<div></div><div>1. Introduction</div><div>Adipose tissue is a highly metabolic and vascularized tissue. In native
tissue, a dense capillary network provides the supply of the residing
cells with nutrients and inspiratory gases and the removal of waste
products. Since the diffusion limit of oxygen is less than 200 µm
(Olive, Vikse, &amp; Trotter, 1992; Thomlinson &amp; Gray, 1955), the centers
of large tissue constructs experience necrosis and volume loss without a
functional vascular network. Consequently, there is an urgent need for
fast vascularization after implantation of adipose tissue implants to
maintain tissue mass and viability. In addition, for the <i>in vitro</i>use of tissue constructs, for example as a testing system, a stable
functional vascular system would be desirable to allow constructs of a
larger size and to maintain comprehensive cell behavior. Furthermore,
such vascularized tissue constructs would allow <i>in vitro</i>investigations regarding the development and therapy of vascular
diseases. The inclusion of a functional vascular system remains one of
the biggest challenges in three- dimensional (3D) tissue engineering
approaches. To date there are several strategies to vascularize
engineered 3D tissue constructs, e.g. functionalized scaffolds,
perfusion bioreactors, co-culture and <i>in vivo</i> approaches (Lovett,
Lee, Edwards, &amp; Kaplan, 2009). Pro-angiogenic factors immobilized in
the scaffold material were found to enhance vascularization (Laschke et
al., 2008; Yoon, Chung, Lee, &amp; Park, 2006). For example, vascular
endothelial growth factor (VEGF) and basic fibroblast growth factor
(bFGF), which are known to induce vessel formation and platelet-derived
growth factor (PDGF) β which supports stabilization of the newly formed
vessels (Gaengel, Genove, Armulik, &amp; Betsholtz, 2009). Different
co-culture systems, using monolayer or spheroid cultures, demonstrated
the spontaneous formation of vascular-like structures (Walser et al.,
2013; Wenz, Tjoeng, Schneider, Kluger, &amp; Borchers, 2018). In
particular, the co-culture of endothelial cells (ECs) with
adipose-derived stem cells (ASCs) showed a beneficial effect on vessel
maturation and sprouting (Verseijden et al., 2012; Volz, Hack, Atzinger,
&amp; Kluger, 2018). ECs have a reciprocal effect on pre-adipocyte
proliferation and differentiation (Aoki, Toda, Sakemi, &amp; Sugihara,
2003). With these techniques, vascularization has been achieved directly<i>in vivo</i> or after implantation of the engineered tissue constructs
(prevascularization) (Laschke, Strohe, et al., 2009; Laschke, Vollmar,
&amp; Menger, 2009). So far there is no successful <i>in vitro</i> approach
with a physiological and functional vascular system, which ensures
adequate stability and reproducibility. In most approaches, some type of
feeder cells are used to support the formation of vascular-like
structures by ECs. This living cellular part impedes a commercial
application due to the difficult handling and storage. In contrast,
lyophilized acellular biomaterials can be stored for long periods of
time. In addition, acellular biomaterials evoke far fewer concerns
regarding their admission for regenerative medicine compared to the
approaches including living cells. However, during dehydration of
natural materials changes in structure and composition may occur. Thus,
it has to be clarified if the processing of the biomaterial affects its
ability to influence cellular behavior. To address this issue, next to
the effect of the wet hydrogel-like form, the effect of the dehydrated
materials on cellular behavior should be investigated.</div><div>A critical requirement for engineering tissue constructs is the use of a
suitable scaffold that provides appropriate biological and
physicochemical properties. The cell surrounding material also plays an
important role in vascularization. There are several synthetic and
natural scaffold materials used for vascularized tissue engineering
approaches, e.g. polylactic acid, polyethylene glycol, collagen or
hyaluronic acid. However, the extracellular matrix (ECM) as the natural
environment of the cells <i>in vivo</i> represents the most
physiological biomaterial. A variety of ECM-hybrid materials and pure
decellularized ECM were investigated towards their ability to support
stem cell differentiation and (neo)vascularization <i>in vivo</i> and<i>in vitro</i> (Adam Young, Bajaj, &amp; Christman, 2014; Badylak,
Freytes, &amp; Gilbert, 2009; L. Flynn, Prestwich, Semple, &amp; Woodhouse,
2009; L. E. Flynn, 2010). All these studies were performed with
decellularized ECM derived from native tissue. For several years,
another source of natural ECM moves to the fore. <i>In vitro</i>generated cell-derived ECM (cdECM) from different cell-types (e.g.
fibroblasts and ASCs) was isolated and used as a biomaterial in a
variety of applications (Lu, Hoshiba, Kawazoe, &amp; Chen, 2011; Lu,
Hoshiba, Kawazoe, Koda, et al., 2011; Sart et al., 2016; Schenke-Layland
et al., 2009; Wolchok &amp; Tresco, 2010). Several studies show that
cdECMs, obtained from different cell-types, can induce adipogenic,
chondrogenic and also osteogenic differentiation of ASCs indicating its
influence on cell fate (Dzobo et al., 2016; Guneta, Loh, &amp; Choong,
2016; Guneta et al., 2017; Guo et al., 2013).</div><div>Our previous study revealed the spontaneous formation of vascular-like
structures by mvECs in co-culture with adipogenic differentiated ASCs
(Volz et al., 2018). In this study, we aimed to analyze whether this
effect has to be attributed to cell-cell or cell-matrix interactions.
Consequently, we investigated the effect of dry and wet cdECM regarding
its ability to support the formation of vascular-like structures by
mvECs. Furthermore, we tested whether there is a difference between ECM
derived from stem cells and adipogenic differentiated cells regarding
their capability to induce vascular-like structure formation.</div><div>2. Materials and Methods</div><div>All research was carried out in accordance with the rules for the
investigation of human subjects as defined in the Declaration of
Helsinki. Patients provided written agreement in compliance with the
Landesärztekammer Baden-Württemberg (F-2012- 078, for normal skin from
elective surgeries).</div><div>2.1. Cell isolation and expansion</div><div>ASCs were isolated from human tissue samples obtained from patients
undergoing plastic surgery (Dr. Ziegler; Klinik Charlottenhaus,
Stuttgart, Germany) as described before (Huber, Borchers, Tovar, &amp;
Kluger, 2016). ASCs were initially seeded at a density of 5x103
cells/cm2 in serum-free Mesenchymal Stem Cell (MSC) Growth Medium
(MSCGM, PELOBiotech) containing 5 % human platelet lysate. ASCs were
used up to passage three.</div><div>Dermal microvascular ECs (mvECs) were isolated from juvenile foreskins
(Dr. Yurttas, Stuttgart, Germany) as described before (Volz, Huber,
Schwandt, &amp; Kluger, 2017). Briefly, dermis was cut into small pieces
and digested in a dispase solution (2 U/ml; Serva Electrophoresis,
Germany) overnight at 4 °C. After the removal of the epidermis, mvECs
were isolated from the dermal layer by incubation with 0.05 % trypsin
in ethylenediaminetetraacetic acid (EDTA; Life Technologies, Germany)
for 40 min at 37 °C and mechanically isolated in mvEC Growth Medium-2
(EGM-2mv; Lonza, Switzerland). For cell expansion, mvECs were seeded
with 5 x 10³ cells/cm². MvECs were used up to passage three.</div><div>2.2. Generation of cell-derived extracellular matrix substrates and ASC
feeder layer</div><div>ASCs were seeded into 8-well chamber slides (ibidi, Germany) and 24-well
plates respectively at a density of 25 x103 cells/cm2 in serum-free
MSCGM containing 5 % human platelet lysate. At confluency, medium was
changed to either serum-containing GM (Dulbecco’s Modified Eagle Medium
(DMEM) with 10 % fetal calf serum (FCS) = scdECM) or adipogenic
differentiation medium (DMEM with 10 % FCS, 1 µg/mL insulin, 1 µM
dexamethasone, 100 µM indomethacin, 500 µM 3-isobutyl-1- methylxanthine
= acdECM) both supplemented with 50 µg/mL Na-L-Ascorbate. The medium was
changed every other day. At day 7, cells were lysed using hypotonic
ammonium hydroxide solution and ECM was washed with ultrapure water. For
dry ECM approaches (= dry), ECM was dried at room temperature (RT) and
for wet ECM approaches (= wet), ECM was stored in ultrapure water until
seeded with mvECs. Cellular substrates (= FL) were seeded with mvECs
without lysis of ASCs (Figure 1).</div><div>2.3 Microscopic pictures and degree of swelling</div><div>Macroscopic pictures of wet cdECM substrates were taken directly after
cell removal. To investigate the water uptake, respectively the degree
of swelling, lyophilized cdECMs was weighed to determine the dry weight
[weight (dry cdECM)]. Subsequently, cdECMs were swollen in
demineralized water for 24h at RT and weighed again [weight (swollen
cdECM)].</div><div>The degree of swelling was calculated as:</div><div><span class="math ltx_Math v1">\(\text{Degree\ of\ swelling\ }\left[\%\right]=\frac{\text{weight\ }\left(\text{swollen\ cdECM}\right)-weight\ (dry\ cdECM)}{weight\ (dry\ cdECM)}\)</span>x 100 (1)</div><div>2.4 Immunofluorescence staining of fibronectin and picro sirius staining
of ECM substrates</div><div>For immunofluorescence (IF) staining of fibronectin, cdECM substrates
were fixed in 4 % paraformaldehyde (Carl Roth, Germany) for 10min
followed by incubation with blocking solution, consisting of 3 % bovine
serum albumin (Biomol, Germany) in 0.1 % Triton X (Sigma Aldrich,
Germany) for 30 min to block unspecific binding sites. Subsequently, the
primary antibody (mouse anti-fibronectin, Santa Cruz, Germany; 1:200)
was incubated for 1h at RT and secondary antibody (anti-mouse Cy3,
Dianova, Germany; 1:250) was incubated for 30 min at RT. Both were
diluted in blocking solution. For the histological picro sirius
staining, fixed cdECM samples were dehydrated and blocked in paraffin.
Histological sections (5 µm) were produced and stained with picro sirius
according to manufacturer’s protocol (Morphisto, Germany). Images were
taken with an Axio Observer microscope and Axiocam 506 mono using the
software ZENblue (Carl Zeiss, Germany).</div><div>2.5 Seeding of mvECs on cell-derived ECM and feeder layer</div><div>Isolated dry and wet cdECM substrates were re-seeded with mvECs at a
density of 1 x 104 cells/cm2 in a defined mvEC adipocyte co-culture
medium 9. For FL approaches mvECs were directly seeded on top of
adipogenic differentiated and undifferentiated ASCs at a density of 1 x
104 cells/ cm2 in defined co-culture medium, developed by us earlier 9.
Cells were cultured for 14 days and the medium was changed every other
day (Figure 1). As a control, all experiments were performed on collagen
I (rat tail; 250 µg/mL in 0.1 % acetic acid) coated tissue culture
polystyrene (COL I) and uncoated tissue culture polystyrene (TC). All
media were supplemented with 1 % penicillin/streptomycin.</div><div>2.6 Cytocompatibility</div><div>Cytocompatibility of the cdECM substrates was shown by the analysis of
lactate dehydrogenase (LDH) in the cell culture supernatant. At day 3
after seeding, an LDH assay (TaKaRa Bio Europe, France) was performed
according to the manufacturer’s instructions. To exclude the remaining
LDH from cell lysis, LDH concentration from supernatant from cdECM
substrates without mvECs was determined. Values were subtracted from the
LDH concentrations measured from mvECs on the different cdECM
substrates. On day 14, live-dead staining was performed to assess the
viability of cultured cells. Before staining the cells were washed in
phosphate-buffered saline (PBS, Biochrom, Germany) and subsequently
treated with staining solution, consisting of 200 ng/ml fluorescein
diacetate (FDA, Sigma Aldrich, Germany) and 20 µg/mL propidium iodide
(PI, Sigma Aldrich, Germany) in DMEM, for 15 min at 37 °C. Finally,
cells were imaged in PBS with calcium and magnesium at RT with an Axio
Observer microscope and an Axiocam 506 mono camera using the software
ZEN (Carl Zeiss, Germany).</div><div>2.7 Immunofluorescence staining of cell-specific proteins</div><div>For IF staining of cell-specific proteins, cells were fixed in 4 %
paraformaldehyde for 10 min and permeabilized for 10 min with 0.1 %
Triton X in PBS. Following, cells were incubated in blocking solution,
consisting of 3 % bovine serum albumin in</div><div>0.1 % Triton X for 30 min to block unspecific binding sites. Primary
antibodies (mouse anti-CD31, 1:50, Dako, Germany; rabbit anti-CD31,
1:200, abcam, GB; goat anti-E-selectin, 1:200, R&amp;D Systems, USA; sheep
anti-thrombomodulin, 1:200, R&amp;D Systems, USA) were diluted in blocking
solution and incubated for 2 h at RT. Secondary antibodies (anti-rabbit
Alexa FluorTM 488, abcam, GB; anti-mouse Cy3, Dianova, Germany; donkey
anti-sheep Alexa FluorTM 647, abcam, GB; donkey anti-goat Alexa FluorTM
594, abcam, GB) were diluted 1:250 incubated 30 min at RT. All
antibodies were diluted in blocking solution.</div><div>2.8 Enzyme-linked immunosorbent assay</div><div>For characterization of cdECM substrates regarding growth factors
composition, substrates were washed 3 days in culture medium. For the
characterization of FL, medium from day 3 was collected. Determination
of growth factors VEGF, bFGF and PDGFβ were performed using
enzyme-linked immunosorbent assays (ELISA) (all PEPROTech, Germany)
according to the manufacturer’s instructions. The converted TMB was read
out at 450 nm with a wavelength correction set at 620 nm (TECAN Saphire
II, Tecan, Switzerland)</div><div>2.9 Statistics</div><div>All experiments were repeatedly performed, using cells from at least
three different biological donors of ECs. The obtained data were
compared by a one-way analysis of variance (ANOVA) with repetitive
measurement and a Tukey post-hoc test using OriginPro 2018b. Statistical
significances were stated as *p ≤ 0.05, very significant as **p ≤ 0.01
and highly significant as ***p ≤ 0.001.</div><div>3. Results</div><div>3.1 Macroscopic pictures and degree of swelling</div><div>Macroscopic pictures show that wet scdECM and acdECM substrates exhibit
a transparent gel-like appearance on the bottom of the petri dish
(Figure 2). Determination of the degree of swelling of the different
cdECM substrates revealed a higher water uptake capacity of acdECM
(2357.6 (± 201.1) %) compared to the scdECM (1624.3 (± 96.4) %). IF
staining of fibronectin revealed smaller pores in the scdECM substrates
compared to the acdECM substrates. Images of the picro sirius staining
showed more densely packed collagen fibers in the scdECM substrate
compared to the acdECM substrate.</div><div>3.2 Acellular and cellular substrates are cytocompatible for mvECs</div><div>Cytocompatibility of the materials was determined by the measurement of
the release of LDH after seeding with mvECs. LDH is an enzyme that is
released during cell death and therefore can be used to quantify
cytotoxicity. LDH release of mvECs seeded onto the different substrates
was determined 3 days after seeding (Figure 3, A). The values of TC were
set as 100 (± 3.5) %. For cdECM substrates, values were normalized to
TC. Results showed no significant increase of released LDH of mvECs when
seeded on COL I coating (89.4 (± 13.8) %), dry scdECM (113.7 (± 31.0)
%); dry acdECM (108.0 (± 29.0) %), wet scdECM (96.3 (± 33.4) %), or
wet acdECM (93.4 (± 29.0) %). For the stem cell and adipogenic
differentiated FL substrates, values were normalized to stem cell FL
approach without mvECs (FL stem cell), which was set as 100 (± 3.3) %.
For adipogenic differentiated FL (FL adipogenic: 157.9 (± 13.4) %)
approach, a higher LDH release was found compared to stem cell FL. As in
the cdECM approaches, no significant increase in LDH release was
observed when mvECs were seeded onto the FL for stem cell and adipogenic
differentiated cells (FL stem +mvECs: 126.1 (± 15.8) %; FL ad +mvECs:
176.8 (± 25.0) %).</div><div>The viability of the mvECs cultured on the different substrates was
assessed on day 14 after seeding with mvECs by live-dead staining with
FDA and PI (Figure 3, B). Results show that mvECs were viable on all
acellular and FL substrates on day 14 and only a few dead cells could be
found.</div><div>3.3 Cell-derived ECM substrates support the formation of vascular-like
structures by mvECs</div><div>To investigate the effect of cdECM substrates on the formation of
vascular-like structures by mvECs, CD31 was visualized by IF staining
(Figure 4). CD31 is a specific endothelial surface protein mainly
localized on cell-cell connections and mainly responsible for the
control of leukocyte transmigration <i>in vivo</i> (Piali et al., 1995).
The staining pattern showed that mvECs grew to a confluent cell layer on
all acellular substrates. The degree of structure formation on the
different substrates was analyzed and quantified using ImageJ on basis
of the CD31 IF images. The formation of vascular-like structures by
mvECs was detected on all tested substrates in contrast to the controls
(TC and COL I) on which no structure formation was observed.
Quantification of structure lengths revealed longer structures for wet
acdECM (433.5 (± 293.1) µm) substrate compared to both scdECM substrates
(dry: 235.9 (± 100.0) µm; wet: 232.9 (± 183.8) µm). Dry acdECM (297.2 (±
149.1) µm) substrates exhibited a slightly but not significantly higher
structure length compared to dry and wet scdECM substrates. Vascular
structure lengths of mvECs cultured on the adipogenic FL (FL adipogenic:
483.5 (± 287.4) µm) were significantly longer than those of all other
approaches except for wet acdECM substrate. Vascular structures on stem
cell FL exhibited an average length per structure of 302.1 (± 168.7) µm.
Another essential criterion for the maturation of a functional vascular
network is the formation of nodes. Therefore, the number of nodes formed
by the mvECs on the different substrates was quantified. No nodes could
be detected on the controls TC and COL I. In adipogenic approaches (dry
acdECM: 1.2 (± 1.1); wet acdECM: 1.7 (±1.2); FL adipogenic: 6.7 (± 3.9))
the number of nodes was higher compared to the stem cell approaches (dry
scdECM: 0.7 (± 0.4); wet scdECM: 0.2 (± 0.6); FL stem cell: 1.7 (± 1.5))
for all substrates. Furthermore, the number of nodes on wet acdECM was
slightly but not significantly higher compared to the dry acdECM and
comparable to stem cell FL. The significantly highest number of nodes
could be observed in the adipogenic FL approach. In sum, on scdECM
substrates, many short structures were identified whereas on acdECM
substrates longer and more branched structures were formed. By
co-culture with the stem cell FL, the mvECs formed islets of a confluent
layer within the ASCs and vascular-like structures sprouting from these
islets were apparent. Long and highly branched vascular-like structures
were formed by mvECs on adipogenic FL.</div><div>3.4 Quantification of pro-angiogenic factors on substrates</div><div>To confine which ECM components are responsible for its pro-angiogenic
effect, the relative concentration of growth factors VEGF, bFGF and
PDGFβ were determined in the supernatant after washing the acellular
substrates for 3 days (Figure 5). Values were normalized to TC.
Determination of VEGF revealed significantly higher concentrations for
dry and wet acdECM substrates compared to all other acellular substrates
(TC: 1 (± 0.2)fold; COL I: 0.9 (± 0.2)fold; dry scdECM: 1 (± 0.1)fold;
dry acdECM: 1.4 (± 0.1)fold; wet scdECM: 1.1 (± 0.1)fold; wet acdECM:
1.5 (± 0.2)fold). For quantification of growth factor concentration on
FL approaches cell culture supernatant from day 3 (according to the 3
days of washing of acellular substrates) was collected. A 10-fold higher
concentration of VEGF can be found on FL approaches with no difference
between stem cell FL and adipogenic FL (FL stem cell: 10.0 (± 0.3)fold;
FL adipogenic: 10.0 (± 0.2)fold). For bFGF significantly higher
concentrations can be found on cdECM substrates compared to controls
(TC: 1 (±0.1)fold; COL I: 1.1 (±0.3)fold; dry scdECM: 2.6 (±0.2)fold;
dry acdECM: 2.2 (±0.2)fold; wet scdECM: 2.4 (±0.1)fold; wet acdECM: 2.5
(±0.2)fold) and on FL approaches higher concentration can be found
compared to all other substrates (FL stem cell: 6.1 (±1.2)fold; FL
adipogenic: 7.0 (±0.7)fold). For PDGFβ a significantly higher
concentration can be found on cdECM substrates (dry scdECM: 3.6 (±
0.7)fold; dry acdECM: 3.5 (± 0.2)fold; wet scdECM: 3.5 (± 0.4)fold; wet
acdECM: 3.3 (± 0.3)fold) compared to the controls TC (1 (± 0.2)fold) and
COL I (0.9 (± 0.5)fold). Between the different cdECM substrates no
difference in PDGFβ concentration can be measured. On FL substrates, a
higher concentration of PDGFβ can be found compared to acellular
substrates but no difference between stem cell and adipogenic
differentiated approach (FL stem: 5.5 (± 0.3)fold; FL ad: 6.3 (±
1.0)fold) was observed.</div><div>3.5 Expression of proteins associated with tube formation in newly
formed vascular structures</div><div>Recent studies showed that the expression of adhesion molecules
E-selectin and thrombomodulin in ECs is associated with the tube
formation of new blood vessels (Oh et al., 2007; Pan et al., 2017).
Therefore, we investigated the expression of these proteins in the newly
formed vascular-like structures (Figure 6). Results of the IF staining
revealed the expression of neither E-selectin nor thrombomodulin in
mvECs cultured on TC or COL I. However, all newly formed vascular-like
structures showed expression of E- selectin and thrombomodulin on all
cdECM substrates. E- selectin and thrombomodulin expression of the
vascular-like structures were comparably high in the FL approaches.
E-selectin and thrombomodulin staining corresponded to the CD31 staining
pattern of the vascular-like structures (Supplementary figure 1).</div><div>4. Discussion</div><div>The implementation of a functional vascular system into an engineered
tissue construct would address one of the major bottlenecks in tissue
engineering and regenerative medicine. In the present study, we aimed to
investigate the supportive effect of cdECM on the self-assembled
formation of vascular-like structures by mvECs for its use as a
biomaterial for adipose tissue engineering compared to the
well-established application of a supportive FL.</div><div>Determination of the degree of swelling revealed higher capacity of
water uptake of the adipogenic ECM compared to stem cell ECM. This
effect can be explained by the higher pore size in adipogenic ECM shown
by fibronectin and collagen staining. The higher pore size of adipogenic
ECM may also be able to enhance the degree of vascular structure
formation by ECs. Chui et al. showed that higher pore size is associated
with a higher degree of neovascularization in an <i>in vitro</i> PEG
hydrogel model (Chiu et al., 2011). Furthermore, Artel et al. proposed
an agent-based model indicating that pores of larger size support
vascularization in a polymer scaffold (Artel, Mehdizadeh, Chiu, Brey, &amp;
Cinar, 2011).</div><div>Analysis of LDH release of mvECs on the substrates revealed no cytotoxic
effects of the cdECM substrates, the FL cells or the controls (COL I
coating and TC). Even on day 14 after seeding, a confluent viable
monolayer of mvECs was observed which indicates a good cytocompatibility
of the cdECM substrates and their possible use in tissue engineering.
For comprehensive toxicological and immunogenic characterization further
analysis is required, e.g. the analysis of the ECM impact on the
metabolic activity of the mvECs and for the intended <i>in vivo</i> use,
biocompatibility of the ECM has to be evaluated.</div><div>Visualization of mvECs on day 14 after seeding by staining of the
specific surface protein CD31 showed the self-assembled formation of
vascular-like structures on all substrates except for the controls COL I
and TC. Structure formation on the adipogenic FL approach was in line
with our previous study 9 as a lower degree of structure formation was
found on the stem cell FL approach. In addition, on dry and wet cdECM
approaches, the degree of structure formation on adipogenic ECM
substrates was higher compared to the corresponding stem cell approach,
which is reflected by longer structures and a higher number of nodes.
The effect of enhanced structure formation on adipogenic substrates
could be explained by the different secretomes of ASCs and
(pre-)adipocytes (Kapur &amp; Katz, 2013). It is well known that ASCs
secrete a broad spectrum of pro-angiogenic proteins and they were often
used as a delivery system of growth factors and cytokines in
vascularization approaches (Kondo et al., 2009; Liu et al., 2011; Moon
et al., 2006; Nakagami, Maeda, Kaneda, Ogihara, &amp; Morishita, 2005;
Rehman et al., 2004). For example, Matusda et al. showed that
conditioned cell culture medium of ASCs positively influenced EC
proliferation and the formation of new vessels <i>in vivo</i> (Matsuda
et al., 2013). During adipogenic differentiation, ASCs secret further
pro-angiogenic factors like leptin. Leptin is known to be upregulated
during adipogenic differentiation and was shown to exhibit a
pro-angiogenic effect itself but also upregulates the secretion of VEGF
(Cao, Brakenhielm, Wahlestedt, Thyberg, &amp; Cao, 2001). By secreting
their specific set of proteins, ASCs and (pre-)adipocytes not only
condition their cell culture medium but also their ECM which we use in
this study as a biomaterial for induction of vascular formation by
mvECs. Thus, cdECM does not only contain a set of specific factors, but
a broad spectrum pro-angiogenic factors with its synergistic effects
needed for the successful formation of vascular structures by ECs.
Especially acdECM induces the formation of vascular-like structures and
seems to be able to stabilize the newly formed structures.</div><div>The two most important pro-angiogenic factors are VEGF and bFGF. Results
revealed higher VEGF concentrations released from acdECM substrates
compared to scdECM approaches. On FL approaches high amounts of VEGF
were found, most likely produced by FL cells. These results are in line
with the degree of vascular-like structure formation. On acdECM
approaches, longer and more branched structures were formed whereas on
FL approaches the highest degree of structure formation occurred.
Determination of the bFGF concentration in the different substrates
revealed a higher concentration from cdECM substrates compared to
controls and the highest bFGF concentration from FL approaches. Both
factors – VEGF and bFGF – are able to induce the formation of new
vascular structures (Marra et al., 2008; Murakami &amp; Simons, 2008;
Nissen et al., 2007; Tomanek, Hansen, &amp; Christensen, 2008). Therefore,
in our study, the induction of the formation of vascular-like structures
may among other events, be attributed to the synergistic effect of
available VEGF and bFGF. We further investigated the amount of
pro-angiogenic factor PDGFβ from cdECM substrates. It is secreted by ECs
during angiogenesis to attract perivascular cells, which stabilize the
newly formed vessels 6. Further, PDGFβ was shown to induce vascular
structure formation by modulating proliferation and tube formation of
ECs (Battegay, Rupp, Iruela-Arispe, Sage, &amp; Pech, 1994). PDGFβ can be
found in all cdECM substrates as well as FL approaches. The PDGFβ
concentration from FL approaches is higher compared to the other
substrates which is in line with the higher degree of structure
formation. <i>In vivo</i> , these growth factors are known to be
partially bound to ECM after their secretion (Ostman, Andersson,
Betsholtz, Westermark, &amp; Heldin, 1991). To date, there are no studies
investigating their binding capacity and protein half-life in <i>in
vitro</i> generated cdECM.</div><div>A critical step in the formation of a new vascular system is the
formation of a lumen in the vascular structure to enable perfusion with
blood i<i>n vivo</i> and culture medium <i>in vitro</i> . Recent studies
show that the adhesion proteins E-selectin and thrombomodulin are
associated with tube formation. <i>In vivo</i> , E- selectin is mainly
contributing to the binding of immune cells by mediating adhesive
interactions of circulating leukocytes with the endothelium (Ley &amp;
Tedder, 1995). Nevertheless, it also plays a role in the homing of
endothelial progenitor cells (EPCs) and therefore promotes
neovascularization. Studies showed that E-selectin potentiates
angiogenesis in ischaemic tissue, by mediating EPC- endothelial
interactions (Oh et al., 2007). During this process of
neovascularization, EPCs are mobilized from the bone marrow into the
circulation and recruited to new sites of vascularization, using cues
that resemble an inflammatory response. Therefore, E-selectin plays a
crucial role in EPC homing and following neovascularization and tube
formation. In this study, we use this protein as an indicator for a
functional vascular structure. IF staining of E-selectin revealed
specific expression of E- selectin almost exclusively on the newly
formed vascular-like structures. Thrombomodulin is a transmembrane
molecule expressed on ECs acting as an anticoagulant (Dahlback &amp;
Villoutreix, 2005; Dittman &amp; Majerus, 1990). The fourth and fifth
region of an epidermal growth factor (EGF)-like region of thrombomodulin
(TME45) was shown to stimulate proliferation of human umbilical vein ECs
and to promote tube formation and angiogenesis (Ikezoe et al., 2017).
Therefore, we used this protein as an indicator for functionality of the
newly formed vascular-like structures. In line with the results obtained
by E-selectin staining, thrombomodulin was found to be expressed mainly
in the mvECs contributing to the structure formation. Thus, we suggest
that newly formed vascular-like structures exhibit promising
characteristics to develop a functional vascular system. The expression
of E-selectin and thrombomodulin and their function in tube formation
and neovascularization <i>in vivo</i> represent promising
characteristics when considering implantation of prevascularized
constructs.</div><div>The chemical composition and physical properties of the ECM are highly
tissue specific. Different components of the ECM, like collagens,
fibronectin and laminin, were investigated towards their ability to
mimic the natural ECM (Huber et al., 2016; Lv, Bu, Kayser, Bausch, &amp;
Li, 2013; Rammelt et al., 2006). However, the highly complex composition
of natural ECM could not be copied by a combination of the different
components. Furthermore, ECM is biocompatible and can be remodeled by
the residing cells (Badylak, 2007). In addition to the biological
activity, like the induction of ASC differentiation (Guneta et al.,
2017) and vascular-like structure formation by ECs shown in this study,
there are several further advantages of cdECM. The amount of autologous
ECM is limited to the donated tissue and allogenic ECM suffers from
donor variability. Furthermore, donors vary in age, gender, BMI and
different donor sites may also influence the chemical and physical
properties which might impact the cell-ECM interaction. In contrast, the
production of cdECM is scalable, as producing cells can be expanded in
advance. Native ECM is usually derived from adult tissue state. AcdECM
however, could be obtained from the developmental state of the tissue
specific cells and therefore may provide more supportive signals for
tissue development and regeneration. These signals can promote the
differentiation of stem cells and support the formation of vascular
structures by ECs. Also scdECM provides the ability to induce
vascular-like structure formation by ECs and can be used for approaches
addressing other tissues. For example evaluating its usability in
vascularized tissue engineering of tissues producing low amounts of ECM
by itself, e.g. liver, would be promising.</div><div>One critical point in the commercial application of biomaterials is the
maintenance of their biological impact after processing and storage. The
most common processing for preserving biomaterials is drying. In this
study, it could be shown that dry cdECM partly maintains its biological
properties regarding the induction of the self-assembled vascular-like
structure formation of ECs with some restrictions. However, drying of
the cdECM would be a conceivable method for improving storage
possibilities when necessary.</div><div>5. Conclusion</div><div>In the present study, we demonstrated that cdECM (as a dry coating and
as a wet hydrogel-like form) is able to induce the self-assembled
formation of vascular-like structures by mvECs and helps to support
their maintenance. Thereby, this study revealed a promising application
of acdECM as a biomaterial in adipose tissue engineering approaches. The
present study clearly confirms acdECM as a promising material for
vascularized adipose tissue engineering by supporting the formation of
vascular structures. In addition, also scdECM provides the ability to
induce vascular-like structure formation and can be used for approaches
addressing other tissues. Further investigations regarding other
lineage-specific cdECMs and the transfer from 2D cell culture to 3D cell
culture should be pursued. Thereby the combination with other materials,
allowing the direct adjustment of geometry and relevant physical
parameters should be addressed.</div><div>Acknowledgements</div><div>This study was financially supported by the Landesgraduiertenförderung
by the Ministry of Science, Research and the Arts (Baden-Württemberg,
Germany) under the program “Intelligent Process and Material
Development in Biomateriomics” (University of Tuebingen and Reutlingen
University).</div><div>Conflicts of interest</div><div>The authors declare no conflict of interest.</div><div>References</div><div>Adam Young, D., Bajaj, V., &amp; Christman, K. L. (2014). Award winner for
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