## 3 Results
### Effects of Soluble and Bound VEGF  
Comparison between the -F+V and +F+V cases, as shown in Figure 2, reveals that the endothelial cells begin morphological changes earlier; however, a cell network could not form afterward if the biogel had no fibronectin. In contrast, although the cell commenced morphological change somewhat later in +F+V, the cells continued to migrate and aggregate into a cellular network. This result indicated that soluble and bound VEGF might have different effects on cell behavior. However, in addition to binding VEGF, fibronectin could bind cells through its integrin-binding domain. To clarify whether it was the cell-binding or VEGF-binding to the fibronectin responsible for the different cell behaviors, we conducted a series of experiments using the synthetic VEGF-blocking-peptide (VBP). The sequences of VBP were designed according to the fibronectin-binding domain of VEGF (exon-7). This peptide was added to the fibronectin-coating wells before adding the HUVEC suspension to shield the growth factor-binding domain of fibronectin. Because the bound VEGF was prevented from forming, the added VEGF would remain in the soluble form. The VPB shielding effect was assessed using fluorescence immunoassay, where a FITC-conjugated anti-VEGF antibody was used to monitor the VEGF. Results are as shown in Figure 3 for four different cell culture conditions: without VBP (-P), with VBP (+P), without VEGF (-V), and with VEGF (+V). The darker fluorescence response in the case of +P+V than in -P+V indicated that VBP could avoid VEGF from biding fibronectin. The two subsets of -P-V and +P-V showed that even in the cases with no adding VEGF in the culture medium, the fluorescence response could still be detected, albeit the responses were weaker than the counterpart cases of -P+V and +P+V, respectively. The results indicated that HUVECS could secrete VEGF. This autocrine signaling explained why HUVECs presented a morphological change in the cases without adding VEGF from outside. However, because the cells experienced rapid cell death in the two cases without VEGF added, adding VEGF was essential to cell viability to prevent the cell un-attachment and cell death during the network developing process. We further tested the VBP effect on cells by changing dose usage. The number of junctions quantitatively indicated the integrity and complexity of the capillary-like network. The results are displayed in Figure 4, which shows HUVECs could differentiate into a network and established an average of 140 junctions per view f  
Figure 3: Bound VEGF location after 6 h of incubation in different cell culture conditions: without VBP (\(-\)P), with VBP (+P), without VEGF (\(-\)V), and with VEGF (+V). A fluorescent signal from the primary antibody against VEGF was detected on the cell membrane and biogel surface. The VBP shielding effect avoided VEGF from binding biogel resulting in the darker VEGF field in both the +P\(-\)V and +P+V cases. or adding a small amount of VBP. The junction number dramatically decreased with the peptide concentration larger than 0.0125 ng/\(\mu\)L, but the cells kept attaching to the biogel. The results showed that the usage of VBP interfered with the regulating effect of VEGF and affected the integrity of network structure but not the cell adhesion. Based on the optimum culturing environment (+F+V), as shown in Figure 2, we tested on HUVECs the stimulating effect of soluble and bound VEGF, respectively, by adding 400 \(\mu\)L DPBS per well with 0.125 ng/\(\mu\)L dissolved VBP. The cell culture images are shown in Figure 5. In the -P situation, i.e., without VBP addition, HUVECs showed the change in morphology and subsequently formed the capillary-like network. In the +P situation, i.e., with VBP addition, HUVECs could not ultimately create the cell network, albeit the cells presenting morphological change earlier than the -P case.