Novel spectroscopic technique for in situ monitoring of collagen fibril alignment in gels

Abstract

Development of collagen fibril alignment in contracting fibroblast-populated and externally tensioned acellular collagen gels was studied using elastic scattering spectroscopy. Spectra of the backscattered light (320-860 nm) were acquired with a 2.75-mm source-detector separation probe placed perpendicular to the gel surface and rotated to achieve different angles to the collagen fibril alignment. Backscatter was isotropic for noncontracted/unloaded gels (disorganized matrix). As gels were contracted/externally loaded (collagen alignment developed), anisotropy of backscatter increased: more backscatter was detected perpendicular than parallel to the direction of the fibril alignment. An "anisotropy factor" (AF) was calculated to characterize this effect as the ratio of backscatter intensities at orthogonal positions. Before contraction (or zero strain) the AF was close to unity at all wavelengths. In contrast, at 72 h, backscatter anisotropy varied from AF(400 nm) = 2.14 +/- 0.29 to AF(700 nm) = 3.04 +/- 0.48. It also increased over threefold up to a strain of 20%. The AF strongly correlated with the contraction time/strain. Different directions of the backscatter were detected in gel zones with known differences in the matrix alignment. Thus, backscatter anisotropy allows in situ nondestructive determination of collagen fibril alignment and quantitative monitoring of its development.

FIGURE 1

(a) Spectra acquisition setup with the optical probe applied to monitor the development of alignment in the central zone of a contracting collagen gel. Probe vertical position was adjusted using a manual rack pinion microscope stage. (b) Noncontracted (0 h) and contracted (72 h) FPCLs. A geometric parameter, D_c, was calculated as a ratio of the gel's width in the central zone during contraction, D, to that of the uncontracted gel, D (0 h): D_c = D/D (0 h).

FIGURE 2

Scheme of the gel and probe application. (a) Position of a gel in the rectangular coordinate system. Direction of stress (either developed during FPCL contraction or externally applied to acellular gels) corresponded to the longitudinal axis of the gel, along the y axis. (b) Positions of the zones of interest: central zone, δ-zone and corner zone, used in monitoring. Dotted profile shows gel waisting over the contraction period. (c) Angular positions of the gel relative to the optical probe used to obtain spatially resolved spectral measurements.

FIGURE 3

SEM images obtained from the central zones of the gels after 0 h (a), 17 h (b), and 72 h (c) of contraction. Bars correspond to 100 μm. Cells are round before contraction (a), then start to elongate parallel with the direction of stress (arrow, b) and eventually produce an aligned matrix (c).

FIGURE 4

Spectra of backscattered light, acquired from the central zone of a noncontracted (a) and 72 h contracted (b) fibroblast populated collagen gel. Probe was placed perpendicular to the gel surface and the gel was rotated through all angles between the gel longitudinal and the probe detection axes. At 0° and 180° positions the detection axis of the probe was parallel to the direction of stress, and at 90° and 270° positions it was perpendicular. Each line represents a mean (n = 3) of spectra acquired at the same position of the probe.

FIGURE 5

Radial diagrams of the intensity of backscattered light at 700 nm from the noncontracted (—•—) and 72 h contracted (- -▪- -) fibroblast populated collagen gels (n = 3).

FIGURE 6

Radial diagrams of the intensity of backscattered light at 700 nm from different zones of a fibroblast populated collagen gel contracted for 72 h: (a) δ-zone, (b) central zone, and (c) corner zone. Each data point represents mean (n = 3) from spectra acquired at the same position of the probe within the same zone of the gel. Schemes next to each diagram illustrate the zones, from which spectra were taken, indicating actual alignment (if any).

FIGURE 7

Time course of gel contraction, shown by D_c, width of the gel in the central zone at time points relative to that at 0 h (- -□- -), and the corresponding backscatter anisotropy registered in the same zone of the gel: AF₄₀₀ (—•—) and AF₇₀₀ (- -○- -). As gel contracted, D_c decreased mirroring the increase in backscatter anisotropy.

FIGURE 8

SEM images obtained from the central zones of acellular gels without (at 0% strain) (a) or with external loading (10% strain). (b) Bars correspond to 5 μm in a and 1 μm in b. The unloaded collagen gel was more hydrated and disorganized in a, whereas after loading the gel was clearly fibrous and aligned in the direction of the applied load indicated by an arrow in b. Small round features in the unstrained gel in a were drying artifacts.

FIGURE 9

Anisotropy of backscattered light as a function of strain, produced by external tensile loading of acellular gels (n = 7). Dotted lines show 95% confidence intervals.