We measured strain in the lateral ligaments of 10 human cadaver ankles while moving the ankle joint and applying stress in a variety of ways. We studied the anterior talofibular, calcaneofibular, posterior talofibular, anterior tibiofibular, and posterior tibiofibular ligaments. Strain measurements in the ligaments were recorded continuously while the ankle was moved from dorsiflexion into plantar flexion. We then repeated measurements while applying inversion, eversion, internal rotation, and external rotation forces. Strain in the anterior talofibular ligament increased when the ankle was moved into greater degrees of plantar flexion, internal rotation, and inversion. Strain in the calcaneofibular ligament increased as the talus was dorsiflexed and inverted. These findings support the concept that the anterior talofibular and calcaneofibular ligaments function together at all positions of ankle flexion to provide lateral ankle stability. We measured maximum strain in the posterior talofibular ligament when the ankle was dorsiflexed and externally rotated. The strain in the anterior and posterior tibiofibular ligaments increased when the ankle was dorsiflexed. External rotation increased strain in the anterior tibiofibular ligament and decreased strain in the posterior tibiofibular ligament. Based upon strain measurements in the lateral ankle ligaments in various ankle joint positions, we believe the anterior talofibular ligament is most likely to tear if the ankle is inverted in plantar flexion and internally rotated. Theoretically, the calcaneofibular ligament tears primarily in inversion if the ankle is dorsiflexed; the anterior tibiofibular ligament tears in dorsiflexion, especially if combined with external rotation; and the posterior tibiofibular ligament tears with extreme dorsiflexion.

The tendon-to-bone attachment (enthesis) is a complex hierarchical tissue that connects stiff bone to compliant tendon. The attachment site at the micrometer scale exhibits gradients in mineral content and collagen orientation, which likely act to minimize stress concentrations. The physiological micromechanics of the attachment thus define resultant performance, but difficulties in sample preparation and mechanical testing at this scale have restricted understanding of structure-mechanical function. Here, microscale beams from entheses of wild type mice and mice with mineral defects were prepared using cryo-focused ion beam milling and pulled to failure using a modified atomic force microscopy system. Micromechanical behavior of tendon-to-bone structures, including elastic modulus, strength, resilience, and toughness, were obtained. Results demonstrated considerably higher mechanical performance at the micrometer length scale compared to the millimeter tissue length scale, describing enthesis material properties without the influence of higher order structural effects such as defects. Micromechanical investigation revealed a decrease in strength in entheses with mineral defects. To further examine structure-mechanical function relationships, local deformation behavior along the tendon-to-bone attachment was determined using local image correlation. A high compliance zone near the mineralized gradient of the attachment was clearly identified and highlighted the lack of correlation between mineral distribution and strain on the low-mineral end of the attachment. This compliant region is proposed to act as an energy absorbing component, limiting catastrophic failure within the tendon-to-bone attachment through higher local deformation. This understanding of tendon-to-bone micromechanics demonstrates the critical role of micrometer scale features in the mechanics of the tissue.

When mechanical factors underlie growth, development, disease or healing, they often function through local regions of tissue where deformation is highly concentrated. Current optical techniques to estimate deformation can lack precision and accuracy in such regions due to challenges in distinguishing a region of concentrated deformation from an error in displacement tracking. Here, we present a simple and general technique for improving the accuracy and precision of strain estimation and an associated technique for distinguishing a concentrated deformation from a tracking error. The strain estimation technique improves accuracy relative to other state-of-theart algorithms by directly estimating strain fields without first estimating displacements, resulting in a very simple method and low computational cost. The technique for identifying local elevation of strain enables for the first time the successful identification of the onset and consequences of local strain concentrating features such as cracks and tears in a highly strained tissue. We apply these new techniques to demonstrate a novel hypothesis in prenatal wound healing. More generally, the analytical methods we have developed provide a simple tool for quantifying the appearance and magnitude of localized deformation from a series of digital images across a broad range of disciplines.

Quantifying dynamic strain fields from time-resolved volumetric medical imaging and microscopy stacks is a pressing need for radiology and mechanobiology. A critical limitation of all existing techniques is regularization: because these volumetric images are inherently noisy, the current strain mapping techniques must impose either displacement regularization and smoothing that sacrifices spatial resolution, or material property assumptions that presuppose a material model, as in hyperelastic warping. Here, we present, validate, and apply the first three-dimensional (3D) method for estimating mechanical strain directly from raw 3D image stacks without either regularization or assumptions about material behavior. We apply the method to high-frequency ultrasound images of mouse hearts to diagnose myocardial infarction. We also apply the method to present the first ever in vivo quantification of elevated strain fields in the heart wall associated with the insertion of the chordae tendinae. The method shows promise for broad application to dynamic medical imaging modalities, including high-frequency ultrasound, tagged magnetic resonance imaging, and confocal fluorescence microscopy.

Current in vivo abdominal aortic aneurysm (AAA) imaging approaches tend to focus on maximum diameter but do not measure three-dimensional (3D) vascular deformation or strain. Complex vessel geometries, heterogeneous wall compositions, and surrounding structures can all influence aortic strain. Improved understanding of complex aortic kinematics has the potential to increase our ability to predict aneurysm expansion and eventual rupture. Here, we describe a method that combines four-dimensional (4D) ultrasound and direct deformation estimation to compute in vivo 3D Green-Lagrange strain in murine angiotensin II-induced suprarenal dissecting aortic aneurysms, a commonly used small animal model. We compared heterogeneous patterns of the maximum, first-component 3D Green-Lagrange strain with vessel composition from mice with varying AAA morphologies. Intramural thrombus and focal breakage in the medial elastin significantly reduced aortic strain. Interestingly, a dissection that was not detected with high-frequency ultrasound also experienced reduced strain, suggesting medial elastin breakage that was later confirmed via histology. These results suggest that in vivo measurements of 3D strain can provide improved insight into aneurysm disease progression. While further work is needed with both preclinical animal models and human imaging studies, this initial murine study indicates that vessel strain should be considered when developing an improved metric for predicting aneurysm growth and rupture.

Functionally graded, mineralized collagen tissues exist at soft-to-hard material attachments throughout the body. However, the details of how collagen and hydroxyapatite mineral (HA) interact are not fully understood, hampering efforts to develop tissue-engineered constructs that can assist with repair of injuries at the attachments of soft tissues to bone. In this study, spatial control of mineralization was achieved in collagen matrices using simulated body fluids (SBFs). Based upon previous observations of poor bonding between reconstituted collagen and HA deposited using SBF, we hypothesized that mineralizing collagen in the presence of fetuin (which inhibits surface mineralization) would lead to more mineral deposition within the scaffold and therefore a greater increase in stiffness and toughness compared with collagen mineralized without fetuin. We tested this hypothesis through integrated synthesis, mechanical testing and modelling of graded, mineralized reconstituted collagen constructs. Results supported the hypothesis, and further suggested that mineralization on the interior of reconstituted collagen constructs, as promoted by fetuin, led to superior bonding between HA and collagen. The results provide us guidance for the development of mineralized collagen scaffolds, with implications for bone and tendon-to-bone tissue engineering.

Partially mineralized fibrous tissue situated between tendon and bone is believed to be tougher than either tendon or bone, possibly serving as a compliant, energy absorptive, protective barrier between the two. This tissue does not reform following surgical repair (e.g., rotator cuff tendon-to-bone re-attachment) and might be a factor in the poor outcomes following such surgeries. Towards our long-term goal of tissue engineered solutions to functional tendon-to-bone re-attachment, we tested the hypotheses that partially mineralized fibrous matrices can derive toughness from mobility of mineral along their fibers, and that in such cases toughness is maximized at levels of mineralization sufficiently low to allow substantial mobility. Nanofibrous electrospun poly(lactic-co-glycolic acid) (PLGA) scaffolds mineralized for prescribed times were fabricated as model systems to test these hypotheses. Tensile tests performed at varying angles relative to the dominant fiber direction confirmed that mineral cross-linked PLGA nanofibers without adhering to them. Peel tests revealed that fracture toughness increased with mineralization time up to a peak value, then subsequently decreased with increasing mineralization time back to the baseline toughness of unmineralized scaffolds. These experimental results were predicted by a theoretical model combining mineral growth kinetics with fracture energetics, suggesting that toughness increased with mineralization time until mineral mobility was attenuated by steric hindrance, then returned to baseline levels following the rigid percolation threshold. Results supported our hypotheses, and motivate further study of the roles of mobile mineral particles in toughening the tendon-to-bone attachment.

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