The Structural Interactions of Molecular and Fibrillar Collagen Type I with Fibronectin and Its Role in the Regulation of Mesenchymal Stem Cell Morphology and Functional Activity

Abstract

The observed differences in the structure of native tissue and tissue formed in vitro cause the loss of functional activity of cells cultured in vitro. The lack of fundamental knowledge about the protein mechanism interactions limits the ability to effectively create in vitro native tissue. Collagen is able to spontaneously assemble into fibrils in vitro, but in vivo, other proteins, for example fibronectin, have a noticeable effect on this process. The molecular or fibrillar structure of collagen plays an equally important role. Therefore, we studied the interaction of the molecular and fibrillar structure of collagen with fibronectin. Atomic force and transmission electron microscopy showed that the presence of fibronectin does not affect the native structure and diameter of collagen fibrils. Confocal microscopy demonstrated that the collagen structure affects the cell morphology. Cells are better spread on molecular collagen compared with cells cultured on fibrillar collagen. Fibronectin promotes the formation of a large number of focal contacts, while in combination with collagen of both forms, its effect is leveled. Thus, understanding the mechanisms of the relationship between the protein structure and composition will effectively manage the creation in vitro of a new tissue with native properties.

Keywords: actin cytoskeleton; cell focal contacts; fibronectin; molecular and fibrillar collagens.

Figure 1

AFM images of molecular and fibrillar collagen with/without fibronectin (FN): (a)—molecular collagen; (b)—fibrillar collagen; (c)—fibronectin; (d)—molecular collagen + fibronectin; (e)—fibrillar collagen + fibronectin. Scale bar 1 µm.

Figure 2

TEM images of fibrillar collagen with/without fibronectin: (a)—fibrillar collagen; (b)—fibrillar collagen + fibronectin. Scale bar 500 nm.

Figure 3

Fourier transform infrared spectra of fibrillar collagen with/without fibronectin.

Figure 4

SEM images of MSCWJ-1 cell line after 1 day cultivation on molecular and fibrillar collagen with/without fibronectin (FN): (a)—control (cover glass), (b)—molecular collagen; (c)—fibrillar collagen; (d)—fibronectin; (e)—molecular collagen + fibronectin; (f)—fibrillar collagen + fibronectin. Scale bar 10 µm.

Figure 5

Fluorescence micrographs of MSCWJ-1s with vinculin (green) and nuclei (blue) stained after one days of cultivation on molecular and fibrillar collagen with/without fibronectin (FN): control (cover glass) (a), molecular collagen (b); fibrillar collagen (c); fibronectin (d); molecular collagen + fibronectin (e); fibrillar collagen + fibronectin (f). Statistics of cell adhesion on molecular and fibrillar collagen with/without fibronectin after 1 day cultivation. (n = 7–12: ****—p < 0.0001 and ***—p < 0.001 compared with the control, ####—p < 0.0001 compared with the fibronectin). Scale bar 50 µm.

Figure 6

Fluorescence micrographs of MSCWJ-1s with actins (red), collagen fibrils (green) and nuclei (blue) stained after one days of cultivation on molecular and fibrillar collagen with/without fibronectin (FN): control (cover glass) (a–c), molecular collagen (d–f); fibrillar collagen (g–i); fibronectin (j–l); molecular collagen + fibronectin (m–o); fibrillar collagen + fibronectin (p–r). Statistics of cell area (spreading) on molecular and fibrillar collagen with/without fibronectin after 1 day cultivation. (n = 7–21: ****—p < 0.0001 compared with the control). Scale bar 50 µm.