Stefanie Krauss

In situ multi-level analysis of viscoelastic deformation mechanisms in tendon collagen

Tendon is a hydrated multi-level fibre composite, in which time-dependent behaviour is well established. Studies indicate significant stress relaxation, considered important for optimising tissue stiffness. However, whilst this behaviour is well documented, the mechanisms associated with the response are largely unknown. This study investigates the sub-structural mechanisms occurring during stress relaxation at both the macro (fibre) and nano (fibril) levels of the tendon hierarchy. Stress relaxation followed a two-stage exponential behaviour, during which structural changes were visible at the fibre and fibril levels. Fibril relaxation and fibre sliding showed a double exponential response, while fibre sliding was clearly the largest contributor to relaxation. The amount of stress relaxation and sub-structural reorganisation increased with increasing load increments, but fibre sliding was consistently the largest contributor to stress relaxation. A simple model of tendon viscoelasticity at the fibril and fibre levels has been developed, capturing this behaviour by serially coupling a Voigt element (collagen fibril), with two Maxwell elements (non-collagenous matrix between fibrils and fibres). This multi-level analysis provides a first step towards understanding how sub-structural interactions contribute to viscoelastic behaviour. It indicates that nano- and micro-scale shearing are significant dissipative mechanisms, and the kinetics of relaxation follows a two-stage exponential decay, well fitted by serially coupled viscoelastic elements.

Inhomogeneous fibril stretching in antler starts after macroscopic yielding: indication for a nanoscale toughening mechanism.

Antler is a unique mineralized tissue, with extraordinary toughness as well as an ability to annually regenerate itself in its entirety. The high fracture resistance enables it to fulfill its biological function as a weapon and defensive guard during combats between deer stags in the rutting season. However, very little is quantitatively understood about the structural origin of the antlers high toughness. We used a unique combination of time-resolved synchrotron small angle X-ray diffraction together with tensile testing of antler cortical tissue under physiologically wet conditions. We measured the deformation at the nanoscale from changes in the meridional diffraction pattern during macroscopic stretch-to-failure tests. Our results show that on average fibrils are strained only half as much as the whole tissue and the fibril strain increases linearly with tissue strain, both during elastic and inelastic deformation. Most remarkably, following macroscopic yielding we observe a straining of some fibrils equal to the macroscopic tissue strain while others are hardly stretched at all, indicating an inhomogeneous fibrillar strain pattern at the nanoscale. This behavior is unlike what occurs in plexiform bovine bone and may explain the extreme toughness of antler compared to normal bone.

Intrafibrillar plasticity through mineral/collagen sliding is the dominant mechanism for the extreme toughness of antler bone

The inelastic deformability of the mineralised matrix in bones is critical to their high toughness, but the nanoscale mechanisms are incompletely understood. Antler is a tough bone type, with a nanostructure composed of mineralised collagen fibrils ∼100nm diameter. We track the fibrillar deformation of antler tissue during cyclic loading using in situ synchrotron small-angle X-ray diffraction (SAXD), finding that residual strain remains in the fibrils after the load was removed. During repeated unloading/reloading cycles, the fibril strain shows minimal hysteresis when plotted as a function of tissue strain, indicating that permanent plastic strain accumulates inside the fibril. We model the tensile response of the mineralised collagen fibril by a two - level staggered model - including both elastic - and inelastic regimes - with debonding between mineral and collagen within fibrils triggering macroscopic inelasticity. In the model, the subsequent frictional sliding at intrafibrillar mineral/collagen interfaces accounts for subsequent inelastic deformation of the tissue in tension. The model is compared to experimental measurements of fibrillar and mineral platelet strain during tensile deformation, measured by in situ synchrotron SAXD and wide-angle X-ray diffraction (WAXD) respectively, as well as macroscopic tissue stress and strain. By fitting the model predictions to experimentally observed parameters like the yield point, elastic modulus and post-yield slope, extremely good agreement is found between the model and experimental data at both the macro- and at the nanoscale. Our results provide strong evidence that intrafibrillar sliding between mineral and collagen leads to permanent plastic strain at both the fibril and the tissue level, and that the energy thus dissipated is a significant factor behind the high toughness of antler bone.

Self-Repair of a Biological Fiber Guided by an Ordered Elastic Framework

Incorporating sacrificial cross-links into polymers represents an exciting new avenue for the development of self-healing materials, but it is unclear to what extent their spatial arrangement is important for this functionality. In this respect, self-healing biological materials, such as mussel byssal threads, can provide important chemical and structural insights. In this study, we employ in situ small-angle X-ray scattering (SAXS) measurements during mechanical deformation to show that byssal threads consist of a partially crystalline protein framework capable of large reversible deformations via unfolding of tightly folded protein domains. The long-range structural order is destroyed by stretching the fiber but reappears rapidly after removal of load. Full mechanical recovery, however, proceeds more slowly, suggesting the presence of strong and slowly reversible sacrificial cross-links. One likely role of the highly ordered elastic framework is to bring sacrificial binding sites back into register upon stress release, facilitating bond reformation and self-repair.

Accelerated Growth Plate Mineralization and Foreshortened Proximal Limb Bones in Fetuin-A Knockout Mice

The plasma protein fetuin-A/alpha2-HS-glycoprotein (genetic symbol Ahsg) is a systemic inhibitor of extraskeletal mineralization, which is best underscored by the excessive mineral deposition found in various tissues of fetuin-A deficient mice on the calcification-prone genetic background DBA/2. Fetuin-A is known to accumulate in the bone matrix thus an effect of fetuin-A on skeletal mineralization is expected. We examined the bones of fetuin-A deficient mice maintained on a C57BL/6 genetic background to avoid bone disease secondary to renal calcification. Here, we show that fetuin-A deficient mice display normal trabecular bone mass in the spine, but increased cortical thickness in the femur. Bone material properties, as well as mineral and collagen characteristics of cortical bone were unaffected by the absence of fetuin-A. In contrast, the long bones especially proximal limb bones were severely stunted in fetuin-A deficient mice compared to wildtype littermates, resulting in increased biomechanical stability of fetuin-A deficient femora in three-point-bending tests. Elevated backscattered electron signal intensities reflected an increased mineral content in the growth plates of fetuin-A deficient long bones, corroborating its physiological role as an inhibitor of excessive mineralization in the growth plate cartilage matrix - a site of vigorous physiological mineralization. We show that in the case of fetuin-A deficiency, active mineralization inhibition is a necessity for proper long bone growth.

Tubular frameworks guiding orderly bone formation in the antler of the red deer (Cervus elaphus)

Deer antler is a bony tissue which re-grows every year after shedding. Growth speed and material properties of this tissue are truly remarkable, making it an interesting model for bone regeneration. Surprisingly, not much is known about the ultrastructure of the calcified tissues and the temporal sequence of their development during antler growth. We use a combination of imaging tools based on light and electron microscopy to characterize antler tissue at various stages of development. We observe that mineralized cartilage is first transformed into a bone framework with low degree of collagen fibril ordering at the micron level. This framework has a honeycomb-like appearance with the cylindrical pores oriented along the main antler axis. Later, this tissue is filled with primary osteons, whose collagen fibrils are mainly oriented along the pores, thus improving the antlers mechanical properties. This strongly suggests that to achieve very fast organ growth it is advantageous to have a longitudinal porous framework as an intermediate step in bone formation. The example of antler shows that geometric features of this framework are crucial, and a tubular geometry with a diameter in the order of hundred micrometers seems to be a good solution for fast framework-mediated bone formation.

Extrafibrillar diffusion and intrafibrillar swelling at the nanoscale are associated with stress relaxation in the soft collagenous matrix tissue of tendons

The mechanical behaviour of hierarchically structured soft biological tissues like tendon and cartilage shows time-dependent properties. The origin of this phenomenon is undoubtedly related to the nano- and microscale levels of structural hierarchy, but the exact mechanism is not known. Understanding this phenomenon could help us understand normal physiological tendon mechanics and how these alter with tendon degeneration, inflammation or disease. Here we measure the micro- and nanoscale structural changes in tendons during stress relaxation, using a multi-scale strain imaging method (combining confocal scanning microscopy and synchrotron small angle X-ray scattering) together with in situ mechanical testing. We tracked both the transverse (fibre swelling) as well as axial (fibre elongation) strain in both the microscale fibres (∼50 μm diameter) and the nanoscale fibrils (∼100 nm diameter). We find that macroscopic stress relaxation is accompanied by a transverse expansion of nano-fibrils together with an (opposite) reduction of diameter in the micron-scale fibres. The expansion of the fibrils at the nanoscale is more than that required for volume conservation, suggesting a stress-induced diffusion of free fluid molecules from the extrafibrillar to intrafibrillar space during stress relaxation. We propose a simple diffusion thinning mechanism whereby the proteoglycans gel layer coating the fibrils releases loosely bound water molecules upon stress-induction. The simultaneous diffusion thinning and associated water diffusion from extra- to intra-fibrillar compartments is proposed to be the driving mechanism for time-dependent behaviour in hierarchical connective tissues like tendon.