Extracellular Matrix Powder Protects Against Bleomycin-Induced Pulmonary Fibrosis

Article (PDF Available)inTissue Engineering Part A 17(21-22):2795-804 · July 2011with28 Reads
DOI: 10.1089/ten.tea.2011.0023 · Source: PubMed
Pulmonary fibrosis refers to a group of lung diseases characterized by inflammation, fibroblast proliferation, and excessive collagen deposition. Although the mechanisms underlying pulmonary fibrosis are poorly understood, current evidence suggests that epithelial injury contributes to the development of fibrosis. Regenerative medicine approaches using extracellular matrix (ECM) scaffolds have been shown to promote site-specific tissue remodeling. This led to the hypothesis that particulate ECM would promote normal tissue repair and attenuate bleomycin-induced pulmonary fibrosis. C57BL/6 mice were treated intratracheally with bleomycin or saline with or without a particulate form of ECM scaffold from porcine urinary bladder matrix (UBM-ECM) or enzymatically digested UBM-ECM. Mice were sacrificed 5 and 14 days after exposure. Compared to control mice, bleomycin-exposed mice had similar increases in inflammation in the bronchoalveolar lavage fluid regardless of UBM-ECM treatment. However, 14 days after exposure, lung histology and collagen levels revealed that mice treated with bleomycin and the particulate or digested UBM-ECM had negligible fibrosis, whereas mice given only bleomycin had marked fibrosis. Administration of the particulate UBM-ECM 24 h after bleomycin exposure also significantly protected against pulmonary injury. In vitro epithelial cell migration and wound healing assays revealed that particulate UBM-ECM promoted epithelial cell chemotaxis and migration. This suggests that promotion of epithelial wound repair may be one mechanism in which UBM-ECM limits pulmonary fibrosis.
Extracellular Matrix Powder Protects Against
Bleomycin-Induced Pulmonary Fibrosis
Michelle L. Manni, Ph.D.,
Caitlin A. Czajka, B.S.,
Tim D. Oury, M.D., Ph.D.,
and Thomas W. Gilbert, Ph.D.
Pulmonary fibrosis refers to a group of lung diseases characterized by inflammation, fibroblast proliferation, and
excessive collagen deposition. Although the mechanisms underlying pulmonary fibrosis are poorly understood,
current evidence suggests that epithelial injury contributes to the development of fibrosis. Regenerative medicine
approaches using extracellular matrix (ECM) scaffolds have been shown to promote site-specific tissue remodeling.
This led to the hypothesis that particulate ECM would promote normal tissue repair and attenuate bleomycin-
induced pulmonary fibrosis. C57BL/6 mice were treated intratracheally with bleomycin or saline with or without a
particulate form of ECM scaffold from porcine urinary bladder matrix (UBM-ECM) or enzymatically digested
UBM-ECM. Mice were sacrificed 5 and 14 days after exposure. Compared to control mice, bleomycin-exposed
mice had similar increases in inflammation in the bronchoalveolar lavage fluid regardless of UBM-ECM treatment.
However, 14 days after exposure, lung histology and collagen levels revealed that mice treated with bleomycin and
the particulate or digested UBM-ECM had negligible fibrosis, whereas mice given only bleomycin had marked
fibrosis. Administration of the particulate UBM-ECM 24 h after bleomycin exposure also significantly protected
against pulmonary injury. In vitro epithelial cell migration and wound healing assays revealed that particulate
UBM-ECM promoted epithelial cell chemotaxis and migration. This suggests that promotion of epithelial wound
repair may be one mechanism in which UBM-ECM limits pulmonary fibrosis.
diopathic pulmonary fibrosis (IPF) is a debilitating
disease characterized by inflammation, fibroblast prolif-
eration, and excessive extracellular matrix (ECM) deposition
in the lung. The pathogenesis of pulmonary fibrosis has been
studied widely in animal models. The most widely used
model to study pulmonary fibrosis in rodents is the bleo-
mycin model.
While the mechanisms for injury are still not
fully understood, it appears that the pathology begins with
epithelial damage followed by activation of alveolar macro-
phages and infiltration of the lung tissue by circulating in-
flammatory cells that release cytokines such as tumor
necrosis factor-a and interleukin-1b.
Ultimately, this
proinflammatory environment leads to the recruitment and
proliferation of fibroblasts/myofibroblasts that produce
transforming growth factor-b
and deposit large quantities
of collagenous, fibrotic tissue. Currently, pulmonary trans-
plantation is the only viable treatment option for patients
with IPF.
New molecular therapies for pulmonary fibrosis
have been suggested and are under investigation, but there is
still a need for alternatives for treatment of IPF.
Biologic scaffolds composed of mammalian ECM have
been shown to promote site-specific remodeling of muscu-
loskeletal, cardiovascular, urogenital, and dermal tissues.
The mechanisms by which these ECM scaffolds promote
tissue remodeling are not fully understood, but appear to
include the presentation of a three-dimensional microenvi-
ronment supportive of cell growth and migration that
transmits biochemical and mechanical cues to the cells,
rapid degradation with subsequent release of small peptide
fragments that possess innate bioactivity (e.g., chemotaxis for
progenitor cells and antibacterial behavior),
and modu-
lation of the host immune response.
ECM scaffolds have recently received attention for treat-
ment of airway injury in several preclinical models. Recent
studies have shown that ECM scaffold materials can prevent
air leakage into the pleural cavity when used as a primary
treatment or when used as reinforcement for a surgical staple
line after partial lung resection.
Repair of the lung with
urinary bladder matrix (UBM) scaffold showed moderately
dense well-organized collagenous tissue formation at the site
of resection without evidence of inflammation, necrosis, or
scarring in the lung.
In addition, UBM has recently been
Departments of
Pathology and
Bioengineering, University of Pittsburgh, Pittsburgh, Pennsylvania.
McGowan Institute for Regenerative Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania.
Department of Surgery, University of Pittsburgh, Pittsburgh, Pennsylvania.
Department of Cardiothoracic Surgery, Children’s Hospital of Pittsburgh, University of Pittsburgh Medical Center, Pittsburgh, Penn-
Volume 17, Numbers 21 and 22, 2011
ª Mary Ann Liebert, Inc.
DOI: 10.1089/ten.tea.2011.0023
shown to promote the formation of site-appropriate pseu-
dostratified, columnar, ciliated epithelium when used for
patch tracheoplasty in a canine model.
Due to its ability to
promote site-specific tissue remodeling in the airway, it was
hypothesized that UBM could attenuate bleomycin-induced
pulmonary fibrosis, which is the focus of the present study.
Further, the results for an intact, lyophilized UBM powder
were compared to an enzymatically digested form of UBM
that would simulate the release of bioactive degradation
products from the intact UBM, thereby allowing an under-
standing of whether the intact morphology of the UBM or
the degradation products are more important to the pre-
vention of pulmonary fibrosis.
Materials and Methods
Preparation of UBM-ECM
The preparation of UBM has been previously described.
Porcine urinary bladders were harvested from market-weight
pigs (*110–130 kg) immediately after sacrifice at an abattoir
and transported to the lab on ice. The urothelial layer of the
bladders was removed by soaking the material in 1 M saline.
The tunica serosa, tunica muscularis externa, tunica submu-
cosa, and most of the muscularis mucosa were mechanically
delaminated from the bladder tissue. The remaining basement
membrane of the tunica epithelialis mucosa and the subjacent
tunica propria were collectively termed UBM. UBM was de-
cellularized and disinfected by immersion in 0.1% (v/v) per-
acetic acid, 4% (v/v) ethanol, and 96% (v/v) deionized water
O) (pH *2.5) for 2 h at room temperature. The material
was then washed twice for 15 min with phosphate-buffered
saline (pH 7.4) and twice for 15 min with diH
After the UBM was decellularized and disinfected, the
scaffold was lyophilized and chopped into small sheets. The
chopped material was then fed through a rotary knife mill. A
#60 screen was used to restrict the collected powder size to
< 250 mm. The powder was then sifted through stainless steel
mesh on a Sonic Sifter to < 75 mm. For all studies, the par-
ticulate material was terminally sterilized with 2 MRad g -
irradiation before use in vivo.
Particulate UBM was also added to 1 mg/mL pepsin
(Sigma) in 0.01 M HCl for a final concentration of 10 mg/mL
UBM suspension. The suspension was mixed on a stir plate
at room temperature for *48 h until no visible pieces of
UBM remained. Pepsin buffer control samples were pre-
pared by mixing the pepsin digestion buffer (1 mg/mL
pepsin in 0.01 M HCl) at room temperature for 48 h.
All animal experiments were reviewed and approved by
the University of Pittsburgh Institutional Animal Care and
Use Committee. All treatments were done intratracheally as
previously described.
Ten-week-old male C57BL/6 mice
(unless otherwise noted) were intratracheally instilled with
0.07 units of bleomycin sulfate (Hospira, Inc) with or without
280 mg particulate UBM or digested UBM to determine the
effect of UBM on the development of fibrosis. Control mice
were treated with 0.9% saline vehicle with and without
280 mg of particulate or digested UBM. Mice were euthanized
either 5 or 14 days after exposure. In a separate experiment,
16-week-old male C57BL/6 wild-type mice were intra-
tracheally treated with bleomycin or saline vehicle and then
given particulate UBM or saline intratracheally 24 h later.
These mice were euthanized 14 days after the initial bleomy-
cin or saline treatment. For both experiments, bronchoalveolar
lavage fluid (BALF) was obtained by the intratracheal instil-
lation and recovery of 0.8 mL of 0.9% saline as previously
Lungs from mice 14 days after exposure were
inflation fixed with 10% buffered formalin and paraffin
embedded for histological analysis of fibrosis.
Bronchoalveolar lavage fluid
Total protein was determined by use of the Coomassie
Plus Protein Assay Reagent (Pierce). Total white blood cell
counts were obtained with a Beckman Z1 Coulter particle
counter (Beckman Coulter). To obtain a differential count,
BALF samples were adhered to glass slides with a cytospin,
stained with DiffQuik, and the numbers of macrophages,
neutrophils, lymphocytes, and eosinophils were counted
under a microscope (at least 200 cells). The remaining BALF
was centrifuged at 200 · g and supernatants were stored at
- 70C. Protein concentration in undiluted BALF was mea-
sured as previously described.
Histology and fibrosis scoring
Standard hematoxylin and eosin staining was performed
on 5-mm-thick lung sections as previously described.
matoxylin and eosin-stained sections were scored as previ-
ously described
by a pathologist (T.D.O.) who was
blinded to sample groups. Individual fields were examined
with a light microscope at · 200 magnification. Briefly, every
field in the entire lung was scored, starting peripherally. To be
counted, each field had to contain alveolar tissue in > 50% of
the field. Scoring in each field was based on the percentage of
alveolar tissue with interstitial fibrosis according to the fol-
lowing scale: 0 = no fibrosis, 1 = up to 25%, 2 = 25%–50%,
3 = 50%–75%, 4 = 75%–100%. The pathological index score was
then reported as a ratio of the sum of all of the scores divided
by the total number of fields counted for each sample.
Picrosirius red collagen staining
Slides stained with picrosirius red were viewed on a Ni-
kon E600 microscope (Nikon) with Nuance digital camera
(CRi). Images were captured using Nuance Imaging System
software. At · 400 magnification, three random images of
the lung parenchyma were captured and saved. Analysis of
the red areas of the slide was completed using ImageJ (NIH)
and the ‘Threshold Colour’ plug-in (courtesy of Gabriel
Images were loaded and colors were isolated
by using the hue histogram filter available in ‘Threshold
Colour.’ Images were subjected to a threshold such that
each nonwhite pixel was turned black and each white pixel
remained white. Then, the number of black pixels in each
image was used to calculate the percentage of the image area
that corresponded to a certain color. The average percentage
was then calculated for the saline, saline + particulate UBM,
bleomycin, and bleomycin + particulate UBM groups.
Toxicity assay
A549 human epithelial cells (ATCC) were cultured in F12K
media supplemented with 10% fetal bovine serum (FBS). A549
cells were seeded on 96-well plates (5000 cells/well). Cells
were serum starved for 4 h before treatment with either 0.02
units of bleomycin, or bleomycin with various amount of
particulate and digested UBM as indicated in serum-free
media. Twenty-four hours after treatment, cell viability was
measured using CellTiter 96 AQ Nonradioactive Assay ac-
cording to manufacturer’s instructions (Promega).
Chemotaxis assay
Responses of epithelial cells to UBM degradation products
were quantitatively evaluated utilizing the Neuro Probe 48-
well microchemotaxis chamber (Neuro Probe). A549 cells
were serum-starved for 14–17 h before experimentation. Based
upon pilot studies to determine the appropriate filter pore
size, 5 mm polycarbonate chemotaxis filters (Neuro Probe,
PFB5) were coated equally on both sides (by immersion) with
0.05mg/mL rat tail collagen I (BD Biosciences) and allowed to
dry before chamber assembly. About 27.5 mLofF12Kmedia
(Cellgro, 10–025-CV), F12K media supplemented with 10%
FBS, 0.5 mg/mL UBM digest, 0.1 mg/mL UBM digest,
0.5 mg/mL Pepsin digest, and 0.1 mg/mL Pepsin digest was
added to the bottom chamber wells. The filter was placed over
the bottom chamber, and the apparatus was assembled ac-
cording to the manufacturer’s instructions. Approximately
30,000 cells were then added to each upper chamber well of
the apparatus, and the chamber was incubated for 4 h at 37C
in a humidified atmosphere in 95% air:5% CO
. Cells re-
maining on the topside of the membrane (i.e., nonmigrated
cells) were removed, and then cells on the bottom side of
the membrane (i.e., migrated cells) were stained with Diff
Quik (Dade AG). The filter was then mounted with Vecta-
shield containing DAPI (Vector Laboratories, H-1200) and
fluorescent images of each conditioned well were captured at
10 · magnification using a Nuance multispectral imaging
system and Nikon microscope. Each experimental condition
was tested in quadruplicate in three independent experiments.
The average number of migrated cells in each experiment was
normalized to the positive control (10% FBS) by calculating
the average percentage of migrated cells as a percentage of the
positive control for each condition.
Wound healing assay
A549 cells cultured in F12K media (Cellgro 10–025-CV)
supplemented with 10% FBS were seeded and grown to
confluence in a six-well plate. Cells were serum starved in
nonsupplemented F12K media overnight before initiation of
the experiment. Straight wounds were created as previously
by scratching vertically with a p-200 micropi-
pette tip. The wounds were then washed and treated with
the following experimental conditions: 0.075 U/mL bleomy-
cin; 0.075 U/mL bleomycin + 0.5 mg/mL particulate UBM;
0.075 U/mL bleomycin + 0.5 mg/mL UBM digest; 0.075 U/
mL bleomycin + 0.5 mg/mL pepsin digest; and 0.5 mg/mL
UBM digest. Images were captured at 0 and 24 h after ad-
dition of treatments. Using ImageJ, average wound widths in
pixels were obtained for each well at each time point.
Statistical analyses
Data were analyzed using GraphPad Prism 5 (Graphpad
Software Inc.). The significance of all quantitative data was
assessed using a one-way analysis of variance with Tukey’s
post-test except for the delayed administration animal study
in which a two-way analysis of variance with a Bonferroni
post-test was used. All values are means ( standard error of
the mean). A p-value < 0.05 was considered to be statistically
Wild-type C57BL/6 mice were treated with bleomycin or
saline vehicle with and without particulate UBM or digested
UBM and sacrificed 5 and 14 days after exposure. There was
an increase in the number of leukocytes within the BALF at
both 5 and 14 days after bleomycin exposure regardless of
UBM treatment (Fig. 1A). The BALF differentials showed a
significant increase in macrophages and modest increases in
neutrophils and lymphocytes in the BALF of mice treated
with bleomycin and bleomycin with either particulate or
digested UBM when compared to controls at both 5 and 14
days after intratracheal instillation (Fig. 1B).
Although UBM treatment did not affect the inflammatory
response to bleomycin, histological analyses of the lungs 14
days after treatment showed a significant reduction in fi-
brosis in the lungs of mice that were co-administered par-
ticulate UBM with bleomycin when compared to the mice
that received bleomycin alone (Fig. 2A). These differences in
fibrosis were quantified through histological scoring (Fig. 2B)
and further confirmed by picrosirius red staining, another
measure of collagen deposition in the lungs (Fig. 2C). Ad-
ministration of UBM alone did not cause any adverse effects
and the lungs were pathologically identical to the saline
controls (Fig. 2A). Similar protective effects were seen for
mice given UBM digest and bleomycin when compared to
mice that only received bleomycin (data not illustrated).
In addition to co-administration, particulate UBM was
also administered after bleomycin exposure to assess its
ability to limit the development of fibrosis after the initial
injury has occurred. To investigate this, 16-week-old male
C57BL/6 wild-type mice were intratracheally treated with
bleomycin or saline vehicle and then given UBM or saline
intratracheally 24 h later. Based on histology of the lungs,
bleomycin-treated mice that received UBM 24 h after injury
developed significantly less fibrosis 14 days later when
compared to bleomycin-treated mice that received saline
control (Fig. 3A). Histological scoring also revealed that
UBM treatment 1 day after bleomycin exposure markedly
limited pulmonary fibrosis in mice (Fig. 3B). In addition to
fibrosis, bleomycin-treated mice that received UBM also had
less protein (Fig. 3C) and lower levels of inflammatory cells
(Fig. 3D) in their BALF than bleomycin-treated mice that
received saline. Cell differentials of the BALF showed that
bleomycin-treated mice that received UBM have significantly
less neutrophils and lymphocytes, but significantly more
macrophages than bleomycin-treated mice that received
saline (Fig. 3E). Although delayed administration of UBM
significantly decreased bleomycin injury in the lung, saline-
treated mice that received UBM 24 h later had small focal
areas of lymphocytic inflammation and interstitial thickening
present in their lungs (not illustrated) and some neutrophils
present in their airspaces (Fig. 3E).
A549 cells were treated with bleomycin and different do-
ses of UBM or digested UBM to verify that the presence of
FIG. 1. Treatment with ECM does not prevent bleomycin-induced leukocyte accumulation. Wild-type mice were admin-
istered bleomycin or saline (vehicle) with and without particulate UBM-ECM or digested UBM-ECM and euthanized 5 and 14
days after intratracheal instillation. All bleomycin-treated mice regardless of UBM-ECM co-administration had increased total
cells in their BALF (A) and specifically significantly more macrophages and modest increases in neutrophils and lymphocytes
when compared to controls (B).*p < 0.05 compared to vehicle control (saline or pepsin digest),
p < 0.05 compared to
ECM control (UBM-ECM o r UBM-ECM Digest) and vehicle control,
p < 0.01 compared to BLM + Pepsin Digest; n = 4–7/
treatm ent/ti mepoint. ECM, extracellular matrix; UBM, u rinary bladder matr ix; BALF, bronchoal veolar lavage fluid.
the ECM scaffold material does not alter the toxicity of the
bleomycin. These results showed that reduction in cell via-
bility caused by bleomycin was not significantly altered due
to bleomycin treatment simultaneous with UBM (Fig. 4A) or
UBM digest (Fig. 4B). Treatment with 5, 10, 25, 50, or 100 mg
of UBM, UBM digest, or pepsin digest alone did not affect
cell viability (representative graph of all ECM components is
shown in Fig. 4C). Similar results were also seen using the
MH-S murine alveolar macrophage cell line (ATCC; data not
Migration of serum-starved A549 cells toward the di-
gested form of UBM approached the migration promoted by
the positive control, 10% FBS (Fig. 5A). The digested UBM at
500 mg/mL concentration showed 94% 35% of the cell mi-
gration observed for the positive control, whereas 100 mg/
mL concentration showed 73% 20% of the cell migration
observed for the positive control. The relative cell migration
toward the pepsin digest controls were 23% 17% and
19% 18% for 500 and 100 mg/mL concentration, respec-
tively. The migration toward both concentrations of pepsin
digest was similar to the F12K serum free media (13% 11%).
At both concentrations, the UBM digest showed significantly
improved migration compared to the pepsin digest control
(Fig. 5A).
Using a wound healing assay, serum-starved A549 cells
were treated with particulate UBM or UBM digest in the
presence of bleomycin to evaluate the ability of ECM to
improve wound repair. UBM digest treatment promoted
wound closure in the presence of bleomycin, whereas the
pepsin digest control did not affect wound width (Fig. 5B).
Particulate UBM treatment showed a trend toward improved
wound closure, but did not significantly reduce wound
width in the presence of bleomycin.
UBM scaffold material prevented bleomycin-induced
pulmonary fibrosis regardless of the form of the material
(particulate or digested). UBM treatment significantly re-
duced the histologic presentation of fibrosis, such that there
was no significant difference between the histologic ap-
pearance of the lungs in animals treated with bleomycin and
FIG. 2. UBM-ECM prevents
bleomycin-induced fibrosis.
Wild-type mice were eutha-
nized 14 days after
intratracheal administration
of bleomycin or saline vehicle
with or without particulate
UBM-ECM. (A) Histological
analyses of the lungs revealed
that UBM-ECM limited pul-
monary fibrosis. (B) Average
histology score was deter-
mined for each group. (C)
Fibrosis was also assessed by
determining the amount of
collagen deposition in the
lungs using picosirius red
staining and is reported as the
area of collagen content per
total area of the field. Similar
results were seen with
co-administration of a
digested form of UBM-ECM.
*p < 0.05 when compared to all
other treatment groups and
p < 0.05 when compared
to saline control; n = 4–7/
treatment group.
UBM as compared to those treated with the saline alone.
More impressively, UBM treatment significantly prevented
the development of bleomycin injury even when adminis-
tered 1 day after bleomycin exposure in mice. Less than 1%
of bleomycin intratracheally instilled in the lungs of mice is
present 24 h after injection.
Thus, UBM can attenuate the
severity of fibrosis even when administered after the bleo-
mycin injury has occurred. Therefore, these novel findings
demonstrate the immense potential for the use of UBM as a
novel therapy for pulmonary fibrosis.
It is clear that the attenuation of fibrosis in response to
UBM was not due to neutralization of the bleomycin when
co-administered with these compounds as cell culture stud-
ies showed that UBM did not prevent cell death. Further,
animals treated with bleomycin and ECM products had the
same increase in the number of inflammatory cells and
similar cellular composition in the BALF as animals treated
with bleomycin alone. These results suggest that UBM lim-
ited bleomycin-induced fibrogenesis and did not do so by
interfering with the direct injurious effects of bleomycin.
FIG. 3. Administration of
UBM-ECM 1 day after bleo-
mycin exposure attenuates
pulmonary injury. Wild-type
mice were intratracheally
treated with bleomycin or
saline control and 24 h later
were given either particulate
UBM-ECM or saline via
intratracheal instillation. Mice
were euthanized 14 days after
bleomycin exposure to assess
injury in the lungs. Histologi-
cal analyses of the lungs by
microscopy (A) and by aver-
age histologic scoring of fi-
brosis (B) revealed that UBM-
ECM limited pulmonary
fibrosis when administered
after bleomycin exposure. In
addition, subsequent treat-
ment with UBM-ECM after
bleomycin exposure also lim-
ited the levels of protein (C)
and total cells (D) present in
the BALF when compared to
bleomycin-treated mice that
were given saline. Differential
counts of the BALF cells were
conducted and there were
significantly more lympho-
cytes and neutrophils,
whereas less macrophages in
the bleomycin-treated mice
that received saline when
compared to bleomycin-
treated mice that received
UBM-ECM (E).*p < 0.05
between treatment groups,
p < 0.05 shows interaction,
p < 0.05 when comparing the
cell type indicated between
the following groups: **Saline
+ UBM-ECM and BLM +
Saline + Saline and
all other
treatment groups; n = 4–5/
treatment group.
The digested form of UBM had the same protective effect
as the lyophilized particulate form in vivo. This suggests that
the composition of the UBM and its degradation products
may be more important to the repair process than the specific
ultrastructure of the particulate form of UBM.
UBM is
known to possess basement membrane structure that is
preserved through the process of making a powder.
presence of a basement membrane structure has been shown
to promote healing in various organs, including the lungs, by
providing guidance for re-epithelialization and separating
the epithelium from the interstitial connective tissue.
motaxis and wound healing assays performed in the present
study showed that the digested form of UBM could promote
migration of airway epithelial cells and also could stimulate
wound healing in the presence of bleomycin. It is possible
that small peptides from the degradation of laminin and
collagen IV in the basement membrane increase the migra-
tion of epithelial cells.
FIG. 4. ECM treatment does not affect bleomycin toxicity.
A549 cells (5000 cells/well) were incubated with 0.02 units of
bleomycin (BLM), BLM with indicated amounts of particulate
UBM-ECM (A) or digested UBM-ECM (B), or ECM (particu-
late or digested UBM-ECM) without BLM (C) for 24 h. Cell
viability was reported as the percentage of viable cells when
compared to media control (mean SEM). *p < 0.05 when
compared to media control. SEM, standard error of the mean.
FIG. 5. UBM-ECM promotes chemotaxis of epithelial cells
and stimulates re-epithelialization. (A) Chemotaxis of A549
cells (30,000 cells/well) was evaluated in response to F12K
media with 10% FBS, serum-free F12K media, 100 and
500 mg/mL UBM-ECM digest, or control pepsin digest. The
average percentage of migrated cells for each condition was
normalized to the average percentage of cells that migrated
toward the media with 10% FBS in each experiment
(mean SEM). *p < 0.05 when compared to 10% FBS. (B)
A549 cell monolayers were wounded and exposed to UBM-
ECM digest, bleomycin (BLM), BLM with particulate or
digested UBM-ECM, or BLM with pepsin digest control.
Wound widths were measured in triplicate for each treat-
ment for three independent experiments. Results were re-
ported as the percentage of change in wound width over 24 h
(mean SEM). *p < 0.05 when compared to UBM-ECM digest
control. FBS, fetal bovine serum.
Many studies have noted that degradation of the UBM is
an important component of the host response that leads to
site-appropriate tissue remodeling. Previous studies have
shown that UBM degradation products recruit progenitor
cells to the site of remodeling,
promote angiogenesis,
and provide bacteriostasis.
On the other hand, studies have
found that the oxidative fragmentation of ECM components,
such as heparan sulfate, collagen, syndecan-1, and hyalur-
onan, influences the development of fibrosis.
As the
UBM scaffold material is a mixture of ECM proteins and
other substances, such as growth factors,
it is likely that
this complex composition of ECM promotes healing that
outweighs the detrimental effects of the proteoglycans and
glycosoaminoglycans degradation products in these ECM-
based materials.
Although this study clearly illustrates that UBM attenu-
ates bleomycin-induced pulmonary fibrosis, the mechanism
by which it does so is still unclear. Current evidence is pre-
sented to suggest that the UBM plays a role in epithelial
repair, but additional studies are warranted to fully under-
stand this phenomenon. The current work also shows that
treatment of UBM with or after bleomycin showed an ele-
vated number of immune cells, predominantly macrophages.
In previous studies in which UBM scaffolds were used to
bridge a defect in the rat abdominal wall, site-specific tissue
repair was associated with a predominately M2 macrophage
phenotype during the first month after surgery.
the lung, the M2 macrophage phenotype has been reported
to be associated with the onset of fibrosis.
Therefore, it is
likely important to fully characterize the immune response to
UBM in this model as it could provide evidence that the
ability for the UBM to modulate the immune response may
be tissue dependent. The phenotype of the macrophages
could also play a role in the matrix metalloproteases that
are present within the lung to degrade the instilled parti-
culate ECM and to participate in remodeling of the lung
Finally, UBM was selected for investigation because it
contains a basement membrane component and has been
shown to be beneficial for other airway applications. Organ-
specific ECM derived from the trachea and lung has recently
been explored for airway repair.
In the lung models, the
acellular lung matrix with its three-dimensional architecture
and composition largely intact promoted site-specific cell
differentiation and retained similar mechanical characteris-
It is possible that an airway-derived ECM may be
even more effective for prevention of fibrosis than a hetero-
topic ECM source due to the organ-specific signaling mole-
cules that would be present within the ECM.
The present study showed that ECM from porcine urinary
bladder can protect against pulmonary fibrosis in a bleo-
mycin model, and that the composition and degradation of
the scaffold is likely more important that the ultrastructure of
the intact scaffold in guiding this response. These findings
also strongly suggest that one mechanism by which UBM
prevents fibrosis is through release of bioactive degradation
products that promote epithelial cell chemotaxis and re-
epithelialization. Future studies will investigate whether
UBM scaffolds also play a role in modulation of the immune
response. In addition, future studies will elucidate whether
UBM can also attenuate the fibrotic response to chronic
environmental irritants, such as asbestos and silica, which
would suggest that it may have broader therapeutic use in
different forms of pulmonary fibrosis.
The authors would like to acknowledge Kristen Agnew
for participating in the animal care for the studies described.
This study was funded by NIH R01 HL63700-09 (T.D.O.),
AHA #0715279U (M.L.M.), and Commonwealth of Penn-
sylvania (T.W.G.).
Disclosure Statement
No competing financial interests exist. T.W.G. serves on
the Scientific Advisory Board for ACell, Inc.
1. Meltzer, E.B., and Noble, P.W. Idiopathic pulmonary fibro-
sis. Orphanet J Rare Dis 3, 8, 2008.
2. Moeller, A., Ask, K., Warburton, D., Gauldie, J., and Kolb,
M. The bleomycin animal model: a useful tool to investigate
treatment options for idiopathic pulmonary fibrosis? Int J
Biochem Cell Biol 40, 362, 2008.
3. Piguet, P.F., Collart, M.A., Grau, G.E., Sappino, A.P., and
Vassalli, P. Requirement of tumour necrosis factor for de-
velopment of silica-induced pulmonary fibrosis. Nature 344,
245, 1990.
4. Piguet, P.F., Kaufman, S., Barazzone, C., Muller, M., Ryffel,
B., and Eugster, H.P. Resistance of TNF/LT alpha double
deficient mice to bleomycin-induced fibrosis. Int J Exp Pa-
thol 78, 43, 1997.
5. Zhang, Y., Lee, T.C., Guillemin, B., Yu, M.C., and Rom, W.N.
Enhanced IL-1 beta and tumor necrosis factor-alpha release
and messenger RNA expression in macrophages from idio-
pathic pulmonary fibrosis or after asbestos exposure. J Im-
munol 150, 4188, 1993.
6. Broekelmann, T.J., Limper, A.H., Colby, T.V., and McDo-
nald, J.A. Transforming growth factor beta 1 is present at
sites of extracellular matrix gene expression in human pul-
monary fibrosis. Proc Natl Acad Sci USA 88, 6642, 1991.
7. Giri, S.N., Hyde, D.M., and Hollinger, M.A. Effect of anti-
body to transforming growth factor beta on bleomycin in-
duced accumulation of lung collagen in mice. Thorax 48,
959, 1993.
8. Khalil, N., Bereznay, O., Sporn, M., and Greenberg, A.H.
Macrophage production of transforming growth factor beta
and fibroblast collagen synthesis in chronic pulmonary in-
flammation. J Exp Med 170, 727, 1989.
9. Korfhagen, T.R., Swantz, R.J., Wert, S.E., McCarty, J.M.,
Kerlakian, C.B., Glasser, S.W., et al. Respiratory epithelial
cell expression of human transforming growth factor-alpha
induces lung fibrosis in transgenic mice. J Clin Invest 93,
1691, 1994.
10. Santana, A., Saxena, B., Noble, N.A., Gold, L.I., and Marshall,
B.C. Increased expression of transforming growth factor beta
isoforms (beta 1, beta 2, beta 3) in bleomycin-induced pul-
monary fibrosis. Am J Respir Cell Mol Biol 13, 34, 1995.
11. Brown, B., Lindberg, K., Reing, J., Stolz, D.B., and Badylak,
S.F. The basement membrane component of biologic scaf-
folds derived from extracellular matrix. Tissue Eng 12, 519,
12. Gilbert, T.W., Stewart-Akers, A.M., Sydeski, J., Nguyen,
T.D., Badylak, S.F., and Woo, S.L.-Y. Gene expression by
fibroblasts seeded on small intestinal submucosa and sub-
jected to cyclic stretching. Tissue Eng 13, 1313, 2007.
13. Beattie, A.J., Gilbert, T.W., Guyot, J.P., Yates, A.J., and
Badylak, S.F. Chemoattraction of progenitor cells by re-
modeling extracellular matrix scaffolds. Tissue Eng Part A
15, 1119, 2009.
14. Brennan, E.P., Reing, J., Chew, D., Myers-Irvin, J.M., Young,
E.J., and Badylak, S.F. Antibacterial activity within degra-
dation products of biological scaffolds composed of extra-
cellular matrix. Tissue Eng 12, 2949, 2006.
15. Brennan, E.P., Tang, X.H., Stewart-Akers, A.M., Gudas, L.J.,
and Badylak, S.F. Chemoattractant activity of degradation
products of fetal and adult skin extracellular matrix for
keratinocyte progenitor cells. J Tissue Eng Regen Med 2,
491, 2008.
16. Gilbert, T.W., Stewart-Akers, A.M., Simmons-Byrd, A., and
Badylak, S.F. Degradation and remodeling of small intesti-
nal submucosa in canine Achilles tendon repair. J Bone Joint
Surg Am 89, 621, 2007.
17. Li, F., Li, W., Johnson, S., Ingram, D., Yoder, M., and Ba-
dylak, S.F. Low-molecular-weight peptides derived from
extracellular matrix as chemoattractants for primary endo-
thelial cells. Endothelium 11, 199, 2004.
18. Badylak, S.F., and Gilbert, T.W. Immune response to bio-
logic scaffold materials. Semin Immunol 20, 109, 2008.
19. Badylak, S.F., Valentin, J.E., Ravindra, A.K., McCabe, G.P.,
and Stewart-Akers, A.M. Macrophage phenotype as a de-
terminant of biologic scaffold remodeling. Tissue Eng Part A
14, 1835, 2008.
20. Brown, B.N., Valentin, J.E., Stewart-Akers, A.M., McCabe,
G.P., and Badylak, S.F. Macrophage phenotype and re-
modeling outcomes in response to biologic scaffolds
with and without a cellular component. Biomaterials 30,
1482, 2009.
21. Downey, D.M., Harre, J.G., and Pratt, J.W. Functional com-
parison of staple line reinforcements in lung resection. Ann
Thorac Surg 82, 1880, 2006.
22. Downey, D.M., Michel, M., Harre, J.G., and Pratt, J.W.
Functional assessment of a new staple line reinforcement in
lung resection. J Surg Res 131, 49, 2006.
23. Gilbert, T.W., Nieponice, A., Spievack, A.R., Holcomb, J.,
Gilbert, S., and Badylak, S.F. Repair of the thoracic wall with
an extracellular matrix scaffold in a canine model. J Surg Res
147, 61, 2008.
24. Gilbert, T.W., Gilbert, S., Madden, M., Reynolds, S.D., and
Badylak, S.F. Morphologic assessment of extracellular ma-
trix scaffolds for patch tracheoplasty in a canine model. Ann
Thorac Surg 86, 967, 2008; discussion 73–74.
25. Gilbert, T.W., Stolz, D.B., Biancaniello, F., Simmons-Byrd,
A., and Badylak, S.F. Production and characterization of
ECM powder: implications for tissue engineering applica-
tions. Biomaterials 26, 1431, 2005.
26. Fattman, C.L., Chu, C.T., Kulich, S.M., Enghild, J.J., and
Oury, T.D. Altered expression of extracellular superoxide
dismutase in mouse lung after bleomycin treatment. Free
Radic Biol Med 31, 1198, 2001.
27. Ramsgaard, L., Englert, J.M., Tobolewski, J., Tomai, L.,
Fattman, C.L., Leme, A.S., et al. The role of the receptor for
advanced glycation end-products in a murine model of sil-
icosis. PLoS One 5, e9604, 2010.
28. Englert, J.M., Hanford, L.E., Kaminski, N., Tobolewski, J.M.,
Tan, R.J., Fattman, C.L., et al. A role for the receptor for
advanced glycation end products in idiopathic pulmonary
fibrosis. Am J Pathol 172, 583, 2008.
29. Fattman, C.L., Tan, R.J., Tobolewski, J.M., and Oury, T.D.
Increased sensitivity to asbestos-induced lung injury in mice
lacking extracellular superoxide dismutase. Free Radic Biol
40, 601, 2006.
30. Gilbert, T.W., Agrawal, V., Gilbert, M.R., Povirk, K.M., Ba-
dylak, S.F., and Rosen, C.A. Liver-derived extracellular
matrix as a biologic scaffold for acute vocal fold repair in a
canine model. Laryngoscope 119, 1856, 2009.
31. Kliment, C.R., Englert, J.M., Gochuico, B.R., Yu, G., Ka-
minski, N., Rosas, I., et al. Oxidative stress alters syndecan-1
distribution in lungs with pulmonary fibrosis. J Biol Chem
284, 3537, 2009.
32. Liang, C.C., Park, A.Y., and Guan, J.L. In vitro scratch assay:
a convenient and inexpensive method for analysis of cell
migration in vitro. Nat Protoc 2, 329, 2007.
33. Lazo, J.S., and Pham, E.T. Pulmonary fate of
H bleomycin
A2 in mice. J Pharmacol Exp Ther 228, 13, 1984.
34. Vracko, R. Basal lamina scaffold-anatomy and significance
for maintenance of orderly tissue structure. Am J Pathol 77,
314, 1974.
35. Reing, J.E., Zhang, L., Myers-Irvin, J., Cordero, K.E., Freytes,
D.O., Heber-Katz, E., et al. Degradation products of extra-
cellular matrix affect cell migration and proliferation. Tissue
Eng Part A 15, 605, 2009.
36. Gao, F., Koenitzer, J.R., Tobolewski, J.M., Jiang, D., Liang, J.,
Noble, P.W., et al. Extracellular superoxide dismutase in-
hibits inflammation by preventing oxidative fragmentation
of hyaluronan. J Biol Chem 283, 6058, 2008.
37. Kliment, C.R., Tobolewski, J.M., Manni, M.L., Tan, R.J., En-
ghild, J., and Oury, T.D. Extracellular superoxide dismutase
protects against matrix degradation of heparan sulfate in the
lung. Antioxid Redox Signal 10, 261, 2008.
38. Petersen, S.V., Oury, T.D., Ostergaard, L., Valnickova, Z.,
Wegrzyn, J., Thogersen, I.B., et al. Extracellular superox-
ide dismutase (EC-SOD) binds to type i collagen and pro-
tects against oxidative fragmentation. J Biol Chem 279,
13705, 2004.
39. Badylak, S.F., Freytes, D.O., and Gilbert, T.W. Extracellular
matrix as a biological scaffold material: structure and func-
tion. Acta Biomater 5, 1, 2009.
40. Hancock, A., Armstrong, L., Gama, R., and Millar, A.
Production of interleukin 13 by alveolar macrophages
from normal and fibrotic lung. Am J Respir Cell Mol Biol 18,
60, 1998.
41. Murray, L.A., Rosada, R., Moreira, A.P., Joshi, A., Kramer,
M.S., Hesson, D.P., et al. Serum amyloid P therapeuti-
cally attenuates murine bleomycin-induced pulmonary
fibrosis via its effects on macrophages. PLoS One 5, e9683,
42. Cortiella, J., Niles, J., Cantu, A., Brettler, A., Pham, A.,
Vargas, G., et al. Influence of acellular natural lung matrix on
murine embryonic stem cell differentiation and tissue for-
mation. Tissue Eng Part A 16, 2565, 2010.
43. Jungebluth, P., Go, T., Asnaghi, A., Bellini, S., Martorell, J.,
Calore, C., et al. Structural and morphologic evaluation of a
novel detergent-enzymatic tissue-engineered tracheal tubu-
lar matrix. J Thorac Cardiovasc Surg 138, 586, 2009.
44. Macchiarini, P., Jungebluth, P., Go, T., Asnaghi, M.A., Rees,
L.E., Cogan, T.A., et al. Clinical transplantation of a tissue-
engineered airway. Lancet 372, 2023, 2008.
45. Ott, H.C., Clippinger, B., Conrad, C., Schuetz, C.,
Pomerantseva, I., Ikonomou, L., et al. Regeneration and
orthotopic transplantation of a bioartificial lung. Nat Med
16, 927, 2010.
46. Petersen, T.H., Calle, E.A., Zhao, L., Lee, E.J., Gui, L., Rar-
edon, M.B., et al. Tissue-engineered lungs for in vivo im-
plantation. Science 329, 538, 2010.
47. Price, A.P., England, K.A., Matson, A.M., Blazar, B.R., and
Panoskaltsis-Mortari, A. Development of a decellularized
lung bioreactor system for bioengineering the lung: the
matrix reloaded. Tissue Eng Part A 16, 2581, 2010.
48. Remlinger, N.T., Czajka, C.A., Juhas, M.E., Vorp, D.A., Stolz,
D.B., Badylak, S.F., et al. Hydrated xenogeneic decellularized
tracheal matrix as a scaffold for tracheal reconstruction.
Biomaterials 31, 3520, 2010.
Address correspondence to:
Thomas W. Gilbert, Ph.D.
McGowan Institute for Regenerative Medicine
University of Pittsburgh
100 Technology Drive, Suite 200
Pittsburgh, PA 15219
E-mail: gilberttw@upmc.edu
Received: January 12, 2011
Accepted: June 17, 2011
Online Publication Date: July 28, 2011
    • "The images were obtained with an x40 objective lens, recorded on a digital camera (DP-71, Olympus) and analyzed using ImageJ W image analysis software (http:// rsbweb.nih.gov/ij/). The analysis methodology was performed according to Manni et al. [35] . Collagen content was calculated as a percentage of the area of each image (24,137μm 2 ; 3,338,208 pixels). "
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