Collagen I self-assembly: revealing the developing structures that generate turbidity

Abstract

Type I collagen gels are routinely used in biophysical studies and bioengineering applications. The structural and mechanical properties of these fibrillar matrices depend on the conditions under which collagen fibrillogenesis proceeds, and developing a fuller understanding of this process will enhance control over gel properties. Turbidity measurements have long been the method of choice for monitoring developing gels, whereas imaging methods are regularly used to visualize fully developed gels. In this study, turbidity and confocal reflectance microscopy (CRM) were simultaneously employed to track collagen fibrillogenesis and reconcile the information reported by the two techniques, with confocal fluorescence microscopy (CFM) used to supplement information about early events in fibrillogenesis. Time-lapse images of 0.5 mg/ml, 1.0 mg/ml, and 2.0 mg/ml acid-solubilized collagen I gels forming at 27°C, 32°C, and 37°C were collected. It was found that in situ turbidity measured in a scanning transmittance configuration was interchangeable with traditional turbidity measurements using a spectrophotometer. CRM and CFM were employed to reveal the structures responsible for the turbidity that develops during collagen self-assembly. Information from CRM and transmittance images was collapsed into straightforward single variables; total intensity in CRM images tracked turbidity development closely for all collagen gels investigated, and the two techniques were similarly sensitive to fibril number and dimension. Complementary CRM, CFM, and in situ turbidity measurements revealed that fibril and network formation occurred before substantial turbidity was present, and the majority of increasing turbidity during collagen self-assembly was due to increasing fibril thickness.

Figure 1

CRM images of collagen gels at plateau and arrest (overlaid insets). Scale bar is 10 μm and is the same for all images. CRM images are unprocessed but are cropped, and each shows a representative portion of the full image.

Figure 2

Comparison of CFM and CRM images at time points of interest. Images are from a representative 1.0 mg/ml collagen sample gelled at 32°C also shown in Video S1. Left panel: The first images collected, 88 s after neutralization. (a) The initial CFM image reveals small isotropic features of moderate intensity against a lower intensity homogeneous background, whereas (b) the earliest CRM image reveals no structures. Right panel: Images at arrest and plateau. (c) The CFM image at t_ARR (5.6 min) reveals many structures. (d) The CRM image at arrest displays structures of low intensity that can be better visualized when (e) contrast is enhanced. (f) The CRM image at plateau (12.5 min) reveals that many structures in the fully developed gel can already be visualized at arrest (as in (c) and (e)). Scale bar is 25 μm for all images.

Figure 3

Traditional turbidity measurements track IST and CRM intensity evolution. At left, turbidity as measured with a spectrophotometer (black open squares), IST (black solid squares), and CRM intensity (red squares) from representative 1.0 mg/ml collagen samples gelled at 37°C. The standard turbidity curve was shifted by one minute as described in the text. At right, images corresponding to dotted lines at time points of interest extracted from the IST curve: (a) initial CRM image, (b) CRM image at t_LAG, (c) CRM image at t_INF, (d) CRM image at t_PL, (e) CRM image beyond plateau, and (f) IST image beyond plateau. Images corresponding to t_ARR (orange line) and t_MS (green line) are shown as insets at left. Scale bar is 25 μm for all images. All images are unprocessed but cropped and show the same representative portion of the full image. To see this figure in color, go online.

Figure 4

Mesh size evolution during fibrillogenesis. (a) Mesh size during gelation of 0.5 mg/ml (squares), 1.0 mg/ml (circles), and 2.0 mg/ml (diamonds) at 27°C. Every other point is shown for clarity. Large symbols indicate t_INF and t_PL as determined from CRM intensity curves. Mesh size at (b) t_INF and (c) t_PL vs. (b) t_INF and (c) t_PL for all gels. Error bars represent standard deviations and are shown when larger than the symbols. To see this figure in color, go online.

Figure 5

Average (a) IST and (b) CRM curves for 0.5 mg/ml (squares), 1.0 mg/ml (circles), and 2.0 mg/ml (diamonds) collagen solutions gelled at 27°C (black), 32°C (red), and 37°C (green). Every other point is shown for clarity. Error bars represent standard deviations. To see this figure in color, go online.

Figure 6

Time points of interest (t_LAG, t_INF, t_PL) and slope of the growth phase (k_G) obtained from CRM versus those obtained from IST: (a) t_LAG, (b) t_INF, (c) t_PL, and (d) k_G. Linear regression was performed with the y-intercept set to zero, yielding slopes of 0.81, 0.94, and 0.96 with R² ≥ 0.98. In (a), if the 27°C 0.5 mg/ml point is excluded from the fit, the slope of the best-fit line is 0.88 and all R² > 0.998. In (d), IST k_G values were normalized by multiplying by final CRM intensity over final IST, yielding k′_G. Error bars are standard deviations and are shown only when larger than the symbols. Colors and symbols for (a–d) are as shown in the legend in (a). To see this figure in color, go online.

Figure 7

(a) The ratio of CRM intensity to concentration (W = CRM_int/c) versus that of IST to concentration (A = IST/c) at t_INF (open symbols) and t_PL (solid symbols). Time points evaluated were determined from the CRM intensity curve for W and the IST curve for A. (b) Ratio of CRM intensity at t_ARR to that at t_PL versus CRM intensity at t_PL. Symbols and colors are as shown in (a). Error bars indicate standard deviations and are shown only when larger than the symbols. To see this figure in color, go online.