Biomaterials 32 (2011) 6729e6736

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Biomaterials
journal homepage: www.elsevier.com/locate/biomaterials

Tailoring the porosity and pore size of electrospun synthetic human elastin
scaffolds for dermal tissue engineering
Jelena Rnjak-Kovacina a, b, Steven G. Wise a, Zhe Li c, Peter K.M. Maitz c, Cara J. Young d, Yiwei Wang c,
Anthony S. Weiss a, *
a
School of Molecular Bioscience, University of Sydney, New South Wales 2006, Australia
b
Now at Department of Biomedical Engineering, School of Engineering, Tufts University, MA 02155, USA
c
Burns Unit, Concord Repatriation General Hospital, New South Wales 2139, Australia
d
Graduate School of Biomedical Engineering, University of New South Wales 2052, Australia

a r t i c l e i n f o a b s t r a c t

Article history: We obtained low and high porosity synthetic human elastin scaffolds by adapting low (1 mL/h) and high
Received 11 March 2011 (3 mL/h) flow rates respectively during electrospinning. Physical, mechanical and biological properties of
Accepted 24 May 2011 these scaffolds were screened to identify the best candidates for the bioengineering of dermal tissue. SHE
Available online 17 June 2011
scaffolds that were electrospun at the higher flow rate presented increased fiber diameter and greater
average pore size and over doubling of overall scaffold porosity. Both types of scaffold displayed Young’s
Keywords:
moduli comparable to that of native elastin, but the high porosity scaffolds possessed higher tensile
Synthetic human elastin
strength. Low and high porosity scaffolds supported early attachment, spreading and proliferation of
Tropoelastin
Dermal substitute
primary dermal fibroblasts, but only high porosity scaffolds supported active cell migration and infil-
Electrospinning tration into the scaffold. High porosity SHE scaffolds promoted cell persistence and scaffold remodeling
Pore size in vitro with only moderate scaffold contraction. The scaffolds persisted for at least 6 weeks in a mouse
Porosity subcutaneous implantation study with fibroblasts on the exterior and infiltrating, evidence of scaffold
remodeling including de novo collagen synthesis and early stage angiogenesis.
Ó 2011 Elsevier Ltd. All rights reserved.

1. Introduction employs a strong electric field to process a polymer solution into

a fibrous construct with highly interconnected pores [6]. However,
Efficacious dermal substitutes assist in the successful treatment the resulting scaffolds have inherently small pore sizes that do not
of large and deep burns [1], as they effectively close the wound and allow for cellular infiltration and ingrowth [11e16] that would be
help to guide cells during granulation tissue formation, fibroblast- essential for the replacement and repair of many tissues, including
driven remodeling, angiogenesis and re-epithelization [2]. the dermis [17e24]. Fibroblasts are the principal cellular component
Collagen-based scaffolds currently dominate the dermal substitute of the skin responsible for dermal homeostasis and repair. Dermal
field but are limited by lack of elasticity, substantial scaffold substitute scaffolds promote fibroblast adhesion, growth and infil-
contraction and scar formation during the repair process [3,4]. tration, which accelerates and enhances dermal and epidermal
Elastin-containing dermal substitutes have the potential to regeneration [17e24].
improve the elasticity and functionality of severe scars by replacing We have previously demonstrated that recombinant human
the missing elastic network or by signaling the up-regulation of tropoelastin [25], the soluble precursor of elastin, can be electrospun
elastic tissue biosynthesis during the wound healing process [5]. into three-dimensional, fibrous and highly elastic scaffolds [26e28]
Electrospinning is a valuable tool in dermal substitute engi- but a lack of cell infiltration was the major factor limiting the
neering as it generates thin, continuous polymer fibers that are potential of these scaffolds as dermal substitutes. Our initial studies
morphologically similar to native ECM fibers, and promote cellular showed that an increase in the electrospinning flow rate results in
interactions leading to new tissue formation [6e10]. Electrospinning the creation of open-weave scaffolds with large pore sizes that allow
for fibroblast infiltration into the scaffold. Here we present an
assessment of the physical, mechanical and biological properties of
* Corresponding author. School of Molecular Bioscience, Biochemistry Building
these highly porous, open-weave elastin-based scaffolds, with
G08, University of Sydney, NSW 2006 Australia. Tel.: þ61 2 9351 3464; fax: þ61 2
9351 5858. emphasis on long-term cell growth and scaffold re-modeling in vitro
E-mail address: [email protected] (A.S. Weiss). and scaffold tolerance and evidence of persistence in vivo.

0142-9612/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biomaterials.2011.05.065
6730 J. Rnjak-Kovacina et al. / Biomaterials 32 (2011) 6729e6736

2. Materials & methods unbound cells were removed by washing twice with PBS prior to further analysis.
Cells were analyzed at 30 min and days 1, 5, 8, 14, 21, 28 and 35 post-seeding.
2.1. Materials

Recombinant human tropoelastin isoform SHELD26A (Synthetic Human Elastin 2.7. Cell visualization and quantification
without domain 26A) corresponding to amino acid residues 27e724 of GenBank
entry AAC98394 (gi 182020) was purified from bacteria on a multi-gram scale as Samples for histological analysis were fixed in 4% (w/v) paraformaldehyde
previously described [29,30]. GMP clinical grade tropoelastin with the same overnight at 4  C and embedded in paraffin. 5 mm sections were deparaffinized in
sequence was sourced from Elastagen Pty Ltd for in vivo work. Primary human xylene and mounted with SlowFade Gold antifade reagent containing DAPI.
dermal fibroblasts were obtained from biopsies of healthy patients undergoing Cell proliferation over 8 days was quantified by examining DAPI-stained nuclei
breast or abdomen reduction surgery at the Concord Repatriation General Hospital in 20 SHE scaffold cross-sections (each at least 50 mm apart) for each time point
(Concord, NSW), in accordance with the approval of the hospital research and ethics (n ¼ 2e3 per treatment/timepoint).
committee. All reagents were purchased from SigmaeAldrich unless specified
otherwise.

2.8. Detection of ECM proteins secreted by cells

2.2. Electrospinning and cross-linking tropoelastin
Electrospun SHE scaffolds (n ¼ 3) were fixed in 4% (w/v) paraformaldehyde and
A 20% (w/v) solution of tropoelastin was dissolved in 1,1,1,3,3,3-hexafluoro- embedded in paraffin. 5 mm sections were deparaffinized in xylene and immuno-
2-propanol (HFP) and loaded into a syringe equipped with a blunt 18 gauge needle. probed for the expression of collagen I and fibronectin. Immunohistochemistry
Flow rates of 1 mL/h and 3 mL/h were modulated using a syringe pump. The needle studies were controlled by comparison with samples that were devoid of cells and
was connected to a 20 kV positive power supply (Gamma High Voltage Research, no primary antibody and isotype controls.
Inc.) and directed at a grounded, 30 mm diameter circular, brass collector at Collagen I target retrieval was achieved by incubation with 100 mM glycine HCl
a collector distance of 20 cm. Electrospun tropoelastin scaffolds were chemically and 50 mM ammonium chloride in 0.1 M Tris Buffer Saline (TBS) containing 0.3 M
cross-linked to stabilize their structures in aqueous environments. Scaffolds were NaCl and 0.05% (v/v) Tween-20 for 30 min at room temperature, followed by
placed in an open stage desiccator and cross-linked by vapor from a separate 25% (v/ incubation in 0.2% (w/v) hyaluronidase in TBS (without Tween-20) at 37  C for
v) aqueous glutaraldehyde (GA) solution. Unreacted GA in the scaffolds was 15 min. Endogenous peroxidase activity was blocked by incubation with 1% (v/v)
quenched by immersion into 0.2 M glycine solution overnight. Scaffolds were then hydrogen peroxide in methanol for 20 min at room temperature. Non-specific
washed repeatedly in PBS. Cross-linked tropoelastin is referred to as synthetic binding was blocked with 10% (v/v) goat serum in TBS for 20 min at room
human elastin (SHE) scaffolds or fibers. temperature. Samples were incubated with polyclonal anti-collagen I antibody
(AbCam) for 1 h at room temperature, followed by Dako EnVision HRP labeled
2.3. Fiber width quantification polymer (anti-rabbit) for 30 min at room temperature. Color was developed with
Dako Liquid DAB þ Substrate chromagen system for 5 min at room temperature
Cross-linked electrospun SHE scaffolds were fixed with 2.5% (w/v) glutaralde- followed by multiple water washes. Samples were counterstained with hematoxylin
hyde in 0.1 M phosphate buffer (PB) for 1 h at room temperature. Samples were and embedded in DPX mountant for Microscopy-10.
washed 3X with PB and post-fixed with 1% (v/v) osmium tetroxide in PB. The Fibronectin target retrieval was achieved by incubation in Dako target retrieval
scaffolds were dehydrated in increasing concentrations of ethanol and dried over- solution (pH 9) for 12 min at 95  C. Endogenous peroxidase activity was blocked by
night in hexamethyldisilazane. Uncross-linked and cross-linked samples were incubation with 1% (v/v) hydrogen peroxide in methanol for 20 min at room
sputter-coated with 20 nm gold and imaged using a Philips XL-30 Scanning Electron temperature. Non-specific binding was blocked with 10% (v/v) goat serum in TBS for
Microscope (SEM). The widths of 10 fibers within an image for a total of 10 images 20 min at room temperature. Samples were incubated with monoclonal anti-
per sample (n ¼ 3 per treatment) were quantified using ImageJ (National Institutes fibronectin antibody (Thermo Scientific) for 1 h at room temperature, followed by
of Health) [31]. Dako EnVision HRP labeled polymer (anti-mouse) for 30 min at room temperature.

2.4. Pore diameter and porosity quantification

2.9. Quantification of scaffold contraction
Pore diameters were quantified from SEM images of electrospun SHE scaffolds.
The diameters of the longest axes in each of 5 pores within an image for a total of 5 High porosity SHE scaffold contraction was quantified by measuring the diam-
images per sample (n ¼ 3 per treatment) were quantified using ImageJ. eter of circular scaffolds seeded with fibroblasts up to 35 days, expressed as
Scaffold porosity was quantified from hematoxylin and eosin (H&E)-stained a percentage of the original scaffold diameter prior to cell seeding (% original scaf-
scaffold cross-sections [32] with modifications. SHE scaffolds were fixed in 10% (w/ fold diameter) (n ¼ 3 per time point).
v) buffered formalin solution and embedded in paraffin. 5 mm sections were
deparaffinized in xylene and stained with H&E. Section images were converted to
binary format using ImageJ, where the fibers appeared black and pores white. The 2.10. Subcutaneous implantation of SHE scaffolds in mice
percentage of the white area relative to the total scaffold section surface area in each
image was defined as the percent porosity. A mouse model of subcutaneous implantation was used to evaluate the toler-
ance, persistence and cellularization of SHE scaffolds. Pathogen-free, female and
2.5. Mechanical properties of SHE scaffolds male BALB/c mice, aged 8 weeks and weighing 21  1.9 g (female)/25  2.7 g (male)
were purchased from the Australian Animal Resources Center. All animals were
Tensile mechanical properties of SHE scaffolds (dimensions 3  1 cm) were acquired, housed and studied under a protocol approved by SSWAHS Animal
tested using an Instron 5543 with a 50 N load cell at a constant strain rate of 3 mm/ Welfare Committee in Sydney, Australia. Each mouse (n ¼ 7) was anesthetized
min until failure (n ¼ 2e3). Samples were immersed in PBS at 37  C during testing. individually by intra-peritoneal injection of a mixture of ketamine (75 mg/ml) and
The Young’s modulus was obtained from the slope of the stress-strain curve xylazine (10 mg/ml) at 0.01 ml/g of body weight. The dorsal hair was shaved and
generated over the linear portion of the strain range after the initial toe region. skin was cleaned with betadine solution and washed with sterile saline. An incision
Tensile strength was defined as the ultimate stress at break. The calculation of strain of about 1 cm in length was made and dissected to create a subcutaneous pouch into
was based on the cross-sectional area of each of the samples. which the SHE scaffold was inserted. The wounds were then closed with 6-0 silk
sutures and covered using IV3000 wound dressings (Smith & Nephew) for 5 days.
2.6. Fibroblast isolation and cell culture Carprofen (5 mg/kg) was given at the time of anesthesia and then on the following
day, post surgery for analgesia. After surgery, each mouse was caged individually for
Human skin dermis was separated from the epidermis by incubation in dispase, the first two days and then two mice per cage thereafter with free access to water
diced into small sections and incubated in 1 mg/mL type I collagenase solution at and food. Skin biopsies were collected for histological analysis at 6 weeks post-
37 C. The digested dermal mixture was filtered through a 100 mm cell strainer to
 implantation.
harvest cells. Dermal fibroblasts up to passage 5 were maintained in Dulbecco’s
Modified Eagle’s Medium (DMEM) supplemented with 10% (v/v) fetal calf serum, 1%
(v/v) L-glutamine and 50 mmol/mL gentamicin. 2.11. Statistical analysis
Fibroblasts (5  104 /cm2) were seeded onto three-dimensional (3D) SHE scaf-
folds or a thin layer of electrospun SHE fibers on glass coverslips in a static cell Data are expressed as mean  standard deviation. Statistically significant
culture system. 3D scaffolds were kept retained in cell culture wells using hollow differences were determined by one- or two-way analysis of variance (ANOVA) and
well inserts for 3 h post-seeding. Scaffolds were then transferred to larger wells with Bonferroni post-test. Statistical significance was accepted at p < 0.05 and indicated
no rings and cell culture media was changed every 2 days. Loosely adherent or in the figures as *(p < 0.05), **(p < 0.01), ***(p < 0.001).
 J. Rnjak-Kovacina et al. / Biomaterials 32 (2011) 6729e6736 6731

Fig. 1. Physical properties of SHE scaffolds electrospun at 1 mL/and 3 mL/h. (A) Tropoelastin fiber diameter distribution, (B) SHE scaffold cross-sections, (C) SHE scaffold porosity and
pore size and (D) SHE scaffold Young’s modulus and tensile strength.
6732 J. Rnjak-Kovacina et al. / Biomaterials 32 (2011) 6729e6736

3. Results on low and high porosity 3D SHE scaffolds were analyzed using
DAPI-stained scaffolds sections, where cell nuclei appeared blue,
3.1. Effect of flow rate on physical properties of electrospun while SHE scaffolds appeared red as a result of elastin auto-
tropoelastin scaffolds fluorescence. Cells seeded on low porosity scaffolds only prolifer-
ated across the scaffold surface, in comparison to cells seeded on
Electrospun tropoelastin fibers displayed flat, ribbon-like high porosity scaffolds which infiltrated the scaffold. At day 1,
morphology when electrospun at 1 mL/h and 3 mL/h. The mean post-seeding cell attachment was confined to the surface of high
fiber diameter increased significantly with increased flow rate porosity scaffolds, but by day 3 post-seeding cells had migrated half
(p < 0.001), at 2.3  0.5 mm when electrospun at 1 mL/h compared way through the scaffold and by day 8 post-seeding they spanned
to 3.2  1.0 mm at 3 mL/h. The fiber diameter distribution was also the entire scaffold and reached the unseeded surface (Fig. 3A).
influenced by the flow rate. At 1 mL/h, most fiber diameters were Cell numbers increased over time on both scaffolds. Cell
2e3 mm but ranged between 1 mm and 4 mm. When tropoelastin numbers on low porosity scaffolds increased 2.1-fold between day
was electrospun at 3 mL/h the fiber diameter distribution was 1 and day 3 post-seeding and 2.5-fold between day 3 and day 8
broader, ranging from 1 mm to 7 mm (Fig. 1A). post-seeding. Cell numbers on high porosity scaffolds increased by
Increasing the flow rate increased the scaffold porosity and pore 1.9-fold between day 1 and day 3 post-seeding and by 1.9 fold
size. SHE scaffolds electrospun at increased flow rates showed an between day 3 and day 8 post-seeding (Fig. 3B).
increase in open space between fibers and a decrease in fiber
density. Scaffold thickness depended on the amount of solution 3.3. Long-term cellular interactions with high porosity SHE
that was electrospun, but SHE scaffolds electrospun at 3 ml/h were scaffolds in vitro
consistently 1.6 times thicker than those at 1 ml/h, when the same
volume of tropoelastin solution was used (Fig. 1B). Scaffolds made Considering superior cell infiltration into high porosity over low
at 1 mL/h and 3 mL/h displayed increasing (p < 0.001) porosities of porosity SHE scaffolds, only high porosity scaffolds were studied
14.5  0.8% and 34.4  1.3% respectively. When tropoelastin was further. Having established cell attachment, infiltration and migra-
electrospun at 1 mL/h, the pore diameters ranged from 1.6 mm to tion on these scaffolds over a relatively short cell culture period (8
21.3 mm with a mean of 8.0  0.2 mm, which was less (p < 0.05) than days), cells were grown for extended periods post-seeding to study
at 3 mL/h with a range of 4.0 mme27.9 mm and mean pore diameter cell persistence, scaffold remodeling and contraction in vitro. Cells
of 11.7  0.1 mm (Fig. 1C). SHE scaffolds electrospun at 1 mL/h are persisted throughout high porosity SHE scaffolds for at least 35 days
therefore referred to as ‘low porosity’ and 3 mL/h as ‘high porosity’. in a static cell culture system (Fig. 4A) and expressed ECM proteins
High porosity SHE scaffolds showed similar elasticity but higher collagen I and fibronectin (Fig. 4B). Collagen I and fibronectin
tensile strength than low porosity scaffolds. The Young’s moduli of expression appeared by day 14 post-seeding, then progressively
low and high porosity SHE scaffolds were comparable at increased over the following 21 days, with the most prominent
219.8  57.8 kPa and 139.9  29.0 kPa respectively (Fig. 1D). expression seen at day 35 post-seeding. An increase in protein
Elongation at break of low porosity scaffolds ranged from 34% to deposition over time was particularly obvious within the scaffold,
65%, compared to 76%e109% for high porosity scaffolds. Low while collagen I and fibronectin on the scaffold surface remained
porosity SHE scaffolds had lower tensile strength (p < 0.001) at high throughout the cell culture period (Fig. 4B).
150.9  7.5 kPa than the high porosity scaffolds at 219.8  3.9 kPa SHE scaffolds retained most (87.1%) of their original size in the
(Fig. 1D). first 14 days post-seeding and contracted slightly more to 68.3% of
their original size by day 35 post-seeding in vitro (Fig. 5). No
3.2. Cellular interactions with low and high porosity SHE scaffolds obvious decrease in scaffold thickness was observed during the 35
in vitro day incubation period (Fig. 4A).

Cell interactions with individual fibers were studied on thin 3.4. High porosity SHE scaffold tolerance, persistence and cellular
layers of fibers electrospun onto glass coverslips at a flow rate of interactions in vivo
1 ml/h (low porosity) or 3 ml/h (high porosity). Fibroblasts attached
to and started spreading on the fibers of low and high porosity High porosity SHE scaffolds were implanted subcutaneously in
scaffolds as early as 30 min post-seeding, with cellular projections mice to study their tolerance and to gauge persistence and cellular
extending along the fibers (Fig. 2). Cell infiltration and proliferation interactions in vivo in this environment. Explanted scaffolds and

Fig. 2. Dermal fibroblast interactions with the fibers of (A) low porosity and (B) high porosity SHE scaffolds.
 J. Rnjak-Kovacina et al. / Biomaterials 32 (2011) 6729e6736 6733

Fig. 3. Dermal fibroblast migration and proliferation on low and high porosity SHE scaffolds over 8 days. (A) SHE scaffold cross-sections showing DAPI-stained cells in blue and
scaffold autofluorescence in red. Scale bars are 200 mm; (B) Quantification of fibroblast proliferation. (For interpretation of the references to colour in this figure legend, the reader is
referred to the web version of this article.)

surrounding skin were stained with H&E and Milligan’s trichrome infiltrated far into the scaffold and were accompanied by some
stains. When stained with H&E, SHE scaffolds appeared bright pink, scaffold breakup (Fig. 6D). Infiltrating fibroblasts deposited collagen
while cells appeared blue. When stained with Milligan’s trichrome, fibers throughout the SHE scaffolds (Fig. 6E). Early stages of angio-
SHE scaffolds appeared magenta and collagen appeared turquoise. genesis were evidenced by small capillaries that formed at the edges
SHE scaffolds persisted for at least 6 weeks post-implantation and of the scaffolds (Fig. 6F).
displayed moderate scaffold degradation and scaffold remodeling.
SHE scaffolds were encapsulated with 5e10 layers of fibroblast cells 4. Discussion
(Fig. 6A) and were accompanied by native mouse collagen, as indi-
cated by turquoise staining around the scaffold (Fig. 6B). No Statistical modeling and experimental data reveal that an
neutrophils or monocytes were found and no other obvious increase in the diameter of electrospun fibers is accompanied by in
immunological response was observed. In contrast, there was a high an increase in the pore size of electrospun scaffolds [15,32e37]. We
level of fibroblast infiltration into the scaffold (Fig. 6C). Some cells have utilized this finding to increase the overall scaffold porosity
6734 J. Rnjak-Kovacina et al. / Biomaterials 32 (2011) 6729e6736

Fig. 4. Long term (up to 35 days post-seeding) in vitro dermal fibroblast growth on high porosity SHE scaffolds; (A) SHE scaffold cross-sections showing DAPI-stained cells in blue
and scaffold autofluorescence in red; (B) IHC staining of type I collagen and fibronectin shown in brown. Scale bars are 50 mm. (For interpretation of the references to colour in this
figure legend, the reader is referred to the web version of this article.)

and pore size of electrospun SHE scaffolds. A strong correlation disagreement with the data obtained here. However, the relation-
between the flow rate at which tropoelastin was electrospun and ship between fiber diameter and elasticity of elastin-based scaf-
the final scaffold porosity and pore size was observed. This rela- folds had never been tested while most comparable studies use
tionship is a result of an increase in the fiber diameter with stiffer synthetic polymers.
increasing flow rate [34]. An increase in the flow rate from 1 mL/h Dermal fibroblasts interacted favorably with high porosity SHE
to 3 mL/h resulted in a 1.4-fold increase in the tropoelastin fiber scaffolds in vitro, displaying early cell attachment and spreading,
diameter, which corresponded to a 1.5-fold increase in the average cell proliferation and active intra-scaffold migration. Fibroblasts
pore size and a 2.3-fold increase in the overall scaffold porosity attached to and spread along the fibers of low and high porosity
(Fig. 1AeC). scaffolds as early as 30 min post-seeding (Fig. 2). Cells proliferated
High porosity SHE scaffolds displayed similar elasticity, but were on low and high porosity SHE scaffolds for at least 8 days post-
significantly stronger than low porosity scaffolds (Fig. 1D). There is seeding (Fig. 3B). Cell proliferation rate was slightly lower on high
no agreement in the electrospinning literature as to the effect of porosity scaffolds compared to low porosity scaffolds in a static cell
increased fiber diameter on the tensile strength properties of culture system, most likely a result of reduced oxygen and nutrients
electrospun scaffolds. Increased fiber diameter has been reported available to infiltrating cells compared to those cells that were
to both increase [32] and decrease [8] tensile strength of electro- restricted to scaffold surfaces [7].
spun scaffolds. The difference in tensile strength properties of low Cells on high porosity SHE scaffolds displayed active cell
and high porosity scaffolds may be a result of the differences in migration rather than passive infiltration. At day 1, post-seeding
fiber diameter, the number of cross-links formed, scaffold porosity cells were only present on the surface of high porosity scaffolds,
or fiber alignment. Most reports suggest that an increase in fiber while on day 3, they migrated w100 mm through the scaffold and
diameter results in increased scaffold stiffness [8,32], which is in by day 8 post-seeding, cells had migrated throughout the entire
scaffold to reach the unseeded surface (Fig. 3A). The even cell
distribution and proliferation on high porosity SHE scaffolds was
encouraging as it indicates that cells interact with these porous
scaffolds in a manner that is conducive to cell growth.
Having established favorable interactions between dermal
fibroblasts and high porosity SHE scaffolds over a relatively short
cell culture period (8 days), cell persistence and scaffold remodel-
ing were investigated over 5 weeks in vitro. Fibroblasts infiltrated
throughout persistent scaffolds and deposited increasing amounts
of collagen type I and fibronectin over the cell culture period
(Fig. 4), demonstrating the cells’ ability to proliferate in and modify
their environment [38].
High porosity SHE scaffolds displayed only moderate scaffold
contraction in this system (Fig. 5), compared to significant contrac-
tion that is typically observed when collagen scaffolds are cultured
with dermal fibroblasts. For example, electrospun collagen scaffolds
contract to less than 50% their original size within the first 7 days of
culture with dermal fibroblasts [39]. The limited contraction found
here may be due to the elasticity of SHE scaffolds or signaling events
initiated by cellular interactions with elastin. Fibroblast differentia-
Fig. 5. High porosity SHE scaffold contraction over a 35 day fibroblast cell culture tion into contractile myofibroblasts is reduced on elastic substrates
period. [40] and elastin may suppress the differentiation of phenotypically
 J. Rnjak-Kovacina et al. / Biomaterials 32 (2011) 6729e6736 6735

Fig. 6. High porosity SHE scaffold subcutaneous implantation in mice. Images (A), (C) and (D) are stained with H&E, while images (B), (E) and (F) are stained with Milligan’s
trichrome. Boundaries of SHE scaffolds are indicated with black dashed lines in images (A), (B) and (D). Black arrowheads point to dermal fibroblasts encapsulating the SHE scaffold
in image (A), collagen deposition encapsulating the SHE scaffold in image (B), native collagen fibers deposited between SHE fibers in image (E) and to newly formed capillaries in
image (F). Scale bars in images A&B are 100 mm and in images CeF are 50 mm.

proliferating fibroblasts into contractile myofibroblasts in vitro [41]. Acknowledgments

In support of this model, the presence of a-elastin in collagen-based
scaffolds has been shown to decrease scaffold stiffness [42] and JR is a recipient of the Australian Postgraduate Award. ASW
modulate collagen contraction in vivo [43,44]. acknowledges grant support from the Australian Research Council,
High porosity SHE scaffolds supported favorable interactions the National Health and Medical Research Council" National Health
with primary dermal fibroblasts in vitro, in particular, cell migration and Medical Research Council and the Defence Health Foundation.
and even distribution of ECM proteins throughout the scaffold in the ASW is Founder and Consultant Scientist at Elastagen Pty Ltd. We
absence of major scaffold contraction. Having established the thank Elastagen Pty Ltd for a gift of GMP clinical grade tropoelastin.
potential of these scaffolds as dermal substitutes in vitro, we sought The authors gratefully acknowledge the contributions of Dr. Jane
to assess their tolerance, and gauge persistence and cell interactive Radford and Ms. Carolynn Glover, Histopathology Laboratory, The
properties in an in vivo model. SHE scaffolds were therefore exam- University of Sydney and Dr. Penny Martens, Graduate School of
ined following subcutaneous implantation in mice. Biomedical Engineering, University of NSW. The authors also
High porosity SHE scaffolds persisted for at least 6 weeks in vivo, acknowledge the facilities as well as scientific and technical assis-
displaying moderate scaffold degradation. SHE scaffolds did not tance from staff in the AMMRF (Australian Microscopy & Micro-
cause any major negative immunological response in mice. They analysis Research Facility) at the Australian Center for Microscopy
were surrounded by a thin layer of dermal fibroblasts which had and Microanalysis, The University of Sydney. We thank Ms. Yannie
apparently secreted a layer of collagen around the scaffolds and Poon & Ms. Kate Nieuwendyk for technical assistance.
provided a reservoir of infiltrating fibroblast cells which contrib-
uted to scaffold degradation and remodeling over the 6 week
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