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-1ß 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. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:269-280, 2017.

Advanced quantitative imaging and biomechanical analyses of periosteal fibers in accelerated bone growth.

PURPOSE: The accepted mechanism explaining the accelerated growth following periosteal resection is that the periosteum serves as a mechanical restraint to restrict physeal growth. To test the veracity of this mechanism we first utilized Second Harmonic Generation (SHG) imaging to measure differences of periosteal fiber alignment at various strains. Additionally, we measured changes in periosteal growth factor transcription. Next we utilized SHG imaging to assess the alignment of the periosteal fibers on the bone both before and after periosteal resection. Based on the currently accepted mechanism, we hypothesized that the periosteal fibers adjacent to the physis should be more aligned (under tension) during growth and become less aligned (more relaxed) following metaphyseal periosteal resection. In addition, we measured the changes in periosteal micro- and macro-scale mechanics. METHODS: 30 seven-week old New Zealand White rabbits were sacrificed. The periosteum was imaged on the bone at five regions using SHG imaging. One centimeter periosteal resections were then performed at the proximal tibial metaphyses. The resected periosteal strips were stretched to different strains in a materials testing system (MTS), fixed, and imaged using SHG microscopy. Collagen fiber alignment at each strain was then determined computationally using CurveAlign. In addition, periosteal strips underwent biomechanical testing in both circumferential and axial directions to determine modulus, failure stress, and failure strain. Relative mRNA expression of growth factors: TGFß-1, -2, -3, Ihh, PTHrP, Gli, and Patched were measured following loading of the periosteal strips at physiological strains in a bioreactor. The periosteum adjacent to the physis of six tibiae was imaged on the bone, before and after, metaphyseal periosteal resection, and fiber alignment was computed. One-way ANOVA statistics were performed on all data. RESULTS: Imaging of the periosteum at different regions of the bone demonstrated complex regional differences in fiber orientation. Increasing periosteal strain on the resected strips increased periosteal fiber alignment (p<0.0001). The only exception to this pattern was the 10% strain on the tibial periosteum, which may indicate fiber rupture at this non-physiologic strain. Periosteal fiber alignment adjacent to the resection became less aligned while those adjacent to the physes remained relatively unchanged before and after periosteal resection. Increasing periosteal strain on the resected strips increased periosteal fiber alignment (p<0.0001). The only exception to this pattern was the 10% strain on the tibial periosteum, which may indicate fiber rupture (and consequent retraction) at this non-physiologic strain. Increasing periosteal strain revealed a significant increase in relative mRNA expression for Ihh, PTHrP, Gli, and Patched, respectively. CONCLUSION: Periosteal fibers adjacent to the growth plate do not appear under tension in the growing limb, and the alignments of these fibers remain unchanged following periosteal resection. SIGNIFICANCE: The results of this study call into question the long-accepted role of the periosteum acting as a simple mechanical tether restricting growth at the physis.

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.

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.

Spatial and frequency-based super-resolution of ultrasound images.

Modern ultrasound systems can output video images containing more spatial and temporal information than still images. Super-resolution techniques can exploit additional information but face two challenges: image registration and complex motion. In addition, information from multiple available frequencies is unexploited. Herein, we utilised these information sources to create better ultrasound images and videos, extending existing technologies for image capture. Spatial and frequency-based super-resolution processing using multiple motion estimation and frequency combination was applied to ultrasound videos of deforming models. Processed images are larger, have greater clarity and detail, and less variability in intensity between frames. Significantly, strain measurements are more accurate and precise than those from raw videos, and have a higher contrast ratio between 'tumour' and 'surrounding tissue' in a phantom model. We attribute improvements to reduced noise and increased resolution in processed images. Our methods can significantly improve quantitative and qualitative assessments of ultrasound images when compared assessments of standard images.

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.

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 tendon's 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.

Ultrasound assessment of ex vivo lung tissue properties using a fluid-filled negative pressure bath.

A relationship between tendon stress and strain and ultrasonic echo intensity has previously been defined in tendons, demonstrating a correlation between tissue stiffness and echo intensity. An analogous relationship between volume-dependent pressure changes and echo intensity changes in inflating lungs would indicate a correlation between lung compliance and echo intensity. Lung compliance is an important metric to diagnose pathologies which affect lung tissue mechanics, such as emphysema and cystic fibrosis. The goal of this study is to demonstrate a correlation between ultrasound echo intensity and lung tissue mechanics in an ex vivo model using a fluid-filled negative pressure bath design which provides a controlled environment for ultrasonic and mechanical measurements. Lungs from 4 male Sprague-Dawley rats were removed and mechanically tested via inflation and deflation in a negative pressure chamber filled with hetastarch. Specific volumes (1, 2, 3, and 4 mL) were removed from the chamber using a syringe to create negative pressure, which resulted in lung inflation. A pressure transducer recorded the pressure around the lungs. From these data, lung compliance was calculated. Ultrasound images were captured through the chamber wall to determine echo intensity (grayscale brightness in the ultrasound image), which was then related to mechanical parameters. Ultrasound images of the lung were successfully captured through the chamber wall with sufficient resolution to deduce echo intensity changes in the lung tissue. Echo intensity (0-255 scale) increased with volumetric changes (18.4 ± 5.5, 22.6 ± 5.1, 26.1 ± 7.5, and 42.9 ± 19.5 for volumetric changes of 1, 2, 3, and 4 mL) in a pattern similar to pressure (-6.8 ± 1.7, -6.8 ± 1.4, -9.4 ± 0.7, and -16.9 ± 6.8 cm H2O for 1, 2, 3, and 4 mL), reflecting changes in lung compliance. Measured rat lung tissue compliance was comparable to reported values from ex vivo lungs (0.178 ± 0.067, 0.378 ± 0.051, 0.427 ± 0.062, and 0.350 ± 0.160 mL/cm H20 for 1, 2, 3, and 4 mL), supporting proof of concept for the experimental method. Changes in echo intensity reflected changes in lung compliance in this ex vivo model, thus, supporting our hypothesis that the stiffness-related changes in echo intensity originally seen in tendon can be similarly detected in lung tissue. The presented ultrasound-based methods allowed measurement of local lung tissue compliance in a controlled environment, however, the methods could be expanded to facilitate both ex vivo and in vivo studies.

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.

Interleukin-1 receptor antagonist modulates inflammation and scarring after ligament injury.

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.

Effect of age and exercise on the viscoelastic properties of rat tail tendon.

Tendon mechanical properties are thought to degrade during aging but improve with exercise. A remaining question is whether exercise in aged animals provides sufficient regenerative, systemic stimulus to restore younger mechanical behaviors. Herein we address that question with tail tendons from aged and exercised rats, which would be subject to systemic effects but not direct loading from the exercise regimen. Twenty-four month old rats underwent one of three treadmill exercise training protocols for 12 months: sedentary (walking at 0° incline for 5 min/day), moderate (running at 0° incline for 30 min/day), or high (running at 4° incline for 30 min/day). A group of 9 month old rats were used to provide an adult control, while a group of 3 month old rats provided a young control. Tendons were harvested at sacrifice and mechanically tested. Results show significant age-dependent differences in modulus, ultimate stress, relaxation rate, and percent relaxation. Relaxation rate was strain-dependent, consistent with nonlinear superposition or Schapery models but not with quasilinear viscoelasticity (QLV). Trends in exercise data suggest that with exercise, tendons assume the elastic character of younger rats (lower elastic modulus and ultimate stress).

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 Young's 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.

Mechanical compromise of partially lacerated flexor tendons.

Tendons function to transmit loads from muscle to move and stabilize joints and absorb impacts. Functionality of lacerated tendons is diminished, however clinical practice often considers surgical repair only after 50% or more of the tendon is lacerated, the "50% rule." Few studies provide mechanical insight into the 50% rule. In this study cyclic and static stress relaxation tests were performed on porcine flexor tendons before and after a 0.5, 1.0, 2.0, or 2.75 mm deep transverse, midsubstance laceration. Elastic and viscoelastic properties, such as maximum stress, change in stress throughout each test, and stiffness, were measured and compared pre- and post-laceration. Nominal stress and stiffness parameters decreased, albeit disproportionately in magnitude, with increasing percent loss of cross-sectional area. Conversely, mean stress at the residual area (determined using remaining intact area at the laceration cross section) exhibited a marked increase in stress concentration beginning at 47.2% laceration using both specified load and constant strain analyses. The marked increase in stress concentration beginning near 50% laceration provides mechanical insight into the 50% rule. Additionally, a drastic decrease in viscoelastic stress parameters after only an 8.2% laceration suggests that time-dependent mechanisms protecting tissues during impact loadings are highly compromised regardless of laceration size.

Temporal healing in rat achilles tendon: ultrasound correlations.

The purpose of this study was to explore whether a new ultrasound-based technique correlates with mechanical and biological metrics that describe the tendon healing. Achilles tendons in 32 rats were unilaterally transected and allowed to heal without repair. At 7, 9, 14, or 29 days post-injury, tendons were collected and examined for healing via ultrasound image analysis, mechanical testing, and immunohistochemistry. Consistent with previous studies, we observe that the healing tendons are mechanically inferior (ultimate stress, ultimate load, and normalized stiffness) and biologically altered (cellular and ECM factors) compared to contralateral controls with an incomplete recovery over healing time. Unique to this study, we report: (1) Echo intensity (defined by gray-scale brightness in the ultrasound image) in the healing tissue is related to stress and normalized stiffness. (2) Elongation to failure is relatively constant so that tissue normalized stiffness is linearly correlated with ultimate stress. Together, 1 and 2 suggest a method to quantify mechanical compromise in healing tendons. (3) The amount and type of collagen in healing tendons associates with their strength and normalized stiffness as well as their ultrasound echo intensity. (4) A significant increase of periostin in the healing tissues suggests an important but unexplored role for this ECM protein in tendon healing.

Strain-induced damage reduces echo intensity changes in tendon during loading.

Tendon functionality is related to its mechanical properties. Tendon damage leads to a reduction in mechanical strength and altered biomechanical behavior, and therefore leads to compromised ability to carry out normal functions such as joint movement and stabilization. Damage can also accumulate in the tissue and lead to failure. A noninvasive method with which to measure such damage potentially could quantify structural compromise from tendon injury and track improvement over time. In this study, tendon mechanics are measured before and after damage is induced by "overstretch" (strain exceeding the elastic limit of the tissue) using a traditional mechanical test system while ultrasonic echo intensity (average gray scale brightness in a B-mode image) is recorded using clinical ultrasound. The diffuse damage caused by overstretch lowered the stress at a given strain in the tissue and decreased viscoelastic response. Overstretch also lowered echo intensity changes during stress relaxation and cyclic testing. As the input strain during overstretch increased, stress levels and echo intensity changes decreased. Also, viscoelastic parameters and time-dependent echo intensity changes were reduced.

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).

Damage mechanics of porcine flexor tendon: mechanical evaluation and modeling.

Porcine flexor tendons underwent cyclic and stress relaxation testing before and after strain exceeding elastic limit ("overstretch") to examine which mechanical parameters undergo changes following subfailure damage. From these data, we developed an "effective strain" damage model (in which the tendon is modeled as if being pulled to a lower strain). Damage was induced at three strain levels to determine the extent to which post-damage parameter changes were affected by overstretch strain level. We found that diffuse damage induced by overstretch decreased elastic and viscoelastic parameters obtained during testing. The stress response of tendon to strain is therefore altered following damage. We next compared the strain-dependent parameter behavior to damage-dependent behavior to determine the effective strain for each parameter. Effects of damage became more pronounced as strain during overstretch increased; following overstretch to 6.5, 9, or 13% strain, effective strain was 2.43 ± 0.33, 1.98 ± 0.3, or 0.88 ± 0.43% strain, respectively. By determining the effective strain and using it to calculate predicted values of post-damage mechanical parameters, it was possible to predict the stress relaxation behavior of tendons with Schapery's nonlinear viscoelastic model. Using this approach, a single parameter predicts both elastic and viscoelastic compromise from known, strain-dependent behaviors.

Time-dependent ultrasound echo changes occur in tendon during viscoelastic testing.

The viscoelastic behavior of tendons has been extensively studied in vitro. A noninvasive method by which to acquire mechanical data would be highly beneficial, as it could lead to the collection of viscoelastic data in vivo. Our lab has previously presented acoustoelasticity as an alternative ultrasound-based method of measuring tendon stress and strain by reporting a relationship between ultrasonic echo intensity (B mode ultrasound image brightness) and mechanical behavior of tendon under pseudoelastic in vitro conditions [Duenwald, S., Kobayashi, H., Frisch, K., Lakes, R., and Vanderby Jr, R., 2011, "Ultrasound Echo is Related to Stress and Strain in Tendon," J. Biomech., 44(3), pp. 424-429]. Viscoelastic properties of the tendons were not examined in that study, so the presence of time-dependent echo intensity changes has not been verified. In this study, porcine flexor tendons were subjected to relaxation and cyclic testing while ultrasonic echo response was recorded. We report that time- and strain history-dependent mechanical properties during viscoelastic testing are manifested in ultrasonic echo intensity changes. We also report that the patterns of the echo intensity changes do not directly mimic the patterns of viscoelastic load changes, but the intensity changed in a repeatable (and therefore predictable) fashion. Although mechanisms need further elucidation, viscoelastic behavior can be anticipated from echo intensity changes. This phenomenon could potentially lead to a more extensive characterization of in vivo tissue behavior.

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.