Cross‐link stabilization does not affect the response of collagen molecules,fibrils, or tendons to tensile overload

We investigated whether immature allysine‐derived cross‐links provide mechanically labile linkages by exploring the effects of immature cross‐link stabilization at three levels of collagen hierarchy: damaged fibril morphology, whole tendon mechanics, and molecular stability. Tendons from the tails of young adult steers were either treated with sodium borohydride (NaBH₄) to stabilize labile cross‐links, exposed only to the buffer used during stabilization treatment, or maintained as untreated controls. One‐half of each tendon was then subjected to five cycles of subrupture overload. Morphologic changes to collagen fibrils resulting from overload were investigated using scanning electron microscopy, and changes in the hydrothermal stability of collagen molecules were assessed using hydrothermal isometric tension testing. NaBH₄ cross‐link stabilization did not affect the response of tendon collagen to tensile overload at any of the three levels of hierarchy studied. Cross‐link stabilization did not prevent the characteristic overload‐induced mode of fibril damage that we term discrete plasticity. Similarly, stabilization did not alter the mechanical response of whole tendons to overload, and did not prevent an overload‐induced thermal destabilization of collagen molecules. Our results indicate that hydrothermally labile cross‐links may not be as mechanically labile as was previously thought. © 2013 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 31:1907–1913, 2013     

Mechanical overload decreases the thermal stability of collagen in an in vitro tensile overload tendon model

Musculoskeletal soft tissue injuries are very common, yet poorly understood. We investigated molecular‐level changes in collagen caused by tensile overload of bovine tail tendons in vitro. Previous investigators concluded that tensile tendon rupture resulted in collagen denaturation, but our study suggests otherwise. Based on contemporary collagen biophysics, we hypothesized that tensile overload would lead to reduced thermal stability without change in the nativity of the molecular conformation. The thermal behavior of collagen from tail tendons ruptured in vitro at two strain rates (0.01 s^?1 and 10 s^?1) was measured by differential scanning calorimetry (DSC). The 1,000‐fold difference in strain rate was used since molecular mechanisms that determine mechanical behavior are thought to be strain rate‐dependent. DSC revealed that the collagen in tensile overloaded tendons was less thermally stable by 3° to 5°C relative to undamaged controls and was not denatured since there was no change in enthalpy of denaturation. The decrease in thermal stability occurred throughout the overloaded regions, independent of rupture site, and was greater in specimens ruptured at the lower strain rate. The deformation mechanism apparently involves disruption of the lattice structure of the collagen fibrils and greatly increases the molecular freedom of the collagen molecules, leading to reduced thermal molecular stability and the previously reported increased proteolysis. This has important implications for understanding soft tissue injuries, disease etiology and treatment, and for developing tissue engineered products with improved durability. © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res     

Stress shielding of patellar tendon: effect on small-diameter collagen fibrils in a rabbit model

The purpose of this study was to assess the effects of stress shielding on the microstructure and ultrastructure of the patellar tendon using 40 mature female Japanese white rabbits. The patellar tendon was completely released from stress by drawing the patella toward the tibial tubercle with a stainless steel wire installed between them. Microstructurally, stress shielding for 3 and 6 weeks increased the number of cells approximately fivefold, to that of the control tendon. Collagen bundles were less well oriented in the stress-shielded tendon than in the control. Ultrastructurally, small collagen fibrils with a diameter of less than 90nm increased in the stress-shielded tendon. The median collagen fibril diameter in 6-week stress-shielded tendon was significantly smaller (P 0.05) than in the control tendon (58.8% of control). The ratio of the total area of collagen fibrils to the whole visualized area in the stress-shielded patellar tendon was significantly smaller at 3 and 6 weeks than that in the control. This study demonstrated that complete stress shielding significantly affects the microstructure and ultrastructure of the patellar tendon     

Collagen V expression is crucial in regional development of the supraspinatus tendon

Manipulations in cell culture and mouse models have demonstrated that reduction of collagen V results in altered fibril structure and matrix assembly. A tissue‐dependent role for collagen V in determining mechanical function was recently established, but its role in determining regional properties has not been addressed. The objective of this study was to define the role(s) of collagen V expression in establishing the site‐specific properties of the supraspinatus tendon. The insertion and midsubstance of tendons from wild type, heterozygous and tendon/ligament‐specific null mice were assessed for crimp morphology, fibril morphology, cell morphology, as well as total collagen and pyridinoline cross‐link (PYD) content. Fibril morphology was altered at the midsubstance of both groups with larger, but fewer, fibrils and no change in cell morphology or collagen compared to the wild type controls. In contrast, a significant disruption of fibril assembly was observed at the insertion site of the null group with the presence of structurally aberrant fibrils. Alterations were also present in cell density and PYD content. Altogether, these results demonstrate that collagen V plays a crucial role in determining region‐specific differences in mouse supraspinatus tendon structure. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:2154–2161, 2016.     

Mechanisms of collagen fibril alignment in tendon injury: From tendon regeneration to artificial tendon

The process by which collagen fibrils are aligned following tendon injury remains unknown. Therefore, we analyzed the process of tendon regeneration by transmission electron microscopy, using a film model method. In mice, the Achilles tendon of medial head was transected. On day 3, after only the proximal end of the transected tendon was placed on film and kept in vivo, a translucent substance containing granules, called tendon gel, was secreted. On day 5, the granules assembled in a loose (L) layer, and coalesced tightly in a dense (D) layer, forming an L‐D‐L layered pattern. On day 10, granules showed high electron density in H layers, which developed into D‐H‐D layers on day 13. The distal end was placed on film to face the proximal end. On day 10, the tendon gel showed a D‐H‐D layer pattern. On day 11, mechanical stress from muscular constriction changed the tendon gel to aligned collagen fibrils (6 ± 2 nm in diameter). Thereafter, the diameter of the fibrils increased. Tendon gel harvested on day 5 or day 10 was pulled manually or by hanging weights (about 0.6 MPa). Aligned collagen fibrils (32 ± 7 nm in diameter) were created by traction using tendon gel harvested on day 10. © 2011 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 29:1944–1950, 2011     

Nanostructure of collagen fibrils in human nucleus pulposus and its correlation with macroscale tissue mechanics

Collagen fibrils are the main structural components of the nucleus pulposus tissue in the intervertebral discs. The structure–property relationship of the nucleus pulposus (NP) tissues is still unclear. We investigated the structure of individual collagen fibrils of the NP and evaluated its correlation with the bulk mechanical properties of the tissue. Collagen fibrils were extracted from the NP of discs retrieved from adolescents during scoliosis correction surgery, and the extracts were confirmed by SDS‐PAGE. The diameters of the individual collagen fibrils were measured through atomic force microscopy, and the compressive mechanical properties of the tissues were evaluated by confined compression. The correlations between the nanoscale morphology of the collagen fibrils and the macroscale mechanical properties of the tissues were evaluated by linear regression. The SDS‐PAGE results showed that the fibril extracts were largely composed of type II collagen. The mean diameter of the collagen fibrils was 92.1 ± 26.54 nm; the mean swelling pressure and compressive modulus of the tissues were 6.15 ± 4.3 kPa and 1.23 ± 0.7 MPa, respectively. The mean fibril diameter had no linear correlation (R² = 0.30) with the swelling pressure of the tissues. However, it had a mild linear correlation with the compressive modulus (p = 0.023, R² = 0.68). This is the first study, to our knowledge, to evaluate the nanostructure of the individual collagen fibrils of the nucleus pulposus and its relationship with macroscale mechanical properties of the NP tissues. © 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:497–502, 2010     

Structural and mechanical effects of in vivo fatigue damage induction on murine tendon

The purpose of this study was to develop and validate an in vivo mouse model of tendon fatigue and use this model to investigate and quantify the physical manifestations of fatigue damage in mouse tendon. Patellar tendons of C57BL/6J mice were fatigue loaded at 2 Hz to three endpoints (4 N peak force per cycle for 1 h, 6 N for 1 h, and 4 N for 2 h), during which hysteresis, tangent stiffness, and peak strain of each cycle were measured. Damage accumulation was then quantified using in situ histology, and each tendon was loaded monotonically to failure. Histological damage increased significantly in all three groups (≥2-fold), and monotonic stiffness decreased significantly in the 6 N, 1 h and 4 N, 2-h groups (~25%), suggesting that damage initially manifests as changes to the collagen structure of the tendon and subsequently as changes to the function. For the fatigue loading protocols used in this study, none of the evaluated real-time parameters from fatigue loading correlated with damage area fraction measured structural damage or monotonic stiffness, suggesting that they are not suited to serve as proxies for damage accumulation. In future studies, this model will be used to compare the biological response of mouse tendon to fatigue damage across genetic strains.