Figure legends
Figure 1: Schematic overview of the study procedure. The
pro-angiogenic potential of different acellular and cellular substrates
was analyzed. ASCs were cultured in growth and adipogenic
differentiation medium respectively for 7 days. For acellular ECM
substrates (cdECM), ASCs were removed and the remaining ECM was dried or
stored under wet conditions. For cellular substrates (FL), ASCs were not
removed. MvECs were seeded onto the different substrates.
Cytocompatibility was determined at day 3 and structure formation was
determined at day 14 of cell culture.
Figure 2: Degree of swelling of ECM substrates and staining of
fibronectin and collagen. A: Macroscopic pictures of cdECM substrates
show a transparent gel-like ECM on the bottom of the petri dish. The
degree of swelling was calculated in percent. Results reveal a higher
swelling rate of acdECM compared to the scdECM (Diameter of petri dish
is 35 mm). B: Fixed cdECM samples were stained for fibronectin and
histological sections of the cdECM samples were stained for collagen
fibers using picro sirius staining. Fibronectin staining revealed
smaller pores in the scdECM substrate compared to the acdECM substrate
(Fibronectin indicated in red; scale bar: 200 µm). Picro sirius staining
showed denser packed collagen fibers (red staining) in the scdECM
compared to the acdECM (Scale bar: 100 µm)
Figure 3: Biocompatibility of the acellular and feeder layer
substrates . 1 x 104 cells/cm2 mvECs were seeded in a defined medium
onto the different substrates. A: Relative LDH release was measured at
day 3 after seeding with mvECs. For acellular substrates values were
normalized to TC. No significant increase in LDH release can be observed
on COL I coating or dry and wet cdECM for both, scdECM and acdECM. For
FL approaches, values were normalized to stem cell FL without mvECs (FL
stem cell). None of the FL approaches (stem cell and adipogenic
differentiated) exhibit a significant increase of released LDH after
seeding of mvECs (FL +mvECs), (n.s. = not significant; ** p ≤ 0.01 ) B:
Live-dead staining (FDA, indicating alive cells displayed in green/PI,
indicating dead cells, displayed in red) was performed at day 14 after
seeding with mvECs. A confluent layer of viable cells was observed in
all approaches. Scale bar represents 200 µm.
Figure 4: Formation of vascular-like structures by mvECs on
cellular and acellular ECM substrates . 1 x 104 mvECs /cm2 were seeded
in defined co-culture medium onto the different substrates and were
cultured for 14 days. Medium was changed three times a week. For
determination of newly formed vascular- like structures, IF staining of
CD31 (indicated in red) was performed at day 14 after seeding with
mvECs. On controls (TC and COL I) a confluent layer of mvECs could be
observed without any structure formation. On cdECM substrates, formation
of vascular-like structures could be observed with the strongest
manifestation on wet acdECM. The highest degree of structure formation
could be observed on adipogenic FL. On stem cell FL cluster formation of
mvECs can be found and a considerably lower degree of structure
formation compared to the adipogenic approach was detected. Length per
structure and number of formed nodes was quantified using ImageJ.
Analysis revealed a significantly higher structure length of mvECs on
wet acdECM substrate compared to dry acdECM and stem cell approaches
(dry and wet scdECM) and comparable to FL approaches. Structure length
on adipogenic FL was significantly higher compared to all approaches
except wet acdECM. On adipogenic FL, a significantly higher number of
nodes can be observed compared to all other approaches. (Scale bar: 200
µm; * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001)
Figure 5: Pro-angiogenic factors concentration on cellular and
acellular ECM substrates . Relative concentration from released
acellular and cellular substrates. For the determination of VEGF, bFGF
and PDGFβ from substrates, supernatant from day 3 was investigated
regarding the concentration of the growth factors using ELISA. For
statistical analysis, values on TC were set as 1 and data were
normalized to TC. For VEGF a significantly higher amount can be found in
acdECM substrates (dry and wet) compared to all other acellular
substrates including controls. FL substrates exhibit a 10-fold higher
concentration of VEGF compared to acellular substrates. For bFGF a
higher concentration can be found in cdECM substrates compared to
controls. On FL approaches, a higher concentration can be found compared
to all other approaches. For PDGFβ a significantly higher (3-fold)
amount can be found in all cdECM substrates compared to the controls TC
and COL I. Between the individual cdECM substrates no difference in
remaining PDGFβ can be found. FL substrates exhibit significantly higher
PDGFβ concentrations compared to acellular substrates. (* p ≤ 0.05; ** p
≤ 0.01; *** p ≤ 0.001).
Figure 6: Expression of E-selectin and thrombomodulin by mvECs
forming vascular-like structures. 1 x 104 mvECs /cm2 were seeded in
defined co-culture medium onto the different substrates and were
cultured for 14 days. Medium was changed three times a week. For both
proteins no specific staining was observed on controls (TC and COL I
coating). For all acellular and cellular substrates, specific staining
of E-selectin and thrombomodulin (both indicated in red) can be found
mainly on the newly formed vascular-like structures. (Scale bar: 200 µm)
Supplementary Figure 1: Expression of CD31 by mvECs. CD31
staining of samples stained for E-selectin. and thrombomodulin. Both
corresponding to Figure 6. (Scale bar: 200µm; green: CD31, white:
Nuclei)