choi2011

Decellularized extracellular matrix derived from human adipose tissue
as a potential scaffold for allograft tissue engineering
Ji Suk Choi,1* Beob Soo Kim,1* Jun Young Kim,2 Jae Dong Kim,1 Young Chan Choi,1
Hyun-Jin Yang,2 Kinam Park,3 Hee Young Lee,2 Yong Woo Cho1
1
Department of Chemical Engineering and Department of Bionanotechnology, Hanyang University, Ansan, Gyeonggi-do 426791, Republic of Korea
2
Kangnam Plastic Surgery Clinic, Seoul 135-120, Republic of Korea
3
Departments of Biomedical Engineering and Pharmaceutics, Purdue University, West Lafayette, Indiana 47907
Received 28 June 2010; revised 4 November 2010; accepted 13 January 2011
Published online 29 March 2011 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jbm.a.33056
Abstract: Decellularized tissues composed of extracellular matrix (ECM) have been clinically used to support the regeneration
of various human tissues and organs. Most decellularized tissues so far have been derived from animals or cadavers. Therefore, despite the many advantages of decellularized tissue,
there are concerns about the potential for immunogenicity and
the possible presence of infectious agents. Herein, we present
a biomaterial composed of ECM derived from human adipose
tissue, the most prevalent, expendable, and safely harvested
tissue in the human body. The ECM was extracted by successive physical, chemical, and enzymatic treatments of human
adipose tissue isolated by liposuction. Cellular components
including nucleic acids were effectively removed without signif-
icant disruption of the morphology or structure of the ECM.
Major ECM components were quantified, including acid/pepsinsoluble collagen, sulfated glycosaminoglycan (GAG), and soluble elastin. In an in vivo experiment using mice, the decellularized ECM graft exhibited good compatibility to surrounding
tissues. Overall results suggest that the decellularized ECM containing biological and chemical cues of native human ECM
could be an ideal scaffold material not only for autologous but
C 2011 Wiley Periodicals, Inc.
also for allograft tissue engineering. V
INTRODUCTION
components that may induce an inflammatory response.14
The ECM-based scaffolds derived from decellularized porcine urinary bladders and SIS have been extensively studied
for xenografts to reconstruct musculoskeletal structures,
cardiovascular tissues, and skin.4,15–17 Decellularized rat
hearts have been studied for the replacement of injured
heart muscle.13 The widespread use of decellularized ECM
scaffolds across many clinical applications is attributed to
their excellent biocompatibility, biodegradability, and bioinductive properties. A number of decellularized allogenic or
xenogenic medical products are now being introduced into
the market and have received regulatory approval for use in
human patients using tissues from human dermis
(AllodermV, LifeCell, Corp.), porcine SIS (SurgiSISV, Cook
Biotech; RestoreV, DePuy Orthopaedics), porcine urinary
bladder (MatriStemV, ACell), and porcine heart valves (SynergraftV, CryoLife). However, it should be noted that most
decellularized tissues have been derived from animals or
cadavers. They may raise some concerns regarding immunogenicity and pathogen transmission.
Herein, we demonstrate that decellularized ECM derived
from human adipose tissue has great potential for use in allograft tissue engineering. Adipose tissue is the most
Biological scaffolds composed of extracellular matrix (ECM)
have been widely used in clinics for the regeneration of various tissues and organs.1,2 ECM has a complex composition
including a variety of bioactive molecules. ECM helps hold
cells together in tissues, regulates dynamic cellular behavior,
and performs protective and supportive functions. The
structural and functional molecules of ECM have not been
fully characterized; however, individual components, such as
collagen, elastin, laminin, fibronectin, and glycosaminoglycans (GAGs), have been isolated and used for many applications.3 ECM-based scaffolds have been harvested from various tissues including small intestinal submucosa (SIS),4
urinary bladder,5 cholecyst,6 blood vessels,7 heart valves,8
skin,9 liver,10 and adipose tissue.11,12
Scaffolds play a major role in tissue engineering strategies. Scaffolds should be designed to include biological and
chemical cues that mimic the native microenvironment.13
The biological scaffolds derived from decellularized tissues
and organs have received significant attention in regenerative medicine. Different methods have been used to decellularized tissues and organs, including physical, chemical, and
enzymatic treatments. The decellularization removes cellular
J Biomed Mater Res Part A: 97A: 292–299, 2011.
Key Words: extracellular matrix, scaffold, tissue engineering,
adipose tissue, allograft
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Additional Supporting Information may be found in the online version of this article.
*These authors contributed equally to this work.
Correspondence to: Y. W. Cho; e-mail: [email protected]
Contract grant sponsor: National Research Foundation of Korea; contract grant numbers: 2009-0075546 and R11-2008-044-02001-0
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C 2011 WILEY PERIODICALS, INC.
V
ORIGINAL ARTICLE
prevalent, expendable, and safely harvested tissue in the
human body.18–20 Adipose tissue contains various ECM components21,22 and secretes a variety of peptides, cytokines,
and complement factors, which regulate numerous cellular
processes including insulin action, energy homeostasis,
inflammation, and cell growth.23 More recently, adipose tissue derived ECM has been under investigation as potential
scaffolds for adipose tissue regeneration strategies.24,25 We
previously reported that ECM-based scaffolds with different
three-dimensional (3D) shapes could be fabricated from
human adipose tissue by simple physical treatments such as
homogenization, centrifugation, and freeze-drying.11,12 Since
the ECM-based scaffolds have been derived from patient’s
own adipose tissue, it may be termed autologous tissue engineering. In this study, we focus on the removal of potential immunogenic components for allograft tissue engineering, that is, the application of ECM-based scaffolds derived
from a donor genetically unrelated to the recipient. Human
adipose tissue obtained by liposuction was decellularized
through a series of successive chemical and enzymatic treatments using sodium chloride (NaCl), sodium dodecyl sulphate (SDS), DNase, and RNase. The removal of potential
immunogenic components was examined by histological and
DNA assays. To explore the feasibility of using decellularized
ECM for allograft, it was injected as a viscous suspension
into mice, and the interaction between graft and host tissue
was assessed by histological and immunofluorescence
staining.
MATERIALS AND METHODS
Preparation of decellularized ECM from human adipose
tissue
Human adipose tissue was obtained with informed consent
from six healthy female donors aged between 20 and 40
years who had undergone liposuction at the Kangnam Plastic Surgery Clinic (Seoul, Korea). Infiltration of saline, liposuction, and centrifugation were performed by a single combined machine (Lipokit, Medikan, Seoul, Korea). The adipose
tissue obtained via liposuction (20 mL) was washed several
times with distilled water to remove blood components. Distilled water (10 mL) was added to the adipose tissue, and
the tissue/water mixture was homogenized at 12,000 rpm
for 5 min at room temperature using a homogenizer (T 18
basic ULTRA-TURAX, IKAV-Werke GmbH & Co. KG Staufen,
Germany). The tissue suspension was centrifuged at 1800
g for 5 min, and the upper oil-containing layer was discarded. The viscous suspension (5 mL) was treated with a
buffered 1M hyperosmolar solution of NaCl (diluted 1:1) for
2 h at 37 C in a shaking water bath (Personal-11EX, TAITEC, Tokyo, Japan). The suspension was centrifuged at 200
g for 5 min at 4 C. After decanting, the residue was rinsed
with distilled water for 24 h at 4 C under gentle shaking.
The medium was replaced every 2 h with fresh distilled
water. Subsequently, the residue was incubated in buffered
0.5% sodium dodecyl sulfate (SDS; Sigma, St. Louis, MO) for
1 h at room temperature in a shaking water bath. The suspension was centrifuged and washed with distilled water
for 24 h at 37 C under gentle shaking. Then, the tissue
products were treated with a mixture of 0.2% DNase (2000
U, Sigma) and 200 lg/mL RNase (Sigma) for 1 h at 37 C.
The suspension of ECM was centrifuged, thoroughly washed
with distilled water, and stored in sterile distilled water at
4 C until further use.
Scanning electron microscopy
ECM specimens were fixed in 2.5% glutaraldehyde for 1 h
at room temperature. After extensive rinsing, each sample
was mounted onto a cover glass and air-dried at room temperature. The surface morphology of ECMs was observed by
scanning electron microscopy (SEM, Hitachi S-4800 FE-SEM,
Japan) after being coated with platinum at an accelerating
voltage of 15 kV.
DNA quantification
DNA was isolated with a commercially available extraction
kit (iNtRON Biotechnology, Korea). The total DNA content
was measured by absorption at 260 nm on a UV–VIS spectrophotometer (Ultrospec 2100 pro, Amersham Biosciences,
Piscataway, NJ). The DNA content was normalized to the initial wet weight of the sample. The total DNA was used as a
template for polymerase chain reaction (PCR) analysis with
primers specific for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (NM002046, forward 50 -GGG CTG CTT TTA
ACT CTG GT-30 , reverse: 50 -GCA GGT TTT TCT AGA CGG-30 ).
Amplification was performed using Taq polymerase as follows: initial denaturation for 5 min at 94 C; followed by 35
cycles of 1 min at 94 C for denaturation, 1 min at 56 C for
annealing, and 7 min at 72 C for extension. PCR products
were separated by electrophoresis in 1.5% agarose gels.
Fluorescence microscopy
ECM specimens were fixed in 4% paraformaldehyde at 4 C
for 1 h. The specimens were embedded in paraffin (Merck,
Darmstadt, Germany) and sectioned at 10-lm thickness.
The sections were deparaffinized, dehydrated through a series of graded ethanol, and stained with acridine orange
(AO) (Sigma) and 4,6-diamidino-2-phenylindole (DAPI)
(Thermo Scientific, Rockford, IL) to identify nuclear components such as DNA and RNA. The stained sections were
examined using a fluorescence microscope (IX81, Olympus
Corporation, Tokyo, Japan).
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Biochemical analyses
Biochemical assays were performed for the quantification of
ECM components such as acid/pepsin-soluble collagen, sulfated GAG, and soluble elastin.26,27 All contents were normalized to the ECM dry weight in microgram. Collagen type
I (rat tail), chondroitin 4-sulfate (bovine trachea), and aelastin (bovine neck) were used as standards for the biochemical assays.
Acid/pepsin-soluble collagen quantification. The content
of acid/pepsin-soluble collagen in ECM was measured using
a Sircol soluble collagen assay kit (Biocolor, Carrickfergus,
Northern Ireland). For extraction of acid/pepsin-soluble collagen, ECM specimens were digested with 0.5M acetic acid
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containing 1% (w/v) pepsin (P7012, Sigma) at room temperature for 24 h.28 The suspension was centrifuged at
10,000 g for 10 min. The supernatant was collected and
incubated with 1-mL Sircol dye reagent for 30 min at room
temperature. The collagen–dye complex was precipitated by
centrifugation at 10,000 g for 10 min and the supernatant
was removed. The pellets were dissolved in 1-mL alkali reagent, and the relative absorbance was measured in a 96well plate at 540 nm using a microplate reader.
Sulfated GAG quantification. The content of sulfated GAG
in ECM was measured using a Blyscan sulfated GAG assay
kit (Biocolor). For extraction of sulfated GAG, ECM specimens were digested with 0.1M phosphate buffer (pH 6.8)
containing 125 lg/mL papain (Sigma), 10 mM cystein
hydrochloride (Sigma), and 2-mM EDTA (Sigma) at 60 C for
48 h. The suspension was centrifuged at 15,000 g for 30
min. The supernatant was collected and incubated with
0.2M sodium citrate buffer (pH 4.8) containing 0.2% (w/v)
cetylpyridinium at 37 C for 2 h.29 After centrifugation at
10,000 g for 10 min, the precipitated sulfated GAG was
dissolved in 2M lithium chloride and reprecipitated by mixing with cold ethanol. Following centrifugation, the pellet
was drained and resuspended in deionized water. The
extracted sulfated GAG was mixed with 1-mL Blyscan dye
and shaken for 30 min. The precipitate was collected by
centrifugation for 5 min and then dissolved in 1 mL dissociation reagent. The absorbance was measured in a 96-well
plate at 656 nm using a microplate reader.
Soluble elastin quantification. The content of soluble elastin in ECM was measured using a Fastin elastin assay kit
(Biocolor). For extraction of soluble elastin, ECM specimens
were hydrolyzed with 0.25M oxalic acid (Sigma) at 100 C
for 1 h. The insoluble residues were separated by centrifugation. The supernatant was collected, and the sediment
underwent an additional extraction under the same conditions.30 The extraction was repeated several times until no
elastin was found in the supernatants. The extracted soluble
elastin was mixed with 1-mL Fastin dye and stirred for 30
min. The precipitate was collected by centrifugation for 10
min and then dissolved in 250-lL dissociation reagent. The
absorbance was measured in a 96-well plate at 513 nm
using a microplate reader.
Histology
Specimens were fixed in 4% paraformaldehyde, embedded
in paraffin, and sliced using a microtome. Sections were
deparaffinized and dehydrated in ethanol. For collagen fiber
staining, sections were first stained by a rapid trichrome
method.31,32 The samples were fixed in Bouin’s solution for
1 h at 56 C and stained with Wiegert’s iron hematoxylin for
10 min. Samples were washed and stained with Gomori’s
trichrome solution for 20 min, then differentiated in a 0.5%
acetic acid solution. The Fullner and Lillie’s orcinol-new
fuchsin method33,34 was used to stain elastic fibers in the
ECM. Samples were stained at 37 C with an orcinol-new
fuchsin working solution for 15 min and dehydrated.
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CHOI ET AL.
FIGURE 1. Schematic representation of the preparation of decellularized
ECM from human adipose tissue. Macroscopic appearances of the adipose tissue product (ECM-1) after the physical treatments, including homogenization and centrifugation, and the decellularized ECM (ECM-2)
after completion of the entire process. [Color figure can be viewed in the
online issue, which is available at wileyonlinelibrary.com.]
Implantation
A viscous suspension (0.5 mL) of ECM was injected subcutaneously into the backs of female Slc/ICR mice (6-week-old)
using an 18-gauge needle. At 7 weeks after the injection,
the mice were sacrificed and the grafts were explanted.
Eight mice per experimental group were analyzed.
DECELLULARIZED ECM DERIVED FROM HUMAN ADIPOSE TISSUE
ORIGINAL ARTICLE
FIGURE 2. SEM images of ECM-1 (A) and ECM-2 (B).
Histology and immunofluorescence staining
After being embedded in OCT compound (Tissue-Tek, Sakura
Finetek, Torrance, CA), the tissue samples were frozen at
70 C. The frozen samples were sliced into 5-lm sections,
fixed in acetone for 10 min at room temperature, washed
with distilled water, and 30% isopropanol to remove the
OCT compounds, and then stained with hematoxylin and eosin (H&E) (Sigma), Prussian Blue (Sigma), anti-CD3, and
anti-CD8 (BioLegend, San Diego, CA) working solutions.
The H&E stain was used to detect cell nuclei in the ECM.
The Prussian Blue solution was prepared by mixing equal
volumes of 4% potassium ferrocyanide and 4% HCl solutions. The Prussian Blue, anti-CD3, and anti-CD8 were used
to detect activated macrophages and T cells. The stained
sections were observed with a fluorescence microscope
(IX81, Olympus Corporation, Tokyo, Japan).
RESULTS
The procedure for preparation of ECM-2 from human adipose tissue and its potential application to allograft tissue
engineering are schematically represented in Figure 1.
Human adipose tissues were obtained from healthy females
who had undergone liposuction. The raw adipose tissue was
first washed with distilled water to remove blood components, and then the tissue/water mixture was thoroughly
homogenized. The resulting suspension was centrifuged to
remove oil components such as lipids. The ECM-1 through
homogenization and centrifugation had a yellowish appearance. Subsequently, it was treated with NaCl and SDS to
remove the cellular membranes and cytoplasmic components. The final step of decellularization, the enzymatic
treatment, degraded nucleic acids such as DNA and RNA. After the whole decellularization process, the ECM-2 had a
whitish appearance. The volume of the ECM-2 extracted
from adipose tissue was approximately 5% of the original
adipose tissue volume.
SEM images showed the surface morphology of ECMs
extracted from human adipose tissue (Fig. 2). Compared
with ECM-1 [Fig. 2(A)], a fibrous structure was more markedly revealed in ECM-2 [Fig. 2(B)]. The presences DNA and
RNA in ECMs were analyzed using quantitative and qualitative methods (Fig. 3). The DNA content of decellularized
JOURNAL OF BIOMEDICAL MATERIALS RESEARCH A | 1 JUN 2011 VOL 97A, ISSUE 3
ECM was significantly decreased after NaCl-SDS-enzyme
treatments [Fig. 3(A)]. The presence of nucleic acids in the
ECM-2 was also assessed with DAPI and AO staining [Fig.
3(B–E)]. Before NaCl-SDS-enzyme treatments, abundant
nucleic acids were apparent, showing positive staining by
DAPI [Fig. 3(B)] and AO [Fig. 3(D)]. However, after the
decellularization procedure, DNA and RNA were barely
detected by DAPI and AO stainings [Fig. 3(C,E)], indicating
the effective removal of nucleic acids.
An almost complete DNA elimination was validated by
electrophoresis [Fig. 3(F)] and GAPDH gene expression [Fig.
3(G)]. There was a considerable amount of DNA before
NaCl-SDS-enzyme treatments, while DNA was barely
detected in the ECM-2. The GAPDH gene was also clearly
expressed in ECM-1, but was not expressed in ECM-2. Thus,
DNA electrophoresis and PCR results demonstrated that the
decellularization significantly reduced the potential immunogenic components in decellularized ECM.
To analyze ECM components after decellularization, biochemical assays [Fig. 4(A–D)] and histological staining [Fig.
4(E–H)] were performed. Both ECM-1 and ECM-2 were rich
in acid/pepsin-soluble collagen and soluble elastin although
there was a slight difference between the donors. A small
amount of sulfated GAG was also found. The SDS-NaCl-enzymatic treatments caused a significant decrease in the two
soluble ECM components as well as the removal of cellular
components. The SDS-NaCl-enzymatic decellularization was
accompanied by decreases of 24% for acid/pepsin-soluble
collagen and 21% for soluble elastin content. Figure 4(E–H)
displays the histological examination of the ECMs. ECM
specimens before and after SDS-NaCl enzymatic treatments
were specifically stained with Gomori’s one-step trichrome
for collagen fibers [Fig. 4(E,F)] and Fullner and Lillie’s orcinol-new fuchsin for elastic fibers [Fig. 4(G,H)]. The histological examination shows that the contents of the two soluble ECM components decreased after decellularization.
To assess in vivo biocompatibility, cell infiltration and
inflammation of host tissue, a viscous suspension of ECM-2
was injected subcutaneously into the backs of Slc/ICR mice.
The injected ECM-2 was easily identified throughout the
whole experimental period. At the end of this period, the
ECM-2 that had adhered to the surrounding tissues was
carefully separated, as shown in Figure 5(A). The gross
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the ECM-2 grafts exhibited good stability and compatibility
with the surrounding tissues. The H&E stain shows that
host cells infiltrated into ECM-1 [Fig. 5(B,D)] and ECM-2
[Fig. 5(C,E)] grafts. Although the host cells were in a compact mass with a surface layer of grafts, a portion of the
host cells infiltrated into both of the grafts. The results of
the histological (Prussian Blue stain) and immunological
(CD3 and CD8) evaluations showed that macrophages and T
cells were present in the ECM-1 graft, whereas they were
hardly detected in the ECM-2 graft (Fig. 6). Thus, the in vivo
FIGURE 3. (A) DNA contents before and after decellularization with
NaCl, SDS, and enzymatic treatments. Columns and error bars represent mean values and standard deviations, respectively. All samples
were normalized to the ECM wet weight. Histological analyses of
ECM-1 (B, D) and ECM-2 (C, E). DAPI staining (B, C). The light blue
color indicates residual nucleic acids. AO staining (D, E). The orange
and green colors indicate residual RNA and DNA, respectively. Scale
bar represents 100 lm. (F) Gel electrophoresis images of DNA isolated from ECM-1 (line 3-5) and ECM-2 (line 6-8). (G) GAPDH expression for the housekeeping gene detected by PCR. Human adiposederived stem cells were used as a positive control for total DNA (line
1) and GAPDH (line 2) genes. [Color figure can be viewed in the
online issue, which is available at wileyonlinelibrary.com.]
volume of the graft was virtually maintained. The grafts
showed no signs of inflammatory response. The top of the
graft was covered by a thin layer of yellow tissue and new
vessels, which were tightly connected to the graft. Overall,
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FIGURE 4. (A–D) Biochemical analysis of ECM components including
acidic/pepsin-soluble collagen, sulfated GAG, and soluble elastin. All
samples were normalized to ECM dry weight. Data are shown
as means 6 standard deviations. Histological observations of ECM-1
(E, G) and ECM-2 (F, H) for collagen (E, F) and elastin (G, H). Scale bar
represents 50 lm. [Color figure can be viewed in the online issue,
which is available at wileyonlinelibrary.com.]
DECELLULARIZED ECM DERIVED FROM HUMAN ADIPOSE TISSUE
ORIGINAL ARTICLE
The goal of decellularization is to remove the immunogenic cellular components while maintaining the biological
activity and mechanical integrity of the ECM. The optimization of the decellularization protocol needs to balance a
reduction in antigenicity with preservation of the ECM
structure. Different types of decellularization have been
widely studied, including physical, chemical, and enzymatic
treatments.14 Generally, the use of these treatments alone is
insufficient to achieve complete decellularization. Hence, the
decellularization protocol should be combined with a physical, chemical and/or enzymatic treatment to increase decellularization efficiency and optimized in terms of various factors such as origin, composition, and density of the tissue.
We first isolated raw ECM (ECM-1) from human adipose tissue by simple homogenization. Then, the ECM-1 was treated
with NaCl, SDS, and enzymes. Human adipose tissue is a
type of loose connective tissue, and it can be easily disrupted by homogenization. However, due to the sticky property of extracted ECM-1 and the tendency of cellular components to adhere to ECM proteins, strong chemical and
enzymatic agents were additionally necessary to facilitate
the removal of cellular remnants. In general, the treatments
with a hyperosmolar solution of NaCl and an ionic detergent
of SDS are known to be effective in obtaining decellularized
scaffolds.35 In studies on the decellularization of tissues or
organs, SDS has been used more extensively than other
chemical reagents such as Triton X-100 and polyethylene
FIGURE 5. (A) Macroscopic appearance of grafts in mice. An aqueous
suspension of ECM-2 (0.5 mL) was injected subcutaneously into the
backs of female Slc/ICR mice (6-week-old, n ¼ 8) under aseptic conditions. After 7 weeks, the mice were sacrificed and the grafts
were explanted. The scale bar represents 0.5 cm. Histological examinations of ECM-1 (B, D) and ECM-2 (C, E) grafts 7 weeks after implantation. Tissues were stained with hematoxylin and eosin. Black
and red scale bars represent 100 and 50 lm, respectively. [Color
figure can be viewed in the online issue, which is available at
wileyonlinelibrary.com.]
test of the ECM-2 confirmed that no significant interactions
between ECM-2 and host tissue were found, demonstrating
that the immunogenic components had been removed.
DISCUSSION
Decellularized tissues derived from various tissues or
organs have been used in human clinical applications. However, there are some limitations to the use of decellularized
tissues, such as the fact that most decellularized tissues
have been isolated from animals or cadavers. In this study,
we explored the feasibility of using decellularized ECM
derived from human adipose tissue. Considering that adipose tissue is the tissue that can be safely obtained from
humans, the ECM extracted from human adipose tissue is
highly promising as an allograft material for tissue engineering and regenerative medicine.
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FIGURE 6. Immunohistological examinations of ECM-1 (A, C, E) and
ECM-2 (B, D, F) grafts 7 weeks after implantation. The Prussian Blue (A,
B), anti-CD3 (C, D), and anti-CD8 (E, F) staining were used to detect activated macrophages and T cells. Arrows denote areas of magnified
images and show the presence of macrophages (A) and T cells (C, E),
respectively. The scale bar represents 100 lm. [Color figure can be
viewed in the online issue, which is available at wileyonlinelibrary.com.]
297
FIGURE 7. Scaffolds prepared from ECM-2 with a variety of macroscopic
shapes. The scale bars represent 1 cm. [Color figure can be viewed in
the online issue, which is available at wileyonlinelibrary.com.]
the stress–strain curve, wheereas the ECM-2 scaffold had a
significantly higher Young’s modulus of 8.312 MPa. The
ECM-2 became stiffer and less extensible after decellularization. Most naturally derived biomaterials have a viscoelastic
property and show a wide range of stiffness values, such as
collagen fibers of cartilage (2–46 MPa), collagen gels of calf
skin (0.002–0.022 MPa), and heart muscles of rats/humans
(0.1–0.5 MPa).44 The results observed in mechanical testing
suggest that the ECM-2 has an appropriate mechanical integrity for use as an allograft material in clinical application.
We also performed in vivo experiments to demonstrate
the effectiveness of ECM-2 in an animal model (Supporting
Information Figs. S2, 5 and 6). We observed that the macroscopic appearance of the ECM-2 grafts was well conserved,
and host cells infiltrated into the graft. Moreover, immunogenic cells including macrophages, CD3 and CD8 T cells
were detected in the ECM-1 graft, whereas the immunogenic
cells were not detected in the ECM-2 graft. Thus, in vivo
results provide evidence that ECM-2 derived from adipose
tissue might provide an ideal scaffold material not only for
autologous grafts but also for allograft tissue engineering.
CONCLUSIONS
glycol, particularly for full removal of cellular components.36–38 The NaCl-SDS-enzyme treatment in our decellularization system was effective at removing cellular components (Fig. 3). However, the chemical or enzymatic reagents
may interact with ECM components such as collagen, GAG,
and elastin, leading to disruption and changes in the composition of the native ECM structure.39,40 Therefore, we measured the contents of major ECM components such as acid/
pepsin-soluble collagen, sulfated GAG, and soluble elastin
using biochemical analysis (Fig. 4). The NaCl-SDS-enzyme
treatment was found to reduce the contents of acid/pepsinsoluble collagen and soluble elastin. However, the ECM-2
in our system exhibited distinct ECM fiber networks
[Fig. 2(B)], implying that the reduction in the ECM components did not translate into a significant disruption of the
native ECM structure.
The fibrous components of ECM in connective tissue act
as a supporting framework of tissue and cells, thus distributing stress forces uniformly in tissues. In particular, sulfated GAG and elastin, as incorporated into collagen fibers
and other fibrous components, provide strength and stiffness to maintain the structures of tissues.41–43 Therefore,
changes in the ECM distribution due to the reduction or
damage in collagen fiber networks and incorporated GAG/
elastin fibers may result in the loss of ECM mechanical function. As shown in Figure 7, the ECM-2 were fabricated into
3D scaffolds with a variety of shapes, such as round dishes,
sheets, microspheres, square molds, films, and hollow tubes.
The 3D scaffolds had a highly porous structure and exhibit
excellent mechanical properties (Supporting Information
Videos 1 and 2). The scaffolds show a highly elastic behavior, even when wet. The ECM-2 scaffolds showed the typical
pattern of hyper-elastic materials (Supporting Information
Fig. S1). The ECM-1 scaffold had a Young’s modulus of
0.566 MPa, as calculated from the initial linear portion of
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CHOI ET AL.
Decellularized ECM was obtained from human adipose tissue through a series of physical, chemical, and enzymatic
treatments. The cellular components were effectively
removed without significant disruption of the morphology
or structure of the ECM. Although the decellularization process led to a loss in some ECM components, the elastic
property of the ECM was maintained. The ECM exhibited
the ability to promote host cell infiltration in animal models
without any sign of immunogenic response. The decellularized ECM could be fabricated into a variety of 3D shapes
with good mechanical properties, making it highly suitable
as a scaffold for tissue engineering.
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