Regulation of corneal fibroblast morphology and collagen reorganization by extracellular matrix mechanical properties

Abstract

Purpose: To investigate how extracellular matrix mechanical properties influence cell and matrix patterning in three-dimensional culture.

Methods: Human corneal fibroblasts were seeded within 30 x 10 mm collagen matrices that were unconstrained (UN), fully constrained (CO) along the long axis by attaching the construct to two immobilized plastic bars, or partially constrained (PC) by allowing linear elastic displacement of one bar. After 24 hours, constructs were labeled with phalloidin and were imaged using fluorescent and reflected light (for collagen) confocal microscopy. Cell morphology and local collagen fibril density and alignment were measured using digital image processing.

Results: Corneal fibroblasts in UN matrices were less elongated (UN < PC < CO; P < 0.05) than those in constrained matrices. Cells were aligned parallel to the long axis in the anisotropic region of constrained matrices but were randomly aligned in unconstrained (isotropic) matrices (UN < PC = CO; P < 0.05). Both the local collagen density and the degree of cell/collagen coalignment were higher in constrained matrices (UN < PC < CO; P < 0.05). In regions of higher cell density, additional bands of aligned collagen were often observed between individual cells.

Conclusions: These data suggest that cell spreading, alignment, and contractile force generation are directly influenced by the mechanical properties of the surrounding extracellular matrix (ECM). Corneal fibroblasts generally align and compact collagen parallel to the axis of greatest ECM stiffness. Mechanical cross-talk between adjacent cells leads to enhancement of matrix reorganization, and results in additional, more complex matrix patterning.

Figure 1

Schematic of the three models used. (A) UN model. (B) PC model. (C) CO model.

Figure 2

Regions selected for imaging and further analysis were obtained from the A- and D-zones of the constructs. Shaded areas represent collagen blocks processed for confocal microscopy. Images were collected from the central area of each region (not at the edges).

Figure 3

Maximum intensity projections demonstrating typical cell morphologies within the A-zone of the three models (UN, PC, CO). The long axis of the construct is horizontal. Note the numerous branching cell processes within the UN model; cells within the PC and CO models are bipolar.

Figure 4

Quantitative analysis of projected cell lengths for the three models studied.

Figure 5

Maximum intensity projections showing cell orientation for the three models (UN, PC, CO) within the two different zones (A-zone and D-zone). The long axis of the construct is horizontal. (A, C, E) Randomly oriented cells within the D-zone for all three models. (B, D, F) Cells within the A-zone. Note that cells in the UN model are randomly oriented (B), whereas cells within the PC and CO models (D, F) are aligned parallel to the long axis of the construct.

Figure 6

Quantitative analysis of cellular alignment for the three models studied. Δθ is the difference between the long axis of the cell and the long axis of the construct (45° = random orientation; 0° = aligned with the long axis).

Figure 7

Representative images of cell-induced matrix reorganization at the ends of cells. F-actin is shown in green, and collagen is shown in red. (A, C, E) Cell-collagen interactions in the D-zone for UN, PC, and CO, respectively. (B, D, F) Cell–collagen interactions in the A-zone for UN, PC, and CO, respectively. The long axis of the construct is horizontal.

Figure 8

OI results from FT analysis are shown for (A) acellular constructs and (B) cellular constructs. OI values are shown at the cell angle.

Figure 9

Summary of local collagen density measurements for UN, PC, and CO models. (A) Collagen density for acellular constructs. (B) Collagen density for cellular constructs.

Figure 10

Normalized collagen density measurements for UN, PC, and CO models.

Figure 11

Interactions between parallel (A) cells within the A-zone and obliquely (B) aligned cells within the D-zone. (A) Band of highly compacted and aligned collagen between the ends of the two cells. (B) When cells were not aligned in parallel, straps of collagen were often observed originating from the sides of the pseudopodial processes (arrows), resulting in the formation of collagen bands at oblique angles from the long axis of the cells (arrowhead).