Journal of Biomechanics 43 (2010) 3183–3190

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Journal of Biomechanics
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Epidermal differentiation governs engineered skin biomechanics

G.C. Ebersole a, P.M. Anderson a, H.M. Powell a,b,n
a
Department of Materials Science and Engineering, The Ohio State University, 116 W. 19th Ave., 243C Fontana Labs, Columbus, OH 43210, USA
b
Department of Biomedical Engineering, The Ohio State University, The Ohio State University, 116 W. 19th Ave., 243C Fontana Labs, Columbus, OH 43210, USA

a r t i c l e in fo abstract

Article history: Engineered skin must be mechanically strong to facilitate surgical application and prevent damage
Accepted 24 July 2010 during the early stages of engraftment. However, the evolution of structural properties during culture,
the relative contributions of the epidermis and dermis, and any correlation with tissue morphogenesis
Keywords: are not well known. These aspects are investigated by assessing the mechanical properties of
Tissue engineering engineered skin (ES) and engineered dermis (ED) during a 21-day culture period, including correlations
Skin with cellular metabolism, cellular organization and epidermal differentiation. During culture, the
Differentiation epidermis differentiates and begins to cornify, as evidenced by immunostaining and surface electrical
Strength capacitance. Tensile testing reveals that the ultimate tensile strength and linear stiffness increase
linearly with time for ES, but are relatively unchanged for ED. ES strength correlates significantly with
epidermal differentiation (po 0.001) and a composite strength model indicates that strength is largely
determined by the epidermis. These data suggest that strategies to improve ES biomechanics should
target the dermis. Additionally, time-dependant changes in average ES strength and percent elongation
can be used to set upper bound limits on mechanical stimulation profiles to avoid tissue damage.
& 2010 Elsevier Ltd. All rights reserved.

1. Introduction cell-seeded collagen constructs were significantly stiffer than their

Tissue engineered skin (ES) offers promise as an alternate
wound healing treatment when sufficient amounts of autograft
are not available, such as in massive burns. Numerous tissue ES
replacements have been created (Ehrenreich and Ruszczak, 2006;
Bello et al., 2001; Eaglstein et al., 1999; Berthod and Damour,
1997; Purdue et al., 1997; Clark et al., 1996; Wainwright et al.,
1996; Cooper et al., 1991; Boyce et al., 1988; Heimbach et al.,
1988) and clinically utilized ES has been shown to reduce the
donor site area required to permanently close wounds, mortality,
and morbidity from scarring (Boyce et al., 2006, 2002, 1995).
Unfortunately, ES, with published values of ultimate tensile
strength between 0.01 and 0.7 MPa (Powell and Boyce, 2009,
2006; Powell et al., 2009; LaFrance et al., 1995), is orders of
magnitude weaker than normal human skin. As a result, it is
difficult to apply surgically and subject to damage by mechanical
shear (Powell et al., 2009; Boyce et al., 2005).
Mechanical stimulation has been widely used to promote the
in vitro formation of engineered tissues such as blood vessels, heart
patches, bone and cartilage (Datta et al., 2006; De Croos et al., 2006;
Isenberg et al., 2006; Freyria et al., 2005; Stegemann and Nerem,
2003). For instance, mechanically stimulated mesenchymal stem
non-stimulated counterparts (Nirmalanandhan et al., 2008). In
fibroblasts on collagen scaffolds, cyclic strain produced increased
proliferation and thicker tissue constructs (Eastwood et al., 1998).
For ES, a 20% static strain during culture significantly increased
ultimate tensile strength and promoted epidermal differentiation
(Powell et al., 2009). Although mechanical stimulation can have
positive effects on engineered tissues, little is known regarding the
optimal magnitude of external mechanical stimuli for skin. Such
optimization requires better understanding and prediction of the
mechanical properties of ES as a function of time.
The goal of this study was to investigate the evolution of
mechanical properties in ES and correlate changes in mechanical
strength and stiffness with biological activity, including differ-
entiation and proliferation. The relative contribution of the
epidermis and dermis to the tissue properties was also investi-
gated. From this data, mathematical relations were developed to
describe the time dependant-behavior of ES. These relations could
be utilized to set load and strain limitations for dynamic
mechanical stimulation of ES.
2. Materials and methods
2.1. Scaffold preparation

n
Corresponding author at: Department of Materials Science and Engineering,
The Ohio State University, 116 W. 19th Ave., 243C Fontana Labs, Columbus, OH
43210, USA. Tel.: +1 614 247 8673; fax: +1 614 292 1537.
E-mail address: powell.299@osu.edu (H.M. Powell).
Electrospun collagen scaffolds were fabricated using a 10% w/v solution of
acid-soluble collagen (SEMED S) in 1,1,1,3,3,3-hexafluoro-2-propanol (HFP, Sigma,
St. Louis, MO). Matrices were spun at a potential of 30 kV onto an 8.5 cm2
grounding plate at a distance of 20 cm. The electrospun scaffolds were physically

0021-9290/$ - see front matter & 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.jbiomech.2010.07.026
3184 G.C. Ebersole et al. / Journal of Biomechanics 43 (2010) 3183–3190
crosslinked by vacuum dehydration at 140 1C for 24 h, then chemically crosslinked
in a solution of 5 mM 1-ethyl-3-3-dimethylaminopropylcarbodiimide hydrochlor-
ide (EDC, Sigma, St. Louis, MO) in 100% ethanol for 24 h (Powell et al., 2008). The
scaffolds were then disinfected in 70% ethanol for 24 h and rinsed thoroughly to
prepare for cell inoculation (Powell et al., 2008).
2.2. Engineered skin
Engineered human skin was prepared on the collagen scaffolds using human
dermal fibroblasts (HF) and epidermal keratinocytes (HK) following a procedure
previously described (Powell and Boyce, 2008). Briefly, fibroblasts were inoculated
onto the scaffolds at a density of 5  105 cells/cm2 and incubated at 37 1C and 5%
CO2 for 1 day. Subsequently, HKs were inoculated onto the cell-scaffold constructs
at a density of 1.0  106 cells/cm2. The day of keratinocyte inoculation corresponds
to day 0 of ES fabrication. The following day, the ES was lifted onto a perforated
stainless steel platform covered by a piece of sterile cotton to form an air–liquid
interface for the ES. The cells were incubated for 21 days with growth media
changed every 48 h.
2.3. Engineered dermis
To independently determine the properties of the dermis and epidermis,
dermal replacements were fabricated following the same protocol for ES with no
epidermal layer added on top of the dermis. HFs were inoculated into collagen
scaffolds at a density of 5  105 cells/cm2 and incubated in a 150 mm tissue
culture dish at 37 1C, 5% CO2 in UCMC 160 medium (Swope et al., 2006). As the
interaction between the fibroblasts and keratinocytes is essential for proper
tissue development, HKs were inoculated onto the bottom of the 150 mm tissue
culture dish at 1  106 cells/cm2 at day 0 to more closely mimic the ES
environment and allow for chemical communication between the two cell types.
The dermal constructs were incubated for 21 days with growth medium
exchanged every 48 h.
2.4. Surface electrical capacitance
Previous studies show that surface electrical capacitance (SEC) is inversely
related to barrier function in skin, and is a convenient, non-destructive measure of
skin surface hydration (Boyce et al., 1996). SEC measurements were collected from
the engineered skin in vitro using the NOVA dermal phase meter (DPM 9003,
NOVA Technology; Portsmouth, NH). On culture days 4, 7, 10, 14, and 21,
measurements were taken from six sites on each piece of ES and the SEC values are
expressed in pF7 standard deviation.
2.5. Cellular metabolism
On days 4, 7, 10, 14, and 21, 4-mm punch biopsies were collected from the ES
(2 punches/graft, 11 grafts per time point) and each was placed into a separate
well of a 24-well plate. A standard MTT assay was performed on the punch
biopsies. Briefly, a sterile MTT solution was made containing 0.5 mg/mL (3-(4,
5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) in phosphate buf-
fered saline (pH 7.4 PBS). The biopsy-containing wells were each filled with 0.5 mL
of the sterile MTT solution and incubated at 37 1C and 5% CO2 for 3 h. After the 3 h
incubation, the MTT solution was aspirated and replaced with 0.5 mL of methoxy
ethanol and agitated at room temperature for 3 h. The amount of MTT–formazan
product released was measured at 590 nm on a microplate reader (SpectraMax
190, Gemini) with values reported as mean optical density 7standard deviation.
2.6. Mechanical testing
Uniaxial tensile tests were performed on each piece of ES and ED on days 4, 7,
10, 14, and 21(ES n¼ 12, 4 ED n ¼5). For each test, 5 mm wide by 40 mm long
strips of tissue (to facilitate a gauge length of 30 mm) were cut from the
engineered tissues and placed into the compression grips of a 100R TestResources
mechanical tester (Shakopee, MN). Specimens were uniaxially loaded to 10% strain
at an elongation rate of 1 mm/s, held at 10% strain for 60 s, returned to the original
length at a rate of  1 mm/s, held for 60 s, then elongated to failure at 2 mm/s.
Ultimate tensile strength (UTS), percent elongation at failure, linear stiffness, and
relaxation rate were calculated from the mechanical testing data. The raw force–
time relaxation data during the 60 s hold at 10% strain were fit to
FðtÞ ¼ At n ð1Þ
where A is the force at t ¼0 and n is the time exponent. Eq. (1) captures
experimental trends from previous skin studies (Corr et al, 2009).
To quantify the mechanical properties of the epidermis alone, the ES was
soaked in a sterile solution of 4.2 U/ml Dispase II (Roche Applied Science, Penzbeg,
Germany) in HBS for 15, 30, and 60 min at 37 1C to facilitate the enzymatic
degradation of the basement membrane. The epidermis was then mechanically
separated using sterile blunt edged forceps. Although, great care was taken during
this process, the engineered epidermis could not be removed without significant
damage and thus its mechanical properties could not be quantified individually.
2.7. Histology
Biopsies for histology from each sample were collected at days 4, 7, 10, 14, and
21, embedded in OCTTM (VWR, West Chester, PA) in cross-section and cryosec-
tioned (n¼ 2 sections per graft per time point). Sections were stained with
hematoxylin and eosin (H&E) and imaged with light microscopy. Bright field
images were collected using Olympus DPController software (Olympus; Valley,
PA) with a total of eleven samples per condition per time point.
To identify markers of basement membrane formation and epidermal
differentiation, cryosections were fixed in methanol for 8 min. After fixation, the
samples were immunostained for human involucrin (Sigma, St. Louis, MO), human
laminin-5 (Santa Cruz Biotechnology, Santa Cruz, CA) and for cellular DNA (DAPI,
Molecular Probes, Eugene, OR). The triple-labeled sections were examined by
confocal microscopy (Olympus Flowview, Center Valley, PA) with a total of 2
biopsies per graft (n¼11) per time point. Total cell number per 20  field of view
was calculated at each time point with a total number of 4 fields of view per graft
(n ¼6) per time point.
2.8. Statistical analyses
All data was analyzed using SigmaStat 3.10 (Systat Software Inc; San Jose, CA).
One way ANOVA with Tukey post-hoc tests were performed with p o 0.05
considered significant. Correlations were determined using Pearson’s correlation
coefficient.
2.9. Composite strength model
The strength of engineered skin was modeled using the standard laminate
composite theory in which the dermis and the epidermis serve as parallel,
mechanically distinct layers (Matthews and Rawlings, 2003):
SðtÞ ¼ sue ðtÞfe ðtÞþ sud ðtÞfd ðtÞ ð2Þ
The quantities sue and sud are the stress in the epidermis and dermis, respectively,
when the tissue reaches the ultimate tensile strength, and fe(t) and fd(t) are the
corresponding epidermal and dermal volume fractions. Thus, Eq. (2) is a statement
of mechanical equilibrium along the in-plane direction of loading. It was applied at
five time points: ti ¼4, 7, 10, 14, and 21 days by replacing S(ti), fe(ti), fd(ti), with
experimentally measured values of the UTS and constituent volume fractions of
engineered skin. The volume fraction data were based on the average of n¼ 20
thickness measurements per histological image per graft using the ImageJ
software (NIH freeware). The dermal stress sud ðti Þ was replaced by Sd(ti), the
experimentally measured strength of engineered dermis at these time points.
Thus, Eq. (2) furnishes sue ðti Þ.
These estimates of sue ðti Þ are viewed as lower bounds to the epidermal
strength Se(ti) for two reasons. First, the epidermis may not be at peak strength
when the UTS of engineered tissue is reached. Second, the substitution
sud ðti Þ ¼ Sd ðti Þ supplies an upper bound to sud ðti Þ, so that Eq. (2) furnishes a lower
bound to sue ðti Þ.
The resulting five values of sue ðti Þ were used to calibrate a four parameter
sigmoidal function (Tichopad et al., 2002):
2D
Ssig
e ðtÞ ¼ A þ ð3Þ
1 þ eðtt0 Þ=t
This function is commonly employed to describe cell activity, such as
keratinocyte differentiation versus [Ca2 + ] (Walker et al., 2006), as well as
intracellular bonding strength (Baumgarter et al., 2000). Specifically, A, D, t0, and
t were chosen to minimize the squared error between the sue ðti Þ values obtained
from Eq. (2) and the predicted Ssig e ðti Þvalues from Eq. (3). The nature of the
sigmoidal function is that Se monotonically increases with time from Se(t5t0)  A
to Se(t0) ¼A + D to Se(t bt0)  A + 2D, where t is the time scale for these transitions.
3. Results
3.1. Cellular organization, viability, and tissue morphogenesis
Histological evaluation of the ES at ti ¼4, 7, 10, 14, and 21 days
showed changes in epidermal organization with time (Fig. 1A–D).
Stratum corneum grew thicker and more apparent with time
(Fig. 1A–D, arrows). At all time points, ES was comprised of a
densely populated epidermis with a continuous epidermal basal
cell layer. The dermis contained a relatively sparse population of
 G.C. Ebersole et al. / Journal of Biomechanics 43 (2010) 3183–3190 3185

fibroblasts in the porous collagen matrix which became thinner
with time (Fig. 1A–D).
Engineered skin barrier formation, as measured indirectly by
surface hydration, showed a distinct drop in surface electrical
capacitance with increasing days in culture (Fig. 2A). At day 4, the
surface of the ES was fully hydrated (no barrier formation) with
SEC values significantly greater than at all other time points
(5515.97279.2 pF). By day 14, the surface of the ES became drier
with ES hydration reaching normal levels of human skin
hydration (Fig. 2A). Additional culture time (21 days) did not
significantly alter surface hydration (Fig. 2A).
Analysis of cellular metabolism via MTT revealed an increase in
metabolism with culture time until day 14 (Fig. 2B). At culture day
21, metabolism was reduced by  14% compared to day 14 but it
was still significantly higher compared to days 4 and 7 (Fig. 2B).
Quantification of cell number per time point also indicated an
increase in total number of cells per field of view with days in
culture reaching a maximum at days 10 and 14 (179717 and
161721 cells/20  field) with a slight decrease in cell number
observed at later time points (145721 cells/20  field at day 21).
Immunostaining for laminin-5, a major protein constituent of the
basement membrane, indicated that basement membrane formed
by day 7 and was maintained throughout the culture period (Fig. 3).
Positive staining for involucrin, a cytoplasmic protein precursor of
the epidermal cornified envelope, was present at days 14 and 21 but
was not observed at day 7 (Fig. 3). Involucrin was present in the day
10 samples; however, staining was diffuse and not as intense
compared to later time points (Fig. 3B).
3.2. Mechanical properties
The tensile tests revealed that ES substantially strengthened
and stiffened over the culture period, with more modest

Fig. 1. Histological sections of engineered skin at culture days: (A) 7, (B) 10, (C) 14, and (D) 21 stained with H&E. Note the clear stratification of the epidermis and dermis at
all time points and the thickening of the stratum corneum with time. By day 21, the scaffolding within the dermis began to degrade. Scale bar ¼ 200 mm.

Fig. 2. (A) Surface electrical capacitance versus days in culture of engineered skin. The lower the surface capacitance the greater the barrier function. (B) Cellular
metabolism of engineered skin versus days in culture.
3186 G.C. Ebersole et al. / Journal of Biomechanics 43 (2010) 3183–3190

variations in the relaxation time exponent and strain to failure. In
particular, the average 21-day strength (6327222 kPa) was 10
times that for day 7 (Table 1, Fig. 4A) and the average 21-day
stiffness (0.077 70.002 N/m) was  7 times that for day 7
(Table 1, Fig. 4B). The average relaxation time exponent decayed
modestly from 0.22 to  0.15 over this period, and the average
strain to failure first increased and then decreased, with the
average 21-day elongation  0.8 times that for day 7 (Table 1).
The associated regression analyses revealed a significant linear
correlation with time for average strength and stiffness, but not
for other mechanical properties (Fig. 4).
Tensile tests reveal that ED did not strengthen or stiffen
over time, in contrast to ES. At day 7, ED and ES had relatively
similar average strength and stiffness but ED values actually

Fig. 3. Immunostaining of engineered skin at days: 7 (A), 10 (B), 14 (C), and 21 (D) for cell nuclei (blue), laminin-5 (green), and involucrin (red). At day 7 (A), the basement
membrane has formed (green) while little staining for involucrin, a marker of epidermal differentiation is observed. Involucrin staining, and thus epidermal differentiation,
becomes more evident as the engineered is incubated for longer periods of time. (For interpretation of the references to colour in this figure legend, the reader is referred to
the web version of this article.)

Table 1
Mechanical properties of engineered skin and engineered dermis determined by uniaxial tensile testing.

Engineered skin Engineered dermis

Days in Strength S (kPa) % Elongation at failure Linear stiffness Strength Sd (kPa) % Elongation at Linear stiffness
culture (N/mm) failure (N/mm)

4 23.8 7 9.6a 47.6 7 17.0b 0.0097 0.003c 52.3 7 7.1d 108.2 7 3.5e 0.0057 001
7 61.4 7 43.9a 83.2 7 33.7 0.0117 0.004c 63.8 7 10.0d 62.5 7 7.2 0.0057 0.001
10 178.1 7 56.6f 107.1 7 41.1g 0.0277 0.009h 59.3 7 3.4d 59.9 7 11.7 0.0047 0.002
14 425.1 7 157.6g 93.1 7 20.7 0.0447 0.009i 48.8 7 7.8d 51.8 7 4.2 0.0037 0.002
21 632.3 7 221.7 69.0 7 23.8 0.0777 0.023 138.5 7 41.3 50.97 13.3 0.0067 0.003

a
p o 0.001 vs. day 10, 14, and 21.
b
po 0.05 vs. day 7, 10, and 14.
c
po 0.01 vs. day 10, 14, and 21.
d
po 0.05 vs. day 21.
e
p o 0.001 vs. All.
f
po 0.001 vs. day 14 and 21.
g
p o0.05 vs. day 21.
h
p o 0.01 vs. 14&21
i
p o0.01 vs. 21
 G.C. Ebersole et al. / Journal of Biomechanics 43 (2010) 3183–3190 3187

Fig. 4. Scatter plot and linear regression lines for mechanical properties of engineered skin (ES) and engineered dermis (ED) as a function of time: (A) ultimate tensile
strength, (B) linear stiffness, and (C) relaxation rate (exponent n from Eq. 1). The relaxation rates could not be determined for engineered skin at days 4 and 7 and the
engineered dermis at all time points because the magnitude of force was too small.

Fig. 5. Engineered skin (ES) strength versus (A) surface electrical capacitance and (B) cellular metabolism. Note the strong correlation between surface capacitance and
strength and relatively weak correlation between metabolism and strength.

decreased from day 7 to day 14 and more than doubled from day Table 2
14 to day 21. The net result was that ED values are only  7% to Experimental values of average fractional thickness of the epidermis (fe) and
dermis (fd) for ES and predicted epidermal strength (Se) from Eq. (2). Also shown
22% of ES values at days 14 and 21 (Table 1, Fig. 4). Average ED
are the average epidermal contribution (feSe) and dermal contribution (fdSd) to the
elongation to failure ranged from 0.3 to 0.7 times that for ES strength S of engineered skin.
between day 7 and day 21. Overall, average elongation to failure
and stiffness of ED did not improve over the 21-day culture Time fe fd r0 e (kPa)a fer0 e (kPa) fdSd (kPa)b
period (p 4 0.05, Table 1). The ED relaxation time exponent t (days)

could not be determined since the ED samples generated 4 0.097 0.04 0.91 70.04  43.8 4 47
insufficient load at 10% strain (Fig. 4B). 7 0.087 0.05 0.92 70.05 92 7 59
10 0.127 0.05 0.88 70.05 1029 123 52
14 0.167 0.08 0.84 70.08 2441 391 41
3.3. Correlation of tissue morphogenesis and viability with 21 0.207 0.19 0.80 70.19 2575 515 111
mechanical properties
a
Derived from Eq. (2) using S and Sd values from Table 1, and fe and fd values
Ultimate tensile strength of ES was plotted against surface from Table 2.
b
Sd values from Table 1.
electrical capacitance and absorbance (Fig. 5) to determine if
mechanical properties correlate with measures of proliferation
and differentiation (Fig. 5). Strength correlated more significantly
derives primarily from the epidermis. Even though the epidermal/
with lower surface electrical capacitance (Fig. 5A) compared to
dermal thickness ratio was  0.2 at day 21 (Table 2), the ES
increased cellular metabolism (Fig. 5B).
strength in Eq. (2) had a contribution sue ðtÞfe ¼ 515 kPa from the
epidermis compared to sud ðtÞfd ¼111 kPa from the dermis
3.4. Composite strength model (Table 2).
A least squares regression line of the sigmoidal function in
Application of the composite strength model (Eq. (2)) showed Eq. (3) was fit to the average sue ðtÞ values at the five time points
that the average epidermal contribution, sue ðtÞfe , increased to a (ti ¼4, 7, 10, 14, and 21 days), with R2 ¼0.99 (Fig. 6). The resulting
21-day value that is  70 times the 7-day value (Table 2). The fitted parameters were A¼  57 kPa, D ¼1316 kPa, t0 ¼10.4 days,
results clearly showed that at day 10 and beyond, ES strength and t ¼ 1.22 days (Table 2). This corresponded to Se(t5t0)
3188 G.C. Ebersole et al. / Journal of Biomechanics 43 (2010) 3183–3190
Fig. 6. (A) Fractional thickness of the epidermis and dermis within engineered skin versus days in culture. (B) Experimental data for average ES strength and ED strength
versus days in culture. Corresponding estimates of epidermal stress, sue at peak ES strength are furnished by the composite strength model (Eq. (2)) and sigmoidal fit is
provided by Eq. (3), with a least squared error R¼ 0.99.
  57 kPa to Se(t0)¼1259 kPa to a saturation strength Se(tbt0)
2575 kPa.
4. Discussion
Epidermal differentiation increased with time in culture as
evidenced by stratification of the epidermis (Fig. 1), decreased
epidermal hydration (Fig. 2A), and positive involucrin staining
(Fig. 3). These data correlated well with several models for ES that
showed skin maturation with time at the air–liquid interface
(Powell et al, 2009; Sun et al., 2005; Pouliot et al., 2002; Butler
et al., 1999).
There are relatively few investigations of ES and ED mechanical
properties (Powell et al., 2009; LaFrance et al., 1995) with no studies
as a function of culture time. The present work demonstrated that
UTS and stiffness increased significantly with time in ES but they
increased more modestly or even degraded with time in ED (Fig. 4A,
Table 1). Structural proteins, including involucrin, loricrin, and
trichohyalin, are produced as ES matures, imparting biomechanical
strength to the epidermis (Candi et al., 2005; Nemes and Steinert,
1999). The absence of these proteins in a less differentiated
epidermis, characteristic of early culture times (Fig. 3), has been
shown to alter murine skin biomechanics and could reduce strength
and stiffness in ES. For example, loricrin-deficient mice are more
susceptible to epidermal damage by mechanical stress (Koch et al.,
2000; Ishida-Yamamoto and Iizuka, 1998).
This study shows that ED stiffness and ductility did not change
significantly with time (Fig. 4A, Table 1). ED strength decreased
from day 7 to day 14 and then approximately tripled in value at
day 21. This increase was likely a result of dermal thinning (Fig. 6A)
and a constant maximum load at failure. These data contrast with
prior studies showing increased dermal thickness, extracellular
matrix production, and cellular proliferation with culture time.
Culture of human dermal fibroblasts on porous collagen scaffolds
showed significant increase in DNA content and a 2-fold increase
in procollagen type-I production over a 2 week culture period
(George et al., 2008). Rat dermal fibroblasts cultured on collgen–
chitosan scaffolds showed steady cell proliferation, IL-6 production,
and ECM deposition (Sun et al., 2009).
However, the present study had different culture conditions. In
prior studies, dermal fibroblasts were cultured on scaffold constructs
without additional cells and in fibroblast growth medium often
containing fetal bovine serum and other growth factors/proteins
that promote proliferation and ECM production (Sun et al., 2009;
George et al., 2008). In contrast, the UCMC 161 medium used to
culture skin (co-culture of fibroblasts and keratinocytes) was
designed to promote epidermal proliferation and differentiation
while maintaining fibroblast viability. Prior studies showing BrdU
staining of ES culture in this medium indicated that the epidermis
was highly proliferative but very few fibroblasts in the dermis
were actively proliferating (Kalyanaraman et al., 2008). As the
culture medium is formulated to maintain fibroblast viability and
promote epidermal proliferation and differentiation, it is not
surprising that dermal strength in our model was lower than
that of studies that utilize DME-based mediums with FBS
supplements where fibroblasts are highly proliferative and
deposit large amounts of ECM.
The present work showed that ES strength increased approxi-
mately linearly with time and epidermal strength fit well to a
sigmoidal function (Fig. 6). The epidermal contribution to ES
strength increased the most with culture time over a period
from t ¼t0  t ¼9.18 to saturation at t¼t0 + t ¼11.62 days (Fig. 6),
whereas the dermal contribution was essentially constant. The
saturation in epidermal strength coincided with the point where
ES surface hydration reached normal human skin values (Fig. 2A)
and proteins of the cornified envelope appeared in the epidermis,
indicating that the epidermis was nearing terminal differentiation
(Fig. 3). The observed plateau in epidermal strength was expected
as the epidermis undergoes significant stratification and differ-
entiation during the culture process, reaching a plateau in
maturation after approximately 12–14 days in culture (Powell
and Boyce, 2008; Powell et al., 2008, 2009). Additionally, the
storage modulus of the epidermis is reported to decrease from the
outermost stratum corneum to the most viable, inner most layer
of the epidermis the stratum basale (Kendall et al., 2007)
supporting the theory that ES at early culture times has not yet
fully differentiated (i.e. it is comprised only of the viable stratum
basale) and would thus be significantly weaker.
These data also provide insight into optimizing profiles for
mechanical stimulation. Engineered tissues are often stimulated
following the principles of functional tissue engineering (FTE)
where engineered organs are subjected to the patterns of activity
and the magnitude of stressors encounters in normal use (Butler
et al., 2000). While this concept has been widely utilized for
engineered tendon (Butler et al., 2000, 2009), our current data
suggest that modifications to this theory are warranted in
engineered organs that undergo significant differentiation, as
 G.C. Ebersole et al. / Journal of Biomechanics 43 (2010) 3183–3190
the ‘‘in use’’ stimulation profiles will likely damage the maturing
tissue.
5. Conclusions
The present study suggests that the mechanical properties of
engineered skin (ES) are highly dependent on culture time and
morphogenesis. At shorter culture times (e.g., 4 and 7 days), the
dermis is largely responsible for the strength of ES but at larger
culture times (e.g., 14 and 21 days), the epidermis is largely
responsible. It is postulated that differences in medium formula-
tion between the present and prior studies affect fibroblast
senescence, so that strategies to improve fibroblast activity and
ECM production could lead to the most significant improvements
to ES biomechanics. Overall, knowledge of how engineered tissue
biomechanics evolve with time will help foster new strategies to
improve these properties and set limits to mechanical stimulation
profiles to prevent tissue damage during culture.
Conflict of interest statement
The authors have no financial or personal conflicts of interest
associated with the presented work.
Acknowledgement
The authors thank Jessica Wolever for assistance with cell
culture and engineered skin fabrication.
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