Fig 2: Stability of FGF-2 aqueous solutions (770 ng/mL) upon lyophilisation and storage. FGF-2 in solutions F1 (water alone), F5 (MC 0.05% w/v and alanine 20 mM) and F6 (MC 0.05% w/v and HSA 1 mg/mL) were lyophilised over 24 h then stored at -4 °C (A), 4 °C (B) or 18 °C (C) for up to 12 months. At defined storage time points, the lyophilised powders were reconstituted with water and the FGF-2 content as determined by ELISA is expressed as a percentage of the baseline FGF-2 content determined in the FGF-2 solutions prior to lyophilisation (mean ± SD, n = 3).

Fig 3: Effect of excipients on the stability of reconstituted FGF-2 aqueous solutions (770 ng/mL) over a 7-day period. FGF-2 in solutions F5 (MC 0.05% w/v and alanine 20 mM) and F6 (MC 0.05% w/v and human serum albumin 1 mg/mL) were lyophilised over 24 h and the dry powders stored at -4 °C (A,B), 4 °C (C,D) or 18 °C (E,F) for up to 12 months. At the defined storage time points, the FGF-2 powders were reconstituted with water, and the resultant solutions were stored for 7 days at 4 °C (A,C,E) or 7 days at 18 °C (B,D,F). The FGF-2 content in the stored solutions was determined by ELISA and expressed as a percentage of the baseline FGF-2 content determined in the FGF-2 solutions prior to lyophilisation (mean ± SD, n = 3).

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.

Fig 5: Stability of aqueous FGF-2 solution to different temperatures. FGF-2 solutions (315 pg/mL) reconstituted from the commercial lyophilised powder were incubated at 4 °C, 25 °C and 37 °C for up to 48 h. Residual FGF-2 content measured using an ELISA kit is expressed as a percentage of baseline FGF-2 content (mean ± SD, n = 4).