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Figures
- Fig. 1.
Alginate:collagen hydrogels support controlled release of therapeutic factors and cell viability in vitro. (A) Bright field micrographs of 2:1, 1:1 or 1:2 mixtures of alginate:collagen hydrogel patches incubated in cell growth medium at 37°C for 1-7 days. Black arrows indicate intact hydrogel pieces. Red arrowheads indicate pieces of degraded hydrogel material, black boxes indicate the region shown in the enlarge image below the overview image. Scale bars: 1000 µm in the first two rows and 500 µm in the third row. (B) Quantification of the mean cumulative degradation (density of degraded hydrogel particles) of the hydrogel patches after 1, 3, 5 and 7 days of incubation. 2:1, 1:1 and 1:2 indicate the relative concentrations of alginate to collagen. Error bars indicate s.e.m. ***P<0.001. n=7. (C) Quantification of the mean absorbance of dyes labeling living versus dead K1000 cells following overnight incubation embedded in 1:1 alginate:collagen hydrogel patches. n=12. (D) ELISA quantification of free hFGF2 in the medium of 1:1 alginate:collagen hydrogel patches containing 106 K1000 cells and incubated for 1-5 days in DMEM growth medium at 37°C. Error bars indicate s.e.m. n=4. (E) Bright field micrographs of 1:1 alginate:collagen hydrogel patches made with PBS or 1000 ng/ml FGF2, 6 days after 106 PAECs were seeded onto their surface and incubated in DMEM growth medium at 37°C. Black arrows point to PAEC tube-like structures. Scale bar: 100 µm. (F) Quantification of the length of tubes as indicated in C. ***P<0.001, n=8. (G) Fluorescent micrographs of DiI-labeled PAECs grown for 2-5 days in DMEM at 37°C on 1:1 alginate:collagen hydrogel patches made with PBS or 1000 ng/ml FGF2. Scale bar: 100 µm. (H) Quantification of the area of the red colonies shown in E. ***P<0.001, n=115, 124, 309, 295 colonies were counted from six images in the 2 days PBS, 2 days FGF-2, 5 days PBS and 5 days FGF-2 groups, respectively.
- Fig. 2.
Synthesis of alginate:collagen microspheres. (A) Schematic representation of the set-up for synthesis of alginate:collagen hydrogel microspheres. (B) Quantification of and correlation between the number of cells encapsulated and diameter of the microspheres synthesized using the 1:1 alginate:collagen polymer mix under various parameters as indicated in Table 1. Black triangles represent the addition of 1.5×106 cells to the 10 ml polymer solution, black circles represent the addition of 3.0×106 cells to the 10 ml synthesis polymer solution. Each value represents the mean of at least 20 individual microspheres from a separate synthesis experiment. (C) Quantification of the mean diameter of spheres synthesized using the 1:1 alginate:collagen polymer mix and different air flow-rates as indicated in Table 1. Each value represents the mean of at least 20 individual microspheres from a separate synthesis experiment. (D) Quantification of the mean diameter of spheres synthesized using the 1:1 alginate:collagen polymer mix and different polymer flow-rates as indicated in Table 1. Each value represents the mean of at least 20 individual microspheres from a separate synthesis experiment. n.s., not significant. (E) Bright field micrographs of spheres containing cells synthesized using air flow-rates of 5 l/min (left) or 10 l/min (right), polymer flow rates of 0.25 l/min (first and third row) or 0.75 l/min (second and fourth row) or using 1.5×106 cells (upper four images) or 3.0×106 cells (lower four images). Scale bars: 50 µm. Dark dots inside the microspheres are indicative of cells.
- Fig. 3.
Release of therapeutic cells or factors from alginate:collagen microspheres. (A) Bright field and fluorescent micrographs of 1:1 alginate:collagen spheres containing DiI-labeled PAECs (red in the image to the right) 1-6 days after synthesis incubated in DMEM growth medium. Red arrows indicate tunnels created from dissolution of collagen and movement of cells through the microsphere. Green arrows indicate DiI-positive PAECs and yellow arrows indicate DiI-negative PAECs. Black box in the middle image indicates the region enlarged in the image to the right. (B) Quantification of the proportion of dark tunnels as indicated with red arrows in A versus the whole microsphere area 6 days after synthesis. n=8. (C) TEM micrographs of closed (left image) or opened (middle and right-most images) microspheres immediately (left image), 24 h (middle image) or 48 h (right image) after synthesis and incubation at 37°C. Scale bars: 100 µm. (D) ELISA quantification of the amount of FGF2 released from microspheres produced with 1:1 alginate:collagen containing 1000 ng/ml FGF2 after incubation in DMEM growth medium for 1-7 days. ***P<0.001, n=4.
- Fig. 4.
In vivo tolerance of alginate:collagen microspheres in mice and therapeutic angiogenesis in zebrafish. (A) Photographs of mice (left and middle image) or excised hydrogel plugs (right image) 5 days after implantation of 105 1:1 alginate:collagen microspheres subcutaneously in 0.1 ml PBS. Scale bar: 500 µm. (B) Confocal micrographs of excised alginate:collagen plugs (left column) or adjacent muscle (middle column) or fat (left column), as shown in A, stained with antibodies against the pan-macrophage marker F4/80 (red, top row), the neutrophil marker Ly6G (green, middle row) or the merged images (bottom row). Scale bar: 50 µm. (C) Confocal micrographs of blood vessels (green) from fli1a:EGFP transgenic zebrafish embryos at 3 days post fertilization implanted with either PBS (left image) or 100 pg FGF-2 (left image) in the periviteline space 24 h prior. White dashed line indicates the sub-intestinal vessels. White arrows indicate FGF2-induced ectopic sprouts. The graph depicts the quantification of ectopic sprouts in PBS or FGF2-implanted zebrafish embryos shown in the images. ***P<0.001, n=12. Scale bar: 200 µm. (D) Confocal micrographs of blood vessels (green) from fli1a:EGFP transgenic zebrafish embryos at 5 days post fertilization implanted with either FGF2-non-producing 3T3-Ras cells (left image) or FGF2-producing K1000 cells (right image) in the periviteline space at 48 h post fertilization. White dashed line indicates the outline of the cell implants. White arrows indicate vessels that have grown into the cell implants. The graph depicts the quantification of vessel density in the cell implants from 3T3-Ras or K1000-baring zebrafish embryos shown in the images. *P<0.05, n=5. Scale bar: 200 µm. (E) Confocal micrographs of blood vessels (green) from fli1a:EGFP transgenic zebrafish embryos at 5 days post fertilization implanted with DiI-labeled PAECs (red) either in DMEM growth medium supplemented with vehicle (PBS, top row) or 100 pg/nl FGF2 (bottom row) in the periviteline space at 48 h post fertilization. White arrows indicate PAECs that have been incorporated into host blood vessels. White box in the images to the left indicate the region enlarged in the images to the right. The graph depicts the quantification of the number of DiI-labeled PAECs incorporated into host GFP-positive vessels in the PBS or FGF2 groups as shown in the images. *P<0.05, n=5. Scale bars: 200 µm, magnified images 50 µm.
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