examined via FDA live cell staining under a florescence microscope on the first and seventh day after seeding (Fig. 7B). The cells within the scaffolds began to spread, proliferate and infiltrate in the 3D structure after cell attachment over time. Yet,the images of infiltration were not captured due to the inability of the light microscope to focus on the Z-axis. Especially FCA/COS1 indicated the highest colour intensity in seventh day culture. Therefore, biological examinations of the scaffolds indicate that COS1 is the best molecular size to induce fibroblast cell proliferation after cross-linking
with FCA. 3.5.2. Cell morphology The morphology of the NHDF-neo cells were observed under SEM and the micrographs of cells on FCA and FCA/COS1 scaffolds are shown in Fig. 7C. After 7 days of incubation, the cells were intensively attached, migrated and grown over the pore surfaces including pore walls and bottom surfaces. The morphology of the
fibroblasts on the FCA/COS1 scaffold’s bottom and the side wall surfaces were mostly flattened and spread well by randomly deposited anchors compared to the pure FCA scaffolds. In addition cells were suspended in the space of the pore by extending pseudopods contact with pore walls. This cell behavior was prominent in FCA/COS1 scaffolds. The promotion in cell behavior in FCA wasmainly induced by the integrin mediated cellularity of collagen [4]. Yet, COS1 promoted a well spread cell morphology in FCA/COS1 scaffolds. These results indicate thatthepositive charges onCOS increasedthe interaction between the negatively charged cell surface and the surface of the scaffolds, which in turn promoted the well spread cell morphology in FCA/COS1 scaffolds.
4. Conclusion
Cross-linking of FC, SA, and COSs using EDC is an effective means of obtaining a synergy of the beneficial properties of all compounds to better mimic the physical, mechanical, and biological properties of skin tissue. A 3D FCA scaffold fabricated via freeze drying and subsequent EDC cross-linking were reinforced by additional COS cross-links.After cross-linking with COSs, all FCA/COS scaffolds were characterized by superior interconnected porous architecture that had pore size ranging between 160 and 260 m and porosity greater than 90%. However, the introduction of COSs notably improved the mechanical property while reducing biodegradation, in line with the MWs of COSs. In addition, excellent water uptake and retention capacities with marginal reductions from FCA/COS1 to FCA/COS4 indicate that FCA/COS scaffolds can be used in the application of skin tissue regeneration. The biological response of the NHDF-neo cells to FCA/COS scaffolds exhibited improved cell adhesion and proliferation than on pure FCA scaffolds, and the FCA/COS1 scaffoldexhibitedthe best cellular compatibility,indicating the cellular induction effect of COSs on the scaffolds. Overall,the comprehensive physicochemical, mechanical, and biological properties suggestthatthe FCA/COS1 scaffold is a superior candidate for skin tissue-engineering application.