Figure 2. VBP Microspheres Sequester VEGF from PC Prepared via Freeze/Thaw.

A: Schematic of platelet activation and subsequent VEGF sequestering to VBP microspheres incubated in PC. B: Multiplexed bead-based ELISA was performed to assess the abundance of 12 pro- angiogenic growth factors in the supernatant after incubation of microspheres with PC. Data was compared to standard curve generated using recombinant growth factors in PBS, and the abundance of each growth factor in supernatant was calculated from the standard curve using 4- PL analysis in GraphPad Prism. ND = Not Detected. Statistical comparisons were made using one-way ANOVA with Fisher’s least significant difference post-hoc test. Statistical significance is denoted for p-value < 0.05 (*) between the conditions indicated in brackets. C: Quantification of VEGF sequestering to VBP microspheres (in % bound VEGF) calculated by subtracting the concentration of VEGF in the no microsphere control (NS) by the concentration of VEGF in each respective microsphere condition and dividing by the total amount of VEGF in the NS control. Statistical comparisons were made using one-way ANOVA and Bonferroni post-hoc test and is denoted for p-value<0.05 relative to Scramble (**) and Blank (*) microspheres. D: VEGF sequestering to dimeric VBP, VBP₂, and dimeric Scramble,_, Scr₂, at varying peptide concentration (presented as % peptide per norbornene group during microsphere synthesis with PEG-NB). % Bound VEGF was calculated as described above. Statistical analysis was performed using two-way ANOVA (peptide identity, peptide concentration, and interaction p-value < 0.05) with post-hoc Bonferroni test denoted for p-value < 0.05 with an asterisk (*) comparing VBP₂ and Scr₂ at each peptide concentration.