Figure 1.
Schematic of experimental principles.
Left) 2D diffraction patterns from tendons are dominated by the meridional Bragg reflections corresponding to ∼67.5 nm and harmonic frequencies of this spacing. Right) This pattern results from the quarter-stagger model of collagen. More simply, this can be seen as “gap” and “overlap” regions with high and low electron dense areas that scatter X-rays as wide periodic slits [63]. Middle) The meridional intensities were integrated over azimuthal (θ) sectors, which resulted in 1-D intensity profiles, I [a.u.] vs. q [nm−1]. The peak positions or correspondingly the distance between peaks) were used to calculate the length of the periodicity (Bragg's law) during tensile experiments. Changes of the peak spacing served then as averaged measure for collagen fibril elongation: absolute deformation (D) and relative fibril strain).
Figure 2.
Sketch of collagen fibril deformation and failure.
A1) Unloaded collagen fibrils. A2) With applied strain (blue arrows), the collagen fibril D-periods increase homogenously and are approximately equally long. This results in sharp peaks of the meridional collagen reflections. A3) Post yielding, the collagen fibrils are deformed more heterogeneously. This resulted in the broader peaks, measured as FWHM. Some AGE samples this heterogeneity resulted in two distinct populations of fibrils, which resulted in peak splitting. B1) Individual collagen fibril from A1 composed of collagen molecules with shown D-periodic pattern. B2) Apart from the molecule elongation the fibril elongation consists of side-by-side sliding of collagen molecules, which results in a decreased overlap (O) region, while the D-period increases (not shown). This decreases the O/D ratio.
Figure 3.
Mechanical characterization of the tendon-AGE model.
Row A: Stress-strain from the first experimental set. Except for the MGO 96 h group, these curves were measured simultaneously with the SAXS experiments. Row B: A selection of material properties extracted from row A: B1) Elastic modulus was obtained in the linear range. Given are: mean (horizontal bar), percentile box (25% and 75%) and ±1 SD. The specificity of the MGO treatment was shown by the addition of L-lysine and L-arginine (LYS-ARG group) to the MGO solution and compared to MGO controls. For ease of reading this control is not shown. B2) Ultimate tensile strength (UTS). B3) Separate stress relaxation experiment (n = 5) was performed with repeated measurements on the same samples and compared to a corresponding control (n = 5). Stress relaxation is reported as: σR(175 s) = 1-σ(175 s)/σ(0 s) [%].
Figure 4.
Results from SAXS experiments.
Row A) Collagen fibril stress) vs. D-period. Normalized collagen fibril stress was calculated using the rule of mixtures. The numerical gradient (slope) of the D-period vs. collagen fibril stress curves was used to measure fibril stiffness. Peak stiffness was used as the parameter for statistical interference testing. The second experimental set (w/o a 24 h MGO group) is not plotted here since over all the stresses were approximately one third lower but with equal group effects (two-way ANOVA: interactions term, p = 0.531). B1) Ratio of the integrated intensities for the 2nd and 3rd order meridional collagen reflections. B2) Relative contribution of overlap (O) region to the D-period (O/D) vs. D-period, when assuming a two-phasic approximation of the D-period electron density [17], [63]. B3) Peak widths (FWHM) from fitted Gaussians measured during the tensile experiments vs. D-period length. Note: The grayed area in the insets represents the range of yield point of the control samples as reference.
Figure 5.
Failure mode of collagen fibrils.
A+B) Diffraction patterns recorded after yielding. A) A control sample. B) A 6 h MGO sample. The corresponding 1D azimuthal averages are indicated by blue dots. The intensity range is scaled to show the full scale from the 3rd diffraction peak. Peaks were fitted to Gaussian functions (red). In some MGO samples the best fit was bimodal Gaussian (green) in account of the peak splitting as the sum of two Gaussians (red). The first peak is not shown. C) Stress-strain curve of a MGO sample that showed peak splitting shortly after yielding (grey horizontal bar). The gradient of the stress-strain curve, which is a local estimate of elastic modulus, is also shown. D-period estimates of the same sample is also shown (3rd reflection), with the loaded and deformed collagen fibrils (♦) and the more relaxed ones (◊).
Figure 6.
Row A) Averaged (±1 SD) tissue stress–time relations from consecutive relaxation increments of size = 0.6% L0: A1) Control samples (n = 10).
A2) 6 h MGO group (n = 10). A3) The corresponding total stress decay. Row B) Collagen fibril relaxation defined as relative length change of the D-period during relaxation steps: B1) Controls B2) 6 h MGO. B3) The corresponding total change in fibril length.
Table 1.
The parameters and upper (UCI) and lower (LCI) 95% confidence intervals that were fitted to the stress and fibril relaxation at comparable fibril elongations.
Figure 7.
Top) The effect of 30 mM MGO on lysine and arginine content on collagen, normalized to collagen mass measured with a hydroxyproline assay and assuming 14% hydroxyproline per collagen.
Averages are given with ±1 SD. Bottom) Effects of the MGO treatment on selected AGEs normalized by collagen content.