Distinctive roles of fibrillar collagen I and collagen III in mediating fibroblast-matrix interaction: A nanoscopic study

Abstract

One major goal in tissue engineering is to create functional materials, mimicking scaffolds in native tissues, to modulate cell function for tissue repair. Collagen is the most abundant structural protein in human body. Though collagen I (COLI) and collagen III (COLIII) are the predominant collagen types in connective tissues and they form stable hybrid fibrils at varied ratios, cell responses to the hybrid matrices are underinvestigated. In this work, we aim to explicate the distinctive roles of COLI and COLIII in fibroblast activation. Unidirectionally aligned COLI, COLIII and COLI-COLIII hybrid nanofibrils were generated via epitaxial growth of collagen on mica. AFM analyses revealed that, with the increase of COLI/COLIII ratio, the fibril width and stiffness increased and the binding affinity of cells to the matrix decreased. A hybrid matrix was found to activate fibroblasts the most effectively, characterized by extensive cell polarization with rigid stress fiber bundles and high α-SMA expression, and by the highest-level of collagen synthesis. It is ascribed to the fine balance between biochemical and biophysical cues achieved on the hybrid matrix. Thus, matrices of aligned COLI-COLIII hybrid fibrils and their derived multifunctional composites can be good candidates of implantation scaffolds for tissue regeneration.

Keywords: Cell-matrix interaction; Collagen I; Collagen III; Collagen synthesis; Fibroblast activation; Nanofibrils.

Figure 1.

Unidirectionally aligned collagen fibrils and the induced cell polarization. (a–e) AFM images of gelatin and collagen fibrils on mica at various COLI/COLIII ratios (bar size: 1 μm): (a) Gelatin; (b) Pure COLI; (c) COLI: COLIII=2:1; (d) COLI: COLIII=1:1; (e) Pure COLIII. The inset of (b) is the high-resolution image illustrating the characteristic collagen D-period. (f–j) Optical images of fibroblasts 24 h post-plating on the respective matrices (bar size: 100 μm). The arrows indicate the direction of fibril alignment.

Figure 2.

Binding force measurements on various matrices. (a) Functionalization of α1β1-integrin on an AFM probe to measure its binding force to a collagen matrix. (b–d) Histograms showing the distribution of binding forces measured between α1β1-integrin and COLI (b), COLIII (c) or a control BSA coated surface (d). Gaussian fitting peaks are shown in (b,c). (e) Histograms showing the distribution of adhesion forces measured between a whole cell and a COLI (black) or COLIII (red) matrix.

Figure 3.

Cytoskeletal structure and local elasticity of fibroblasts on various matrices. (a–d) Immunostaining of α-SMA (green) and nuclei (blue) of fibroblasts on various matrices. Bar size: 56 μm on (b); 25 μm on the rest. (e–l) Corresponding AFM topographic (e–h) and amplitude (i–l) images of fibroblasts on various matrices. (m–p) E-maps derived from the force maps collected at the same regions of the AFM images in (e–l). Bar size: (e, i, m) 26 μm; (f, j, n) 20 μm; (g, k, o) 25 μm; (h, i, p) 20 μm.

Figure 4.

Gene expression profiles of fibroblasts grown on various matrices. (a) COLI, COLIII and ACTA2 expressions in cells on gelatin, COLI, COLIII and COLI-COLIII (2:1) matrices; (b) COLI/COLIII ratio for cells on each matrix type.