Sarah Duenwald-Kuehl

Relationship between tendon stiffness and failure: a metaanalysis

Tendon is a highly specialized, hierarchical tissue designed to transfer forces from muscle to bone; complex viscoelastic and anisotropic behaviors have been extensively characterized for specific subsets of tendons. Reported mechanical data consistently show a pseudoelastic, stress-vs.-strain behavior with a linear slope after an initial toe region. Many studies report a linear, elastic modulus, or Youngs modulus (hereafter called elastic modulus) and ultimate stress for their tendon specimens. Individually, these studies are unable to provide a broader, interstudy understanding of tendon mechanical behavior. Herein we present a metaanalysis of pooled mechanical data from a representative sample of tendons from different species. These data include healthy tendons and those altered by injury and healing, genetic modification, allograft preparation, mechanical environment, and age. Fifty studies were selected and analyzed. Despite a wide range of mechanical properties between and within species, elastic modulus and ultimate stress are highly correlated (R(2) = 0.785), suggesting that tendon failure is highly strain-dependent. Furthermore, this relationship was observed to be predictable over controlled ranges of elastic moduli, as would be typical of any individual species. With the knowledge gained through this metaanalysis, noninvasive tools could measure elastic modulus in vivo and reasonably predict ultimate stress (or structural compromise) for diseased or injured tendon.

Tenogenic differentiation of human induced pluripotent stem cell-derived mesenchymal stem cells dictated by properties of braided submicron fibrous scaffolds

Tendon and ligament (T/L) engineering is a growing area of research with potential to address the inadequacies of current T/L defect treatments. Our group previously developed braided submicron fibrous scaffolds (BSMFSs) and demonstrated the viability of BSMFSs for T/L tissue engineering. The objective of this study was to investigate the effect of fiber chemistry and braiding angle on BSMFS mechanical properties and in turn, tenogenic differentiation of human induced pluripotent stem cell-derived mesenchymal stem cells (hiPSC-MSCs) seeded on BSMFSs subjected to cyclic tensile stimulation in the absence of tenogenic medium. By varying fiber chemistry and/or braiding angle, BSMFSs with a range of mechanical properties were produced. We found that fiber chemistry dictated cell adhesion while braiding angle dictated the tissue-specific lineage commitment of hiPSC-MSCs. Scaffolds braided with large angles better supported hiPSC-MSC tenogenic differentiation as evidenced by the production of T/L-associated markers, downregulation of osteogenic markers, and expression of fibroblast-like, spindle cell morphology compared to scaffolds braided with small angles. Our results demonstrate the importance of substrate properties and mechanical stimulation on tenogenic differentiation. These results also demonstrate the versatility of BSMFSs and the potential of hiPSC-MSCs for T/L tissue engineering.

Quantification of collagen organization using fractal dimensions and Fourier transforms

Collagen fibers and fibrils that comprise tendons and ligaments are disrupted or damaged during injury. Fibrillogenesis during healing produces a matrix that is initially quite disorganized, but remodels over time to resemble, but not replicate, the original roughly parallel microstructure. Quantification of these changes is traditionally a laborious and subjective task. In this work we applied two automated techniques, fast Fourier transformation (FFT) and fractal dimension analysis (FA) to quantify the organization of collagen fibers or fibrils. Using multi-photon images of collagen fibers obtained from rat ligament we showed that for healing ligaments, FA differentiates more clearly between the different time-points during healing. Using scanning electron microscopy images of overstretched porcine flexor tendon, we showed that combining FFT and FA measures distinguishes the damaged and undamaged groups more clearly than either method separately.

Tendon Strain Measurements With Dynamic Ultrasound Images: Evaluation of Digital Image Correlation

Strain is an essential metric in tissue mechanics. Strains and strain distributions during functional loads can help identify damaged and pathologic regions as well as quantify functional compromise. Noninvasive strain measurement in vivo is difficult to perform. The goal of this in vitro study is to determine the efficacy of digital image correlation (DIC) methods to measure strain in B-mode ultrasound images. The Achilles tendons of eight male Wistar rats were removed and mechanically cycled between 0 and 1% strain. Three cine video images were captured for each specimen: (1) optical video for manual tracking of optical markers; (2) optical video for DIC tracking of optical surface markers; and (3) ultrasound video for DIC tracking of image texture within the tissue. All three imaging modalities were similarly able to measure tendon strain during cyclic testing. Manual/ImageJ-based strain values linearly correlated with DIC (optical marker)-based strain values for all eight tendons with a slope of 0.970. DIC (optical marker)-based strain values linearly correlated with DIC (ultrasound texture)-based strain values for all eight tendons with a slope of 1.003. Strain measurement using DIC was as accurate as manual image tracking methods, and DIC tracking was equally accurate when tracking ultrasound texture as when tracking optical markers. This study supports the use of DIC to calculate strains directly from the texture present in standard B-mode ultrasound images and supports the use of DIC for in vivo strain measurement using ultrasound images without additional markers, either artificially placed (for optical tracking) or anatomically in view (i.e., bony landmarks and/or muscle-tendon junctions).

Immune modulation with primed mesenchymal stem cells delivered via biodegradable scaffold to repair an Achilles tendon segmental defect

Tendon healing is a complex coordinated series of events resulting in protracted recovery, limited regeneration, and scar formation. Mesenchymal stem cell (MSC) therapy has shown promise as a new technology to enhance soft tissue and bone healing. A challenge with MSC therapy involves the ability to consistently control the inflammatory response and subsequent healing. Previous studies suggest that preconditioning MSCs with inflammatory cytokines, such as IFN‐γ, TNF‐α, and IL‐1β may accelerate cutaneous wound closure. The objective of this study was to therefore elucidate these effects in tendon. That is, the in vivo healing effects of TNF‐α primed MSCs were studied using a rat Achilles segmental defect model. Rat Achilles tendons were subjected to a unilateral 3 mm segmental defect and repaired with either a PLG scaffold alone, MSC‐seeded PLG scaffold, or TNF‐α‐primed MSC‐seeded PLG scaffold. Achilles tendons were analyzed at 2 and 4 weeks post‐injury. In vivo, MSCs, regardless of priming, increased IL‐10 production and reduced the inflammatory factor, IL‐1α. Primed MSCs reduced IL‐12 production and the number of M1 macrophages, as well as increased the percent of M2 macrophages, and synthesis of the anti‐inflammatory factor IL‐4. Primed MSC treatment also increased the concentration of type I procollagen in the healing tissue and increased failure stress of the tendon 4 weeks post‐injury. Taken together delivery of TNF‐α primed MSCs via 3D PLG scaffold modulated macrophage polarization and cytokine production to further accentuate the more regenerative MSC‐induced healing response.

Interleukin-1 Receptor Antagonist Modulates Inflammation and Scarring after Ligament Injury

Abstract Ligaments have limited regenerative potential and as a consequence, repair is protracted and results in a mechanically inferior tissue more scar-like than native ligament. We previously reported that a single injection of interleukin-1 receptor antagonist (IL-1Ra) delivered at the time of injury, decreased the number of M2 macrophage-associated inflammatory cytokines. Based on these results, we hypothesized that IL-1Ra administered after injury and closer to peak inflammation (as would occur clinically), would more effectively decrease inflammation and thereby improve healing. Since IL-1Ra has a short half-life, we also investigated the effect of multiple injections. The objective of this study was to elucidate healing of a medial collateral ligament (MCL) with either a single IL-1Ra injection delivered one day after injury or with multiple injections of IL-1Ra on days 1, 2, 3, and 4. One day after MCL injury, rats received either single or multiple injections of IL-1Ra or PBS. Tissue was then collected at days 5 and 11. Both single and multiple IL-1Ra injections reduced inflammatory cytokines, but did not change mechanical behavior. A single injection of IL-1Ra also reduced the number of myofibroblasts and increased type I procollagen. Multiple IL-1Ra doses provided no additive response and, in fact, reduced the M2 macrophages. Based on these results, a single dose of IL-1Ra was better at reducing the MCL-derived inflammatory cytokines compared to multiple injections. The changes in type I procollagen and myofibroblasts further suggest a single injection of IL-1Ra enhanced repair of the ligament but not sufficiently to improve functional behavior.

Shear load transfer in high and low stress tendons

BACKGROUND Tendon is an integral part of joint movement and stability, as it functions to transmit load from muscle to bone. It has an anisotropic, fibrous hierarchical structure that is generally loaded in the direction of its fibers/fascicles. Internal load distributions are altered when joint motion rotates an insertion site or when local damage disrupts fibers/fascicles, potentially causing inter-fiber (or inter-fascicular) shear. Tendons with different microstructures (helical versus linear) may redistribute loads differently. METHOD OF APPROACH This study explored how shear redistributes axial loads in rat tail tendon (low stress tendons with linear microstructure) and porcine flexor tendon (high stress with helical microstructure) by creating lacerations on opposite sides of the tendon, ranging from about 20% to 60% of the tendon width, to create various magnitudes of shear. Differences in fascicular orientation were quantified using polarized light microscopy. RESULTS AND CONCLUSIONS Unexpectedly, both tendon types maintained about 20% of pre-laceration stress values after overlapping cuts of 60% of tendon width (no intact fibers end to end) suggesting that shear stress transfer can contribute more to overall tendon strength and stiffness than previously reported. All structural parameters for both tendon types decreased linearly with increasing laceration depth. The tail tendon had a more rapid decline in post-laceration elastic stress and modulus parameters as well as a more linear and less tightly packed fascicular structure, suggesting that positional tendons may be less well suited to redistribute loads via a shear mechanism.

Evaluation of Global Load Sharing and Shear-Lag Models to Describe Mechanical Behavior in Partially Lacerated Tendons

The mechanical effect of a partial thickness tear or laceration of a tendon is analytically modeled under various assumptions and results are compared with previous experimental data from porcine flexor tendons. Among several fibril-level models considered, a shear-lag model that incorporates fibril-matrix interaction and a fibril-fibril interaction defined by the contact area of the interposed matrix best matched published data for tendons with shallow cuts (less than 50% of the cross-sectional area). Application of this model to the case of many disrupted fibrils is based on linear superposition and is most successful when more fibrils are incorporated into the model. An equally distributed load sharing model for the fraction of remaining intact fibrils was inadequate in that it overestimates the strength for a cut less than half of the tendons cross-sectional area. In a broader sense, results imply that shear-lag contributes significantly to the general mechanical behavior of tendons when axial loads are nonuniformly distributed over a cross section, although the predominant hierarchical level and microstructural mediators for this behavior require further inquiry.

Reproducibility and feasibility of acoustoelastography in the superficial digital flexor tendons of clinically normal horses

OBJECTIVE To evaluate the feasibility and repeatability of in vivo measurement of stiffness gradients by means of acoustoelastography in the superficial digital flexor tendons (SDFTs) of clinically normal horses. ANIMALS 15 clinically normal horses. PROCEDURES For each horse, stiffness gradient index and dispersion values for SDFTs in both forelimbs were evaluated in longitudinal orientation by use of acoustoelastography at 3 sites (5, 10, and 15 cm distal to the accessory carpal bone) by 2 observers; for each observer, data were acquired twice per site. The left forelimb was always scanned before the right forelimb. Lifting of the contralateral forelimb with the carpus flexed during image acquisition resulted in the required SDFT deformation in the evaluated limb. Interobserver repeatability, intraobserver repeatability, and right-to-left limb symmetry for stiffness gradient index and dispersion values were evaluated. RESULTS Stiffness gradient index and dispersion values for SDFTs at different locations as well as effects of age or sex did not differ significantly among the 15 horses. Interclass correlation coefficients for interobserver repeatability, intraobserver repeatability, and limb symmetry revealed good to excellent agreement (intraclass correlation coefficients, > 0.74). CONCLUSIONS AND CLINICAL RELEVANCE Results indicated that acoustoelastography is a feasible and repeatable technique for measuring stiffness gradients in SDFTs in clinically normal horses, and could potentially be used to compare healthy and diseased tendon states.

Crown Preservation of the Mandibular First Molar Tooth Impacts the Strength and Stiffness of Three Non-Invasive Jaw Fracture Repair Constructs in Dogs

Repairing mandibular body fractures presents unique challenges not encountered when repairing long bones. Large tooth roots and the presence of the inferior alveolar neurovascular bundle limit safe placement for many types of orthopedic implants. Use of non-invasive fracture repair methods have increasingly become popular and have proven safe and effective at achieving bone healing. Non-invasive fixation constructs have not been tested in dogs using cantilevered bending. Furthermore, non-invasive fracture repair constructs have not been tested at the location of a common fracture location – the mandibular first molar tooth (M1). The objectives of this study were to test the strength and stiffness of three non-invasive mandibular fracture repair constructs and to characterize the impact that tooth crown preservation has on fixation strength for fractures occurring at the M1 location. Specimens were assigned to three treatment groups: (1) composite only, (2) interdental wiring and composite (IWC), and (3) transmucosal fixation screw and composite. For each pair of mandibles, one mandible received crown amputation at the alveolar margin to simulate the effect of crown loss on fixation strength and stiffness. Regardless of the status of crown presence, IWC demonstrated the greatest bending stiffness and load to failure. With the crown removed, IWC was significantly stronger compared to other treatments. All fixation constructs were stiffer when the tooth crown was preserved. In fractures at this location, retaining the tooth crown of M1 significantly increases stiffness of interdental wiring with composite and transmucosal screw with composite constructs. If the crown of M1 was removed, IWC was significantly stronger than the other two forms of fixation.