Research Article

www.acsami.org

Cross-linked Collagen Hydrogel Matrix Resisting Contraction To

Facilitate Full-Thickness Skin Equivalents
Christian Lotz,†,# Freia F. Schmid,‡,# Eva Oechsle,‡ Michael G. Monaghan,§,∥,⊥ Heike Walles,†,‡
and Florian Groeber-Becker*,‡
†
Department of Tissue Engineering & Regenerative Medicine (TERM), University Hospital Würzburg, Würzburg 97070, Germany
‡
Translational Center Würzburg ‘Regenerative Therapies in Oncology and Musculoskeletal Diseases’, Würzburg Branch of the
Fraunhofer Institute for Interfacial Engineering and Biotechnology, Würzburg 97070, Germany
§
Department of Cell and Tissue Engineering, Fraunhofer Institute for Interfacial Engineering and Biotechnology, Stuttgart 70569,
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Germany
∥
Trinity Centre for Bioengineering, Trinity Biomedical Sciences Institute, and ⊥Department of Mechanical and Manufacturing
Engineering, School of Engineering, Trinity College Dublin, Dublin 2, Ireland
*
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S Supporting Information

ABSTRACT: Full-thickness skin equivalents are gathering

increased interest as skin grafts for the treatment of large skin
defects or chronic wounds or as nonanimal test platforms.
However, their fibroblast-mediated contraction and poor
mechanical stability lead to disadvantages toward their
reproducibility and applicability in vitro and in vivo. To
overcome these pitfalls, we aimed to chemically cross-link the
dermal layer of a full-thickness skin model composed of a
collagen type I hydrogel. Using a noncytotoxic four-arm
succinimidyl glutarate polyethylene glycol (PEG-SG), cross-
linking could be achieved in cell seeded collagen hydrogels. A concentration of 0.5 mg of PEG-SG/mg of collagen led to a
viability comparable to non-cross-linked collagen hydrogels and no increased release of intracellular lactate dehydrogenase.
Cross-linked collagen hydrogels were more mechanically stable and less prone to enzymatic degradation via collagenase when
compared with non-cross-linked collagen hydrogels. Remarkably, during 21 days, cross-linked collagen hydrogels maintain their
initial surface area, whereas standard dermal models contracted up to 50%. Finally, full-thickness skin equivalents were generated
by seeding human epidermal keratinocytes on the surface of the equivalents and culturing these equivalents at an air−liquid
interface. Immunohistochemical stainings of the cross-linked model revealed well-defined epidermal layers including an intact
stratum corneum and a dermal part with homogeneously distributed human dermal fibroblasts. These results indicate that cross-
linking of collagen with PEG-SG reduces contraction of collagen hydrogels and thus increases the applicability of these models as
an additional tool for efficacy and safety assessment or a new generation of skin grafts.
KEYWORDS: regenerative medicine, tissue engineering, collagen, cross-linking, alternatives to animal testing, skin grafts

■ INTRODUCTION
The skin is the primary interface between the human body and
the environment it encounters. Hence, it can be prone to
extensive tissue damage and is exposed to numerous chemicals
that can lead to adverse health effects. If the integrity of skin
becomes impaired, plain gauze is the most commonly used
wound dressing in hospitals for small injuries that only effect
the epidermis or just superficial parts of the dermis. For larger
or deeper wounds, the biological mechanism for wound closure
changes from regeneration to contraction of the wound,
resulting in the formation of scar tissue and loss of function.
Full-thickness injuries with a surface area larger than 1 cm2
require a skin graft to prevent extensive scarring.1 The current
“gold standard” for skin replacement is an autologous full- or
split-thickness skin graft.2,3 However, this method is limited by
the availability of healthy tissue and inflicts secondary wounds
and additional pain for the patient. Therefore, tissue-engineered
skin equivalents are being developed as skin substitute in
regenerative medicine.4−6
Aside from clinical applications, tissue-engineered skin
models can be used as preclinical test models for risk and
efficacy assessment as an alternative to traditional animal
models. Species-specific variances in skin architecture and
metabolism have led to an increasing demand for alternatives to
animal models. Additionally, international and European
regulations have become more stringent regarding product
safety and the use of animal-free alternative test methods.7
Therefore, numerous efforts have been attempted to provide
Received: March 21, 2017
Accepted: May 30, 2017
Published: May 30, 2017

© 2017 American Chemical Society 20417 DOI: 10.1021/acsami.7b04017

ACS Appl. Mater. Interfaces 2017, 9, 20417−20425
ACS Applied Materials & Interfaces Research Article

Figure 1. Material properties of collagen hydrogels and PEG-SG cross-linked collagen hydrogels. (A) Schematic of collagen cross-linking reaction
with PEG-SG. (B) Optical properties of hydrogels. Macroscopic images of non-cross-linked collagen hydrogels (CHG) and cross-linked collagen
hydrogels with PEG-SG50 (CHG SG-PEG50) and PEG-SG100 (CHG PEG-SG100) (bottom). Transmission of different collagen hydrogels and
collagen solution were compared in an absorption scan from 500 to 800 nm (top). Data are shown as mean values with standard deviation as error
bars. (Mean ± SD; n = 5, technical replicates.) (C) Mechanical characterization of collagen hydrogels. Mechanical stability of collagen hydrogels
shown as compression E-modulus. The compression E-modulus was calculated as the ratio of stress to strain in the linear region of stress−strain
curves in compression tests. (D) Resistance to enzymatic degradation of collagen hydrogels. Cross-linked collagen hydrogels (CHG PEG-SG50 and
CHG PEG-SG100) were generated and treated with collagenase for 14 h. Untreated collagen hydrogels were used as control. Data is shown as mean
values with standard deviation as error bars. Statistically relevant differences against the CHG control group are indicated with stars (**P < 0.01,
***P < 0.001, n = 3 independent experiments). (E) Ultrastructural analysis by transmission (TEM) and scanning electron microscopy (SEM) of
acellular cross-linked and non-cross-linked hydrogels. White arrows indicating a typical collagen fibril banded structure in collagen hydrogels (CHG).
A few fibril banded structures for cross-linked collagen hydrogels with PG-SG50 (CHG PG-SG50) were visible. For collagen hydrogels cross-linked
with PG-SG100 (CHG PG-SG100) thin fibril-like structures of approximately 5 nm were detectable. Scale bar indicates 280 nm. SEM visualized the
fibril network of acellular cross-linked and non-cross-linked collagen hydrogels. Non-cross-linked collagen hydrogels (CHG) showed a homogeneous
network of regular fibrils. Few fibril-like structures were recognizable for cross-linked collagen hydrogels with PEG-SG50 (CHG PG-SG50), indicated
by white arrows. No fibrils occurred in cross-linked collagen hydrogels with PEG-SG100 (CHG PG-SG100). Scale bar indicates 1 μm.

alternatives to animal models for dermal toxicity testing in
recent years.8 Currently, two different versions of artificial skin
models are available. Reconstructed human epidermis consists
exclusively of a multilayered epidermal construct, whereas full-
thickness skin models constitute an epidermis grown upon a
dermal layer.9 Although reconstructed human epidermis is a
promising tool for toxicity testing, the lack of a dermal part
limits the applicability of the models. Previous studies have
shown that the crosstalk between the epithelium and the
connective tissue regulates skin morphology and homeostasis.10
Additionally, there is experimental evidence that metabolic
activity is reduced in the absence of a dermis.11,12 With regard
to the assessment of toxicological endpoints that require an
interaction with cutaneous enzymes, for example, genotoxicity,
this lack of influential crosstalk can be problematic since it
decreases the accuracy of test methods based on reconstructed
human epidermis considerably.13 Furthermore, skin grafts
benefit from a dermal layer for wound healing. The dermal
layer increases the chance of graft integration up to 75% and
provides growth factors and cytokines regulating wound
healing.14
Hence, to improve upon recapitulating human skin for in
vivo and in vitro application, the use of tissue-engineered skin
including both skin layers is highly desirable. A key element of
20418
full-thickness equivalents is the matrix that is used to generate
the dermal layer. Since the most abundant proteins within the
dermis are collagen type I and type III, most skin models are
generated using collagen hydrogels.15−17 However, a major
pitfall of collagen-based hydrogels is their susceptibility to
fibroblast-mediated contraction during culture that results in an
epidermal layer that lacks attachment to the insert wall.18 In a
similar way, skin substitutes can contract and degrade, reducing
their efficacy and engraftment chances.
Thus, hydrogel contraction impedes the standardization of
full-thickness skin models and their reliable application for
toxicity testing and the clinical application as skin implants.
Several attempts have been made to overcome the contract-
ibility of collagen hydrogels containing human dermal
fibroblasts. Besides physical modification of collagen hydrogels
by plastic compression,19 the chemical cross-linking of collagen
has yielded reduced collagen contraction.20,21 Because of the
cytotoxic properties of most cross-linking agents such as
glutaraldehyde and 1-ethyl-3-(3-(dimethylamino)propyl) car-
bodiimide, the cross-linking process is usually performed prior
to cell seeding, after which remnants of the cross-linking
reaction are washed out to facilitate cell culture.22,23 However,
these approaches limit the creation of homogeneous scaffolds
that can be used in in vitro models and clinical applications. To
DOI: 10.1021/acsami.7b04017
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ACS Applied Materials & Interfaces
create a cross-linked collagen hydrogel that can serve as a
matrix for supporting dermal layer of full-thickness skin models,
a cross-linker that can be directly incorporated into the matrix
during cell seeding prior to hydrogel gelation is needed.
Previously, such cross-linking approaches have included the use
of microbial transglutaminase and multiarm polyethylene glycol
polymers of varying molecular weights, branching, and
functional terminations.24−26 In this study we explore the use
of a four-arm polyethylene glycol with succinimydil glutarate-
terminated branches (PEG-SG). Previously, PEG-SG was
investigated as a cross-linking agent to cross-link collagen
hydrogels during fibroblast incorporation delivering exogenous
microRNA.26
This study hypothesizes that chemically cross-linking a
collagen hydrogel harboring fibroblasts can be achieved using
PEG-SG without the induction of cytotoxic effects and that
such a chemically modified matrix elicits a reduction of
fibroblast-mediated contraction. Cross-linked collagen hydro-
gels were generated and evaluated in regard to their chemical
and mechanical stability, ultrastructure, viability, and contrac-
tion. Finally, cross-linked full-thickness skin equivalents were
generated to demonstrate the capacity of the cross-linked
collagen to facilitate skin tissue formation.
■
MATERIAL AND METHODS
Cell Isolation. Human dermal fibroblasts and human epidermal
keratinocytes were isolated from foreskin biopsies of 2−5 year old
donors with approval of the local ethics committee (approval number
IGBZSF-2012-078) and after the confirmed consent of their guardians
using a well-established protocol described previously.27 Briefly,
biopsies were washed, minced, and digested with Dispase (Life
Technologies, Germany) to dissociate the epidermis from the dermis.
Thereafter, the epidermis was trypsinized (Life Technologies) and the
dermis was digested with collagenase (Serva, Germany) to generate
single-cell suspensions.
Cross-linking of Collagen Using Four-Armed Polyethylene
Glycol Succinimidyl Glutarate and Generation of Dermal
Equivalents. To generate cross-linked collagen hydrogels, type I rat
tail collagen at 6 mg/mL in 0.1% acetic acid was mixed with gel
neutralization solution (75 mM 4-(2-hydroxyethyl)piperazine-1-
ethanesulfonic acid , 48 mM glucose, 268 mM NaCl; Sigma-Aldrich,
Germany) at a 1:1 volumetric ratio. For cross-linking, different
concentrations of PEG-SG (molecular weight 10 000; JenKem
Technology, USA) were additionally added to the gel neutralization
solution. Therefore, the desired amount of PEG-SG was solubilized in
ice cold gel neutralization solution and subsequently mixed with the
collagen solution for 30 s at room temperature. The amount of PEG-
SG was based upon the molar ratio of free collagen primary amine
groups and the n-hydroxysuccinimide ester of PEG-SG. With the four-
arm structure of PEG-SG, theoretically, one expects 1 mol of PEG-SG
to react with 4 mol of free amines available in collagen type I.
Therefore, 1 mg of collagen was mixed with 0.5 mg or 1 mg of PEG-
SG, resulting in a theoretical cross-linking of 50% or 100% of all free
amine groups. A detailed calculation of the molar ratio of collagen
primary amine groups and the n-hydroxysuccinimide ester of PEG-SG
can be found in the Supporting Information. Following, a PEG-SG
concentration theoretically sufficient to cross-link 50% of all free
amines will be termed PEG-SG50 and a PEG-SG concentration that
can theoretically cross-link all free amines will be termed PEG-SG100.
After the gel neutralization solution was mixed with PEG-SG and
collagen type I solution, the hydrogels were incubated for 10−15 min
at 37 °C for completion of gelation. For the generation of dermal
equivalents human dermal fibroblasts were added to the gel
neutralization solution in the second passage of culture, resulting in
a final concentration of 1 × 105 cells/mL in the dermal equivalent. A
schematic of the cross-linking reaction is depicted in Figure 1A. Non-
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Research Article
cross-linked collagen hydrogels were generated in a similar manner,
but without the addition of the cross-linker.
Transparency of Collagen Hydrogels. The optical density of
200 μL of acellular cross-linked, non-cross-linked hydrogels and
nonassembled collagen solutions was determined by an absorption
scan from 500 to 800 nm in a 96-well plate using a spectrophotometer
(Infinite 200M; Tecan, Switzerland). Transmission was calculated as a
ratio of the incident light and absorbed light.
Scanning Electron Microscopy (SEM). Hydrogels were fixed
with 2.5% glutaraldehyde in 50 mM cacodylate buffer for 48 h at 4 °C.
Samples were dehydrated in a series of increasing acetone
concentrations, dried by critical point drying, sputtered with gold−
palladium, and examined with a field emission SEM JEOL JSM-7500F
(JEOL, Germany).
Transmission Electron Microscopy (TEM). Hydrogels were
fixed at 4 °C for 48 h in 2.5% glutaraldehyde in 50 mM sodium
cacodylate buffer and postfixed using 2% osmium tetroxide for 2 h at 4
°C. Ultrathin sections were stained with 0.5% aqueous uranyl acetate,
dehydrated with ethanol, and embedded in Epon 812. Sections were
inspected with a TEM JEOL JEM-2100 microscope (JEOL,
Germany).
Mechanical Characterization of Hydrogels. The mechanical
properties of the hydrogels were assessed by performing a
compression test with a materials testing machine (Zwick/Roell
Z005; Zwick GmbH & Co. KG, Germany) comprising a 2.5 kN load
cell. Data were recorded by using the testing software testXpert II
(Zwick GmbH & Co. KG, Germany). After preparation, the hydrogels
were incubated for 24 h at 37 °C. A cylindrical hydrogel sample with a
diameter of 1.5 mm and a height of 10 mm was placed between the
plates of the instrument. For the compression test a constant traverse
speed of 1 mm/min was conducted until a gap of 2 mm between
traverse and crosshead was reached. A stress−strain curve was plotted
during the measurement. The elastic compressive E-modulus of each
sample was evaluated as a ratio of the stress and strain in the linear
area of the stress−strain curve.
Collagenase Assay. Resistance of the hydrogels to enzymatic
digestion was evaluated using a collagenase assay. Hydrogels with a
volume of 500 μL were incubated for 1 h in 1 mL of 0.1 M Tris−HCl
(pH 7.4), containing 50 mM CaCl2 (Sigma-Aldrich, Germany) at 37
°C. Subsequently, the Tris−HCl solution was substituted for 1 mL of
bacterial collagenase solution (10 units/mg of collagenase type IV in
0.1 M Tris−HCl; Life Technologies). After incubation for 14 h at 37
°C, the enzymatic reaction was halted by the addition of 1 mL of 0.25
M ethylenediaminetetraacetic acid. Following vacuum dehydration, the
remaining mass of the hydrogels was determined and normalized to
the remaining mass of nondigested hydrogels.
Viability Assessment of Dermal Equivalents. The viability of
cross-linked and non-cross-linked hydrogels seeded with 1 × 105 cells/
mL of human dermal fibroblasts after 21 days of culture was quantified
by using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromid
(MTT) assay. Cell-seeded hydrogels were placed into 3 mL of MTT
solution (1 g/mL MTT; Sigma-Aldrich). After incubation for 24 h at
37 °C, the MTT solution was discarded and the resulting precipitate
solubilized in 3 mL of 2-propanol for 24 h at 4 °C. Subsequently, the
solubilized formazan salt was quantified by measuring the absorbance
at 570 nm using a spectrophotometer (Infinite 200M; Tecan).
Additionally, the presence of lactate dehydrogenase (LDH),
indicative of cell lysis, was measured by quantifying the enzyme
activity of LDH using a commercially available cytotoxicity detection
kit (Cytotoxicity Detection Kit PLUS, Roche, Germany). Cross-linked
and non-cross-linked hydrogels seeded with 1 × 105 cells/mL of
fibroblasts were prepared and cultured for 24 h. As a positive control,
hydrogels treated with a 1% Triton X-100 solution for 3 h at 37 °C
were included in the analysis. For quantification of LDH, 100 μL of
freshly prepared reaction mixture was added to 100 μL of the
supernatant of each sample in an optically clear 96-well flat bottom
microplate. After 30 min, reduction of a tetrazolium salt to a reddish
formazan product indicative for LDH activity was quantified by
measuring the absorbance at 492 nm using a spectrophotometer
(Infinite 200M; Tecan).
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ACS Applied Materials & Interfaces
Contraction of Dermal Equivalents. To examine the capability
of human dermal fibroblasts to contract hydrogels, a contraction study
was performed for 21 days. After generation of cross-linked and non-
cross-linked hydrogels with 2 × 105 cells/mL, the hydrogels were kept
at 37 °C in 95% humidity and 5% CO2 atmosphere. Culture medium
was replaced twice a week. Surface area was analyzed macroscopically
after 1, 6, 9, 12, 14, and 21 days of culture.
Generation of Full-Thickness Skin Models. Skin models were
generated on a polycarbonate membrane of respective cell culture
inserts (diameter 0.59 cm2, pore size 8 μm; Brand, Germany). Human
dermal fibroblasts were resuspended in gel neutralization solution
according to the generation of the dermal part with or without PEG-
SG. A volume of 150 μL of collagen and 150 μL of gel neutralization
solution per insert was used, resulting in a fibroblast density of 1 × 105
cells/mL. After incubation at 37 °C for 24 h in Dulbecco’s modified
Eagle’s medium (Invitrogen, Germany) supplemented with 10% fetal
calf serum (Lonza Group Ltd., Switzerland), human epidermal
keratinocytes were seeded onto dermal equivalents. Keratinocytes
were applied with a cell density of 5 × 105 cells/cm2, in 150 μL of
EpiLife supplemented with 0.2% v/v bovine pituitary extract, 1 μg/mL
recombinant human insulin-like growth factor-I, 0.18 μg/mL hydro-
cortisone, 5 μg/mL bovine transferrin, 0.2 ng/mL human epidermal
growth factor (all from Life Technologies), and 1.5 mM CaCl2
(Sigma-Aldrich). To ensure sufficient nutrient supply to the skin
models, inserts were cultured in 6.5 mL of medium per row in custom
6-well plates (Brand). Medium was changed after 24 h to EpiLife air−
liquid interface medium,28 additionally containing 73 μg/mL L-
ascorbic acid 2-phosphate and 10 ng/mL keratinocyte growth factor
(both Sigma-Aldrich). Medium was replaced by fresh air−liquid
interface medium 3 times per week and models were cultured at 37 °C
and 5% CO2 in a humidified incubator. Full-thickness skin models
were used for testing after 19 days of airlift culture.
Histological Analysis. After the respective culture periods, cell-
seeded hydrogels were fixed in Bouin’s fixation agent (Sigma-Aldrich)
for 1 h, washed in tap water for 2 h, and embedded in paraffin.
Subsequently, histological cross sections of 3 μm were generated. For
an overview of general histological features, tissue slides were stained
with hematoxylin and eosin (H&E). Immunohistochemical staining
was used to visualize specific epitopes in native skin and skin
equivalents. Immunohistochemical staining was performed with the
Dako EnVisionTM Kit (Dako, Denmark) according to the
manufacturer’s protocol. After endogenous peroxidase was blocked
with 3% H2O2 for 5 min, the primary antibody cytokeratin 14 (dilution
of 1:500; Sigma-Aldrich), cytokeratin 10 (dilution of 1:200; Dako,
Denmark), filaggrin (dilution of 1:100; Biomol GmbH, Germany),
involucrin (dilution of 1:150; Acris Antibodies GmbH, Germany),
vimentin (dilution of 1:1000; Abcam, United Kingdom), Ki67
(dilution of 1:150; Dako), was applied and incubated at 4 °C
overnight. One drop of horseradish peroxidase-coupled secondary
antibody was added per section and the slides were incubated for 30
min at room temperature. The sections were covered with substrate
solution for 4−7 min at room temperature until a macroscopic staining
was observable. To visualize the cell structure, counterstaining with
hematoxylin was performed. After incubation in 2-propanol, the
sections were covered with Isomount 2000 (Labonord, France).
Determination of Proliferation Index. To determine prolifer-
ative human epidermal keratinocytes in skin equivalents, cross sections
were immunohistochemically stained with an antibody against Ki67.
The proliferation index of keratinocytes in the stratum basale was
determined by the number of Ki67 positive nuclei in a total number of
100 basal cells, which was set to 100%. Three different sections of each
sample were evaluated.
Statistical Analysis. Statistical analysis was performed using one
way ANOVA with Dunnett’s post-test using the appropriate control in
each experiment as a reference. Values of p < 0.05 were considered to
be significant.
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■ RESULTS
Cross-linking with PEG-SG Influences Transparency,
Improves Mechanical Properties, and Prolongs Collage-
nase-Mediated Degradation. After complete gelation,
macroscopic images of hydrogels revealed that the collagen
hydrogels were opaque, whereas cross-linking with PEG-SG led
to transparent hydrogels (Figure 1B). To quantify the change
in optical properties, the transmission rates at wavelength
between 500 and 800 nm of unassembled collagen solutions,
collagen hydrogels and cross-linked collagen hydrogels were
evaluated (Figure 1B). For unassembled collagen solutions and
cross-linked collagen hydrogels, the transmission rates remain
constant at wavelength between 500 and 800 nm, whereas non-
cross-linked hydrogels showed an increasing transmission with
rising wavelength. Since transmission rates differ most at 500
nm, this wavelength was chosen to compare optical properties.
Here, the transmission rate of non-cross-linked collagen
hydrogels was 23.8 ± 0.3%, whereas the cross-linked collagen
hydrogels and the collagen solution had statistically significant
higher transmission rates of 88.4 ± 1.2% for PEG-SG50, 89.6 ±
0.3% for PEG-SG100, and 78.5 ± 1.6% for the collagen solution.
To assess the influence of the cross-linking process on
mechanical properties of collagen hydrogels, the compression
E-modulus of the hydrogels was determined (Figure 1C).
When PEG-SG50 was employed, the E-modulus increased
significantly from 315 Pa for the non-cross-linked control up to
557 Pa. An E-modulus of 1225 Pa was reached for PEG-SG100.
Since the introduction of chemical cross-links in collagen
hydrogels can render collagen fibrils less susceptible to
enzymatic attack, a collagenase digestion assay was employed
to assess enzymatic resistance. The used collagenase catalyzes
the hydrolytic cleavage of collagen in both the single-chain and
the triple-helix conformation.29 To evaluate collagenase
digestion, collagen hydrogels with and without cross-linking
were generated and digested with collagenase for 14 h (Figure
1D). Collagen hydrogels that were not cross-linked degraded at
a statistically significant faster rate and were reduced to 3 ± 2%
of the initial mass after 14 h. In contrast, collagen hydrogels
subjected to PEG-SG50 kept 35 ± 9% of the initial mass. An
even higher stability was obtained for PEG-SG100 with 60 ± 5%
of the remaining mass.
Cross-linking Alters Collagen Assembly. To gain insight
into the ultrastructure of cross-linked and non-cross-linked
collagen hydrogels, SEM and TEM were performed. TEM
images revealed densely packed collagen fibrils in non-cross-
linked collagen hydrogels, showing the typical banding formed
by the unidirectional staggered arrangement of collagen
molecules.30 In collagen hydrogels cross-linked with PEG-
SG50 only few densely packed collagen fibrils remained. For
PEG-SG100 no typical collagen fibril morphology occurred; thin
fibril-like structures of approximately 5 nm were formed that
seemed to be homogeneously distributed and finely branched.
The microstructure of collagen was also examined using SEM
(Figure 1E). Collagen fibrils in non-cross-linked collagen
hydrogels appeared as a homogeneous network of thin and
regular fibrils of approximately 70 nm thickness. In contrast, a
different ultrastructure was visible in the PEG-SG50-treated
collagen hydrogels that did not show the typical fibril structure
and only few collagen fibrils were identifiable. The cross-linking
with PEG-SG100 led to hydrogels with no fibril structures and
the mesh size of the hydrogels appeared denser in comparison
to that of PEG-SG50.
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Cell Viability Depends on PEG-SG Concentration. To

assess if cross-linking with PEG-SG has an effect on cell
viability, cross-linked collagen hydrogels were evaluated using a
MTT assay in which output readings were normalized to non-
cross-linked collagen hydrogels. After 21 days of culture, PEG-
SG50 led to a viability of 110.9 ± 10.7%, whereas the viability
was reduced significantly to 60.2 ± 6.5% for the PEG-SG100
group (Figure 2A). Additionally, the presence of lysed cells was
assessed by quantifying LDH in the cell culture medium.31
Non-cross-linked collagen hydrogels treated with a 1% Triton
solution were used as positive control. No significant
differences were detectable between the level of LDH in non-
cross-linked collagen hydrogels (1.1 [a.u.]), collagen hydrogels
cross-linked with PEG-SG50 (1.1 [a.u.]), and collagen hydrogels
cross-linked with PEG-SG 100 (1.1 [a.u.]). However, a
significantly higher absorption rate of 1.9 [a.u.] was observed
for the positive control, demonstrating the validity of the assay
(Figure 2B).
Cross-linking of Collagen Hydrogels with PEG-SG
Reduced Fibroblast-Mediated Contraction Compared to
Non-cross-linked Hydrogels. To assess the capability of
cross-linked collagen hydrogels to resist cell-mediated con-
traction, collagen hydrogels containing human dermal fibro-
blasts were generated. The surface area of the constructs was
monitored periodically for 21 days (Figure 2C). Non-cross-
linked collagen hydrogels with and without human dermal
fibroblasts were used as controls. During the culture period,
non-cross-linked collagen hydrogels contracted continuously to Figure 2. Biological evaluation of collagen hydrogels and PEG-SG
49.9 ± 8.9% of the initial hydrogel area. In contrast to these cross-linked collagen hydrogels. (A) Viability in dermal equivalents.
findings, significantly less shrinkage was seen in the PEG-SG50 Tissue viability was assessed via MTT in cross-linked collagen
and PEG-SG100 groups whereby a surface area of 99.2 ± 3.1% hydrogels with PEG-SG50 (CHG SG-PEG50) and PEG-SG100 (CHG
and 99.0 ± 1.8% was retained, respectively (Figure 2D). PEG-SG100) seeded with human dermal fibroblasts after 21 days of
Cross-linked Full-Thickness Skin Equivalents Are culture. The values were normalized to non-cross-linked collagen
Capable of Recapitulating the Morphology of Human hydrogels, which were arbitrary set to 100%. Data are shown as mean
Skin. Because of the reduced viability of fibroblasts in collagen values with standard deviation as error bars. A statistically relevant
difference between the experimental groups is indicated using stars
hydrogels cross-linked with PEG-SG100, only the lower PEG- (***P < 0.001, n = 3 independent experiments). (B) Lactate
SG50 concentration was used to generate dermal equivalents for dehydrogenase (LDH) release in dermal equivalents. For the
the construction of full-thickness skin equivalents. The basic assessment of cytotoxicity a LDH assay was performed of non-cross-
histological architecture of the models was visualized by H&E linked collagen hydrogels (CHG) or collagen hydrogels cross-linked
staining with native human skin as a comparison. All in vitro with PEG-SG50 (CHG PEG-SG50) and PEG-SG100 (CHG PEG-SG100)
skin models based on both cross-linked and non-cross-linked seeded with human dermal fibroblasts after 1 day in culture. As a
hydrogels formed a multilayered epidermis at the air−liquid positive control, collagen hydrogels were treated for 2 h with Triton X-
interface (Figure 3A). The overall epidermal architecture 100 1% (CHG + Triton X-100). Data are depicted as mean values with
closely resembled that of human skin. The skin models and standard deviation as error bars. Statistically relevant differences
against the CHG control group are indicated with stars (**P < 0.01,
native skin consisted of 6−8 living cell layers containing a well-
***P < 0.001, n = 3 independent experiments). (C) Contraction study
defined stratum basale, stratum granulosum, and a stratum of dermal equivalents. The contraction of non-cross-linked collagen
spinosum. Human epidermal keratinocytes in the stratum hydrogels (CHG) or cross-linked collagen hydrogels with PEG-SG50
basale had a uniform cuboidal morphology typical for human (CHG PEG-SG50) and PEG-SG100 (CHG PEG-SG100) seeded with
epidermal keratinocytes in the basal layer. In all skin equivalents human dermal fibroblasts was monitored over 21 days by macro-
the cells in the stratum granulosum were flat with some scopically measuring the surface area. As a control, acellular collagen
anuclear cells. Moreover, the cross-linked and non-cross-linked hydrogels (acellular) were included in the study. (D) Contraction of
full-thickness skin equivalents had a stratum corneum dermal equivalents at day 21 of culture. Measured surface area of non-
comparable to human skin. A difference between human skin cross-linked collagen hydrogels (CHG) or cross-linked collagen
and in vitro skin models is the thickness of the stratum hydrogels with PEG-SG50 (CHG PEG-SG50) and PEG-SG100 (CHG
PEG-SG100) seeded with human dermal fibroblasts after 21 days of
spinosum, which has a regular thickness in reconstructed skin culture. Data is shown as mean values with standard deviation as error
models. In contrast, human skin shows an irregular thickness bars. Statistically relevant differences against the CHG control group
because of the dermal papillae. are indicated with stars (**P < 0.01, ***P < 0.001, n = 3 independent
After the analysis of basic morphological features, cross- experiments).
linked and non-cross-linked full-thickness skin equivalents were
compared using immunohistochemistry, again with human skin
as a direct comparison. The epidermal layers were characterized protein involucrin, and interfilamentous protein filaggrin
by four differentiation markers including the intermediate (Figure 3A). To visualize human dermal fibroblasts, vimentin
filaments cytokeratin 10 and cytokeratin 14, the structural was used.
20421 DOI: 10.1021/acsami.7b04017
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ACS Applied Materials & Interfaces Research Article

Figure 3. Histological analysis of the cross-linked full-thickness skin equivalent. (A) Histological and immunohistological staining. Hematoxylin and
eosin (H&E) staining of cross-linked and non-cross-linked full-thickness skin equivalents (FTSE PEG-SG50, FTSE) and human skin (hSkin). Cross-
linked (FTSE PEG-SG50) and non-cross-linked (FTSE) full-thickness skin equivalents as well as human skin (hSkin) were stained for the markers
cytokeratin 14 (CK 14), cytokeratin 10 (CK 10), involucrin, filaggrin, and vimentin. The cross-linked (FTSE PEG-SG50) and non-cross-linked
(FTSE) full-thickness skin equivalents show histological features comparable to human skin. (B) Proliferation index of the epidermal layer.
Determination of the proliferation index of Ki67 in the stratum basale of the epidermal part of cross-linked and non-cross-linked full-thickness skin
equivalents (FTSE PEG-SG50, FTSE) and human skin (hSkin) was done by staining with Ki67 after 3 weeks of culture. The percentage of Ki67-
positive cells in these cultures was determined by counting the number of Ki67-positive cells among the total number of basal cells. (Mean ± SD; n =
3 independent experiments; one-way ANOVA). Hematoxylin was used for counterstaining of cell nuclei. Scale bar 50 μm.

Similar to human skin, cytokeratin 14 is most prominent in

the basal layers of the skin equivalents, whereas cytokeratin 10
as part of the late intermediate filament network was present in
the suprabasal layers. Furthermore, the cross-linked and non-
cross-linked full-thickness skin equivalents showed the same
localization for involucrin as human skin. Although filaggrin was
present in the granular layers of skin equivalents, the staining
was considerably weaker than that in human skin. Immunohis-
tochemical staining of vimentin showed a comparable
distribution of human dermal fibroblasts in the dermal part of
the full-thickness skin equivalents and the dermis of the native
skin. However, the cell density of human dermal fibroblasts in
cross-linked skin equivalents seemed to be lower than that in
non-cross-linked skin equivalents and native skin. To further
characterize the epidermal layer, the proliferation index of
human epidermal keratinocytes was analyzed by calculating the
number of Ki67-positive stained cells in the stratum basale of
the tissue sections. The cross-linked full-thickness skin
equivalents and the human skin showed comparable prolifer-
ation rates with 21.1% and 18.9% Ki67-positive basal cells. The
proliferation index of the non-cross-linked full-thickness skin
equivalents was 14.5%, lower than that in human skin (Figure
3B). However, the differences reached no statistical signifi-
cance.
20422
■ DISCUSSION
To date, no optimal strategy has been developed that allows for
the accurate mimicking of the properties of human skin and
thus for the generation of optimal skin substitutes for skin
grafting or skin models for preclinical testing. As collagen is the
most abundant molecule within the skin and has remarkable
mechanical and biological properties, it is among the most
promising materials for skin tissue engineering. However, the
establishment of collagen-based full-thickness skin equivalents
as in vitro test systems or skin grafts is facing significant
challenges due to a considerable fibroblast-mediated contrac-
tion.32 To stabilize the collagen network of current collagen
hydrogels, one possibility is to introduce additional molecular
bonds between the collagen fibrils via different chemical cross-
linkers. In this study, we aimed to improve the long-term
stability and mechanical properties of collagen hydrogels by
using the nontoxic chemical cross-linker PEG-SG to obviate a
negative impact on cellular viability. The n-hydroxysuccinimide
ester groups of the PEG-SG react with free amine groups of the
collagen. By formation of stable links between different collagen
fibrils, the collagen structure is strengthened and less prone to
cellular remodeling. Confirming this hypothesis, the cross-
linked hydrogels showed increasing mechanical stability with
higher PEG-SG concentration. Another effect of chemical
cross-linking is increased resistance to enzymatic degradation
by collagenase, which was also the case in this study.26,28,33 One
possible explanation for this might be that collagenase cleavage
DOI: 10.1021/acsami.7b04017
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ACS Applied Materials & Interfaces
sites are more effectively masked in the cross-linked collagen
samples.33 This means that a skin graft based on a cross-linked
collagen hydrogel degrades more slowly and has therefore more
time to connect with the native skin tissue. Hence, the chances
of a successful implantation are improved.
Interestingly, an increased transparency was noticeable for all
cross-linked collagen hydrogels when compared to that of non-
cross-linked collagen hydrogels. This would enable a better
inspection of the wound regeneration after skin replacement or
live cell imaging in in vitro models. Furthermore, this could be
interesting for other tissues models such as cornea, which need
a transparent collagen-based scaffold. However, turbidity of
collagen is reported to be proportional to the amount of
fibrillary structures; the alteration in transparency might
indicate a disrupted fibril formation.34,35 To confirm this
hypothesis, the hydrogels were further studied by transmission
and scanning electron microscopy. Both methods showed a
decreased amount of collagen fibrils dependent on the
concentration of PEG-SG. In contrast, non-cross-linked
collagen hydrogels showed a homogeneous network of fibrils
with 70 nm thickness and the typical D-periodic banded
pattern.34 This strengthens the hypothesis that PEG-SG
inhibits fibril formation of collagen during the pH-induced
solidification of the dissolved peptide fragments. The
phenomenon was already described by Yunoki and Matsuda,
who suggested the introduction of intrafibrillar cross-linking
during fibril formation.36 Here both nonfibrous collagen
formed by premature cross-linking and collagen fibrils are
present and form a double network structure (Figure 1A).
After the chemical and mechanical characterization of the
cross-linked hydrogels, the cellular response to the cross-linking
process was assessed. As the cross-linking reaction is conducted
with the presence of embedded fibroblasts, a thorough analysis
had to exclude a possible cytotoxic effect of PEG-SG. The
viability of fibroblasts in collagen hydrogels cross-linked with
PEG-SG50 was similar to the non-cross-linked control.
However, the viability in collagen hydrogels cross-linked with
PEG-SG100 was significantly decreased. To verify whether this
reduced viability is caused by cell death or a diminished
proliferating rate, an LDH assay indicative of cellular rupture
was conducted.37 Since the LDH concentration did not
significantly increase in cross-linked collagen hydrogels, PEG-
SG rather seems to inhibit the proliferation of dermal
fibroblasts within the gels than has acute toxic effects.
To investigate the hypothesis that long-term stable non-
contracting hydrogels can be generated by chemically cross-
linking using PEG-SG, a contraction study was performed using
fibroblast seeded collagen hydrogels. In contrast to non-cross-
linked collagen hydrogels, both concentrations of PEG-SG were
sufficient to almost completely hinder contraction. Previous
studies have described tractive forces exerted by human dermal
fibroblasts attached to collagen fibrils as a mechanism for
collagen contraction in vitro.18,38−42 Cross-linking with PEG-
SG increased the compression E-modulus. This could indicate a
more sterically rigid molecular network, rendering the cells
unable to contract the hydrogel. Furthermore, taking into
account that cross-linked collagen hydrogels formed less fibrils
as visualized in the SEM and TEM images, fewer cells can exert
force to contract the hydrogels.
After the evaluation of the effects of PEG-SG cross-linking on
the dermal part, the cross-linked collagen hydrogels were used
to generate full-thickness skin equivalents. In these experiments
we focused on the PEG-SG50 concentration that theoretically
20423
Research Article
cross-links 50% of free amines. This concentration led to the
same positive effect on the contraction but still enabled
proliferation of dermal fibroblasts comparable to standard
collagen hydrogels. All in vitro skin models formed a
multilayered epidermis with architecture and marker local-
ization closely resembling human skin. However, filaggrin was
more weakly stained in both full-thickness skin equivalents
compared to human skin. This might be due to the shorter
maturation time of engineered skin compared to the in vivo
situation.
Hence, the cross-linking had no observable effect on
epidermal differentiation or the formation of the cornified
layer of tissue-engineered skin. In addition to the differentiation
markers, the proliferation rate of human epidermal keratino-
cytes was investigated by quantifying the number of Ki76-
positive human epidermal keratinocytes in the stratum basale.
Native skin had a proliferation index of 19% similar to cross-
linked full-thickness skin equivalents. These findings are similar
to other studies, where 15% Ki67-positive basal keratinocytes in
full-thickness skin equivalents were found.43
The results of this study suggest that cross-linking with PEG-
SG can be used to generate collagen-based full-thickness skin
equivalents that do not contract during tissue maturation. This
leads to a reproducible generation of full-thickness skin
equivalents for in vitro testing or clinical application. Wounds
can be completely covered without a shrinkage of the wound
dressing that can impede the connection to the native skin. Yet,
to elucidate the full potential for in vitro and in vivo
applications, a functional comparison with the state of the art
systems is envisioned. For in vitro testing, to the best of our
knowledge, a similar result can be achieved only by using
different scaffold materials such as fibrin, which rather
resembles a wound-associated environment than skin in its
healthy state.44 Alternatively, collagen sponges can be employed
to yield standardized skin models. However, such models are
not suitable for culture in trans-well-inserts to create a culture at
the air−liquid interface that is a critical factor for epidermal
differentiation. Additionally, these models do not recreate the
hydrogel character of connective tissue in vivo.20
■ CONCLUSION
Taken together, the cross-linking with PEG-SG can be finely
tuned to gain enhanced mechanical properties compared to
unmodified collagen hydrogels. In this regard, a ratio of 0.125
mol of PEG-SG per mol of free amine groups in the collagen
that is sufficient to theoretically cross-link 50% of the free
amines in the collagen shows the best compromise between
fibroblast proliferation and inhibition of contraction. Thus,
cross-linked collagen hydrogels might provide a new bio-
material platform to generate standardized tissue equivalents
for other tissues that are also hampered by cell-mediated
contraction. Besides the application as refined in vitro models
for preclinical testing, this biomaterial platform might also be
applicable to generate new tissue grafts for clinical applications.
■
ASSOCIATED CONTENT
* Supporting Information
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acsami.7b04017.
Calculation of the molar ratio of collagen primary amine
groups and the N-hydroxysuccinimide ester of PEG-SG.
Tables 1 and 2 show the molecule processing of collagen
DOI: 10.1021/acsami.7b04017
ACS Appl. Mater. Interfaces 2017, 9, 20417−20425
ACS Applied Materials & Interfaces
alpha 1 and alpha 2 chain. Table 3 summarizes the
composition of cross-linked hydrogels (PDF)
■
AUTHOR INFORMATION
Corresponding Author
*Address: Standardized In-Vitro-Test Systems, Translational
Center Würzburg, ‘Regenerative therapies in oncology and
musculoskelettal diseases’, Würzburg Branch of the Fraunhofer
Institute Interfacial Engineering and Biotechnology (IGB),
Röntgenring 11, 97070 Würzburg, Germany. Phone: 0049 931
31 86669. E-mail: florian.groeber@igb.fraunhofer.de.
ORCID
Florian Groeber-Becker: 0000-0003-0062-289X
Author Contributions
#
Both authors contributed equally. The manuscript was written
through contributions of all authors. All authors have given
approval to the final version of the manuscript.
Funding
The authors thank the Bavarian state and the Fraunhofer-
Gesellschaft for supporting this work (VI/3-6622/453/12).
Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS
The authors thank Prof. Dr. Georg Krohne (Division of
Electron Microscopy; Biocenter of the University of Würzburg)
kindly for performing the electron microscopic analysis.
■
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