Acta Biomaterialia 54 (2017) 333–344

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Acta Biomaterialia
journal homepage: www.elsevier.com/locate/actabiomat

Full length article

Human lung fibroblast-derived matrix facilitates vascular

morphogenesis in 3D environment and enhances skin wound healing
Ping Du a, Muhammad Suhaeri a,b, Sang Su Ha a,b, Seung Ja Oh a,b, Sang-Heon Kim a,b, Kwideok Park a,b,⇑
a
Center for Biomaterials, Korea Institute of Science and Technology (KIST), Seoul 02792, Republic of Korea
b
Dept. of Biomedical Engineering, Korea University of Science and Technology (UST), Daejeon 34113, Republic of Korea

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

Article history: Extracellular matrix (ECM) is crucial to many aspects of vascular morphogenesis and maintenance of vas-
Received 22 September 2016 culature function. Currently the recapitulation of angiogenic ECM microenvironment is still challenging,
Received in revised form 23 March 2017 due mainly to its diverse components and complex organization. Here we investigate the angiogenic
Accepted 24 March 2017
potential of human lung fibroblast-derived matrix (hFDM) in creating a three-dimensional (3D) vascular
Available online 27 March 2017
construct. hFDM was obtained via decellularization of in vitro cultured human lung fibroblasts and ana-
lyzed via immunofluorescence staining and ELISA, which detect multiple ECM macromolecules and
Keywords:
angiogenic growth factors (GFs). Human umbilical vein endothelial cells (HUVECs) morphology was more
Human lung fibroblast-derived matrix
(hFDM)
elongated and better proliferative on hFDM than on gelatin-coated substrate. To prepare 3D construct,
Collagen hydrogel hFDM is collected, quantitatively analyzed, and incorporated in collagen hydrogel (Col) with HUVECs.
Human umbilical vein endothelial cells Capillary-like structure (CLS) formation at 7 day was significantly better with the groups containing
(HUVECs) Vascular morphogenesis higher doses of hFDM compared to the Col group (control). Moreover, the group (Col/hFDM/GFs) with
Skin wound healing both hFDM and angiogenic GFs (VEGF, bFGF, SDF-1) showed the synergistic activity on CLS formation
and found much larger capillary lumen diameters with time. Further analysis of hFDM via angiogenesis
antibody array kit reveals abundant biochemical cues, such as angiogenesis-related cytokines, GFs, and
proteolytic enzymes. Significantly up-regulated expression of VE-cadherin and ECM-specific integrin sub-
units was also noticed in Col/hFDM/GFs. In addition, transplantation of Col/hFMD/GFs with HUVECs in
skin wound model presents more effective re-epithelialization, many regenerated hair follicles, better
transplanted cells viability, and advanced neovascularization. We believe that current system is a very
promising platform for 3D vasculature construction in vitro and for cell delivery toward therapeutic
applications in vivo.

Statement of Significance

Functional 3D vasculature construction in vitro is still challenging due to the difficulty of recapitulating
the complex angiogenic extracellular matrix (ECM) environment. Herein, we present a simple and prac-
tical method to create an angiogenic 3D environment via incorporation of human lung fibroblast-derived
matrix (hFDM) into collagen hydrogel. We found that hFDM offers a significantly improved angiogenic
microenvironment for HUVECs on 2D substrates and in 3D construct. A synergistic effect of hFDM and
angiogenic growth factors has been well confirmed in 3D condition. The prevascularized 3D collagen con-
structs also facilitate skin wound healing. We believe that current system should be a convenient and
powerful platform in engineering 3D vasculature in vitro, and in delivering cells for therapeutic purposes
in vivo.
Ó 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

1. Introduction

Neovascularization that includes vasculogenesis and

⇑ Corresponding author at: Center for Biomaterials, Korea Institute of Science and
Technology (KIST), Seoul 02792, Republic of Korea.
E-mail address: kpark@kist.re.kr (K. Park).
angiogenesis is a complex process that involves different cell types,
multiple extracellular matrix (ECM) components, and growth
factors (GFs) [1,2]. Promotion of vascularization is not only critical

http://dx.doi.org/10.1016/j.actbio.2017.03.035
1742-7061/Ó 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
334 P. Du et al. / Acta Biomaterialia 54 (2017) 333–344
for construction of viable engineered tissue constructs in vitro, but
also important for the treatment of various pathological
conditions, i.e., wound, ulcer, myocardial infarction, and peripheral
vascular diseases [3–5]. Upon the significance of vascularization in
tissue engineering, many approaches have been adopted to create
three-dimensional (3D) vasculatures. Common strategies for vas-
culature construction are mostly relied on materials, GFs, and cell
types. Natural hydrogels, such as fibrin [6], collagen, and MatrigelÒ
are very promising materials for 3D vasculature formation [7].
However, these materials have a weak mechanical property and
are easily degradable or deformed. To overcome these shortcom-
ings, synthetic polymer materials were frequently modified using
fibronectin (Fn), collagen (Col), laminin (Ln), or other ECM-like
components to enhance cell-matrix interactions that would trigger
specific signaling pathways for angiogenesis [8]. Diverse angio-
genic GFs, i.e., vascular endothelial growth factor (VEGF), basic
fibroblast growth factor (bFGF), angiopoietin, and stromal derived
factor (SDF)-a1 have also been employed to promote vascular cell
survival, proliferation and vascular morphogenesis [9,10]. Selec-
tion of proper cell types, such as endothelial and perivascular cells,
or human mesenchymal stem cells are other strategies to create a
fully vascularized construct [11].
Previously mentioned approaches of neovascularization are
appreciable but still far short of recapitulating the in vivo process
of vasculature formation. One of the critical issues is to establish
a natural angiogenic ECM microenvironment in 3D condition.
ECM is a physical entity of the extracellular domain that is mainly
comprised of macromolecular proteins, polysaccharides, and other
constituents. ECM includes structural elements (Col, fibrillins, fibu-
lins and elastin), cell adhesive proteins (Fn, Ln, vitronectin, and
tenascins), proteoglycans (chondroitin sulfate and heparin sulfate),
non-proteoglycans, and matrix metalloproteinase [12,13]. Bio-
chemical cues in ECM are also important in initiating and in regu-
lating vascular cells morphogenesis. ECM is known as a reservoir of
diverse GFs. Among many ECM candidates, we have been investi-
gating cell-derived ECM (CDM) as an analog of natural ECM
in vivo. In fact, CDM shares similar constituents with endothelial
basement membrane (BM) through which vascular cells interact
with pericytes or smooth muscle cells [14]. For this reason, CDM
have been utilized to investigate a fundamental of ECs–matrix
interactions that may lead to a neovascular or pathological path-
way [15]. For example, studies have demonstrated that CDMs from
human lung fibroblast [16] or from a co-culture of dermal fibrob-
lasts with breast cancer-cell line [17] supported human umbilical
vein endothelial cells (HUVECs) vascular morphogenesis in vitro.
Other reports have suggested the promotion of ECs sprouting and
lumen formation in the presence of fibroblasts via deposition of
ECM components, and via secretion of angiogenic GFs and prote-
olytic enzymes [18–20]. Our previous studies reported that mouse
fibroblast-derived matrix (FDM) and preosteoblast-derived matrix
(PDM) are an excellent 2D platform for HUVECs adhesion, prolifer-
ation, and vascular morphogenesis, compared to Fn- or gelatin-
coated substrates [21]. Interestingly chondrocyte-derived matrix
was very poor in the formation of capillary-like structure. These
studies set a good example in demonstrating the importance of
providing HUVECs with appropriate ECM niche for vascular mor-
phogenesis. Vascular structure on these 2D environments is how-
ever stable only for a very short period (less than 3 days). And
there are practical issues due to dimensional limitation of 2D envi-
ronment. Accordingly, advanced strategies combining 3D construct
and CDM are necessary to create more stable and robust vascula-
tures for a longer period time.
In this study, we hypothesize that ECM obtained from decellu-
larized human lung fibroblasts can facilitate HUVECs vasculogene-
sis in 3D environment. To test our hypothesis, we first identified
major ECM macromolecules and angiogenic GFs within hFDM by
immunofluorescence and ELISA, respectively. The effect of hFDM
on HUVECs vascular morphogenesis was comprehensively investi-
gated for up to 7 days in 3D constructs that were built incorporat-
ing HUVECs and different doses of hFDM or angiogenic GFs into
collagen hydrogel. Moreover, to figure out the underlying mecha-
nism of significantly advanced HUVECs vasculogenesis, an angio-
genic factors profiling of hFDM was carried out, along with the
study of gene expression related to cell-ECM interactions (inte-
grins) and cell-cell contacts (VE-cadherins). In addition, therapeu-
tic effect of such 3D constructs was evaluated using murine skin
wound model. To our best knowledge, this study is the first report
that clearly demonstrates the pro-angiogenic effects of hFDM in 3D
environment on HUVECs vascular morphogenesis in vitro and ther-
apeutic efficacy in vivo. Current system should be a convenient and
an effective platform in engineering 3D vasculature of angiogenic
model in vitro, and in delivering cells for therapeutic purposes.
2. Materials and method
2.1. Preparation of human fibroblasts-derived matrix (hFDM)
Human lung fibroblasts (hFB) (WI-38; ATCCÒ-CCL-75TM, Manas-
sas, VA) were cultured in Dulbecco’s Modified Eagle’s Medium
(DMEM) supplemented with 10% fetal bovine serum (FBS),
100 U/mL penicillin, and 100 mg/mL streptomycin (Invitrogen,
Carlsbad, CA). For the preparation of hFDM, hFB are seeded at a
density of 2
104/cm2 onto cell culture dishes (100 mm diameter)
or gelatin-coated glass coverslips, and cultivated for 7–10 days
until confluence. The culture medium was supplemented with
50 mg/mL ascorbic acid 2-phosphate (A4403; Sigma) to enhance
cell-derived ECM deposition. The medium was changed every 2
or 3 days. For decellularization, the confluent cells were treated
briefly with a detergent solution containing 0.25% Triton X-100
and 50 mM NH4OH (Sigma-Aldrich) [22]. After the samples were
washed with phosphate buffered saline (PBS), the mixture of
50 U/mL DNase I and 2.5 mL/mL RNase A (Invitrogen) was added
and incubated at 37 °C for 1–2 h. The resultant decellularized
ECM was named hFDM. The hFDM on the culture dishes were used
as prepared or collected in corning tubes with gentle pipetting. All
the hFDMs were stored at 4 °C prior to further use. Meanwhile
hFDM on gelatin-coated coverglass was gently washed with PBS
and used as a control for comparative analysis on 2D condition.
2.2. Compositional analysis of hFDM
2.2.1. BCA assay
To quantitatively determine the total protein contents on hFDM
before and after decellularization, samples prepared in 100 mm
diameter Petri dish were analyzed using BCA assay kit (23225;
Thermo Fisher Scientific). After several rinsing with PBS, hFB sam-
ples before and after decellularization were solubilized in RIPA cell
lysis buffer (R0278; Sigma) with EDTA-free protease inhibitors
cocktail (78415 and 1862495; Thermo Scientific), and then col-
lected in the 1.7 mL centrifuge tubes, respectively. They were then
suspended using pipette, rocked gently for 30 min at 2–8 °C, and
centrifuged at 14,000g for 5 min. The supernatants were trans-
ferred into a new tube and subjected to BCA assay immediately
or stored at 80 °C as aliquots. Total protein amount of each sam-
ple was normalized to the surface area of culture dishes and thus
the decellularized hFDM concentration was denoted as mg/cm2.
2.2.2. Enzyme linked immunoassay (ELISA)
To determine the amount of GFs in hFDM (before and after
decellularization), the same samples obtained from Section 2.2.1
were analyzed using VEGF ELISA kit (DVE00; R&D system) and
 P. Du et al. / Acta Biomaterialia 54 (2017) 333–344
human FGF-basic ELISA development kit (900-K08; Peprotech),
respectively. The concentration of two GFs was also normalized
to the surface area of the culture dishes and therefore each GF
amount is denoted as pg/cm2 [23].
2.2.3. Angiogenesis antibody array
Angiogenesis related factors were screened using a sandwich
immunoassay array kit (ARY007; Proteome ProfilerTM Human
Angiogenesis Array Kit, R&D system) following the manufacturer’s
instructions. In brief, antibody-coated membrane was first blocked
with supplied block buffer and then protein sample (300 mg hFDM)
was added, along with certain amount of detection antibody in
array buffer to make a total volume 1.5 mL. They were incubated
overnight at 2–8 °C on a rocking platform shaker. On the next
day, the membrane was first washed with 1x wash buffer 3 times
(10 min each time), and then incubated with diluted streptavidin-
HRP for 30 min at room temperature on the shaker. After 3 times
rinsing for 10 min each time, membrane was reacted with 1 mL
prepared Chemi Reagent mix for 4 min. Chemiluminescence was
examined via LAS 3000 imaging system (Fuji), and the intensity
of the positive dots was recorded using Multi gauge v3.0 software
(Fuji Film). Quantitative data are obtained after each dot was nor-
malized on a scale ranging from negative control (0%) to positive
control value (100%).
2.3. Huvecs culture, adhesion, and proliferation on 2D substrates
Human umbilical vein endothelial cells (HUVECs) (passage 3 to
6) (C2517A; Lonza, Walkersville, MD) are cultured in endothelial
cell growth medium (EGM-2 BulletKit, CC-3162) at 37 °C under
5% CO2 humidified atmosphere. For cell adhesion and proliferation,
HUVECs suspended in EGM-2 medium or endothelial cell basal
medium (EBM-2) (CC-3156; no GFs, no cytokines, no other supple-
ments) are seeded on the gelatin-coated coverslips (control) and
hFDM in 12-well plates (5
104 cells/well) (n = 4, each group),
respectively. Cell attachment at 3 h and cell proliferation at 24
and 72 h on these substrates were examined in different medium
conditions using cell counting kit-8 (CCK-8) (CK04; Dojindo, Japan)
assay. Briefly, 10% CCK-8 solution diluted in culture medium was
added to each sample and incubated at 37 °C for 2 h. Aliquots of
each sample (100 mL) were transferred to 96-well plate and the
absorbance was measured at 450 nm wavelength using multiskan
microplate reader (Thermo Scientific, Rockford, IL). Cell
proliferation assay was performed in triplicate. In addition,
HUVECs morphology was visualized via CD31 and F-actin
immunofluorescence (IF) staining or examined under phase-
contrast microscope. Moreover, to investigate cell-matrix interac-
tions on two different substrates, HUVECs cultured in EGM-2 or
EBM-2 medium (n = 4, each group) for 24 h were fixed and sub-
jected to IF staining of integrin a5, Fn, and F-actin, respectively.
2.4. Construction of HUVECs 3D culture model
The obtained hFDM from Section 2.1 was re-suspended in 5 mL
autoclaved distilled water (DW) by moderate agitation. After cen-
trifugation at 3000 rpm, the supernatant was removed and this
process was repeated twice, then a mass of hFDM was collected.
To test hFDM effect on HUVECs morphogenesis in 3D environment,
different amounts of hFDM were incorporated into collagen hydro-
gel (Col) to have final concentrations of hFDM at 0, 100, 250 and
500 mg/mL, where they were designated as 0 hFDM, 100 hFDM,
250 hFDM, and 500 hFDM, respectively. A final concentration of
Col at 2.5 mg/mL was prepared [24] using rat tail collagen type I
(354236; CorningÒ) diluted in 1
M199 (11150059) and
10
M199 medium (11825015; GibcoTM) with phenol red. The pH
was adjusted to 7.4 using 1 N NaOH (Sigma) and the final cell
335
density is 2
106 cell/mL. All the procedures were carried out on
ice. Once mixed thoroughly, 30 mL mixture of HUVECs, Col, and dif-
ferent doses of hFDM was loaded into 96-well half-well plate
(3696; Corning) (n = 6, each group) and incubated at 37 °C for
30 min until polymerization. After then, 80 mL EGM-2 medium
was added into each sample. HUVECs morphology in four test
groups was observed at different time points. To visualize different
doses of hFDM, hFDM was incorporated into Col without HUVECs
and stained by Fn. And for a vasculature formation within the con-
structs, samples were fixed on day 3 and 7, and subjected to F-actin
and DAPI staining.
2.5. Incorporation of hFDM and angiogenic growth factors in 3D
constructs
To investigate a synergistic effect of both hFDM and growth fac-
tors (GFs), 3D constructs containing HUVECs (2
106 cell/mL) and
angiogenic GFs were prepared as follows: Col alone (Col), Col with
GFs (Col/GFs), Col with hFDM (Col/hFDM), and Col with both hFDM
and GFs (Col/hFDM/GFs). The GFs are human vascular endothelial
growth factor (VEGF) (cyt-260, Prospec, Israel), recombinant
human fibroblast growth factor basic-2 (bFGF) (cyt-218), and
recombinant human stromal cell-derived factor-1 alpha (SDF-1a)
(chm-262). The final concentration of all three GFs was 200 ng/
mL [25], respectively and hFDM was used at 250 mg/mL. These con-
structs were cultivated for up to 7 days. The same protocols were
applied for the analysis as described in the Section 2.4.
2.6. Immunofluorescence (IF) staining
For hFB, hFDM, and HUVECs staining on 2D substrates, samples
on glass coverslips were fixed in 4% paraformaldehyde (pH 7.4) for
10 min and washed by PBS three times. They were permeabilized
in 0.2% Triton-X 100 for 5 min, blocked with 3% bovine serum albu-
min (BSA) for 1 h, then incubated with primary antibodies over-
night at 4 °C, washed again with PBS, and incubated with
secondary antibodies for 1 h at room temperature. These samples
were washed three times and mounted onto microscope slides
using vectashiedÒ mounting medium with DAPI (H-1200; Vector
Laboratories, INC., CA) for nucleic labeling. There is little chance
of non-specific binding of the secondary antibody after IF staining
(Fig. S1). For capillary-like structure (CLS) observation in 3D con-
structs, the same protocol was applied as mentioned before but
more extensive washing and longer incubation with 0.2% Triton-
X 100 for 30 min and 3% BSA for 2 h. DAPI (46190; thermo scien-
tific) was co-stained with the samples to label nuclei. Once the
staining procedures were completed, samples were washed thor-
oughly and transferred to coverglass bottom dishes (200350; SPL
Life Sciences). Fluorescence images of CLS in the 3D constructs
were taken using a confocal microscope (LSM 700, Carl Zeiss) with
consecutive z-stack images at 400 magnifications (20–80 iterations
with an interval thickness of 1 mm). To identify the luminal struc-
ture and capillary thickness, 3D reconstruction of CLS was per-
formed via Imaris software (Imaris 7.7.2). The CLS formation was
quantitatively evaluated via the number of segments, total capil-
lary length, respectively. The number of ECs per 100 lm capillary
length was also determined using Imaris software, based on the
DAPI-positive stains of capillary networks. In addition, the number
of ECs per capillary diameter was counted using the cross-section
images of lumen space.
Primary antibodies and dilution ratios used in this study are
summarized as follows: mouse anti-Fn (SC-8422; Santa Cruz
Biotechnology, 1: 200), rabbit anti-Fn (SC-9068, 1: 200), goat
anti-Ln (SC-16590, 1: 100), rabbit polyclonal anti-collagen type I
(ab34710, 1: 300; Abcam), rabbit polyclonal anti-CD31 (ab28364,
1: 100), and mouse anti-a-smooth muscle actin (A2547;
336 P. Du et al. / Acta Biomaterialia 54 (2017) 333–344
Sigma-Aldrich, 1: 100). Secondary antibodies are Alexa FluorÒ 488
F(ab0 ) fragment of goat anti-mouse IgG (A-11017), Dylight 488 of
goat polyclonal to rabbit IgG-H&L (ab96899), Alexa FluorÒ 594-
conjugated donkey anti-goat IgG (Jackson Immunoresearch, West
Grove, PA), and Alexa FluorÒ 647-conjugated goat polyclonal to
mouse IgG-H&L (ab150115). All the secondary antibodies were
diluted in 1% BSA at 1: 500. F-actin cytoskeleton of HUVECs was
labeled by tetramethylrhodamine isothiocyanate (TRITC)-
conjugated phalloidin (A415; Life Technologies, 1: 200).
2.7. Real-time quantitative polymerase chain reaction (q-PCR)
Total mRNA of HUVECs cultured in the 3D constructs of Col, Col/
GFs, Col/hFDM, and Col/hFDM/GFs was extracted at day 1, 3, and 5
(n = 4), respectively using RNeasy kit (Qiagen) following the man-
ufacturer’s instruction. Concentration and purity of the isolated
mRNA were determined using a NanoDrop ND-1000 spectropho-
tometer (Thermo Fisher Scientific, Wilmington, DE). 500 ng mRNA
per sample was added to PrimeScriptTM RT reagent Kit (RR037A;
Takara) to a total volume of 10 mL for cDNA synthesis at 37 °C for
15 min and RTase inactivation at 85 °C for 5 s. The cDNA product
(1 mL), 10 pmol of each reverse and forward primer in 2 mL sterile
DW, 10 mL of SYBR Green real-time PCR mix (RR420A; Takara),
0.4 mL of ROX reference dye, and 6.6 mL DW were mixed in PCR
reaction tubes (Applied biosystems) to a total volume of 20 mL.
The reaction reagents were mixed thoroughly, centrifuged briefly,
and then placed in the q-PCR instrument (7500 Real-Time PCR Sys-
tem; Applied biosystems), where the reaction conditions are initial
denaturation and DNA polymerase activation at 95 °C for 5 min,
followed by 40 cycles of denaturation at 95 °C for 10 s, and anneal-
ing/extension at 58 °C for 34 s. Target genes, their primer
sequences, and melting curve analysis are summarized in the
supplementary data (Fig. S2). The sequences of primer sets of
VE-cadherin and integrin subunits are designed using the primer
express 3.0 software (Applied Biosystems) based on human gene
sequences from GeneBank. Glyceraldehyde 3-phosphate dehydro-
genase (GAPDH) is used as a housekeeping gene.
2.8. Animal study: Mouse excisional wound splinting model
Animal study was performed in accordance with the Korea
Institute of Science and Technology Animal Care and Use Commit-
tee Guidelines. 8-week-old male BALB/C nude mice were housed
and cared in a temperature- and humidity-controlled animal room.
5 mm full thickness wounds were created symmetrically on the
dorsal skin using sterile disposable biopsy punches (33–35, Inte-
gra) after induced anesthesia via intraperitoneal injection of Zoletil
and Rompun (1 mL/kg) as previously described [26]. Mice were
randomized into four groups treated with different samples,
respectively (n = 3 animals per group, 2 wounds per animal):
HUVECs in Col (Col), HUVECs in Col with GFs (Col/GFs), HUVECs
in Col with hFDM (Col/hFDM), and HUVECs in Col with hFDM
and GFs (Col/hFDM/GFs). hFDM and GFs followed the same con-
centrations as shown in the Section 2.5. Before transplantation,
HUVECs were pre-labeled using a red fluorescent cell tracker
(PKH-26, Sigma). 3D constructs (200 mL per sample) were prepared
in 48-well plate with HUVECs density of 2
106 cells/mL. All the
samples were cultured in EGM-2 medium for 3 days to allow vas-
culatures formation before implantation. To prevent a self-healing
by wound contraction, all the wounds post-transplantation were
splinted by silicone 5 mm diameter washer (Invitrogen), secured
with tissue adhesive glue (1469SB; 3 M VetbondTM), and treated
with an interrupted black silk 5–0 suture (SK526; Teleflex Medical
OEM, Korea). Wounds were covered with Tegaderm sterile dress-
ing (3 M Healthcare, St Paul, MN) and subsequently with self-
adhering elastic bandages (3 M Healthcare), which were changed
every 2 or 3 days. Digital photographs were taken to record the
wound healing process during 14 days. Wound area is quantified
using ImageJ software (NIH, Bethesda, MD) in which wound heal-
ing is expressed as a ratio of the wound area divided by the initial
wound area at specific time points.
2.9. Histological and IF staining
After transplantation for 14 days, samples in wound areas were
harvested, cut into halves, and fixed in 4% paraformaldehyde. Half
tissue was embedded into optimum cutting medium (OCT) for
cryosection to identify the transplanted HUVECs. The other half
was embedded in paraffin wax, and sliced into 6 mm thick sections
for Hematoxylin and Eosin (H&E) and Masson’s Trichrome staining,
respectively. For paraffin slices staining, the sections were first
deparaffinized, rehydrated, and then microwaved in 10 mM citric
acid buffer (pH 6.0) for antigen retrieval. Histological images were
obtained under a light microscope (Zeiss Axio Vert.A1, Germany).
IF stainings of angiogenic vessel markers (CD31, a-SMA) were also
performed using the same procedures as described in the
Section 2.6.
2.10. Statistical analysis
Statistical analysis of current data was performed using one-
way analysis of variance (ANOVA), with Tukey’s post hoc multiple
comparisons (GraphPad Prism 5). All the data represent the mean
value and standard deviation. Statistically significant difference
was marked as ⁄(p < 0.05), ⁄⁄(p < 0.01), or ⁄⁄⁄(p < 0.001).
3. Results
3.1. hFDM composition and biochemical factors
According to the IF staining of hFDM before and after decellular-
ization, macromolecular ECM components (Fn, Ln, and Col I) were
detected in both samples and they showed a fibrillar structure
(Fig. 1A). The clearance of nuclei was also confirmed in decellular-
ized hFDM (Fig. 1A, bottom). Based on BCA assay, while total pro-
tein content of hFDM (before decellularization) was
8.36 ± 1.64 mg/cm2, that of hFDM (after decellularization) was
2.73 ± 0.55 mg/cm2 (Fig. 1B). Regarding the source of measured pro-
teins, it is notable that previous results indicate that proteins con-
tained in the FBS are barely adsorbed onto the hFDM. From
quantitative analysis of GFs, 43.27 ± 9.46 pg/cm2 VEGF and
190.02 ± 11.27 pg/cm2 bFGF were measured before decellulariza-
tion, whereas 6.36 ± 0.73 pg/cm2 VEGF and 22.73 ± 2.36 pg/cm2
bFGF were found in hFDM (Fig. 1C and D). These results suggest
that significant gap of proteins and GFs amount is mainly due to
the loss of intracellular components during decellularization pro-
cess. hFDM mass collected from cell culture dishes was used for
biochemical analysis. This hFDM mass is also utilized later in build-
ing a 3D construct that is composed of HUVECs, hFDM, and colla-
gen hydrogel (Col).
3.2. Huvecs morphology and proliferation on hFDM
Upon the identification of multiple ECM macromolecules (Fn,
Col, Ln) and angiogenic GFs (VEGF and bFGF), HUVECs behavior
on 2D hFDM was examined, along with the test of gelatin-coated
coverslips. Cultured in endothelial growth medium (EGM-2;
marked ‘‘ + ”), HUVECs on gelatin-coated substrates exhibit a nor-
mal cobble stone like morphology on day 2, whereas these on
hFDM show an elongated morphology (Fig. 2A and inset). Cultured
in endothelial basal medium (EBM-2; marked ‘‘”) without any
 P. Du et al. / Acta Biomaterialia 54 (2017) 333–344 337

Fig. 1. hFDM characterization. Macromolecular ECM components (Col I, Fn, Ln) of human fibroblast (hFB) before or after decellularization were identified by IF staining (A).
Scale bar is 100 mm. Once total protein contents of hFB before or after decellularization were measured using BCA assay, the normalized protein amounts are indicated as mg/
cm2 (B). Total VEGF and bFGF contents were quantified via Elisa and the normalized values of VEGF (C) and bFGF (D) are marked in pg/cm2. Each experiment was performed in
triplicate. Statistically significant difference is denoted as **(p < 0.01) or ***(p < 0.001).

Fig. 2. HUVECs morphology and proliferation on 2D substrates. Phase-contrast images show HUVECs morphology on gelatin-coated coverslips and hFDM at 48 h in EGM-2
medium (A) and in EBM-2 medium (B), respectively (Scale bar = 200 mm). Insets are HUVECs co-stained via CD 31(green) and F-actin (red) at 24 h (Scale bar = 50 mm). HUVECs
morphology is quantitatively compared by measuring cell aspect ratio using image J software (C). Cell proliferation assay at 3, 24 and 72 h, respectively (D). HUVECs cultured
in EGM-2 (with supplements) is denoted (+), and in EBM-2 medium (without any supplements) is denoted (). Statistically significant difference is indicated as *(p < 0.05),
**
(p < 0.01) or ***(p < 0.001). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
338 P. Du et al. / Acta Biomaterialia 54 (2017) 333–344

supplements, HUVECs on gelatin have an abnormal morphology
with much lesser cell density, but those on hFDM show more elon-
gated cell morphology than those on hFDM in EGM-2 (Fig. 2B and
inset). When HUVECs morphology is quantified by measuring cell
aspect ratio using image J, it showed the largest aspect ratio at
hFDM () condition (Fig. 2C). Furthermore, when HUVECs prolifer-
ation in different medium conditions were compared, cells number
on each substrate was similar at the early time point (3 h). How-
ever, HUVECs proliferation on hFDM was better than that on gela-
tin in both EGM-2 (+) and EBM-2 () medium at 24 and 72 h,
respectively (Fig. 2D). Significant increase of cells number on hFDM
in EBM-2 medium within 24 h but little change of cells number on
gelatin in the same condition was notable.
3.3. Observation of cell-matrix interactions
Cell-matrix interactions play a very critical role in regulating
HUVECs vascular network formation. Fn is one of the most impor-
tant ECM components and ECs actively interact with surrounding
environment by either breaking down existing ECM or secreting
new Fn [27]. Therefore, Fn staining of hFDM and F-actin staining
of HUVECs can visualize how matrix fibers of hFDM interact with
HUVECs. HUVECs appeared to be embedded inside the matrix
fibers of hFDM as indicated via z-stack confocal microscope images
and showed largely elongated cell morphology (Fig. 3A). More
elongated cell morphology was noticed on hFDM in EBM-2 med-
ium than in EGM-2 medium, but a hollow luminal structure was
undetectable in both conditions. In addition, it was notable that
newly synthesized Fn by HUVECs was found on gelatin-coated sub-
strate at 24 h and more positive signals of Fn were observed in
EGM-2 medium than in EBM-2 medium (Fig. 3B). HUVECs can also
synthesize new Fn on the hFDM but the difference of Fn itself syn-
thesized on either gelatin or hFDM is unknown at this time.
3.4. Huvecs vascular morphogenesis in 3D construct: Effect of hFDM
dose
The HUVECs behaviors on 2D hFDM demonstrate its supportive
role for HUVECs adhesion, proliferation, and capillary-like mor-
phology. Next step is the examination of hFDM effect in 3D envi-
ronment by combining different doses of hFDM with Col
(Fig. 4A). As compared with Col itself, incorporation of hFDM pro-
moted HUVECs to form a capillary-like structure (CLS) in 3D con-
struct as shown by F-actin staining at 3 days (Fig. 4B). This effect
was even more obvious at 7 days, showing that higher doses of
hFDM were more effective in CLS formation in general (Fig. 4B, bot-
tom). The presence of hFDM in 3D construct (no HUVECs) is visu-
alized via Fn staining (Fig. 4B, inset). The CLS formation is
quantitatively compared via two parameters: number of segments
and total segment length. On day 3, the number of segments in all
hFDM-incorporated groups is significantly higher than that with-
out hFDM (Col) and that of 250 hFDM and 500 hFDM group is
about twice that of 100 hFDM (Fig. 4C). This trend was similar at
7 days. The total capillary length of hFDM-incorporated constructs
was more than five times longer than Col on day 3, and the trend
was maintained until day 7 (Fig. 4D). The quantitative difference
between 250 hFDM and 500 hFDM is not statistically significant.
When the extent of CLS formation in a larger scale at 7 days was
further examined by tile-scanned large dimensional images (mm
scale), they supported more active CLS formation with higher dose
of hFDM and showed the homogeneous distribution of CLS through
the whole 3D construct (Fig. S3). In addition, the stability of CLS
with time is very critical and thus each group was stained using
toluidine blue O on day 14 (Fig. S4). The results showed that few
CLS was detected in Col group, whereas CLS in hFDM-
incorporated constructs was maintained for 14 days and the den-
sity of CLS was directly correlated with hFDM doses.
3.5. Huvecs vascular morphogenesis in 3D construct: Effect of hFDM
and GFs
Since the results in 3.4 indicated similar effects between
250 hFDM and 500 hFDM, we selected 250 hFDM as an optimal
dose and combined it with angiogenic GFs to investigate their syn-
ergistic activities on vascular morphogenesis in 3D construct. On
day 3, comparable level of CLS formation was observed between
Col/GFs and Col/hFDM, and the highest level was found in Col/
hFDM/GFs. The same trend was witnessed at 7 days but Col/hFDM
showed much more effectiveness than Col/GFs (Fig. 5A). When seg-
ments number and total capillary length were quantitatively eval-
uated on day 3, Col/GFs, Col/hFDM, and Col/hFDM/GFs were
significantly better in CLS formation than Col, and the best was

Fig. 3. Cell-matrix interactions. 3D reconstructed confocal microscope z-stack images (24 h) exhibit that HUVECs are embedded in the matrix fibers of hFDM in EGM-2 and
EBM-2 medium, respectively. hFDM was stained using IF of Fn (green), and HUVECs were via F-actin (red) and DAPI (blue) staining (A). Cell-matrix interactions on gelatin-
coated substrate and hFDM in different medium conditions were visualized at 24 h via Fn staining (green), and HUVECs were stained by F-actin (magenta) (B). Scale bar is
50 lm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
 P. Du et al. / Acta Biomaterialia 54 (2017) 333–344 339

Fig. 4. hFDM dose effect on HUVECs vasculogenesis in 3D constructs. Schematic shows HUVECs incorporated in Col with or without hFDM (A). HUVECs morphology on day 3
and 7 was visualized by F-actin (red), DAPI (blue) staining, and hFDM was detected via IF of Fn (green) (inset) (B). The Fn signal in the inset is derived from hFDM, not HUVECs
and indicates the presence of hFDM and its distribution in the construct. CLS formation in each group was quantitatively compared using two parameters: number of
segments (C) and total capillary lengths (D). CLS formation is progressively improved upon hFDM-dose dependent manner. Scale bar is 200 mm. Statistically significant
difference among the test groups is indicated as *(p < 0.05), **(p < 0.01) or ***(p < 0.001).

Fig. 5. Effect of both hFDM and GFs on HUVECs vasculogenesis in 3D constructs. HUVECs morphology on day 3 and 7 was examined using F-actin (red), DAPI (blue) staining,
and the presence of hFDM was spotted via IF of Fn (green) (A). Scale bar is 200 mm. Insets are the schematic of hFDM and/or GFs and HUVECs incorporated in Col. CLS
formation in each group was quantitatively compared via two parameters: number of segments (B) and total capillary length (C). Statistically significant differences are
marked as *(p < 0.05), **(p < 0.01) or ***(p < 0.001). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Col/hFDM/GFs (Fig. 5B and C). The decreasing trend of total length than that of Col and Col/GFs. The difference between Col/
capillary length was obvious on day 7, especially with the hFDM and Col/hFDM/GFs was also statistically significant on day
GFs-containing groups, Col/GFs and Col/hFDM/GFs. And the 7. Although the exact mechanism is unclear at this time, it is likely
hFDM-containing groups were much better with total capillary that hFDM plays a role in stabilizing the bioactivity of GFs
340 P. Du et al. / Acta Biomaterialia 54 (2017) 333–344

compared to Col/GFs group that lacks hFDM and thus loses GFs
efficacy faster. In addition, more analysis of DAPI-positive stains
of Fig. 5A shows that there was a significant increase of ECs num-
ber per 100 lm capillary length for Col/hFDM/GFs at 7 day com-
pared to that on day 3 (Fig. S5).
improving the lumen diameter with time. In addition when the
capillary luminal space was visualized by cross-sectioned 3D
reconstructed confocal microscope images (Fig. S6A), there is little
change of the cell number per capillary diameter with time except
the Col group (Fig. S6B).

3.6. hFDM and GFs significantly increase capillary lumen diameter 3.7. Angiogenic profiling of hFDM

To better understand the quality of capillary structure, IF
images of CLS were taken under confocal microscope at higher res-
olution. Accumulated z-stack images exhibit different level of cap-
illary structures on each group (Fig. 6A). When the diameter of
lumen was quantitatively analyzed, the diameter in both Col/hFDM
(8.46 ± 2.65 mm) and Col/hFDM/GFs (8.52 ± 2.03 mm) was signifi-
cantly larger than that of either Col (5.23 ± 3.26 mm) or Col/GFs
(6.06 ± 1.74 mm) at 3 days (Fig. 6B). It was interesting to see that
the luminal structure on day 7 was dramatically regressed in the
Col group but that of Col/hFDM/GFs was still sustainable and the
size even increased to a maximum diameter of 68.05 mm (Fig. 6C),
much better than that of Col/GFs and Col/hFDM. The average
lumen diameter at 7 days in Col/hFDM/GFs is 27.78 ± 16.61 mm,
which is three times larger than that at 3 days. This may explain
the results of Col/hFDM/GFs (Fig. 5C) that showed significantly
reduced total capillary lengths at 7 days. The lumen diameters in
Col/GFs and Col/hFDM slightly decreased with time. This result
suggests that the combined hFDM and GFs can be synergistic in
stimulating capillary remodeling and accordingly significantly
Upon the confirmation of solid angiogenic potential of hFDM,
we have investigated the hFDM further using 55 angiogenesis-
related antibodies array kit (Fig. 7A). According to the intensity of
positive dots, most abundant angiogenesis-related cytokines
detected in hFDM are coagulation factor III, dipeptidyl peptidase
IV (DPPIV), FGF-basic, hepatocyte growth factors (HGF), serpin E1
(plasminogen activator inhibitor-1), tissue inhibitor of
metalloproteinase-1 (TIMP-1), thrombospondin-1 (TSP-1), uroki-
nase plasminogen activator (uPA). The intensity of these dots is
more than 50% of the positive signals (Fig. 7B). The relatively abun-
dant components are amphiregulin, endostatin, FGF-acidic, FGF-7,
VEGF and their intensity is 30 to 50% to the positive ones. The assay
results demonstrate the angiogenic activity of hFDM and strongly
support the early data in this study.
3.8. VE-cadherin and integrin receptors expression
Cell-cell contacts between ECs are crucial for vasculature for-
mation. VE-cadherin is one of the cell-cell adhesion molecules that

Fig. 6. hFDM and GFs enhance capillary lumen diameters with time. CLS on day 3 and 7 was visualized via F-actin (red) and DAPI (blue) staining, and the entity of hFDM was
identified via IF of Fn (green) (A). Scale bar is 50 mm. CLS lumen diameters in each group were quantitatively determined on day 3 (B) and 7 (C), respectively. Statistically
significant difference is indicated as *(p < 0.05), **(p < 0.01) or ***(p < 0.001). (For interpretation of the references to colour in this figure legend, the reader is referred to the web
version of this article.)
 P. Du et al. / Acta Biomaterialia 54 (2017) 333–344 341

Fig. 7. Angiogenesis related factors analysis. Angiogenesis-related factors in hFDM were analyzed using a sandwich-based human angiogenesis antibody array kit (A). The
positive signals (individual dots) were normalized to the dot intensity of negative (0%; F23 and F24) or positive control values (100%; A1, A2, A23, A24, F1, and F2) (B). The
quantitative data are obtained from three biological replicates.

play a crucial role in the control of vasculature permeability and
integrity [28]. When the gene expression level of VE-cadherin in
each group was examined, it was intriguing that VE-cadherin
expression steadily increased with time only in Col/hFDM/GFs
group (Fig. 8A). While the level of VE-cadherin was similar to each
other on day 1, it gradually climbed for Col/hFDM/GFs and reached
the level to which the difference was statistically significant at
5 days compared with the other groups. This result provides an
implication related to the establishment of cell-cell contacts for
the creation and maintenance of vasculatures. Upon the results
of larger capillary lumen formation in Col/hFDM/GFs on day 7
(Fig. 6C), it is plausible to reason that VE-cadherin may have
involved in the process of vasculatures remodeling to reshape
CLS with larger diameters [29]. To investigate whether macro-
molecular proteins in hFDM are related to HUVECs vasculogenesis,
expression of specific integrin receptors was monitored with time.
On day 1, all the integrin subunits a1, 2, 3, 5, 6 and b1 presented a
similar expression level among four test groups (Fig. S7). However
the integrin receptors of Ln, nidogen, Col IV and Fn are significantly
up-regulated with Col/hFDM and Col/hFDM/GFs on day 5 (Fig. 8B).
This result suggests a possibility that macromolecular components
in hFDM may actively interact with counterpart integrin receptors
in promoting HUVECs vasculogenesis by up-regulation of the
related integrin receptors.
3.9. Therapeutic effect of pre-vascularized 3D construct for skin wound
Previously tested 3D constructs with HUVECs are examined for
their therapeutic effect via skin wound model. The wounds treated
by Col/GFs, Col/hFDM, and Col/hFDM/GFs appeared to have a fully
recovered skin at 14 days whereas those treated by Col exhibited
an incomplete recovery (Fig. 9A). When the extent of wound heal-
ing was quantitatively evaluated by wound occupied area (%) on
day 3, 5, 7 and 14, it presented that Col/hFDM/GFs was faster
and more effective in wound healing compared to the other groups
(Fig. 9B). As the wound regeneration was further investigated by

Fig. 8. Gene expression analysis of VE-cadherin and integrins in 3D constructs. VE-cadherin expression was examined with HUVECs cultured in different 3D constructs on day
1, 3, and 5 (A). Integrin subunits, a1, a2, a3, a5, a6, and b1 expression level was also compared on day 5 (B). Statistically significant differences are marked as *(p < 0.05),
**
(p < 0.01) or ***(p < 0.001).
342 P. Du et al. / Acta Biomaterialia 54 (2017) 333–344

Fig. 9. Therapeutic effect of transplanted 3D constructs in skin wound model. Gross images exhibit skin wound healing process during 14 days (A). Initial wound size is 5 mm.
Wound size is quantitatively analyzed at different time points (B). Skin regeneration in the wound area was evaluated by H&E staining on day 14 (C). Scale bar is 500 mm.
Arrow heads (blue) indicate the neoepidermis and asterisks (blue) denote regenerated hair follicles. Arrows (red) mark the border of initial wound area. Moreover,
arrowheads (blue) indicate the neo-epidermis and this area is shown in a higher magnification (D). Scale bar is 100 mm. Statistically significant differences are marked as
*
(p < 0.05), **(p < 0.01) or ***(p < 0.001).

H&E staining, the wound treated with Col was poorly
re-epithelialized but the one treated by Col/hFDM/GFs formed a
smooth epidermis, with many regenerated hair follicles that were
barely detectable in Col/GFs and Col/hFDM (Fig. 9C). Higher magni-
fication images in the neo-epidermis areas support this finding
(Fig. 9D). In addition, Masson’s Trichrome staining also confirmed
the advanced therapeutic effect of Col/hFDM/GFs with higher col-
lagen deposition, and signs of neovascularization via the presence
of erythrocytes (Fig. S8). Moreover, the transplanted 3D constructs
in the wound model was further evaluated for neovascularization
via immunostaining of angiogenic markers: CD31 and a-SMA.
The results show that neovessels were detected in all the groups
but they were more abundant in Col/hFDM/GFs group (Fig. 10A).
As the blood vessel density (#/mm2) and wound occupied area
(%) were quantitatively analyzed, higher vessel density was obvi-
ous in the wounds treated by Col/hFDM (54 ± 25) and Col/hFDM/
GFs (58 ± 23) better than Col (26 ± 12) or Col/GFs (33 ± 10)
(Fig. 10B). A similar trend was observed with blood vessels occu-
pied area, in which Col/hFDM/GFs showed much larger percentage
than the other groups (Fig. 10C). To investigate the potential mech-
anism of Col/hFDM/GFs on such a therapeutic efficacy in the
wound area, the HUVECs labeled via PKH-26 fluorescent dye before
transplantation are tracked down at14 day. While PKH-26 labeled
HUVECs are observed in all the groups, the most positive signals
were noticed in both Col/hFDM and Col/hFDM/GFs (Fig. S9). In
addition, neovessel stained by a-SMA (green) was also detected
in Col/hFDM/GFs group. However a direct link between trans-
planted HUVECs and host vessels was unidentified.
4. Discussion
The extracellular microenvironments of vascular cells contain a
variety of instructive cues. Each component has a specific role in
regulating cell behaviors and in maintaining a vascular system.
Numerous biochemical cues and biomaterials have been used to
construct angiogenic 3D environments. In this study, when hFDM
was selected and intensively investigated for its angiogenic poten-
tial, the overall results suggested solid angiogenic activities of
hFDM in 3D environment. According to the compositional and bio-
chemical analysis, hFDM reserves multiple GFs, proteolytic
enzymes, and ECM macromolecules, all of which play a crucial role
in regulating vascular cells behavior. The combination of hFDM and
GFs demonstrates significant improvements of HUVECs vascular
morphogenesis, more robust CLS formation, and much effective
wound healing in vivo.
With an effort to understand critical factors that would stimu-
late HUVECs vasculogenic activity, the role of each component in
hFDM needs to be addressed. First of all, according to the quantifi-
cation of GFs contents in hFDM (Fig. 1C and D), the highest dose of
hFDM (500 mg/mL) is estimated to contain about 1.2 ng/mL VEGF
and 4.2 ng/mL bFGF, respectively. Compared with the soluble GFs
(VEGF, bFGF, and SDF-1a) concentrations (200 ng/mL, each) used
in this study, the amount of those GFs in hFDM seems negligible
to be effective. Other biochemical factors as shown in Fig. 7, how-
ever may explain HUVECs vasculogenesis in 3D construct. Among
these factors, FGF-basic, FGF-acidic, FGF-7, VEGF, HGF are very
important proangiogenic GFs [30]. Coagulation factor III, known
as a tissue factor maintains the vascular integrity of tissue by ini-
tiating the coagulation cascade after vessel injury [31]. Amphireg-
ulin is an EGF family GFs, highly expressed in mammary epithelial
cells, and clinical studies have suggested it plays some roles in
human cancer progression [32]. Both TSP-1 and endostatin are
reported as anti-angiogenic agents [33,34].
In addition, we postulate that proteolytic enzymes in hFDM
may have played a certain role in facilitating HUVECs migration
to form vasculatures via ECM remodeling. Proteolytic ECM degra-
dation by stromal cells has shown to increase neovascularization
by either releasing sequestered angiogenic factors or providing
physical guidance cues [35]. Current data of human angiogenesis
 P. Du et al. / Acta Biomaterialia 54 (2017) 333–344 343

Fig. 10. Neovascularization of transplanted 3D constructs in skin wound model. New vessel formation in the wound areas was investigated via CD31 and a-SMA staining,
respectively (A). Neovascularization was quantitatively evaluated using vessels density per mm2 (B) and vessels occupied area in percentage (C). Scale bar is 50 mm.
Statistically significant differences are marked as *(p < 0.05), **(p < 0.01) or ***(p < 0.001).

antibody array demonstrate that DPPIV and uPA are the most
abundant enzymes in hFDM (Fig. 7). Previous study found out that
DPPIV and uPA was implicated in remodeling of ECM to enhance
cell adhesion, proliferation and migration [36,37]. In addition,
TIMP-1 and serpin E1 are also proteolytic enzymes involved in
the angiogenic processes via modulation of ECM remodeling. If this
hypothesis is true, human lung fibroblasts (hFB) conditioned med-
ium (CM) may have some effects on HUVECs vasculogenesis since
human lung fibroblasts secrete these enzymes into culture med-
ium. To test this hypothesis, HUVECs were cultured in Col, with
the addition of CM inside or outside the Col or both sides. Com-
pared with the Col group, CM did not show any positive effects
on HUVECs vasculogenic behaviors on day 2 (Fig. S10). This result
suggests that proteolytic enzymes in CM are insufficient or inactive
to develop a capillary formation. It would be very interesting to fig-
ure out the most active constituent in the hFDM. Our previous
work examined 3D construct containing Fn (instead of hFDM),
GFs, Col, and HUVECs but we could not observe a nicely formed
capillary structure (data not shown).
The macromolecular components of hFDM can also improve
cell-matrix interactions and concurrently trigger angiogenic sig-
naling. Besides cell-cell contacts, cell adhesion to specific compo-
nents of ECM is considered critical for vascular morphogenesis
[38]. Many studies suggest that ECs-matrix coupling through the
cell surface receptors initiates the matrix-integrin-cytoskeletal sig-
nal transduction, which causes simultaneous angiogenic sprouting
[39]. Specific interactions between extracellular components and
integrins are essential in ECs vascular morphogenesis in Col [40].
The major integrins responsible for such interactions are a1b1
(Ln/ collagen IV), a2b1 (Col I), a3b1 (nidogen/Ln), a5b1 (Fn), and
a6b1 (Ln) [41]. In particular, binding between Fn and its receptor
integrin a5b1 is an important step in the angiogenesis process of
ECs [42,43]. Higher integrin a5 expressions of HUVECs on 2D
hFDM (Fig. 3B) support the crucial role of Fn in promoting
HUVECs adhesion, proliferation, and vascular morphogenesis. The
up-regulation of integrin subunit genes of HUVECs in Col/hFDM/
GFs (Fig. 8B) suggests a possibility that the macromolecular com-
ponents in hFDM may actively interact with counterpart integrin
receptors during HUVECs vasculogenesis. Interestingly, much
higher expression level of integrin a3 in Col/hFDM/GFs than Col/
GFs suggests the positive effect of interactions between integrin
a3 and integrin a3 specific ECM molecules (nidogen and laminin)
and this may play a role in facilitating vasculature formation and
remodeling into large vessels in 3D construct. The presence of
nidogen was not confirmed in this study.
Currently we have demonstrated the great potential of hFDM as
a key constituent of angiogenic 3D construct in vitro. For in vivo
study, we find that Col/hFDM/GFs group with the highest level of
cell engraftment promotes skin wound healing much better than
other groups. This result is partly explained by an indirect, para-
crine effect of transplanted cells as reported in the previous litera-
tures [44,45]. Angiogenic factors secreted by the transplanted
HUVECs, and those contained in hFDM can play a major role in
encouraging neovessel formation in the wound area and thus
result in the improvement of wound healing. Regarding the impor-
tance of functional vessel formation in vivo, however current study
is unable to find direct evidences whether the transplanted
HUVECs are linked to the host vasculature. We consider some
other strategies to improve our system by delivering more number
of ECs or adding perivascular cells. Since perivascular cells and
multiple BM macromolecules stabilize native blood vessels, cur-
rent platform lacks such cell type. For more mature and functional
vasculature construction, HUVECs and pericytes co-culture system
can be a next stop. Further in-depth study including hFDM-
mediated intracellular signaling should shed a light on the mecha-
nism of HUVECs vasculogenesis in 3D environment. hFDM can find
various applications with other biomaterials to develop a new plat-
form for functional vasculature formation.
344 P. Du et al. / Acta Biomaterialia 54 (2017) 333–344
5. Conclusions
In summary, one of the major findings of this study is that
hFDM is not just an assembly of ECM macromolecules but contains
angiogenesis-related factors, such as GFs, proteolytic enzymes, and
others. Therefore, hFDM offers a great angiogenic microenviron-
ment for HUVECs vascular morphogenesis in 3D environment.
Incorporation of GFs and hFDM into 3D construct supports their
synergistic activities via much advanced angiogenic parameters
and significantly enlarged capillary diameters with time for up to
7 days. In addition, up-regulated gene expression of cell-cell adher-
ent molecule, VE-cadherin and ECM specific integrin receptors are
also notable, especially with Col/hFDM/GFs. Another benefit of Col/
hFMD/GFs is certified via skin wound model that shows effective
re-epithelialization, nearly complete regeneration of hair follicles,
better transplanted cells viability, and much improved neovascu-
larization. We believe that current platform is very promising in
the construction of 3D vasculature in vitro and as a cell delivery
carrier for therapeutic applications in vivo.
Acknowledgements
This work was supported by National Research Foundation of
Korea (NRF) grant (No. 2015R1 A2A2A04004469) from the Min-
istry of Science, ICT and Future Planning, and by Korea Health
Industry Development Institute (KHIDI), funded by the Ministry
of Health and Welfare (HI16C0133), Republic of Korea.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.actbio.2017.03.
035.
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