Advanced glycation end-product cross-linking inhibits biomechanical plasticity and characteristic failure morphology of native tendon

Abstract

Advanced glycation end-products (AGEs) are formed in vivo from the nonenzymatic reaction between sugars and proteins. AGEs accumulate in long-lived tissues like tendons, cross-linking neighboring collagen molecules, and are in part complicit in connective tissue pathologies experienced in aging and with diabetes. We have previously described discrete plasticity: a characteristic form of nanoscale collagen fibril damage consisting of serial fibril kinking and collagen denaturation that occurs in some mechanically overloaded tendons. We suspect that this failure mechanism may be an adaptive trait of collagen fibrils and have published evidence that inflammatory cells may be able to recognize and digest the denatured collagen produced by overload. In this study, we treated bovine tail tendons with ribose to simulate long-term AGE cross-linking in vitro. We hypothesized that a high degree of cross-linking would inhibit the intermolecular sliding thought to be necessary for discrete plasticity to occur. Tendons were mechanically overloaded, and properties were investigated by differential scanning calorimetry and scanning election microscopy. Ribose cross-linking treatment altered the mechanical response of tendons after the yield point, significantly decreasing postyield extensibility and strain energy capacity before rupture. Coincident with altered mechanics, ribose cross-linking completely inhibited the discrete plasticity failure mechanism of tendon. Our results suggest that discrete plasticity, which may be an important physiological mechanism, becomes pathologically disabled by the formation of AGE cross-links in aging and diabetes. NEW & NOTEWORTHY We have previously shown that mechanically overloaded collagen fibrils in mammalian tendons accrue nanoscaled damage. This includes development of a characteristic kinking morphology within a shell of denatured collagen: discrete plasticity. Here, using a ribose-incubation model, we show that advanced glycation end-product cross-linking associated with aging and diabetes completely inhibits this mechanism. Since discrete plasticity appears to cue cellular remodeling, this result has important implications for diabetic tendinopathy.

Keywords: advanced glycation end-products cross-linking; diabetes; discrete plasticity; mechanical damage; tendon overload and injury.

Fig. 1.

Representative subrupture overload stress-strain curves for an untreated tendon sample and matched-pair ribose cross-linked sample. For clarity, only cycles 1–3, 6, and 10 are shown. The untreated sample shows a characteristically longer postyield (plastic) region, with more energy dissipation after the yield point.

Fig. 2.

A and B: stress-strain curves for the 11th subrupture overload cycle are shown for 2 samples from the same tendon: one untreated (A) and the other after ribose cross-linking (B). A: in the untreated sample, the slope of the stress-strain curve gradually goes to zero. B: ribose cross-linking decreased plasticity. Instead of the stress-strain curve gradually going to zero, load suddenly drops, indicative of fiber rupture within the sample.

Fig. 3.

Mechanical properties calculated for each overload cycle included: linear modulus (E), yield stress and strain (σ_y, ε_y), maximum stress and corresponding strain (σ_u, ε_u), postyield strain (ε_PY), resilience, strain energy density, postyield strain energy density (PY Energy), hysteresis (full area between loading and unloading curves), and postyield hysteresis (PY Hysteresis; portion of the hysteresis loop after the yield point). The curve shown is the first overload cycle for a ribose cross-linked tendon.

Fig. 4.

Representative differential scanning calorimetry endotherms for the study’s 4 sample groups. For untreated bovine tail tendon, mechanical overload caused substantial molecular packing disruption, shifting the endotherm to the left and broadening it significantly. While unloaded tendon showed marginal stabilization following ribose cross-linking, ribose cross-linking substantially altered the tendon response to overload, eliminating most of the change seen in untreated tendon.

Fig. 5.

This representative image shows the color change caused in bovine tail tendon by the 28-day incubation with ribose used to induce advanced glycation end-product cross-links.

Fig. 6.

As assessed by differential scanning calorimetry, the effect of mechanical overload on the molecular packing structure of tendon was significantly altered by ribose cross-linking, with ribose cross-linking inhibiting much of the overload-induced destabilization seen in native tendon. A: onset temperature (T_onset). B: peak temperature (T_peak). C: full-width at half-maximum (FWHM). *P < 0.05, **P < 0.005, ***P < 0.0005.

Fig. 7.

Typical collagen fibril ultrastructures under scanning election microscopy. A: untreated, unloaded tendon samples contained straight fibrils with clear D-banding. B: untreated, overloaded samples contained large regions of fibrils that had experienced discrete plasticity, with fibrils exhibiting the characteristic longitudinally repeating kinked morphology. The sample shown here underwent 15 cycles of subrupture overload. For the first overload cycle, the yield and maximum stresses were 20 and 26 MPa, respectively. C: ribose cross-linking totally inhibited overload-induced discrete plasticity. In tendon that had been ribose-cross-linked, overload had little effect on the fibrils. While occasional sites of fibril damage were identified (Fig. 8C), most fibrils retained the straight D-banded appearance typical of unloaded samples. The sample shown here underwent 10 cycles of subrupture overload. For the first overload cycle, the yield and maximum stresses were 29 and 31 MPa, respectively.

Fig. 8.

A–C: representative scanning election micrographs of the most-damaged fibrils found in untreated and overloaded (A and B) and ribose-cross-linked and overloaded (C) tendon collagen. A and B: sites of extreme fibril damage were easily found in untreated samples, with fibrils having a high linear density of discrete plasticity kinking, complete absence of D-banding, and visible subfibril structure (arrows). The sample shown here underwent 15 cycles of subrupture overload. For the first overload cycle, the yield and maximum stresses were 28 and 44 MPa, respectively. C: in contrast, the most severe fibril damage seen in tendon that was ribose-cross-linked before overload was some limited fibril buckling that was highly localized, not extending for more than a few hundred nanometers along the length of fibrils. Such fibrils retained their D-banding, indicating that the buckles had formed with minimal disruption to molecular packing, consistent with differential scanning calorimetry results. The sample shown here underwent 10 cycles of subrupture overload. For the first overload cycle, the yield and maximum stresses were 24 and 30 MPa, respectively.