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.

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.

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.

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.

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).

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.

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.