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)