A specialized protocol for mechanical testing of isolated networks of type II collagen.

The mechanical responses of most soft biological tissues rely heavily on networks of collagen fibers, thus quantifying the mechanics of both individual collagen fibers and networks of these fibers advances understanding of biological tissues in health and disease. The mechanics of type I collagen are well-studied and quantified. Yet no data exist on the tensile mechanical responses of individual type II collagen fibers nor of isolated networks comprised of type II collagen. We aimed to establish methods to facilitate studies of networked and individual type II collagen fibers within the native networked structure, specifically to establish best practices for isolating and mechanically testing type II collagen networks in tension. We systematically investigated mechanical tests of networks of type II collagen undergoing uniaxial extension, and quantified ranges for each of the important variables to help ensure that the experiment itself does not affect the measured mechanical parameters. Specifically we determined both the specimen (establishing networks of isolated collagen, the footprint and thickness of the specimen) and the mechanical test (both the device and the strain rate) to establish a repeatable and practical protocol. Mechanical testing of isolated networks of type II collagen fibers leveraging this protocol will lead to better understanding of the mechanics both of these networks and of the individual fibers. Such understanding may aid in developing and testing therapeutics, understanding inter-constituent interactions (and their roles in bulk-tissue biomechanics), investigating mechanical/biochemical modifications to networked type II collagen, and proposing, calibrating, and validating constitutive models for finite element analyses.

Cartilage and collagen mechanics under large-strain shear within in vivo and at supraphysiogical temperatures.

Human joints, particularly those of extremities, experience a significant range of temperatures in vivo. Joint temperature influences the mechanics of both joint and cartilage, and the mechanics of cartilage can affect the temperature of both joint and cartilage. Thermal treatments and tissue repairs, such as thermal chondroplasty, and ex vivo tissue engineering may also expose cartilage to supraphysiological temperatures. Furthermore, although cartilage undergoes principally compressive loads in vivo, shear strain plays a significant role at larger compressive strains. Thus, we aimed to determine whether and how the bulk mechanical responses of cartilage undergoing large-strain shear change (1) within the range of temperatures relevant in vivo, and (2) both during and after supraphysiological thermal treatments. We completed large-strain shear tests (10 and 15%) at four thermal conditions: 24∘C and 40∘C to span the in vivo range, and 70∘C and 24∘C repeated after 70∘C to explore mechanics during and after potential treatments. We calculated the bulk mechanical responses (strain-energy dissipation densities, peak-to-peak shear stresses, and peak-effective shear moduli) as of function of temperature and used statistical methods to probe significant differences. To probe the mechanisms underlying differences we assessed specimens, principally the type II collagen, with imaging (second harmonic generation and transmission electron microscopies, and histology) and assessed the temperature-dependent mechanics of type II collagen molecules within cartilage using steered molecular dynamics simulations. Our results suggest that the bulk mechanical responses of cartilage depend significantly on temperature both within the in vivo range and at supraphysiological temperatures, showing significant reductions in all mechanical measures with increasing temperature. Using imaging and simulations we determined that one underlying mechanism explaining our results may be changes in the molecular deformation profiles of collagen molecules versus temperature, likely compounded at larger length scales. These new insights into the mechanics of cartilage and collagen may suggest new treatment targets for damaged or osteoarthritic cartilage.

The zonal evolution of collagen-network morphology quantified in early osteoarthritic grades of human cartilage.

Objective: We aimed to directly quantify the zone-specific evolution in morphology of collagen fibers and networks in human cartilage during the progression of early osteoarthritis. Collagen fibers exhibit depth-dependent orientations and diameters crucial to their mechanical roles. Cartilage degenerates in osteoarthritis, affecting the morphology of the collagen network and ultimately the intra-tissue mechanics. Design: We obtained specimens of human cartilage from healthy human knees ( n = 3 ) and from total knee arthroplasties ( n = 5 ). We utilized TEM and custom image analyses to visualize and quantify distributions in principal orientation, dispersion (about the principal orientation), and diameter of collagen fibers in the early grades of OA within each through-thickness zone. We then used histological and statistical analyses to probe for significant changes in the zone-specific evolution in collagen-network morphology as a function of Osteoarthritis Research Society International (OARSI) grade. Results: Dispersion in the alignment of collagen fibers increased with progression of early OA in both the superficial and deep zones, and decreased in the middle zone, while principal orientation did not change significantly. The non-normal and right-skewed distributions in fiber diameters did not evolve with the progression of OA. Conclusions: We provide the research community with quantitative data (1) on the through-thickness morphology of collagen in healthy cartilage and (2) on the evolution of through-thickness morphology of collagen with progressing early OA. Such quantitative data facilitate an improved mechanistic understanding of the progression of OA, and may facilitate identifying image-based biomarkers and treatment targets, and ultimately finding clinical interventions for OA.