**4.3 Adhesion ratio and cell morphology**

The adhesion activity of L929 cells on Fol-8Col/PLGA scaffold is shown in Figure 15. After 6 hours of cultivation, the adhesion ratio of L929 cells on Fol-8Col/PLGA achieved 62±2.3% while that on PLGA is 45±3.4%. This can be attributed to the effect of the Fol-8Col, which is a hydrophilic compared to PLGA molecule; where in the RGD sequences in Fol-8Col molecule has been proven effective at improving cell adhesion. [66] The result shows that the adhesion ratio (87.6±2.5%) of L929 cells on Fol-8Col/PLGA scaffold after 24 hours cultivation remains higher than that on neat PLGA (68±1.8%).

**4.2 Cytotoxicity assay and live/dead cell staining**

118 Advances in Nanofibers

The cytocompatibility test of murine fibroblast L929 on Fol-8Col/PLGA emulsion electrospun fibrous scaffold was performed to investigate its potential for tissue engineering application. Here we chose the fibrous scaffold prepared from the electrospinning dope of Fol-8Col aqueous 5 wt.%, PLGA 10 wt.%, C/T 75:25. Collagen/PLGA fibrous mat (morphological data not shown) produced under the same electrospinning condition were tested as control. Figure 14 shows that LDH release after 24 hours' cultivation on all the samples was approximately 10% percent without significant difference (p>0.05). From the live/dead fluorescence micro‐ graphs, the majority of live cells incubated on Fol-8Col/PLGA scaffold can be observed with stretching spindle shaped morphology. The cytotoxicity assays indicate that the L929 cells have

comparable viability on Fol-8Col/PLGA scaffold as that on Collagen/PLGA scaffold.

**Figure 14.** L929 cell LDH leakage results and fluorescence micrographs of Calcein AM/PI-stained L929 cells with live cells fluorescing green and dead cells fluorescing red after 24 h culture on the Fol-8Col/PLGA and collagen/PLGA as a

The adhesion activity of L929 cells on Fol-8Col/PLGA scaffold is shown in Figure 15. After 6 hours of cultivation, the adhesion ratio of L929 cells on Fol-8Col/PLGA achieved 62±2.3% while that on PLGA is 45±3.4%. This can be attributed to the effect of the Fol-8Col, which is a hydrophilic compared to PLGA molecule; where in the RGD sequences in Fol-8Col molecule has been proven effective at improving cell adhesion. [66] The result shows that the adhesion

control. Fibers are electrospun at 5 wt% protein aqueous content, C/T 75/25, and 10wt% PLGA.

**4.3 Adhesion ratio and cell morphology**

The immunocytofluorescence staining for nuclei, vinculin, and filamentous actin of L929 cell after 24 hours adhesion shows that L929 cells on both scaffolds exhibited obvious stretching spindle morphology. The filament bundles (green stain) are oriented in a parallel direction following the main cellular axis. A comparatively intensive vinculin signals (red) was inves‐ tigated on the higher magnificent (630x) image of cells on Fol-8Col/PLGA scaffolds compared to that on PLGA. Our previous results have proved that the biocompatibility of Fol-8Col matrix to L929 cell line is higher than that of the native collagen. [67] The immunocytofluorescence staining results on Fol-8Col/PLGA scaffold in this section is consistent with the adhesion ratio analysis, showing that good cytocompatibility has been preserved after the emulsion electro‐ spinning process.

**Figure 15.** Adhesion ratio and fluorescent staining of F-actin (green), vinculin (red), and cell nuclei (blue) for L929 cells after 24 h culture on a Fol-8Col/PLGA fibrous scaffold (electrospun from 5 wt% Fol-8Col aqueous content, C/T 75/25, and 10wt% PLGA). A neat PLGA fibrous scaffold was used as a control.

Previous studies have addressed the issue of preserving the biomolecular activity through various methods. [68, 69] Our previous research on Fol-8Col has shown that it has excellent biocompatibility to L929 cell line. [4] The results in Figure 14 and 15, show that the cytocom‐ patibility of encapsulated Fol-8Col released from Fol-8Col/PLGA fibrous scaffold to L929 cells were preserved. The effectiveness of SPAN80 in the formation of W/O emulsion is thought to facilitate the production of core-sheath structure via the electrospinning process. The coresheath structure lessens the possibility of the exposure of Fol-8Col to the harsh organic solvents and thus protects Fol-8Col from activity loss. We assumed that the collagen derivate sequences, RGD sequences, and the triple-helix have been preserved in Fol-8Col, which is an essential element of its bioactivity.

**Acknowledgements**

**Author details**

University, Ueda, Japan

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Nano Fusion Technology Research Group, Faculty of Textile Science & Technology, Shinshu

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