4. Discussion

The above data creates a novel prospective view on how scaffold fibers may interact with growing cells. It is well known that developing cells are greatly influenced by their environment: matrix anisotropy impacts cell phenotype and matrix stiffness affects cell migration, proliferation and differentiation [37]. The distribution of focal adhesion sites defines the actin network structure and thus impacts cell morphology. Generally, when cells are interacting with a scaffold matrix (either for planar substrate, nanofibrous mats or inside a 3D matrix) they are supposed to lie down on or touch neighboring elements of the scaffold. In this work we are adding one important course in which the cell may develop an interaction with a polymer fiber, enveloping it and extending along it. This spatial pattern of cell-fiber interaction leads to several important consequences which need to be considered. First, it is mechanical adhesion. Although, the fiber is relatively thin, the fact that it is pushed inside the cell makes the surface of the contact area much larger than if the cell would simply lie down on the fiber-like structure. This, in fact, means that the entire fiber surface is available for mechanical adhesion. Second, in the case of the smart scaffold releasing biologically active substances, molecules from the polymer fibers placed inside the cells will not dissipate entirely in the tissue bathing solution, but will rather diffuse directly from the fiber through the cell membrane. Obviously, this makes the efficient concentration much higher as leakage through the nanoscale gap connecting the fiber with the outer world would be negligible. Third, in the case of the conducting scaffold, immersion of the fiber in the cell makes the actual electrical contact of the cells possible, provided that the outer part of the polymer fibers is electrically isolated from the surrounding ionic solution.

As for the precise mechanism of the cell-fiber attachment, it might be somewhat different from what is observed when cells attach to the surface of solid substrate by forming adhesion micro-domains. Obviously, adhesion micro-domains must be formed in the cell-fiber interface by lateral segregation of receptors (integrin in the present case). The clusters of integrin are mechanically stabilized by coupling of the integrin to the actin cortex. The physical basis of the adhesion induced domain formation has been clarified by studies of biomimetic models [38]. However, when a fiber is pushed deep inside a cell, at the gap, leading to the extracellular space, the cell meets itself and a cell-cell interface appears. Also, one can suggest that direct covering of the fibers by fibronectin results in a stronger attachment because of the additional Van

der Waals forces, and we do not know the relative contribution of the latter and attachment, mediated by the RGD motifs. In order to avoid such artefacts the fiber surfaces can be covered with soft hydrogels [39]. It would be interesting to study whether the electrospun fibers could be bio-functionalized in a similar ways as large flat supports by using techniques described in [39]. While this issue creates a direction for further study, it does not detract from the significance of the observed effect: the fibronectin coating is widely used in cell scaffolds and enveloping configuration gives a number of new important features to the scaffold.

Comparing adhesion of cardiomyocytes and fibroblasts to nanofibers we should note that as it was shown in the previous studies, fibroblasts on the suspended polymer fibers form several focal adhesion clusters (FAC) depending on the number of fibers on which they are placed [35]. In the simplest case of a spindleshaped fibroblast, 2 FACs are formed on a single fiber, which stretch the cell in the longitudinal direction, as shown in Figs. 2(c), 3(c). Stretching is limited by the size of the nucleus, which is relatively rigid compared to other cell components and is less susceptible to deformation. With more fibers, more than 2 FAC are formed. In each place of adhesion, forces arise that stretch the cell [40]. In addition, the actin cytoskeleton attempts to reduce the area of the cell membrane, minimizing contact with fibers, so absorbing fibers is not energetically beneficial [36]. Thus, the fibroblasts on suspended fibers are under tension and may envelope the fibers only in the locations of the FAC, where a cell is capable to produce forces able to overcome resistance of actin cortex. In contrast to fibroblasts that have several sites with FAC, integrins are present in cardiomyocytes in costamers - structures that connect sarcomeres to the substrate [41,42]. Thereby, myocytes have much more attachment points to the substrate (each Z-disk, next to the sarcolemma, is attached to the extracellular matrix), that stabilize areas of cell-ECM interaction. When a cardiomyocyte develops on suspended fibers, the myofibrils begin to attach and align with the fiber, trying to maximize the area of interaction with the substrate. As a result, the fiber appears in the middle of the myofibril, despite the fact that it is not energetically beneficial.

5. Conclusion

The high resolution study of the filamentous structures formed by cardiac cells interacting with polymer nanofibers revealed that cardiomyocytes and fibroblasts have different behavior on such type of scaffolds. The study was performed using three independent methods: confocal laser scanning microscopy (CLSM),

scanning probe nanotomography (SPNT), and transmission electron microscopy (TEM). It was demonstrated that in the sparse nanofiber meshes, having a cell-fiber filamentous structure, cardiomyocytes most often create a striking ‘‘sheath” structure, enveloping fiber and, thus, making the contact zone much greater than it was expected to be based on the focal attachment model. In contrast, fibroblasts tend to extend along the fiber through formation of some focal adhesion sites.

Acknowledgements

show our gratitude to T. Starodubtseva for text correction. The work was supported by the Russian Ministry of Education and Science of the Russian Federation grant (state task) 6.9906.2017/ BCh.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, in the online version, at https://doi.org/10.1016/j.actbio.2017.12.031.

References

We thank Dr. N. Agladze and V. Tsvelaya for the help with tissue culture. We thank G. Semenova, V. Peshenko and S. Khutsyan for the help with transmission electron microscopy. We also like to

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