures. Conflict of interest statement The authors confirm that there are no conflicts of interest.
Acknowledgments This work was partially funded by the Center for Information Technology Research in the Interest of Society (CITRIS), University of California, Berkeley. The authors thank Y. Seo and D.W. Gao from the School of Medicine, University of California, San Francisco, for the donation of the American Yorkshire porcine samples, Professor S. Li from the Bioengineering Department, University of California, Berkeley, for helpful discussions, and F. Yuan for assistance in histology staining.
Appendix A. Supplementary materials Supplementary information associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.jbio mech.2013.10.020. References
Fig. 5. Optical microscope images of skin samples (American Yorkshire) obtained after 1 h from testing with a blunt indenter of radius equal to 12.5 mm for a maximum displacement equal to (a) 60 and (b) 500 mm.
Table 1 Mechanical properties of individual skin layers and whole skin extracted from nano/microindentation experiments. Material a
Stratum corneum Viable epidermisb Dermisb Skinb,c a b c
Reduced elastic modulus Er (GPa)
Hardness H (MPa)
0.87 7 0.42 0.217 0.05 0.001917 0.00088 0.003777 0.0017
15.6 710.0 1.30 70.5 0.85 70.45 1.43 70.58
Sharp indenter (R¼ 1 mm). Blunt indenter (R ¼20 mm). hc,max 4100 mm.
layers on irreversible skin deformation is of paramount importance to skin repair. The results of this study provide insight into the mechanical behavior of skin due to indentation loading, which is of particular importance to minimally invasive procedures relying on effective penetration of the stratum corneum, such as transdermal drug delivery, local tissue and gene delivery, and
Agache, P.G., Monneur, C., Leveque, J.L., De Rigal, J., 1980. Mechanical properties and Young's modulus of human skin in vivo. Arch. Dermatol. Res. 269, 221–232. Agner, T., Serup, J., 1990. Sodium lauryl sulphate for irritant patch testing – a dose– response study using bioengineering methods for determination of skin irritation. J. Invest. Dermatol. 95, 543–547. Alexander, H., Cook, T.H., 1977. Accounting for natural tension in the mechanical testing of human skin. J. Invest. Dermatol. 69, 310–314. Archer, C.B., 2010. Functions of the skin. In: Burns, T., Breathnach, S., Cox, N., Griffiths, C. (Eds.), Rook's Textbook of Dermatology, eighth edition, Willey-Blackwell, Oxford, UK, pp. 4.1–4.11. Auriol, F., Vaillant, L., Machet, L., Diridollou, S., Lorette, G., 1993. Effects of shorttime hydration on skin extensibility. Acta Derm.-Venereol. 73, 344–347. Berardesca, E., Borroni, G., Gabba, P., Borlone, R., Rabbiosi, G., 1986. Evidence for elastic changes in aged skin revealed in an in vivo extensometric study at low loads. Bioeng. Skin 2, 261–270. Bhattacharya, A.K., Nix, W.D., 1988. Analysis of elastic and plastic deformation associated with indentation testing of thin films on substrates. Int. J. Solids Struct. 24, 1287–1298. Brazzelli, V., Borroni, G., Vignoli, G.P., Rabbiosi, G., Cavagnino, A., Berardesca, E., 1994. Effects of fluid volume changes during hemodialysis on the biophysical parameters of the skin. Dermatology 188, 113–116. Candi, E., Schmidt, R., Melino, G., 2005. The cornified envelope: a model of cell death in the skin. Nature Rev.: Mol. Cell Biol. 6, 328–340. Cooper, E.R., Missel, P.J., Hannon, D.P., Albright, G.B., 1985. Mechanical properties of dry, normal, and glycerol-treated skin as measured by the gas-bearing electrodynamometer. J. Soc. Cosmet. Chem. 36, 335–348. Cua, A.B., Wilhelm, K.-P., Maibach, I.H., 1990. Elastic properties of human skin: relation to age, sex, and anatomical region. Arch. Dermatol. Res. 282, 283–288. Dikstein, S., Hartzshtark, A., Bercovici, P., 1984. The dependence of low-pressure indentation, slackness, and surface pH on age in forehead skin of women. J. Soc. Cosmet. Chem. 35, 221–228. Diridollou, S., Berson, M., Vabre, V., Black, D., Karlsson, B., Auriol, F., Gregoire, J.M., Yvon, C., Vaillant, L., Gall, Y., Patat, F., 1998. An in vivo method for measuring the mechanical properties of the skin using ultrasound. Ultrasound Med. Biol. 24, 215–224. Diridollou, S., Patat, F., Gens, F., Vaillant, L., Black, D., Lagarde, J.M., Gall, Y., Berson, M., 2000. In vivo model of the mechanical properties of the human skin under suction. Skin Res. Technol. 6, 214–221. Falanga, V., Bucalo, B., 1993. Use of a durometer to assess skin hardness. J. Am. Acad. Dermatol. 29, 47–51. Finlay, B., 1971. The torsional characteristics of human skin in vivo. Biomed. Eng. 6, 567–573. Grahame, R., 1969. Elasticity of human skin in vivo. Ann. Phys. Med. 10, 130–136. Grahame, R., Holt, P.J.L., 1969. The influence of ageing on the in vivo elasticity of human skin. Gerontologia 15, 121–139. Gunner, C.W., Hutton, W.C., Burlin, T.E., 1979. The mechanical properties of skin in vivo – a portable hand-held extensometer. Br. J. Dermatol. 100, 161–163. Hendriks, F.M., Brokken, D., Oomens, C.W.J., Bader, D.L., Baaijens, F.P.T., 2006. The relative contributions of different skin layers to the mechanical behavior of human skin in vivo using suction experiments. Med. Eng. Phys. 28, 259–266.
1192
T. Jee, K. Komvopoulos / Journal of Biomechanics 47 (2014) 1186–1192
Kalis, B., De Rigal, J., Léonard, F., Lévêque, J.L., Riche, O., Le Corre, Y., De Lacharriere, O., 1990. In vivo study of scleroderma by non-invasive techniques. Br. J. Dermatol. 122, 785–791. Leveque, J.L., de Rigal, J., Agache, P.G., Monneur, C., 1980. Influence of ageing on the in vivo extensibility of human skin at a low stress. Arch. Dermatol. Res. 269, 127–135. McGrath, J.A., Uitto, J., 2010. Anatomy and organization of human skin. In: Burns, T., Breathnach, S., Cox, N., Griffiths, C. (Eds.), Rook's Textbook of Dermatology, eighth edition, Willey-Blackwell, Oxford, UK, pp. 3.1–3.53. Nicolopoulos, C.S., Giannoudis, P.V., Glaros, K.D., Barbenel, J.C., 1998. In vitro study of the failure of skin surface after influence of hydration and preconditioning. Arch. Dermatol. Res. 290, 638–640. Ohura, T., Sugihara, T., Honda, K., 1980. Postoperative evaluation in plastic surgery using the bio-skin tension meter. Ann. Plast. Surg. 5, 74–82. Park, A.C., Baddiel, C.B., 1972. Rheology of stratum corneum—I: a molecular interpretation of the stress–strain curve. J. Soc. Cosmet. Chem. 23, 3–12.
Peck, S.M., Glick, A.W., 1956. A new method for measuring the hardness of keratin. J. Soc. Cosmet. Chem. 7, 530–540. Salter, D.C., McArthur, H.C., Crosse, J.E., Dickens, A.D., 1993. Skin mechanics measured in vivo using torsion: a new and accurate model more sensitive to age, sex and moisturizing treatment. Int. J. Cosmet. Sci. 15, 200–218. Sanders, R., 1973. Torsional elasticity of human skin in vivo. Pflügers Arch. 342, 255–260. Schmook, F.P., Meingassner, J.G., Billich, A., 2001. Comparison of human skin or epidermis models with human and animal skin in in-vitro percutaneous absorption. Int. J. Pharm. 215, 51–56. Sugihara, T., Ohura, T., Homma, K., Igawa, H.H., 1991. The extensibility in human skin: variation according to age and site. Br. J. Plast. Surg. 44, 418–422. Warren, R., Gartstein, V., Kligman, A.M., Montagna, W., Allendorf, R.A., Ridder, G.M., 1991. Age, sunlight, and facial skin: a histologic and quantitative study. J. Am. Acad. Dermatol. 25, 751–760.
