Hydration and nanomechanical changes in collagen fibrils bearing advanced glycation end-products

Abstract

Accumulation of advanced glycation end-products (AGEs) in biological tissues occurs as a consequence of normal ageing and pathology. Most biological tissues are composed of considerable amounts of collagen, with collagen fibrils being the most abundant form. Collagen fibrils are the smallest discernible structural elements of load-bearing tissues and as such, they are of high biomechanical importance. The low turnover of collagen cause AGEs to accumulate within the collagen fibrils with normal ageing as well as in pathologies. We hypothesized that collagen fibrils bearing AGEs have altered hydration and mechanical properties. To this end, we employed atomic force and Brillouin light scattering microscopy to measure the extent of hydration as well as the transverse elastic properties of collagen fibrils treated with ribose. We find that hydration is different in collagen fibrils bearing AGEs and this is directly related to their mechanical properties. Collagen fibrils treated with ribose showed increased hydration levels and decreased transverse stiffness compared to controlled samples. Our results show that BLS and AFM yield complementary evidence on the effect of hydration on the nanomechanical properties of collagen fibrils.

Fig. 1

(A). Overview height topography scan of collagen fibrils on a glass slide showing a cross-section profile (inset, scale bars correspond to inset only) and examples of the selected collagen fibrils (rectangles, A). (A, 1-3) AFM height topography scans in air of the three selected collagen fibrils. (B). Characteristic D-periodicity with which collagen fibrils are distinguishable from other proteins. (C). The overview scan in PBS and (C, 1-3) the force volume maps of the individual collagen fibrils. (D). Characteristic force-indentation curve from the crest of the collagen fibril. In both air and PBS, the scan size of the overview images was 20 μm x 20 μm and that of the individual collagen fibrils was 1.5 μm x 8 μm.

Fig. 2

Fibril height measurements from the height channel of the atomic force microscopy image or force-volume map. A. Height topography from the force-volume map constructed at contact point (zero force). The highest points, i.e. the crest of the collagen fibril, are highlighted with red points. The green line across the collagen fibril indicates the location selected as an example cross-section shown in (B). B. Line profile showing the estimation of fibril height. The fibril height was defined as the difference between the average baseline height and the maximum height (indicated with a red point) per line profile. The average height of the collagen fibril shown in A was 238.2 ± 5.2 nm (average ± standard deviation).

Fig. 3

Sketch of the microscope setup used for BLS measurements. Inset shows an example of the projected BLS spectra as measured on the EM CCD camera.

Fig. 4

Spatial maps of ω_B and corresponding widefield transmitted light images. (Field of view for widefield images is 45 μm x 45 μm).

Fig. 5

A. and B. Normalized height profiles to the maximum height in air from a control and ribose-treated collagen fibril, respectively. C. Swelling, i.e. fold-increase in collagen fibril height, for control and ribose-treated. D. Indentation modulus values from control and ribose-treated collagen fibrils.

Fig. 6

Characteristic least-squares data fit for tokes Peaks of ribose treated sample.

Fig. 7

(A): Plot of average frequency shift (ω_B) for the two observed Brillouin scattering peaks in control (black solid-line) and ribose-treated (red, dashed-line) collagen fibrils during dehydration. B. Example Brillouin light spectra at 120 min of dehydration for the control (top, black) and ribose-treated collagen fibrils (bottom, red).

Fig. 8

(A): Sketch showing two different phonon wavevectors that can be coupled to with high NA objective. (B) [left]: BLS frequency shift ω_B of the higher frequency peak during dehydration (inset: corrected FHWM of peak after deconvolution). [Right]: Corresponding change in the relative peak intensity, I_B. (C): Same as (A), but for the lower frequency peak.