## 3 Results and Discussion
### Biological Investigation
#### 3.4.2 Scratch Test  
As mentioned, the in vitro scratch test is a cheap, easy, and well-developed method to measure cell migration in vitro [62; 63]. The in vitro scratch test is useful to evaluate the migration of cells after the creation of a gap on confluent monolayer. Such test mimics cell migration _in vivo_ and it is advantageous to assess through cell-cell interaction and cell interaction with ECM the regulation of cell migration [91]. Additionally, the scratch test uses inexpensive and common supplies found in most laboratories of cell culturing, thus, it is the simplest method to analyze the migration rate of cells in vitro.  
To assess the effect PCL and PCL/bioactive glass composite electrospun mats on NIH 3T3 cells, the closure of the gap in monolayers of mouse embryonic fibroblast was investigated. The scratches in monolayers of cells were created by the tip of a 200-uL pipette and the closure of such scratches was measured at 24 h and 48 h. At 24 h, a significant closure in terms of wound-healing rate (%) (measured as cell migration rate) was registered for PCL_BG10 and PCL_BGZn, compared to neat PCL and PCL_45S5.  
At 48 h, the cell migration rate (%) of PCL/bioactive glass composite electrospun mats was higher compared to that measured for neat PCL (Figure 9). 45S5, BGMS10, and BGMS_2 Zn influenced positively the closure of the scratch on the confluent mouse embryonic fibroblast monolayer. The reason could be ascribed to ions released by different BGs to stimulate specific signaling pathways promoting cellular response [92]. For instance, 45S5 release Ca and Si ions that contribute to the formation of HCA and are involved in specific signaling pathways [93; 94]. It has been reported that Ca ions are involved in the regulation of Golgi, mitochondrial shuttling, and endosomal and vesicular fusion events etc., [95]; thus, Ca ions influence every aspect of cell life and death [96]. Additionally, Si ions showed a strong stimulatory effect on ALP activity [31]. Furthermore, Sr ions in BGMS10 and BGMS_2 Zn have been shown to significantly enhance the expression of ang-1 and VEGF compared to Si ions [31]. Additionally, PCL_BGZn composite mats contain Zn ions that have been shown to stimulate protein synthesis and to enhance ALP activity as presented in [40]. Free intracellular Zn level influences signaling pathway and stability [97] by altering the affinity between ligand binding to receptor [98]. The intracellular zinc has shown to activate MAPK/ERK and Akt pathways which improve cell proliferation and migration [99]. Furthermore, the release of Zn into the cytoplasm seems to be ROS-induced. Key immune functions are activated by both the increase of ROS level and the Zn signal [100]. Therefore, the high wound-healing rate (%) of PCL/bioactive glass composite electrospun mats (Figure 9) could be attributed to the release of ions which have been shown to improve cell adhesion and proliferation by stimulating specific cellular responses.  
Figure 9: Wound-healing rate % (measured as cell migration rate %) 24 h and 48 h after the introduction of the “scratch” in confluent monolayers of NIH 3T3 cells in contact with eluates of PCL, PCL_45S5, PCL_BG10, and PCL_BGZn composite electrospun mats. **_p_\(<\) 0.05.