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Biomaterials 27 (2006) 785795

Mechanical properties of human stratum corneum: Effects of

temperature, hydration, and chemical treatment

Kenneth S. Wua, William W. van Osdolb, Reinhold H. Dauskardtc,

aDepartment of Mechanical Engineering, Stanford University, Stanford, CA 94305, USAbALZA Corporation, Mountain View, CA 94039, USA

cDepartment of Materials Science and Engineering, Stanford University, Stanford, 416 Escondido Mall, Bldg 550, Rm 550G, CA 94305-2205, USA

Received 4 March 2005; accepted 24 June 2005

Available online 10 August 2005

Abstract

An in vitro mechanics approach to quantify the intercellular delamination energy and mechanical behavior of isolated human

stratum corneum (SC) in a direction perpendicular to the skin surface is presented. The effects of temperature, hydration, and a

chloroformmethanol treatment to remove intercellular lipids were explored. The delamination energy for debonding of cells within

the SC layer was found to be sensitive to the moisture content of the tissue and to the test temperature. Delamination energies for

untreated stratum corneum were measured in the range of 18J/m2 depending on test temperature. Fully hydrated specimen

energies decreased with increasing temperature, while room-humidity-hydrated specimens exhibited more constant values of 24 J/

m2. Lipid-extracted specimens exhibited higher delamination energies of12J/m2, with values decreasing to 4J/m2 with increasingtest temperature. The peak separation stress decreased with increasing temperature and hydration, but lipid-extracted specimens

exhibited higher peak stresses than untreated controls. The delaminated surfaces revealed an intercellular failure path with no

evidence of tearing or fracture of cells. The highly anisotropic mechanical behavior of the SC is discussed in relation to the

underlying SC structure.

r 2005 Elsevier Ltd. All rights reserved.

Keywords: Mechanical properties; Fracture toughness; Epithelial cell; Stratum corneum; Tissue treatment

1. Introduction

The layered construction of skin has components that

possess mechanical properties needed to accommodate

intrinsic and imposed mechanical stresses, abrasions and

penetration of foreign objects under variable ambient

moisture and temperature conditions. It must also haveother physical properties that resist the presence of toxic

environmental chemicals, pathogens, and radiation [1].

More severe environmental conditions may result from

occlusion of the skin via application of adhesive

dressings or transdermal drug delivery patches which

can locally elevate moisture content and affect mechan-

ical behavior. The detailed cellular and intercellular

structures of the outermost layer of the epidermis, the

stratum corneum (SC), have been widely studied [25].However, relatively few studies have examined the

mechanical and fracture properties of SC to determine

their dependence on tissue treatment and environmental

conditions[611].In particular, surprisingly few studies

have reported on the mechanical and delamination

properties of the SC in the direction normal to the skin

surface[1215].

The SC consists of layered anucleated cells that

mature and subsequently detach in the natural renewing

ARTICLE IN PRESS

www.elsevier.com/locate/biomaterials

0142-9612/$- see front matter r 2005 Elsevier Ltd. All rights reserved.

doi:10.1016/j.biomaterials.2005.06.019

Abbreviations: SC, stratum corneum; RH, relative humidity; FH,

fully hydrated; RHH, room-humidity hydrated; SS, stress separation;

DCB, double-cantilever beam; CMT, chloroformmethanol treated;

SEM, standard error of the meanCorresponding author. Tel.: +1 650725 0679;

fax: +1 650725 4034.

E-mail address: [email protected] (R.H. Dauskardt).
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process of desquamation. The disk-shaped SC cells, or

corneocytes, composed largely of aligned keratin fila-

ments, have been likened to bricks bound together by a

lipid-rich mortar [16]. Intercellular lipids have been

identified as the primary pathway for chemical diffusion

and as the barrier to water permeability through the SC

layer [1720]. In addition to the intercellular lipids,degraded desmosomal protein junctions, or corneo-

somes, at the cell boundaries are central to SC cohesion

and renewal, and their degradation is necessary for

desquamation[2126]. Ultrastructural studies of the SC

have suggested that increased moisture content is

associated with elevated desmosome degradation and

disruption of intercellular lipid structures, which affect

both mechanical properties and permeability [25,27].

Changes in delamination properties of SC with

environmental conditioning have been simply demon-

strated by a cellophane tape-stripping technique. SC

hydrated for 24 h prior to tape stripping permits cells to

be removed more easily, requiring fewer applications of

tape to fully strip the SC from the epidermis [12].

Another approach involved use of a cohesograph to

measure SC cohesive strength[13]. Similar devices have

been used to examine the bond strength of materials

applied to the skin, such as adhesive dressings [14].

While these techniques provide relative measures of

SC delamination strength, several inherent problems

exist that make the results qualitative and unreliable.

Most significantly, the delamination of SC adhered to

underlying tissue leads to a combined measurement of

both SC and substrate properties. Additionally, the SC

loading is nonuniform and can result in highly variableresults. In the case of the cohesograph, the force

necessary to pull the SC apart may be affected

substantially by the deformation behavior of the under-

lying tissue substrate. The tape-stripping technique

effectively peels the cell layers apart but, similar to the

peel adhesion test for thin films on elastic substrates,

provides qualitative results that are difficult to quantify

[28]. There is clearly a need for quantitative test

techniques to measure cohesion and strength properties

of SC accurately and reproducibly.

In the present study, a quantitative in vitro experi-

mental mechanics approach to examine the SC inter-cellular delamination energy and out-of-plane

mechanical behavior is presented. The delamination

energy of human SC was examined as a function of

selected testing temperatures and moisture precondi-

tioning treatments and related to the underlying cellular

structure. For comparison, human SC delipidized with

a chloroformmethanol treatment was examined in the

same manner. While some of the treatments and

temperatures are nonphysiological, they provide insight

into the microstructural mechanisms of SC cohesion and

help to isolate the role of individual components of the

SC such as intercellular lipids. SC delamination energy

was quantified in terms of the energy required to

propagate a debond through the SC layer and defined

in terms of the strain energy release rate G, measured in

units of J/m2. Stress-separation (SS) tests were per-

formed to measure the out-of-plane mechanical beha-

vior of the SC. Resulting failure surface morphologies

were examined with scanning electron microscopy toprovide an indication of the delamination mechanisms.

The delamination energy and strength property depen-

dence on temperature and moisture are rationalized in

terms of the underlying SC cell structure and inter-

cellular lipids.

2. Materials and methods

2.1. Tissue preparation

Human cadaver SC tissue was isolated for these experiments

from three female Caucasian donors, 7688 years of age, from

the thigh, abdomen or lower back. For each study as detailed

in the following sections, comparative tests were performed on

the same donor tissue to avoid variability between donors. The

SC tissue was separated from the underlying epidermis via a

trypsin enzymatic digest, then stored at 4 1C in a fully hydrated

(FH) state on water-moistened filter paper (Grade 595

General-Purpose Filter Paper, Schleicher & Schuell Micro-

Science GmbH, Dassel, Germany). For the untreated SC, two

sets of tissue were prepared. One set consisted of SC that was

FH at 100% relative humidity (RH) by storing on water-

moistened filter paper, and the other was room-humidity

hydrated (RHH) at 45% RH. Both sets were allowed to

equilibrate for at least 24 h. These initial hydration and testingconditions represent significantly different equilibrium SC

water contents corresponding to 300400% wt/wt and5%wt/wt SC water content, respectively[29,30].The thickness of

untreated RHH SC was measured with a micrometer to be

between 1535mm. The FH SC thickness was observed to

increase by 30% similar to values reported by others [31].

Additional tissue,60 60mm2, was delipidized with a 120-min 30 mL chloroform:methanol (2:1 by volume) soak with

two subsequent 30 min 30 mL water rinses. The SC thickness

was not observed to change significantly with treatment.

Chemically treated SC for SS tests was treated similarly, but

with a shortened 60-min chloroformmethanol soak. The

different treatment time is not believed to influence the lipid

extraction significantly. Studies with a similar chloroformmethanol treatment have shown that the majority of lipids are

removed within the first 15 min[32]. The treated SC was either

fabricated into FH test specimens immediately after treatment

or allowed to dehydrate in an ambient 45% RH environmentsimilar to the untreated SC.

2.2. Delamination energy measurements

The delamination energy of the SC tissue was examined

using fracture mechanics techniques developed to measure the

adhesive properties of highly viscoelastic pressure-sensitive

adhesives [33]. Similar techniques have been employed to

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measure the adhesive properties of polymer bone cements and

polymer layers in elastic substrates [3436]. The technique

involves sandwiching the SC between two elastic polycarbo-

nate substrates (Hyzods GP, Sheffield Plastics Inc., Sheffield,

MA) with a cyanoacrylate adhesive (Instant Krazyglue Gel,

Elmers Products Inc., Columbus, OH) to form fracture-

mechanics-based double-cantilever beam (DCB) specimens

(Fig. 1(a)). Cyanoacrylate adhesive polymerization is readilyinitiated by the presence of small amounts of water on the

bonding surfaces limiting the adhesive to the SC exterior. The

transparent polycarbonate beams facilitate optical inspection

of the inner sandwich structure during specimen preparation

and testing. To enable the use of linear elastic fracture

mechanics to determine the strain energy release rates,

substrate dimensions were chosen to ensure purely elastic

deformation of the substrates during testing [33,34].

To fabricate the specimens, a thin layer of cyanoacrylate

adhesive was applied to one face of a nominally

40 10 2.88 mm3 polycarbonate substrate, leaving a 710-mm region of the beam end uncoated. The substrate was

pressed against the SC on the filter paper backing and a scalpelwas used to cut around the substrate to detach the adhered SC

from surrounding tissue. In the case of RHH SC, the

freestanding film was prepared similarly with a sheet of paper

as a backing. To form the final sandwich structure, another

substrate coated with adhesive in the same manner was pressed

against the SC face of the complimentary beam with adhesive-

free ends aligned. Excess adhesive along the sandwich edges

was removed with a scalpel to ensure that the two halves of the

sandwich structure were bound together by SC only.

DCB specimens containing either untreated or chloroform

methanol-treated (CMT) SC were placed in an environmental

chamber (Model LH-6, Associated Environmental Systems,

Ayer, MA) at selected temperature (10, 25, 75 1C) and RH

(45, 85% RH) conditions and allowed to equilibrate for

10 min. Tests involving FH specimens were conducted in an85% RH environment with RHH specimens examined at 45%

RH unless specified otherwise. The specimens were loaded via

attached loading tabs to propagate a debond within the SC

layers. The specimens were tested in a custom-built mechanical

test system with a computer-controlled DC servoelectric

actuator operated in displacement control. Tests were per-formed at a constant displacement rate of 2 mm/s. Correspond-

ing loads were measured using a 222 N load cell. The

delamination length, a, was measured from recorded load

displacement,PD, and their elastic compliance relationship:

C DP

23

a 0:64h 3E0I

, (1)

where I bh3=12, and C is the specimen compliance, Pis theload, D=2 is the corresponding displacement of each beamfrom its original position at the loading point, E0 E=1 n2is the plane strain Youngs modulus for the polycarbonate, n is

Poissons ratio, I is the area moment of inertia, b is the

polycarbonate substrate width, and h is the height of eachpolycarbonate beam.

By measuring the critical load, P, and the delamination

length, a, at incipient crack extension, the delamination

resistance, Gc, was determined from critical values of the

strain energy release rate, G[33,34,37]:

G 12P2a2

b2h3E0 1

ffiffiffi5

p

2

h

a 1

2

h

a

2 !. (2)

Multiple delamination energies, Gc, were measured for each

DCB specimen by recording the critical loads, Pc, and

associated delamination lengths, a, during delamination

extension (Fig. 2). For the present specimens, the values of

E and n for the polycarbonate were 2.379 GPa and 0.38,respectively. Given the thin film nature of the SC compared to

the massive polycarbonate substrate, the contribution of the

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StratumCorneum

P

Delamination

a

P

Polycarbonate

Polycarbonate

b

h

h

b

h

P

P

StratumCorneum

Polycarbonate

b

h

(a)

(b)

Fig. 1. Fracture mechanics specimen geometries. (a) DCB geometry

illustrating relevant loading parameters (P,D), delamination extension

as measured from loading axis points (a) and relevant specimen

dimensions (b 10mm, h 2:88 mm, length 40 mm). (b) SS speci-men configuration showing loading axis and relevant dimensions

(b

10mm, h

2:88mm).

0 10 200.0

1.0

2.0

3.0

Delamination

Energy,

Gc

(J/m2)

Crack Extension, a (mm)

Fig. 2. Typical delamination energy as a function of delamination

extension. Illustration of the variation in delamination energy (Gc) as a

function of crack length (Da) for a RHH (45% RH) SC specimentested in a DCB configuration at 10 1C, 45% RH.

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elastic strain energy in the SC layer can be ignored in the

analysis[28].

2.3. Stress-separation measurements

Structures containing SC sandwiched between polycarbo-

nate substrates were fabricated in a similar manner to the DCB

specimens (Fig. 1(b)). The nominal substrate dimensions were

10 10 2.88 mm3. The specimens were loaded normal to theSC face via attached loading tabs in an environmental

chamber with controlled temperature and humidity.

SS measurements were performed under the same condi-

tions and SC hydrations as those for the DCB experiments.

Testing was conducted at a constant displacement rate of

1mm/s, yielding a machine compliance-corrected initial strain

rate of0.007 s1 in the SC for an SC thickness of 10 mm. Thespecimens were allowed to equilibrate for20 min prior totesting.

2.4. Scanning electron microscopy

Both DCB and SS specimens were examined after mechan-

ical testing using a scanning electron microscope (Hitachi

S-2500, Hitachi, Tokyo, Japan) to characterize the fracture

surface morphologies. Selected specimens were allowed to dry

in ambient conditions (25 1C, 45% RH), gold or gold-palladium coated, then examined in the electron microscope

operated at 15 kV. Multiple specimens from each testing

condition were inspected to ensure representative character-

ization.

2.5. Statistical analysis

Delamination energies are presented as mean 71.96 standard error of the mean (SEM) in which the mean values

reported are expected to fall within these bounds with 95%

confidence. On average, n 38 for each test condition.Delamination energies were compared using the Wilcoxon

test for independent samples. Statistical significance was set at

1% or 5% as specified in the figures. SS measurements are

presented as mean7standard deviation (SD). Further statis-

tical analysis was not performed due to the small sample sizes

for each test condition (n 324).

3. Results

3.1. Quantifying delamination energies

The delamination energy, Gc, measured as a function

of the delamination length, Da, for a specimen contain-

ing RHH SC is shown inFig. 2. The data represent a so-

called delamination resistance curve, and were ob-

tained from testing of a single DCB specimen. The

variability in the measured Gcvalues as seen in the curve

was predominantly related to inhomogeneities in the SC

along the DCB length. Typically, four cantilever speci-

mens were tested for each condition, each yielding

approximately 712 data points. All data points

(navg 38) for a given test condition were subsequentlyaveraged to produce the delamination resistance values,

Gc, represented in Figs. 3 and 4. Note that the

delamination tip strain rates do not vary significantlyas a function ofDa during such tests, as determined by

modeling and experimental examination of similar tests

on pressure-sensitive adhesives [38]. This reduces the

effects of varying strain rate on the measured delamina-

tion energies.

3.2. Delamination energy variation with temperature and

hydration

Delamination energy values, Gc, for specimens con-

taining RHH and FH SC measured at selected

temperatures are presented inFigs. 3(a) and 4(a). Each

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00.0

1.0

2.0

3.0

4.0

5.0

6.0

+

++*

*

DelaminationEnergy,

Gc

(J/m2)

Temperature (C)

0.0

0.2

0.4

0.6

0.8

1.0

45% R.H.

100% R.H.

PeakStress,

(

MPa)

Temperature (C)

10 20 30 40 50 60 70 80

0 10 20 30 40 50 60 70 80

45% R.H.

100% R.H.

(a)

(b)

Fig. 3. The variation in delamination energy and peak stress with

temperature and hydration for untreated SC. (a) Delamination energy

values from DCB experiments performed at selected temperatures and

hydrations (Po0:01 except *Po0:05, +P40:05). (b) Peak stressesfrom SS experiments similarly performed at selected temperatures and

hydrations. Error bars: (A) mean71.96 SEM and (B) mean7SD.


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plot represents data from a single SC donor to mitigate

tissue specimen variations. SS experiments were per-

formed on the same SC as that in Fig. 3(a). The resultsare shown in Fig. 3(b). Further DCB tests were

performed on the same SC as that in Fig. 4(a) after

chloroformmethanol treatment. These results are pre-

sented inFig. 4(a). With increasing test temperature, the

untreated FH specimens exhibited markedly lower

delamination energies. In contrast, the RHH specimen

energies were not statistically different with increasing

test temperature, except between 10 and 25 1C for results

shown in Fig. 4(a). Note that the Gc values for the

untreated 45% RH specimens in Fig. 3(a) were

significantly higher than the untreated 45% RH speci-

mens of Fig. 4(a). The reasons for these differences

remain unclear. One possible explanation is that SC

specimens from different donors possess variable

properties that elicit different delamination energy

measurements.

3.3. Comparison to stress-separation results

The results of SS tests performed on RHH and FH

specimens from the same SC as that represented in

Fig. 3(a) and under the same environmental testing

conditions are presented in Fig. 3(b). Typically, 34

measurements were taken at each testing condition, with

each measurement requiring one specimen. SS tests

resulted in specimen loading up to a peak stress, after

which the stress decayed rapidly with continued

displacement. Peak stress values obtained from the SS

curves are shown in Fig. 3(b). The peak stresses were

observed to decrease with increasing hydration and

testing temperature, exhibiting similar behavior to the

delamination energy trends seen for the DCB specimens.

3.4. Comparison to delipidized stratum corneum

Delamination in the CMT SC specimens exhibited

markedly different behavior compared to the untreated

specimens. The corresponding data are shown in

Fig. 4(a). Most notably, at each test temperature the

measured delamination energies were significantly high-

er than those of their untreated counterparts and

proportionally smaller differences were observed be-

tween the delamination energies of the RHH and FH

specimens. In contrast to the untreated specimens,statistically significant decreases in Gc values were

observed for the highest test temperature compared

to the lower test temperatures regardless of initial

hydration.

A comparison between untreated and CMT SS

specimens reveals similar trends to those observed

during delamination testing. Delipidized SS specimens

exhibited generally higher peak stresses compared to

their untreated counterparts as shown inFig. 4(b). The

significant scatter in the untreated control specimens in

Fig. 4(b) was not characteristic of most of the SS tests

performed. These specimens possessed the same loadingcharacteristics with loading to a peak stress, followed by

significant load decreases with increasing displacement.

Unlike the control group, the treated specimen peak

stresses exhibited the same trend with testing tempera-

ture regardless of hydration.

3.5. Examination of delamination surface morphologies

Representative scanning electron micrographs of the

fracture surfaces of DCB specimens containing FH and

RHH hydrated SC tested at 25 1C are shown inFig. 5.

Similar micrographs were obtained for such specimens

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0.0

4.0

8.0

12.0

16.0

****

++++

+

+*

*

DelaminationEnergy,

Gc

(J/m2)

Temperature (C)

45% R.H.

100% R.H.

CMT 45% R.H.

CMT 100% R.H.

0.0

0.5

1.0

1.5

2.0

45% R.H.

100% R.H.

CMT 45% R.H.

CMT 100% R.H.

PeakStress,

(

MPa)

Temperature (C)

0 10 20 30 40 50 60 70 80

0 10 20 30 40 50 60 70 80

(a)

(b)

Fig. 4. Comparison of untreated and chloroformmethanol-extracted

SC. (a) Delamination energy measured in DCB tests performed

on untreated and CMT SC at selected temperature (10, 25, 75 1C)

and hydration (45, 100% RH) (Po0:01 except *Po0:05,+,++,z,**P40:05). (b) Peak stress data from SS measurementsconducted at the same temperatures and hydrations for both untreated

and CMT SC. Data offset for clarity. Error bars: (a) mean71.96-

SEM and (b) mean7SD.


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at each temperature. For the FH specimens, individual

SC cells are difficult to discern and surface features

were characterized by tortuous surface undulations

(Fig. 5(a)). The RHH specimen micrograph reveals a

different surface morphology compared to FH fracture

surfaces, showing partial pull-up of individual SC cells.

This produced fracture surface roughness characterized

by individual cellular features (Fig. 5(b)). The micro-

graphs of delipidized specimens revealed the same

difference in fracture surface appearance between

RHH and FH specimens. Fracture surfaces of SS

specimens yielded morphologies similar to those seen

in the DCB specimens in which the RHH specimens

exhibited SC cell pull-up, while the FH specimens lacked

similar cellular features.

4. Discussion

4.1. Origin of delamination energies

As far as fracture properties of materials are

concerned, the SC delamination energy values are

comparatively low. Polymer layers bonded weakly to

elastic substrates may exhibit similar values, but with

more strongly covalently bonded interfaces, fracture

energies increase significantly above 10 J/m2 [35,36,39].

In most of these cases, plastic deformation at the cracktip contributes significantly to the delamination fracture

energy. During delamination of the SC layer, energy is

dissipated by separation of intercellular boundaries,

plastic deformation of the SC layer, and by the work

done during cell pull-up.

To determine the extent of possible plastic deforma-

tion near the crack tip, plastic zone radius estimates, rp,

were obtained using the well-known plane strain plastic

zone expression:

rp 16p

GcE0

s2ys

, (3)

where the plane strain modulus, E0, and the yieldstrength, sys, are for the SC layer. The values ofE

0 wereobtained from the SS initial loading slopes and found to

be in the range 125MPa. We note that there isconsiderable experimental uncertainty in the values ofE0

estimated. These are due to inherent difficulties in

determining the through thickness strain in the thin SC

layer, and values should be treated accordingly. Thesysvalues were taken as the peak stresses in the SS tests as

no obvious yielding was apparent prior to the peak. The

resulting plastic zone size estimates were 212mm,

corresponding to an average of 25% of the original

SC thickness, suggesting that only a fraction of the SC

thickness may have undergone plastic deformation. For

all of the tissue conditions examined, the failure path

occurred between corneocytes. Given the averagecorneocyte thickness of0.51mm, multiple layers ofcells on either side of the delamination may have

undergone plastic deformation. However, energy dis-

sipation by plastic deformation in a plastic zone

represents only one possible energy dissipation mechan-

ism. Alternatively, the cohesive properties of the cell

boundaries themselves may dominate the energy dis-

sipation with minimal general plastic deformation. This

has been substantiated by studies of graded delamina-

tion properties in which delamination energies increased

towards the lower SC as upper layers were removed[40].

Plasticity-dominated dissipation would suggest theopposite trend of decreasing delamination energy with

decreasing SC thickness.

Examination of the delamination surfaces of the DCB

and SS specimens supports the idea that changes in the

intercellular cohesive properties play a central role in

determining delamination energy. The scanning electron

micrographs reveal significant differences between the

RHH and FH specimens (Fig. 5), with both untreated

and delipidized SC showing similar topographic char-

acteristics. Notably, cell pull-up was present in the RHH

specimens (Fig. 5(b)), and similar results were observed

for the CMT specimens. The FH specimen delamination

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Fig. 5. DCB specimen delamination surface morphology. Scanning electron micrographs of DCB specimens containing (a) FH (100% RH), and (b)

RHH (45% RH) SC. Specimens tested at 25 1C corresponding to delamination energies reported inFig. 4(a). Images representative of DCB specimen

delamination surfaces regardless of treatment.


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surfaces presented no obvious cell pull-up and indivi-

dual corneocytes were not easily discernible (Fig. 5(a)).

Similar features were observed for CMT SC.

Consistent with the small variations inGcvalues with

temperature for RHH-untreated specimens (Figs. 3(a)

and 4(a)), corresponding fracture surface morphologies

exhibited no discernible differences in cell pull-updensity with temperature. However, no obvious mor-

phological changes were observed for the FH-untreated

specimens despite large variations in Gc values with

temperature (Figs. 3(a) and 4(a)). These observations

suggest that the delamination energy must be more

intimately tied to the cohesive properties of the

intercellular boundaries of the SC and not to the failure

morphology. This notion is supported by the similar

trends exhibited by the peak stress data. Similar to the

untreated specimens, the delipidized SC fracture surface

morphologies remained unchanged as a function of

temperature for each hydration condition despite

significantGc variations with temperature.

4.2. Environmental effects on SC structure

The marginal changes in delamination energy and

peak cohesive strength with increasing test temperature

exhibited by the untreated RHH SC compared to that of

the FH specimens indicate that SC cohesive strength

depends strongly on initial hydration. The commonly

observed failure of SC between corneocytes highlights

the relevance of the intercellular space to SC fracture

properties[22]. In a simplistic model, SC cells are viewed

as highly keratinized bricks surrounded by a lipidmortar (Fig. 6) [16]. This notion led to the proposal

that SC cohesive behavior is largely influenced by the

intercellular lipid characteristics[16,41,42,].

Additional studies probing the effect of hydration and

chemical treatment provide further insights. The micro-

structure of FH human SC has been examined using

freeze-fracture electron microscopy to reveal the swel-

ling of corneocytes, as well as the presence of water

pools in the intercellular spaces [30,43]. Other studies

have indicated that water disrupts the lipid lamellae to

varying degrees and that intercellular corneosomes

become degraded with time in the presence of water

[25,27,30]. Hydration also has been shown to influencelamellar lipid spacing in hairless mouse SC [44].

Examination of the affect of hydration on the lipid

orthorhombic to hexagonal phase transition tempera-

ture near 35 1C has revealed small decreases in transition

temperature with increasing hydration, suggesting that

hydration helps to fluidize the SC lipids [30]. However,

the precise effects of water on lipid ordering within the

lamellae remain undetermined with current evidence

suggesting that the lipids remain relatively unperturbed

by changes in SC hydration[30,45,46,]. The separation

of corneocyte interfaces by the presence of intercellular

water, particularly in conjunction with increases in

temperature, may be the cause of decreases in delamina-

tion energy and peak stress values for FH untreated SC

as seen inFigs. 3 and 4.

While the precise effects of hydration on lipid

ordering remain unclear, temperature has been observed

to affect the ordering of intercellular lipids by inducing

lipid disorder and phase changes. Specifically, heating-

induced alkyl-chain disordering of porcine SC lipids has

been correlated with increased permeability of water

through the SC membrane [47]. In addition, transmis-

sion electron diffraction has revealed gradual changes in

human SC lipid organization from an orthorhombic to

hexagonal to fluid phase when heated from 20 to 90 1C,representing changes toward a more disordered state

[48,49]. Differential scanning calorimetry of human SC

reveals major transitions near 70 and 80 1C, which have

been deduced to reflect intercellular lamellar lipid

melting then subsequent dissociation of lipidprotein

complexes formed at corneocyte envelope interfaces,

respectively [5052]. Despite such transitions, abrupt

changes in delamination energy with temperature are

not necessarily expected due to a competition between

decreased lipid cohesion and increased plastic dissipa-

tion from greater lipid fluidity. The lack of substantial

changes in delamination energy for RHH specimens asseen inFigs. 3(a) and 4(a)as well as unpublished results

examining a larger temperature range from 10 to 90 1C

further corroborate this notion.

The lack of drastic changes in delamination energies

and peak stress values for the RHH SC compared to

FH SC indicate that lipid disordering is not enough to

weaken the intercellular structure significantly (Figs. 3

and 4). Increased hydration in concert with increasing

temperature seems to play a key role in reducing

delamination energy and peak stress values. In patho-

logic skin states including atopic dermatitis and lamellar

ichthyosis, impaired barrier properties and a prevalence

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Fig. 6. Illustration of SC delamination front and microstructural

components. Schematic of the SC during delamination, illustrating

intercellular failure and showing cellular structure, including aligned

keratin filaments as well as lipid intercellular space with corneosomes.


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of hexagonal and fluid lipid phases suggest that these

lipid regimes may be associated with higher diffusive

mobility of substances through the SC [49]. At inter-

mediate temperatures where multiple phases coexist,

certain substances exhibit increased SC penetration

attributed to diffusion-enhancing phase boundaries

[45,49,53]. As temperature is increased, the lipid phasetransitions likely enable additional diffusion paths that

allow water molecules to penetrate and separate

interfaces otherwise inaccessible at lower temperatures

consistent with our observed decreases in delamination

energy for FH SC and the lack of large changes for the

RHH SC.

Given the implied significance of the lipid structures

on the cohesive behavior of the SC, removal of the

intercellular lipids remains important to understanding

their role in SC delamination properties. The inter-

cellular domain has been noted as a pathway for

chemical diffusion, as observed in transepidermal water

loss experiments and other chemical species tests

[17,20,54,55]. Studies have shown that isolated and fully

delipidized non-plantar SC cells require lipids for

aggregation and proper cohesion [42]. Furthermore,

mechanical tests on individual SC cells have yielded

substantially higher modulus values compared to

coherent SC tissue, highlighting the effect of intercel-

lular constituents on SC mechanical behavior[56]. Still,

understanding of the contribution of the intercellular

lipids to the cohesive strength of the SC remains

incomplete [54,57,58]. The chloroformmethanol lipid

extractions used in the present study have been noted to

remove key lipids believed to be responsible forregulation of desquamation, in particular, cholesterol

sulfate, whose excess is partly responsible for ichthyosis

[5860]. However, such chemical treatment does not

lead to cell dissociation as observed here and by others

[60]. Indeed, the measured delamination energy for the

treated specimens was substantially higher compared to

the untreated controls (Fig. 4(a)). SS tests on the treated

specimens also resulted in higher peak stresses (Fig.

4(b)). Similar observations have been noted by others

using less quantitative cohesometry techniques[22].

The effects of chloroformmethanol lipid extraction

on the SC reveal marked modifications to the inter-cellular space. Treatment with similar chloroform

methanol solutions has shown via thin-layer chromato-

graphy that the majority of the lipid lamellae are leached

from the specimen[32]. The treatment results in intimate

contact between unextracted lipids covalently bound to

the cornified envelopes of adjacent corneocytes and

seemingly leaves corneosomes unaltered[22,6164]. The

increased cohesion of delipidized SC has been attributed

to the interaction between remaining lipids in opposing

corneocyte envelopes and postulated to result from

interdigitation of the opposing envelope lipids [64,65].

Supporting this notion, extraction of the lamellae and

envelope lipids leads to dissociation of SC tissue into

individual cells [42]. While lipid extraction significantly

increased delamination energies compared to those of

untreated SC, the resulting fracture surfaces exhibited

similar features dependent on initial tissue hydration

(Fig. 5). Even with these differences in failure surface

morphologies, the chemically treated RHH and FHspecimens possessed delamination energy values and

peak stresses proportionately more similar than their

untreated counterparts, further highlighting the impor-

tance of intercellular lipids on SC cohesive properties

(Fig. 4).

In addition to intercellular lipid contributions to SC

integrity, the corneosome protein linkages between cells

are known to play a critical role in SC cohesion.

Corneosome degradation is accelerated with increasing

SC hydration [25]. Chapman et al. have shown that

adhesion testing on porcine SC reveals a gradient in SC

intercellular cohesion with progressively weaker bond-

ing from the interior toward the more superficial SC

layers[22]. These results were correlated with a gradient

in corneosome areal density, in which the number of

corneosomes between adjacent SC layers, or nonper-

ipheral corneosomes, was observed to decrease progres-

sively from the inner to outer cell layers[21,22]. Similar

observations have been made in human SC [26].

However, the DCB test results reveal more complex

trends than simply that increased hydration leads to

decreased cohesion (Figs. 3 and 4). It is likely that the

delamination energies are not correlated highly with

expected corneosome cohesive contributions due to the

location of the delamination in the few outer layers ofthe SC, as determined by examination of the graded

properties through the thickness of the SC[40].

4.3. Comparison of out-of-plane to in-plane mechanical

studies of stratum corneum

The reported in-plane properties of SC are signifi-

cantly different from the out-of-plane behavior pre-

sented in this study. These differences reveal the highly

anisotropic nature of the SC composite. With respect to

elastic behavior, in-plane tensile tests on newborn rat SC

led to measurements of decreasing tensile moduliranging from8800 to 12 MPa with increasing humid-ity, while similar tests on human SC revealed moduli

decreasing from80 to 20 MPa, which we calculatedassuming a SC thickness of 10 mm since only force, not

stress, data were reported[6,9]. Our own initial in-plane

tensile studies have yielded moduli decreasing from

1000 to 5 MPa with increasing hydration. Similardecreases in modulus for rat SC have been reported with

increasing temperature [9]. These variations have been

associated with the in-plane orientation of the inter-

mediate filaments in the keratinized corneocytes and the

substitution of existing proteinprotein hydrogen bonds

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with water-mediated bonding to facilitate greater fiber

mobility[7,9,66]. Associated changes in the intercellular

lipids with temperature and hydration may also affect

in-plane tensile moduli. In FH SC tensile tests,

corneocytes were observed to slide past each other,

highlighting the importance of intercellular components.

Compared to our human SC in-plane modulusmeasurements from1000 to 5 MPa with increasinghydration, initial out-of-plane modulus measurements

yielded values in the range of 125 MPa, substantiallylower than those of the in-plane values. Interestingly,

while SC lipid transitions have been observed to be

largely hydration independent, in the present study

mechanical properties are sensitive to the presence of

water to facilitate decreases in structural integrity and

cohesive strength, albeit for different reasons out-of-

plane and in-plane[30,45,46,66].

The out-of-plane SC peak cohesive strengths were

also found to be substantially reduced compared to in-

plane values. For human SC, in-plane strength values

have been reported to decrease from 22 to 5 MPa whentesting in 0100% RH environments with ether-treated

specimens yielded elevated strengths ranging from25to 19 MPa in the same environments [6]. Our prelimin-

ary in-plane tensile measurements gave similar results

with strengths ranging from18 to 2 MPa in testingenvironments of 10100% RH. In contrast, the out-of-

plane strengths are significantly lower in the range

0.10.8 MPa (Figs. 3(b) and 4(b)). The CMT SCspecimens exhibited somewhat higher out-of-plane

strength values of

0.41.4 MPa, but still significantly

lower than in-plane values. The lower out-of-planestrength values are not unexpected, given the continual

renewal of the SC which sheds corneocytes perpendi-

cular to the skin surface.

Only limited work to measure in-plane fracture

properties of human SC as a function of varying

environmental conditions has been reported. Mean

fracture energies of 3600 J/m2 for 76% RH-condi-tioned SC specimens have been measured in a tearing

configuration [10]. Subsequent work suggested that

delamination energy increases with increasing hydration

[11]. This trend was not observed in the delamination

results of the present study. The in-plane fracture energyis expected to be higher than that of the out-of-plane

direction, where lower cohesive values of18 J/m2 areneeded to facilitate natural desquamation. The magni-

tude of the difference is, however, surprising. Peripheral

corneosomes that help to connect the cells in-plane may

contribute to the high toughness values. Note, however,

that the in-plane tearing energies reported above were

calculated assuming linear elastic behavior. Viscoplastic

behavior of the SC, particularly at higher hydrations,

and the unconstrained nature of the tearing configura-

tion suggest that the reported in-plane fracture energies

should be treated with caution.

5. Conclusions

In conclusion, a fracture-mechanics-based approach

using DCB specimens has been developed to examine

quantitatively the delamination properties of human SC.

In conjunction with out-of-plane SS measurements, a

decrease in delamination energy with increasing testtemperature was observed in FH (100% RH) SC tissue.

Little effect of testing temperature was observed for

RHH (45% RH) SC tissue. The observed decrease in

delamination energy was associated with a reduction in

SC cohesive strength. In comparison, delamination

energy values of CMT specimens exhibited little hydra-

tion dependence and were higher than those of

untreated controls, as explained by corneocyte envelope

interactions. Further exploration is necessary to under-

stand the microstructural changes that occur with

delipidization, particularly in conjunction with tempera-

ture variation. Initial measurements of modulus reveal

that SC stiffness in this mode is much lower than that

reported in-plane. From comparisons to in-plane

experiments, the fracture energy and modulus data

reveal that a simple bricks and mortal model requires

refinement to explain the highly anisotropic mechanical

behavior exhibited by SC.

Acknowledgements

The authors would like to thank Dr. Jay Audett and

the ALZA Corporation for funding in support of the

research, and Ms. Nieves Crisologo and Ms. MrudulaPatel from ALZA Corporation for their assistance in

providing the stratum corneum. K.W. was supported by

a Stanford Graduate Fellowship.

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