## 4 Discussion  
Previous experiments primarily focused on the VEGF amount added to the culture environment, lacking the discussion on the two states of VEGF [7; 34]. Less attention was paid to telling the different effects of soluble and bound VEGF. The hypothesis that the formation of capillary-like networks is only regulated by the bound VEGF cannot fit all experimental data [35]. In theoretical modeling, on the other hand, capillary-like networks can be formed by using both soluble-VEGF-based or bound-VEGF-based mathematical models, and a consensus is still absent [36; 37; 38]. Moreover, although many in vitro capillaries culturing experiments have been successful, problems remained in the in vivo trials. Once the artificial scaffold and tissue are transplanted into the human body, the VEGF binding  
Figure 5: HUVECs distribution with and without VBP addition. In the -P case, which means without the VBP addition, HUVECs showed the change in morphology at 12 h with some cells remaining in the cobblestone shape and subsequently formed the capillary-like networks at 18 h. In the +P case, which means with the VBP addition, most HUVECs showed the morphological change at 12 h; however, the network could not form afterward. Figure 6: Junction number, segment number, and total segment length of the capillary-like network. The higher value of these three indicators, the more integrity of the network. All experiments were repeated five times. Data are presented as mean SD. The Student’s t-test was performed for statistical evaluation: * = \(p\) < 0.05, ** = \(p\) < 0.01, and ns = non-significant. The asterisk above the -P+V charts presents the \(p\)-value between the \(-\)P+V and +P+V cases at the same observation time. to the scaffold will gradually be consumed or decomposed over time. How to regularly provide an appropriate amount of VEGF becomes a difficult challenge [39; 40]. A high initial dosage strategy is not adopted because the excessively high VEGF concentration will cause excessive proliferation of capillaries and potential carcinogenic risks [41]. Transplanting biomaterials, such as fibronectin, that can initially capture and gradually release VEGF, seems feasible, while it is essential to precisely predict the dynamic balance between soluble and bound VEGF [12; 33]. Previous experiments showed that parts of the cells with morphological change to elongated shape are connected end-to-end to form the cords in the capillary-like network, while those without morphological change aggregate and create a cobblestone arrangement [34; 42]. The same phenomena were found in our results (+F+V in Figure 2 and -P in Figure 5). Because the animal-extracted materials such as Matrigel and ECGS used in literature are originally rich in VEGF, previous studies lacked the discussion on the respective effects of soluble and bound form. We filled this gap by building a VEGF-free environment, which allowed us to explore the VEGF effects. We chose the type I collagen as substrate ingredient, which had been proven beneficial for HUVECs survival and biogel remodeling [7; 10], and treated with the thin fibronectin layer coated on collagen surface, which provided VEGF-binding areas, and further enhanced HUVECs attachment and morphology [12; 32]. By comparing the cases with and without adding VEGF, we discussed the different roles soluble and bound VEGF played in forming capillary-like networks. The exogenous VEGF tests on HUVECs showed that more cells underwent a morphological change in the \(-\)F+V condition within 12 h of incubation than the standard +F+V case. Since in the \(-\)F+V case there was virtually no VEGF binding to fibronectin, the added VEGF remains in the soluble form; the results indicated a higher concentration of soluble VEGF induced the cell morphological change earlier. The primary response of endothelial cells to the stimulation of soluble VEGF was the shape change from round to elongated shape.