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The aim of the current study is to demonstrate the feasibility of radiofrequency (RF) pulses generated via an optimal control (OC) algorithm to perform magnetic resonance elastography (MRE) and quantify the mechanical properties of materials with very short transverse relaxation times (T2 < 5 ms) for the first time. OC theory applied to MRE provides RF pulses that bring isochromats from the equilibrium state to a fixed target state, which corresponds to the phase pattern of a conventional MRE acquisition. Such RF pulses applied with a constant gradient allow to simultaneously perform slice selection and motion encoding in the slice direction. Unlike conventional MRE, no additional motion-encoding gradients (MEGs) are needed, enabling shorter echo times. OC pulses were implemented both in turbo spin echo (OC rapid acquisition with refocused echoes [RARE]) and ultrashort echo time (OC UTE) sequences to compare their motion-encoding efficiency with the conventional MEG encoding (classical MEG MRE). MRE experiments were carried out on agar phantoms with very short T2 values and on an ex vivo bovine tendon. Magnitude images, wave field images, phase-to-noise ratio (PNR), and shear storage modulus maps were compared between OC RARE, OC UTE, and classical MEG MRE in samples with different T2 values. Shear storage modulus values of the agar phantoms were in agreement with values found in the literature, and that of the bovine tendon was corroborated with rheometry measurements. Only the OC sequences could encode motion in very short T2 samples, and only OC UTE sequences yielded magnitude images enabling proper visualization of short T2 samples and tissues. The OC UTE sequence produced the best PNRs, demonstrating its ability to perform anatomical and mechanical characterization. Its success warrants in vivo confirmation in further studies., (© 2024 John Wiley & Sons Ltd.)

Background: Robot-assisted microsurgery (RAMS) is gradually becoming the preferred method for some delicate surgical procedures. However, the lack of haptic feedback reduces the safety of the surgery. Surgeons are unable to feel the grasping force between surgical instruments and the patient's tissues, which can easily lead to grasping failure or tissue damage., Methods: This paper proposes a tendon-driven grasping force feedback mechanism, consisting of a follower hand and a leader hand, to address the lack of grasping force feedback in flexible surgical robots. Considering the friction in the tendon transmission process, a grasping force estimation model is established for the follower hand. The admittance control model is designed for force/position control of the leader hand., Results: Through experimental validation, it has been confirmed that the grasping force sensing range of the follower hand is 0.5-5 N, with a sensing accuracy of 0.3 N. The leader hand is capable of providing feedback forces in the range of 0-5 N, with a static force accuracy of 0.1 N., Conclusions: The designed mechanism and control strategy can provide the grasping force feedback function. Future work will focus on improving force feedback performance., Trial Registration: This research has no clinical trials., (© 2024 John Wiley & Sons Ltd.)

Tendon allograft and xenograft processing often involves one or more steps of freezing and thawing. As failure strength is an important graft consideration, this study aimed to evaluate effects on failure properties when varying freeze-thaw conditions. Kangaroo tendons, a potential xenograft source, were used to evaluate changes in ultimate tensile strength (UTS), failure strain and elastic modulus after exposure to different freezer-storage temperatures (-20°C vs. -80°C), storage durations (1, 3, 6, 9, or 12 months), number of freeze-thaw cycles (1, 2, 3, 4, 5, or 10), or freeze-thaw temperature ranges (including freezing in liquid nitrogen to thawing at 37°C). Tendons stored for 6 or more months had significantly increased UTS and elastic modulus compared with 1 or 3 months of storage. This increase occurred irrespective of the freezing temperature (-20°C vs. -80°C) or the number of freeze-thaw cycles (1 vs. 10). In contrast, UTS, failure strain and the elastic modulus were no different between storage temperatures, number of freeze-thaw cycles and multiple freeze-thaw cycles across a range of freeze and thaw temperatures. Common freeze-thaw protocols did not negatively affect failure properties, providing flexibility for graft testing, storage, transportation and decellularisation procedures. However, the change in properties with the overall storage duration has implications for assessing the consistent performance of grafts stored for short versus extended periods of time (<6 months vs. >6 months), and the interpretation of data obtained from tissues of varying or unknown storage durations., (© 2024 The Authors. Journal of Orthopaedic Research® published by Wiley Periodicals LLC on behalf of Orthopaedic Research Society.)

Tendons transmit the muscle contraction forces to bones and drive joint movement throughout life. While extensive research have indicated the essentiality of mechanical forces on tendon development, a comprehensive understanding of the fundamental role of mechanical forces still needs to be impaerted. This scoping review aimed to summarize the current knowledge about the role of mechanical forces during the tendon developmental phase. The electronic database search using PubMed, performed in May 2023, yielded 651 articles, of which 16 met the prespecified inclusion criteria. We summarized and divided the methods to reduce the mechanical force into three groups: loss of muscle, muscle dysfunction, and weight-bearing regulation. In contrast, there were few studies to analyze the increased mechanical force model. Most studies suggested that mechanical force has some roles in tendon development in the embryo to postnatal phase. However, we identified species variability and methodological heterogeneity to modulate mechanical force. To establish a comprehensive understanding, methodological commonality to modulate the mechanical force is needed in this field. Additionally, summarizing chronological changes in developmental processes across animal species helps to understand the essence of developmental tendon mechanobiology. We expect that the findings summarized in the current review serve as a groundwork for future study in the fields of tendon developmantal biology and mechanobiology., (© 2023 The Authors. Developmental Dynamics published by Wiley Periodicals LLC on behalf of American Association for Anatomy.)

Rotator cuff tendinopathy has a multifactorial etiology, with both aging and external compression found to influence disease progression. However, tendon's response to these factors is still poorly understood and in vivo animal models make it difficult to decouple these effects. Therefore, we developed an explant culture model that allows us to directly apply compression to tendons and then observe their biological responses. Using this model, we applied a single acute compressive injury to C57BL/6J flexor digitorum longus tendon explants and observed changes in viability, metabolic activity, matrix composition, matrix biosynthesis, matrix structure, gene expression, and mechanical properties. We hypothesized that a single acute compressive load would result in an injury response in tendon and that this effect would be amplified in aged tendons. We found that young tendons had increased matrix turnover with a decrease in small leucine-rich proteoglycans, increase in compression-resistant proteoglycan aggrecan, increase in collagen synthesis, and an upregulation of collagen-degrading MMP-9. Aged tendons lacked any of these adaptive responses and instead had decreased metabolic activity and collagen synthesis. This implies that aged tendons lack the adaptation mechanisms required to return to homeostasis, and therefore are at greater risk for compression-induced injury. Overall, we present a novel compressive injury model that demonstrates lasting age-dependent changes and has the potential to examine the long-term response of tendon to a variety of compressive loading conditions., (© 2023 Orthopaedic Research Society.)

The ability of the median nerve (MN) to adapt in response to altered carpal tunnel conditions is important to mitigate compressive stress on the nerve. We assessed changes in MN deformation and position throughout the entire time course of hand force exertions. Fourteen right-handed participants ramped up force from 0% to 50% of maximal voluntary force (MVF) before ramping force back down in three different hand force exertion tasks (pulp pinch, chuck pinch, power grip). Pinch and grip forces were measured with a digital dynamometer, which were time synchronized with transverse carpal tunnel images obtained via ultrasound. Ultrasound images were extracted in 10% increments between 0% and 50% MVF while ramping force up (loading phase) and down (unloading phase). MN deformation and position relative to the flexor digitorum superficialis tendon of the long finger were assessed in concert. During loading, the nerve became more circular while displacing dorsally and ulnarly. These changes primarily occurred at the beginning of the hand force exertions while ramping force up from 0% to 20%, with very little change between 20% and 50% MVF. Interestingly, deformation and position changes during loading were not completely reversed during unloading while ramping force down. These findings indicate an initial reorganization of carpal tunnel structures. Mirrored changes in nerve deformation and position may also reflect strain-related characteristics of adjoining subsynovial connective tissue. Regardless, time-varying changes in nerve deformation and position appear to be an important accommodative mechanism in the healthy carpal tunnel in response to gripping and pinching tasks., (© 2023 The Authors. Journal of Orthopaedic Research® published by Wiley Periodicals LLC on behalf of Orthopaedic Research Society.)

Tendons are unique dense connective tissues with discrete zones having specific structure and function. They are juxtaposed with other tissues (e.g., bone, muscle, and fat) with different compositional, structural, and mechanical properties. Additionally, tendon properties change drastically with growth and development, disease, aging, and injury. Consequently, there are unique challenges to performing high quality histological assessment of this tissue. To address this need, histological assessment was one of the breakout session topics at the 2022 Orthopaedic Research Society (ORS) Tendon Conference hosted at the University of Pennsylvania. The purpose of the breakout session was to discuss needs from members of the ORS Tendon Section related to histological procedures, data presentation, knowledge dissemination, and guidelines for future work. Therefore, this review provides a brief overview of the outcomes of this discussion and provides a set of guidelines, based on the perspectives from our laboratories, for histological assessment to assist researchers in their quest to utilize these techniques to enhance the outcomes and interpretations of their studies., (© 2023 Orthopaedic Research Society.)

The wide variety of cell and tissue culture systems used to study and engineer tendons can make it difficult to choose the best approach and "optimal" culture conditions to test a given hypothesis. Therefore, a breakout session was organized at the 2022 ORS Tendon Section Meeting that focused on establishing a set of guidelines for conducting cell and tissue culture studies of tendon. This paper summarizes the outcomes of that discussion and presents recommendations for future studies. In the case of studying tendon cell behavior, cell and tissue culture systems are reductionist models in which the culture conditions should be strictly defined to approximate the in vivo condition as closely as possible. In contrast, for tissue engineering tendon replacements, the culture conditions do not need to replicate native tendon, but the outcome measures for success should be narrowly defined for the specific clinical application. Common recommendations for both applications are that researchers should perform a baseline phenotypic characterization of the cells that are ultimately used for experimentation. For models of tendon cell behavior, culture conditions should be well justified by existing literature and meticulously reported, tissue explant viability should be assessed, and comparisons to in vivo conditions should be made to determine baseline physiological relevance. For tissue engineering applications, the functional/structural/compositional outcome targets should be defined by the specific tendons they seek to replace, with key biologic and material properties prioritized for construct assessment. Lastly, when engineering tendon replacements, researchers should utilize clinically approved cGMP materials to facilitate clinical translation., (© 2023 The Authors. Journal of Orthopaedic Research® published by Wiley Periodicals LLC on behalf of Orthopaedic Research Society.)

Tendons are critical for the biomechanical function of joints. Tendons connect muscles to bones and allow for the transmission of muscle forces to facilitate joint motion. Therefore, characterizing the tensile mechanical properties of tendons is important for the assessment of functional tendon health and efficacy of treatments for acute and chronic injuries. In this guidelines paper, we review methodological considerations, testing protocols, and key outcome measures for mechanical testing of tendons. The goal of the paper is to present a simple set of guidelines to the nonexpert seeking to perform tendon mechanical tests. The suggested approaches provide rigorous and consistent methodologies for standardized biomechanical characterization of tendon and reporting requirements across laboratories., (© 2023 Orthopaedic Research Society.)

Tissue level properties are commonly studied using histological stains assessed with qualitative scoring methods. As qualitative evaluation is typically insensitive, quantitative analysis provides additional information about pathological mechanisms, but cannot capture structural heterogeneity across cell subpopulations. However, molecular analyses of cell and nuclear behavior have identified that cell and more recently also nuclear shape are highly associated with cell function and malfunction. This study combined a Visually Aided Morpho-Phenotyping Image Recognition analysis that automatically segments cells based on their shape with an added capacity to further discriminate between cells in certain protein-rich extracellular matrix regions. We used tendon as a model system given the enormous changes in organization and cell and nuclear shape they undergo during aging and injury. Our results uncover that multiple shape modes of nuclei exist during maturity and aging in rat tendon and that distinct subgroups of cell nuclei shapes exist in proteoglycan-rich regions during aging. With injury, several immunomarkers (αSMA, CD31, CD146) were associated with more rounded shape modes. In human tendons, the cell nuclei at sites of injury were found to be more rounded relative to uninjured tissues. To conclude, the tendon tissue changes occurring during aging and injury could be associated with a variation in cell nuclear morphology and the appearance of various region-specific subpopulations. Thus, the methodologies developed allow for a deeper understanding of cell heterogeneity during tendon aging and injury and may be extended to study further clinical applications., (© 2023 Orthopaedic Research Society.)

The small leucine-rich proteoglycans, decorin and biglycan, are minor components of the tendon extracellular matrix that regulate fibrillogenesis and matrix assembly. Our study objective was to define the temporal roles of decorin and biglycan during tendon healing using inducible knockout mice to include genetic knockdown at specific phases of healing: time of injury, the proliferative phase, and the remodeling phase. We hypothesized that knockdown of decorin or biglycan would adversely affect tendon healing, and that by prescribing the timing of knockdown, we could elucidate the temporal roles of these proteins during healing. Contrary to our hypothesis, decorin knockdown did not affect tendon healing. However, when biglycan was knocked down, either alone or coupled with decorin, tendon modulus was increased relative to wild-type mice, and this finding was consistent among all induction timepoints. At 6 weeks postinjury, we observed increased expression of genes associated with the extracellular matrix and growth factor signaling in the biglycan knockdown and compound decorin-biglycan knockdown tendons. Interestingly, these groups demonstrated opposing trends in gene expression as a function of knockdown-induction timepoint, highlighting distinct temporal roles for decorin and biglycan. In summary, this study finds that biglycan plays multiple functions throughout tendon healing, with the most impactful, detrimental role likely occurring during late-stage healing. Statement of clinical importance: This study helps to define the molecular factors that regulate tendon healing, which may aid in the development of new clinical therapies., (© 2023 Orthopaedic Research Society.)

The subsynovial connective tissue is an integral component of flexor tendon gliding in the carpal tunnel, which is strained during longitudinal tendon displacement. We tested the effects of repetition frequency and finger load on flexor tendon function throughout active finger movement. Eleven participants performed metacarpophalangeal joint flexion/extension of the long finger cyclically at three repetition frequencies (0.75, 1.00, 1.25 Hz) and two finger loads (3.5, 7 N). Relative displacement between the flexor digitorum superficialis tendon and subsynovial connective tissue was assessed as the shear-strain index with color ultrasound throughout the entire time history of finger flexion and extension. In addition, long finger joint angles were measured with electrogoniometry while flexor digitorum superficialis and extensor digitorum muscle activities were measured with fine-wire electromyography to characterize the finger movements. The shear-strain index increased with greater finger flexion (p = 0.001), representing higher relative displacement between tendon and subsynovial connective tissue; however, no changes were observed throughout finger extension. The shear-strain index also increased with higher repetition frequencies (p = 0.013) and finger loads (p = 0.029), further modulating time-dependent effects during finger flexion versus extension. Using ultrasound, we characterized the time-dependent response of the shear-strain index, in vivo, providing valuable data on flexor tendon function during active finger movement. Our results infer greater subsynovial connective tissue strain and shear during repetitive and forceful finger movements. Future research characterizing time-dependent effects in carpal tunnel syndrome patients may further elucidate the relations between subsynovial connective tissue function, damage, and carpal tunnel syndrome., (© 2023 The Authors. Journal of Orthopaedic Research® published by Wiley Periodicals LLC on behalf of Orthopaedic Research Society.)

Enriched in glycolytic enzymes, paucicellular and hypovascular intrasynovial flexor tendons fail to mount an effective healing response after injury and repair. In contrast, well-vascularized extrasynovial flexor tendons possess high levels of oxidative phosphorylation (OXPHOS) enzymes and have a markedly improved healing capacity. This study was designed to compare the metabolic profiles of the two types of tendons and to evaluate the impact of metabolic reprogramming on early intrasynovial tendon healing in a clinically relevant canine model. Results showed that healthy intrasynovial tendons expressed higher levels of PDK1 and GAPDH and lower levels of SCX and IGF1 than did extrasynovial tendons. PDK1 encodes a subtype of pyruvate dehydrogenase kinase (PDK) that inhibits OXPHOS. Consistently, ATP production via glycolysis was favored in intrasynovial tendon cells whereas OXPHOS was the preferred pathway in extrasynovial tendon cells. Inhibition of glycolysis in vitro increased SCX expression in intrasynovial tendon cells. Therefore, dichloroacetate (DCA), a PDK1 inhibitor, was used in vivo to shift intrasynovial tendon ATP production from glycolysis to OXPHOS. Oral DCA administration reduced serum lactate concentration and increased acetyl-CoA content in repaired intrasynovial tendons and led to reduced TLR4 and IL1B and increased IGF1, SCX, and TGFB3 expressions in treated intrasynovial tendons compared to controls. Immunohistochemistry staining with anti-Ki67 and anti-CD31 antibodies revealed marked increases in cellularity and neovascularization in treated intrasynovial tendons. Clinical significance: The findings of this experiment indicate that improved gene expression and histological outcomes can be achieved by regulating glucose metabolism in the early stages following intrasynovial tendon repair., (© 2022 Orthopaedic Research Society. Published by Wiley Periodicals LLC.)

Decorin and biglycan are two small leucine-rich proteoglycans (SLRPs) that regulate collagen fibrillogenesis and extracellular matrix assembly in tendon. The objective of this study was to determine the individual roles of these molecules in maintaining the structural and mechanical properties of tendon during homeostasis in mature mice. We hypothesized that knockdown of decorin in mature tendons would result in detrimental changes to tendon structure and mechanics while knockdown of biglycan would have a minor effect on these parameters. To achieve this objective, we created tamoxifen-inducible mouse knockdown models targeting decorin or biglycan inactivation. This enables the evaluation of the roles of these SLRPs in mature tendon without the abnormal tendon development caused by conventional knockout models. Contrary to our hypothesis, knockdown of decorin resulted in minor alterations to tendon structure and no changes to mechanics while knockdown of biglycan resulted in broad changes to tendon structure and mechanics. Specifically, knockdown of biglycan resulted in reduced insertion modulus, maximum stress, dynamic modulus, stress relaxation, and increased collagen fiber realignment during loading. Knockdown of decorin and biglycan produced similar changes to tendon microstructure by increasing the collagen fibril diameter relative to wild-type controls. Biglycan knockdown also decreased the cell nuclear aspect ratio, indicating a more spindle-like nuclear shape. Overall, the extensive changes to tendon structure and mechanics after knockdown of biglycan, but not decorin, provides evidence that biglycan plays a major role in the maintenance of tendon structure and mechanics in mature mice during homeostasis., (© 2022 Orthopaedic Research Society. Published by Wiley Periodicals LLC.)

Mechanical stress is involved in the onset of sports-related enthesopathy. Although the amount of exercise undertaken is a recognized problem during disease onset, changes in muscle contraction type are also involved in the increase in mechanical stress during exercise. This study aimed to clarify the effects of increased mechanical stress associated with muscle contraction type and amount of exercise on enthesis. Twenty mice underwent treadmill exercise, and the muscle contraction type and overall load during exercise were adjusted by varying the angle and speed conditions. Histological analysis was used to the cross-sectional area of the muscle; area of the enthesis fibrocartilage (FC), and expression of inflammation-, degeneration-, and calcification-related factors in the FC area. In addition, the volume and structure of the bone and FC area were examined using microcomputer imaging. Molecular biological analysis was conducted to compare relative expression levels of inflammation and cytokine-related factors in tendons. The Overuse group, which increased the amount of exercise, showed no significant differences in parameters compared to the sedentary mice (Control group). The mice subjected to slow-speed downhill running (Misuse group) showed pathological changes compared to the Control and Overuse groups, despite the small amount of exercise. Thus, the enthesis FC area may be altered by local mechanical stress that would be increased by eccentric muscle contraction rather than by mechanical stress that increases with the overall amount of exercise. Clinical Significance: The muscle contraction type might be more involved in the onset of sports-related enthesopathy rather than the amount of exercise., (© 2021 Orthopaedic Research Society. Published by Wiley Periodicals LLC.)

Rapid and functional bone-tendon (B-T) healing remains a difficulty in clinical practice. Tissue engineering has emerged as a promising strategy to address this problem. However, the majority of tissue engineering scaffolds are loaded with stem cells to enhance the regenerability in B-T healing, which is complicated and inconvenient for clinical application. Accordingly, developing a cell-free scaffold with chemotactic function and chondrogenic inducibility may be an effective approach. In this study, a collagen affinity peptide derived from the A3 domain of von Willebrand factor (a hemostasis factor) was fused into the C-terminal of a stromal cell-derived factor-1α (SDF-1α) to synthesize a recombinant SDF-1α capable of binding collagen and chemotactic activity. The recombinant SDF-1α was then tethered on the collagen fibers of a book-shaped acellular fibrocartilage scaffold (BAFS), thus fabricating a novel scaffold (C-SDF-1α/BAFS) with chemotactic function and chondrogenic inducibility. In vitro tests determined that this scaffold was noncytotoxic and biomimetic, could attract stem cells migrating to the scaffold using sustainably released C-SDF-1α, and inducedthe interacting stem cells down the chondrogenic lineage. In vivo, the C-SDF-1α/BAFS significantly enhanced the B-T healing in a rabbit partial patellectomy model, as shown by the larger cartilaginous metaplasia region, better fibrocartilage regeneration, additional bone formation, and improved biomechanical properties. Therefore, the findings of the study demonstrate that the C-SDF-1α/BAFS could potentially be applied for B-T healing., (© 2020 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.)

Tendons are specialized matrix-rich connective tissues that transmit forces from muscle to bone and are essential for movement. As tissues that frequently transfer large mechanical loads, tendons are commonly injured in patients of all ages. Following injury, mammalian tendons heal poorly through a slow process that forms disorganized fibrotic scar tissue with inferior biomechanical function. Current treatments are limited and patients can be left with a weaker tendon that is likely to rerupture and an increased chance of developing degenerative conditions. More effective, alternative treatments are needed. However, our current understanding of tendon biology remains limited. Here, we emphasize why expanding our knowledge of tendon development, healing, and regeneration is imperative for advancing tendon regenerative medicine. We provide a comprehensive review of the current mechanisms governing tendon development and healing and further highlight recent work in regenerative tendon models including the neonatal mouse and zebrafish. Importantly, we discuss how present and future discoveries can be applied to both augment current treatments and design novel strategies to treat tendon injuries., (© 2020 American Association of Anatomists.)

Bioreactors are powerful tools with the potential to model tissue development and disease in vitro. For nearly four decades, bioreactors have been used to create tendon and ligament tissue-engineered constructs in order to define basic mechanisms of cell function, extracellular matrix deposition, tissue organization, injury, and tissue remodeling. This review provides a historical perspective of tendon and ligament bioreactors and their contributions to this advancing field. First, we demonstrate the need for bioreactors to improve understanding of tendon and ligament function and dysfunction. Next, we detail the history and evolution of bioreactor development and design from simple stretching of explants to fabrication and stimulation of two- and three-dimensional constructs. Then, we demonstrate how research using tendon and ligament bioreactors has led to pivotal basic science and tissue-engineering discoveries. Finally, we provide guidance for new basic, applied, and clinical research utilizing these valuable systems, recognizing that fundamental knowledge of cell-cell and cell-matrix interactions combined with appropriate mechanical and chemical stimulation of constructs could ultimately lead to functional tendon and ligament repairs in the coming decades., (© 2020 Orthopaedic Research Society. Published by Wiley Periodicals LLC.)

Extracellular matrix (ECM) determines the physiological function of all tissues, including musculoskeletal tissues. In tendon, ECM provides overall tissue architecture, which is tailored to match the biomechanical requirements of their physiological function, that is, force transmission from muscle to bone. Tendon ECM also constitutes the microenvironment that allows tendon-resident cells to maintain their phenotype and that transmits biomechanical forces from the macro-level to the micro-level. The structure and function of adult tendons is largely determined by the hierarchical organization of collagen type I fibrils. However, non-collagenous ECM proteins such as small leucine-rich proteoglycans (SLRPs), ADAMTS proteases, and cross-linking enzymes play critical roles in collagen fibrillogenesis and guide the hierarchical bundling of collagen fibrils into tendon fascicles. Other non-collagenous ECM proteins such as the less abundant collagens, fibrillins, or elastin, contribute to tendon formation or determine some of their biomechanical properties. The interfascicular matrix or endotenon and the outer layer of tendons, the epi- and paratenon, includes collagens and non-collagenous ECM proteins, but their function is less well understood. The ECM proteins in the epi- and paratenon may provide the appropriate microenvironment to maintain the identity of distinct tendon cell populations that are thought to play a role during repair processes after injury. The aim of this review is to provide an overview of the role of non-collagenous ECM proteins and less abundant collagens in tendon development and homeostasis. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 38:23-35, 2020., (© 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.)

Ligament and tendon injuries are common problems in orthopedics. There is a need for treatments that can expedite nonoperative healing or improve the efficacy of surgical repair or reconstruction of ligaments and tendons. Successful biologically-based attempts at repair and reconstruction would require a thorough understanding of normal tendon and ligament healing. The inflammatory, proliferative, and remodeling phases, and the cells involved in tendon and ligament healing will be reviewed. Then, current research efforts focusing on biologically-based treatments of ligament and tendon injuries will be summarized, with a focus on stem cells endogenous to tendons and ligaments. Statement of clinical significance: This paper details mechanisms of ligament and tendon healing, as well as attempts to apply stem cells to ligament and tendon healing. Understanding of these topics could lead to more efficacious therapies to treat ligament and tendon injuries. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 38:7-12, 2020., (© 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.)

Tendon injuries are very common and disrupt the transmission of forces from muscle to bone, leading to impaired function and quality of life. Successful restoration of tendon function after injury is a challenging clinical problem due to the pathological, scar-mediated manner in which the tendons heal. Currently, there are no standard treatments to modulate scar tissue formation and improve tendon healing. A major limitation to the identification of therapeutic candidates has been the reliance on terminal endpoint metrics of healing in pre-clinical studies, which require a large number of animals and result in destruction of the tissue. To address this limitation, we have identified quantification of scar tissue volume (STV) from ultrasound (US) imaging as a longitudinal, non-invasive metric of tendon healing. STV was strongly correlated with established endpoint metrics of gliding function including gliding resistance and metatarsophalangeal (MTP) flexion angle. However, no associations were observed between STV and structural or material properties. To define the sensitivity of STV to identify differences between functionally discrete tendon healing phenotypes, we utilized S100a4 haploinsufficient mice (S100a4 GFP/+ ), which heal with improved gliding function relative to wild-type (WT) littermates. A significant decrease in STV was observed in S100a4 GFP/+ repairs, relative to WT at day 14. Taken together, these data suggest US quantification of STV as a means to facilitate the rapid screening of biological and pharmacological interventions to improve tendon healing, and identify promising therapeutic targets, in an efficient, cost-effective manner. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:2476-2485, 2019., (© 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.)

Dorso-ventral oscillations of cetacean caudal flukes generate lift-based thrust for swimming. Movements of the flukes are actuated by epaxial and hypaxial muscles through caudal tendons inserting onto vertebrate in the peduncle. To determine if the caudal tendons in the peduncle affect the flexibility of the flukes, we must understand how the tendons from axial muscles insert onto the caudal vertebrate. The purpose of this study was to provide a detailed description of the various tendons within the cetacean peduncle with regard to their role in swimming and flexibility. Dissection of the peduncle and flukes of multiple odontocete species showed that there were two distinct epaxial tendon sets within the peduncle: (1) extensor caudae medialis tendon (ECM) and (2) extensor caudae lateralis tendon (ECL). There is one distinct hypaxial tendon set, the medial tendon of the hypaxialis lumborum (MHL). The ECM and MHL tendons inserted serially onto caudal vertebrae while the ECL inserted exclusively onto the terminal vertebrae posterior to the fluke insertion. It is typical that tendons insert onto bone, however, the connection to the core fibrous layer of the flukes suggests an element of active control of the flexibility of the flukes via the axial muscles. Tension from muscular contraction transmitted through the tendons could affect both spanwise and chordwise flexibility. Changing flexibility could modulate thrust and efficiency over an extended operation range of swimming speeds in cetaceans., (© 2019 Wiley Periodicals, Inc.)

The effects of the stochasticity of collagen-related structural properties on the biomechanical properties of tendons and ligaments are investigated in this study. The tissue mechanics is modeled by means of a macroscale constitutive model based on a multiscale structural approach. This rationale allows to introduce model parameters directly associated with tissue structural and biochemical features, opening to physically motivated parametric studies. Variance and density-based global sensitivity analyses are employed, together with the quantification of output uncertainty due to stochastic variations of parameters. Novel insights on tissue structure-mechanics relationship are provided, quantifying the dependence between mechanical output quantities on specific collagen-related structural features. Moreover, the uncertainty quantification shows that model predictions provided by the multiscale structural approach are reliable with respect to inevitable uncertainties in tissue structure. Addressing rat tail tendons, the use of average values in tissue properties returns a constitutive response that fits well-available experimental data, and it is robust with respect to parameter stochasticity., (© 2019 John Wiley & Sons, Ltd.)

Extracellular matrix (ECM) is responsible for tendon strength and elasticity. Healed tendon ECM lacks structural integrity, leading to reinjury. Porcine urinary bladder matrix (UBM) provides a scaffold and source of bioactive proteins to improve tissue healing, but has received limited attention for treating tendon injuries. The objective of this study was to evaluate the ability of UBM to induce matrix organization and tenogenesis using a novel in vitro model. We hypothesized that addition of UBM to tendon ECM hydrogels would improve matrix organization and cell differentiation. Hydrogels seeded with bone marrow cells (n = 6 adult horses) were cast using rat tail tendon ECM ± UBM, fixed under static tension and harvested at 7 and 21 days for construct contraction, cell viability, histology, biochemistry, and gene expression. By day 7, UBM constructs contracted significantly from baseline, whereas control constructs did not. Both control and UBM constructs contracted significantly by day 21. In both groups, cells remained viable over time and changed from round and randomly oriented to elongated along lines of tension with visible compaction of the ECM. There were no differences over time or between treatments for nuclear aspect ratio, DNA, or glycosaminoglycan content. Decorin, matrix metalloproteinase 13, and scleraxis expression increased significantly over time, but not in response to UBM treatment. Mohawk expression was constant over time. Cartilage oligomeric matrix protein expression decreased over time in both groups. Using a novel ECM hydrogel model, substantial matrix organization and cell differentiation occurred; however, the addition of UBM failed to induce greater matrix organization than tendon ECM alone. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:1848-1859, 2019., (© 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.)

Tendinopathy, degeneration of the tendon that leads to pain and dysfunction, is common in both sports and occupational settings, but multi-scale mechanisms for tendinopathy are still unknown. We recently showed that micro-scale sliding (shear) is responsible for both load transfer and damage mechanisms in the rat tail tendon; however, the rat tail tendon is a specialized non-load-bearing tendon, and thus the load transfer and damage mechanisms are still unknown for load-bearing tendons. The objective of this study was to investigate the load transfer and damage mechanisms of load-bearing tendons using the rat plantaris tendon. We demonstrated that micro-scale sliding is a key component for both mechanisms in the plantaris tendon, similar to the tail tendon. Namely, the micro-scale sliding was correlated with applied strain, demonstrating that load was transferred via micro-scale sliding in the plantaris and tail tendons. In addition, while the micro-scale strain fully recovered, the micro-scale sliding was non-recoverable and strain-dependent, and correlated with tissue-scale mechanical parameters. When the applied strain was normalized, the % magnitudes of non-recoverable sliding was similar between the plantaris and tail tendons. Statement of clinical significance: Understanding the mechanisms responsible for the pathogenesis and progression of tendinopathy can improve prevention and rehabilitation strategies and guide therapies and the design of engineered constructs. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:1827-1837, 2019., (© 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.)

Adipose-derived stromal vascular fraction (SVF) is a heterogeneous population of cells that yields a homogeneous population of plastic-adherent adipose tissue-derived stromal cells (ASC) when culture-expanded. SVF and ASC have been used clinically to improve tendon healing, yet their mechanism of action is not fully elucidated. The objective of this study was to investigate the potential for ASC to act as trophic mediators for tendon healing. Flexor digitorum superficialis tendons and adipose tissue were harvested from adult horses to obtain SVF, ASC, and tenocytes. Growth factor gene expression was quantified in SVF and ASC in serial passages and growth factors were quantified in ASC-conditioned medium (CM). Microchemotaxis assays were performed using ASC-CM. Tenocytes were grown in co-culture with autologous ASC or allogeneic SVF. Gene expression for insulin-like growth factor 1 (IGF-1), stromal cell-derived factor-1α (SDF-1α), transforming growth factor-β1 (TGF-β1) and TGF-β3 was significantly higher in SVF compared to ASC. Concentrations were significantly increased in ASC-CM compared to controls for IGF-1 (4-fold) and SDF-1α (6-fold). Medium conditioned by ASC induced significant cell migration in a dose-dependent manner. Gene expression for collagen types I and III, decorin, and cartilage oligomeric matrix protein was modestly, but significantly increased following co-culture of tenocytes with autologous ASC. Our findings support the ability of SVF and ASC to act as trophic mediators in tendon healing, particularly through chemotaxis, which stands to critically impact the intrinsic healing response. In vivo studies to further delineate the potential for SVF and/or ASC to improve tendon healing are warranted. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:1429-1439, 2019., (© 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.)

The use of Adipose-Derived Stem Cells (ADSC) has been presented as a new alternative for tendon reconstruction. Have been admitted that ADSCs are related to better outcomes when used in tendon healing. This research was designed to apply the potential of ADSCs in tendon healing. Flexor digitorum superficialis tendon lesion was performed on both legs of eleven New Zealand rabbits and them, at the same time, treated as follows: Suture alone (Group III - Suture, n:10), suture associated with ADSC (Group IV - Suture + ADSC, n:10) or without suture (Group II - SHAN, n:2). At four weeks after the tendon surgery, the animal was euthanized, and the tendon evaluated (biomechanically and macroscopically). We used 5 additional New Zealand rabbits in the control group "Group I - Control, n:10". In the macroscopic evaluation, the group with ADSC presented a more homogeneous gross morphology compared with the group III. Biomechanical testing showed a lower ultimate tensile load, stiffness and a higher cross-sectional area in the group III and IV compared with the control group. The group with ADSC showed a greater ultimate tensile load, a larger cross-sectional area and bigger deformation at the ultimate tensile load when compared to the group without ADSC. In general terms, the use of ADSCs in tendon healing have biomechanical advantages compared to the non-use of ADSCs at 4 weeks after surgery. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:1281-1286, 2019., (© 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.)

Tendon and ligament injuries are a leading cause of healthcare visits with significant impact in terms of economic cost and reduced quality of life. To date, reparative strategies remain largely restricted to conservative treatment or surgical repair. However, these therapies fail to restore native tendon structure and function; thus, the tissue may re-rupture or degenerate with time. To improve tendon healing, one promising strategy may be harnessing the innate potential of resident tendon stem/progenitor cells (TSPCs) to guide tenogenic regeneration. In this review, we outline recent advances in the identification and characterization of putative TSPC populations, and discuss biochemical, biomechanical, and biomaterial methods employed for their culture and differentiation. Finally, we identify limitations in our current understanding of TSPC biology, key challenges for their use, and potential therapeutic strategies to inform cell-based tendon repair. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:1270-1280, 2019., (© 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.)

The function of ligaments and tendons is to support and transmit loads applied to the musculoskeletal system. These tissues are often able to perform their function for many decades; however, connective tissue disease and injury can compromise ligament and tendon integrity. A range of protein and non-protein constituents, combined in a complex structural hierarchy from the collagen molecule to the tissue and covering nanometer to centimeter length scales, govern tissue function, and impart characteristic non-linear material behavior. This review summarizes the structure of ligaments and tendons, the roles of their constituent components for load transfer across the hierarchy of structure, and the current understanding of how damage occurs in these tissues. Disease and injury can alter the constituent make-up and structural organization of ligaments and tendons, affecting tissue function, while also providing insight to the role and interactions of individual constituents. The studies and techniques presented here have helped to understand the relationship between tissue constituents and the physical mechanisms (e.g., stretching, sliding) that govern material behavior at and between length scales. In recent years, new techniques have been developed to probe ever smaller length scales and may help to elucidate mechanisms of load transfer and damage and the molecular constituents involved in the in the earliest stages of ligament and tendon damage. A detailed understanding of load transfer and damage from the molecular to the tissue level may elucidate targets for the treatment of connective tissue diseases and inform practice to prevent and rehabilitate ligament and tendon injuries. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:3093-3104, 2018., (© 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.)

One of the suspected deleterious effects of androgenic-anabolic steroids (AAS) is the increased risk for tendon rupture. However, investigations to date have produced inconsistent results and it is still unclear how AAS influence tendons. A systematic review of the literature was conducted to identify studies that have investigated the mechanical, structural, or biologic effects that AAS have on tendons. In total, 18 highly heterogeneous studies were identified. Small animal studies made up the vast majority of published research, and contradictory results were reported frequently. All of the included studies focused on the potential deleterious effects that AAS have on tendon, which is striking given the recent use of AAS in patients following tendon injury. Rather than providing strong evidence for or against the use of AAS, this review highlights the need for additional research. Future studies investigating the use of AAS as a possible treatment for tendon injury/pathology are supported by reports suggesting that AAS may counteract the irreparable structural/functional changes that occur in the musculotendinous unit following rotator cuff tears, as well as studies suggesting that the purported deleterious effects on tendon may be transient. Other possible areas for future research are discussed in the context of key findings that may have implications for the therapeutic application of AAS. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:2830-2841, 2018., (© 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.)

Recent growth factor, cell, and scaffold-based experimental interventions for intrasynovial flexor tendon repair have demonstrated therapeutic potential in rodent models. However, these approaches have not achieved consistent functional improvements in large animal trials due to deleterious inflammatory reactions to delivery materials and insufficient induction of targeted biological healing responses. In this study, we achieved porous suture-based sustained delivery of connective tissue growth factor (CTGF) into flexor tendons in a clinically relevant canine model. Repairs with CTGF-laden sutures were mechanically competent and did not show any evidence of adhesions or other negative inflammatory reactions based on histology, gene expression, or proteomics analyses at 14 days following repair. CTGF-laden sutures induced local cellular infiltration and a significant biological response immediately adjacent to the suture, including histological signs of angiogenesis and collagen deposition. There were no evident widespread biological effects throughout the tendon substance. There were significant differences in gene expression of the macrophage marker CD163 and anti-apoptotic factor BCL2L1; however, these differences were not corroborated by proteomics analysis. In summary, this study provided encouraging evidence of sustained delivery of biologically active CTGF from porous sutures without signs of a negative inflammatory reaction. With the development of a safe and effective method for generating a positive local biological response, future studies can explore additional methods for enhancing intrasynovial tendon repair. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:2052-2063, 2018., (© 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.)

Appropriate mechanical load is essential for tendon homeostasis and optimal tissue function. Due to technical challenges in achieving physiological mechanical loads in experimental tendon model systems, the research community still lacks well-characterized models of tissue homeostasis and physiological relevance. Toward this urgent goal, we present and characterize a novel ex vivo murine tail tendon explant model. Mouse tail tendon fascicles were extracted and cultured for 6 days in a load-deprived environment or in a custom-designed bioreactor applying low magnitude mechanical load (intermittent cycles to 1% strain, at 1 Hz) in serum-free tissue culture. Cells remained viable, as did collagen structure and mechanical properties in all tested conditions. Cell morphology in mechanically loaded tendon explants approximated native tendon, whereas load-deprived tendons lost their native cell morphology. These losses were reflected in altered gene expression, with mechanical loading tending to maintain tendon specific and matrix remodeling genes phenotypic of native tissue. We conclude from this study that ex vivo load deprivation of murine tendon in minimal culture medium results in a degenerative-like phenotype. We further conclude that onset of tissue degeneration can be suppressed by low-magnitude mechanical loading. Thus a minimal explant culture model featuring serum-free medium with low mechanical loads seems to provide a useful foundation for further investigations. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:1383-1390, 2018., (© 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.)

The enthesis, or interface between bone and soft tissues such as ligament and tendon, is prone to injury and often does not heal, even post surgical intervention. Interface tissue engineering represents an integrative strategy for regenerating the native enthesis by functionally connecting soft and hard tissues and thereby improving clinical outcome. This review focuses on integrative and cell-instructive scaffold designs that target the healing of the two most commonly injured soft tissue-bone junctions: tendon-bone interface (e.g., rotator cuff) and ligament-bone interface (e.g., anterior cruciate ligament). The inherent connectivity between soft and hard tissues is instrumental for musculoskeletal motion and is therefore a key design criterion for soft tissue regeneration. To this end, scaffold design for soft tissue regeneration have progressed from single tissue systems to the emerging focus on pre-integrated and functional composite tissue units. Specifically, a multifaceted, bioinspired approach has been pursued wherein scaffolds are tailored to stimulate relevant cell responses using spatially patterned structural and chemical cues, growth factors, and/or mechanical stimulation. Moreover, current efforts to elucidate the essential scaffold design criteria via strategic biomimicry are emphasized as these will reduce complexity in composite tissue regeneration and ease the related burden for clinical translation. These innovative studies underscore the clinical relevance of engineering connective tissue integration and have broader impact in the formation of complex tissues and total joint regeneration. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:1069-1077, 2018., (© 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.)

Tendons are the connective tissue responsible for transferring force from muscles to bones. A key factor in tendon development, maturation, repair, and degradation is its biomechanical environment. Understanding tendon mechanobiology is essential for the development of injury prevention strategies, rehabilitation protocols and potentially novel treatments in tendon injury and degeneration. Despite the simple overall loading on tendon tissue, cells within the tissue in vivo experience a much more complex mechanical environment including tension, compression and shear forces. This creates a substantial challenge in the establishment of in vitro loading models of the tendon. This article reviews multiple loading models used for the study of tendon mechanobiology and summarizes the main findings. Although impressive progress has been achieved in the functionality and mimicry of in vitro loading models, an ideal platform is yet to be developed. Multidisciplinary approaches and collaborations will be the key to unveiling the tendon mechanobiology. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:566-575, 2018., (© 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.)

Tendons are unique in the sense that they are constantly subjected to large mechanical loads and that they contain tendon-specific cells, including tenocytes and tendon stem/progenitor cells. The responses of these cells to mechanical loads can be anabolic or catabolic and as a result, change the biological properties of the tendon itself that may be beneficial or detrimental. On the other hand, aging also induces aberrant changes in cellular expression of various genes and production of various types of matrix proteins in the tendon, and consequently lead to tendon degeneration and impaired healing in aging tendons; both could be improved by moderate physiological mechanical loading such as treadmill running. This article gives an overview on the mechanobiology research of young and aging animal tendons using treadmill running model. The challenges in such treadmill running studies are also discussed. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:557-565, 2018., (© 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.)

Current rotator cuff repair commonly involves the use of single or double row suture techniques, and despite successful outcomes, failure rates continue to range from 20 to 95%. Failure to regenerate native biomechanical properties at the enthesis is thought to contribute to failure rates. Thus, the need for technologies that improve structural healing of the enthesis after rotator cuff repair is imperative. To address this issue, our lab has previously demonstrated enthesis regeneration using a tissue-engineered graft approach in a sheep anterior cruciate ligament (ACL) repair model. We hypothesized that our tissue-engineered graft designed for ACL repair also will be effective in rotator cuff repair. The goal of this study was to test the efficacy of our Engineered Tissue Graft for Rotator Cuff (ETG-RC) in a rotator cuff tear model in sheep and compare this novel graft technology to the commonly used double row suture repair technique. Following a 6-month recovery, the grafted and contralateral shoulders were removed, imaged using X-ray, and tested biomechanically. Additionally, the infraspinatus muscle, myotendinous junction, enthesis, and humeral head were preserved for histological analysis of muscle, tendon, and enthesis structure. Our results showed that our ETC-RCs reached 31% of the native tendon tangent modulus, which was a modest, non-significant, 11% increase over that of the suture-only repairs. However, the histological analysis showed the regeneration of a native-like enthesis in the ETG-RC-repaired animals. This advanced structural healing may improve over longer times and may diminish recurrence rates of rotator cuff tears and lead to better clinical outcomes. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:289-299, 2018., (© 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.)

The purpose of this review is to provide a brief overview of bioreactor-based culture systems as alternatives to conventional two- and three-dimensional counterparts. The role, challenges, and future aspirations of bioreactors in the musculoskeletal field (e.g., cartilage, intervertebral disc, tendon, and bone) are discussed. Bioreactors, by recapitulating physiological processes, can be used effectively as part of the initial in vitro screening, reducing that way the number of animal required for preclinical assessment, complying with the 3R principles and, in most cases, allowing working with human tissues. The clinical significance of bioreactors is that, by providing more physiologically relevant conditions to customarily used two- and three-dimensional cultures, they hold the potential to provide a testing platform that is more predictable of a whole tissue response, thereby facilitating the screening of treatments before the initiation of clinical trials. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:10-21, 2018., (© 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.)

Aging is an important factor in disrupted homeostasis of many tissues. While an increased incidence of tendinopathy and tendon rupture are observed with aging, it is unclear whether this is due to progressive changes in tendon cell function and mechanics over time, or an impaired repair reaction from aged tendons in response to insult or injury. In the present study, we examined changes in the mechanical properties of Flexor Digitorum Longus (FDL), Flexor Carpi Ulnaris (FCU), and tail fascicles in both male and female C57Bl/6 mice between 3 and 27 months of age to better understand the effects of sex and age on tendon homeostasis. No change in max load at failure was observed in any group over the course of aging, although there were significant decreases in toe and linear stiffness in female mice from 3 to 15 months, and 3 to 27 months. No changes in cell proliferation were observed with aging, although an observable decrease in cellularity occurred in 31-month old tendons. Given that aging did not dramatically alter tendon mechanical homeostasis we hypothesized that a disruption in tendon homeostasis, via acute injury would result in an impaired healing response. Significant decreases in max load, stiffness, and yield load were observed in repairs of 22-month old mice, relative to 4-month old mice. No changes in cell proliferation were observed between young and aged, however, a dramatic loss of bridging collagen extracellular matrix was observed in aged repairs suggest that matrix production, but not cell proliferation leads to impaired tendon healing with aging. Results © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:2716-2724, 2017., (© 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.)

Tendons play a critical role in the transmission of forces between muscles and bones, and chronic tendon injuries and diseases are among the leading causes of musculoskeletal disability. Little is known about sex-based differences in tendon structure and function. Our objective was to evaluate the mechanical properties, biochemical composition, transcriptome, and cellular activity of plantarflexor tendons from 4 month old male and female C57BL/6 mice using in vitro biomechanics, mass spectrometry-based proteomics, genome-wide expression profiling, and cell culture techniques. While the Achilles tendons of male mice were approximately 6% larger than female mice (p < 0.05), the cell density of female mice was around 19% greater than males (p < 0.05). No significant differences in mechanical properties (p > 0.05) of plantaris tendons were observed. Mass spectrometry proteomics analysis revealed no significant difference between sexes in the abundance of major extracellular matrix (ECM) proteins such as collagen types I (p = 0.30) and III (p = 0.68), but female mice had approximately twofold elevations (p < 0.05) in less abundant ECM proteins such as fibronectin, periostin, and tenascin C. The transcriptome of male and female tendons differed by only 1%. In vitro, neither the sex of the serum that fibroblasts were cultured in, nor the sex of the ECM in which they were embedded, had profound effects on the expression of collagen and cell proliferation genes. Our results indicate that while male mice expectedly had larger tendons, male and female tendons have very similar mechanical properties and biochemical composition, with small increases in some ECM proteins and proteoglycans evident in female tendons. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:2117-2126, 2017., (© 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.)

Collagen fibrils in tendon are believed to be discontinuous and transfer tensile loads through shear forces generated during interfibrillar sliding. However, the structures that transmit these interfibrillar forces are unknown. Various extrafibrillar tissue components (e.g., glycosaminoglycans, collagens XII and XIV) have been suggested to transmit interfibrillar loads by bridging collagen fibrils. Alternatively, collagen fibrils may interact directly through physical fusions and interfibrillar branching. The objective of this study was to test whether extrafibrillar proteins are necessary to transmit load between collagen fibrils or if interfibrillar load transfer is accomplished directly by the fibrils themselves. Trypsin digestions were used to remove a broad spectrum of extrafibrillar proteins and measure their contribution to the multiscale mechanics of rat tail tendon fascicles. Additionally, images obtained from serial block-face scanning electron microscopy were used to determine the three-dimensional fibrillar organization in tendon fascicles and identify any potential interfibrillar interactions. While trypsin successfully removed several extrafibrillar tissue components, there was no change in the macroscale fascicle mechanics or fibril:tissue strain ratio. Furthermore, the imaging data suggested that a network of smaller diameter fibrils (<150 nm) wind around and fuse with their neighboring larger diameter fibrils. These findings demonstrate that interfibrillar load transfer is not supported by extrafibrillar tissue components and support the hypothesis that collagen fibrils are capable of transmitting loads themselves. Conclusively determining how fibrils bear load within tendon is critical for identifying the mechanisms that impair tissue function with degeneration and for restoring tissue properties via cell-mediated regeneration or engineered tissue replacements. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:2127-2134, 2017., (© 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.)

Tendon's primary function is a mechanical link between muscle and bone. The hierarchical structure of tendon and specific compositional constituents are believed to be critical for proper mechanical function. With increased appreciation for tendon importance and the development of various technological advances, this review paper summarizes recent experimental approaches that have been used to study multiscale tendon mechanics, includes an overview of studies that have evaluated the role of specific tissue constituents, and also proposes challenges/opportunities facing tendon study. Tendon has been demonstrated to have specific structural characteristics (e.g., multi-level hierarchy, crimp pattern, helix) and complex mechanical properties (e.g., non-linearity, anisotropy, viscoelasticity). Physical mechanisms including uncrimping, fiber sliding, and collagen reorganization have been shown to govern tendon mechanical responses under both static and dynamic loading. Several tendon constituents with relatively small quantities have been suggested to play a role in its mechanics, although some results are conflicting. Further research should be performed to understand the interplay and communication of tendon mechanical properties across levels of the hierarchical structure, and further show how each of these components contribute to tendon mechanics. The studies summarized and discussed in this review have helped elucidate important aspects of multiscale tendon mechanics, which is a prerequisite for analyzing stress/strain transfer between multiple scales and identifying key principles of mechanotransduction. This information could further facilitate interpreting the functional diversity of tendons from different species, different locations, and even different developmental stages, and then better understand and identify fundamental concepts related to tendon degeneration, disease, and healing. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:1353-1365, 2017., (© 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.)

Rotator cuff tendon tears are one of the most common shoulder pathologies, especially in the aging population. Due to a poor healing response and degenerative changes associated with aging, rotator cuff repair failure remains common. Although cell-based therapies to augment rotator cuff repair appear promising, it is unknown whether the success of such a therapy is age-dependent. We hypothesized that autologous cell therapy would improve tendon-to-bone healing across age groups, with autologous juvenile cells realizing the greatest benefit. In this study, juvenile, adult, and aged rats underwent bilateral supraspinatus tendon repair with augmentation of one shoulder with autologous tendon-derived cell-seeded polycaprolactone scaffolds. At 8 weeks, shoulders treated with cells in both juvenile and aged animals exhibited increased cellularity, increased collagen organization, and improved mechanical properties. No changes between treated and control limbs were seen in adult rats. These findings suggest that cell delivery during supraspinatus repair initiates earlier matrix remodeling in juvenile and aged animals. This may be due to the relative "equilibrium" of adult tendon tissue with regards to catabolic and anabolic processes, contrasted with actively growing juvenile tendons and degenerative aged tendons. This study demonstrates the potential for autologous cell-seeded scaffolds to improve repairs in both the juvenile and aged population. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:1250-1257, 2017., (© 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.)

Collagen crimp morphology is thought to contribute to the material behavior of tendons and may reflect the local mechanobiological environment of tendon cells. Following loss of collagen tension in tendons, tenocytes initiate a contraction response that shortens tendon length which, in turn, may alter crimp patterns. We hypothesized that changes in the crimp pattern of tendons are the result of cell-based contractions which are governed by relative tautness/laxity of the collagen matrix. To determine the relationship between crimp pattern and tensional homeostasis, rat tail tendon fascicles (RTTfs) were either allowed to freely contract or placed in clamps with 10% laxity for 7 days. The freely contracting RTTfs showed a significant decrease in percent crimp length on both day 5 (3.66%) and day 7 (7.70%). This decrease in crimp length significantly correlated with the decrease in freely contracting RTTf length. Clamped RTTfs demonstrated a significant decrease in percent crimp length on day 5 (1.7%), but no significant difference in percent crimp length on day 7 (0.57%). The results demonstrate that the tendon crimp pattern appears to be under cellular control and is a reflection of the local mechanobiological environment of the extracellular matrix. The ability of tenocytes to actively alter the crimp pattern of collagen fibers also suggests that tenocytes can influence the viscoelastic properties of tendon. Understanding the interactions between tenocytes and their extracellular matrix may lead to further insight into the role tendon cells play in maintaining tendon heath and homeostasis. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:573-579, 2017., (© 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.)

Tenocytes express primary cilia, which elongate when tendon is maintained in the absence of biomechanical load. Previous work indicates differences in the morphology and metabolism of the tenocytes in the tendon fascicular matrix (FM) and the inter-fascicular matrix (IFM). This study tests the hypothesis that primary cilia in these two regions respond differently to stress deprivation and that this is associated with differences in the biomechanical degradation of the extracellular matrix. Rat tail tendon fascicles were examined over a 7-day period of either stress deprivation or static load. Seven days of stress deprivation induced cilia elongation in both regions. However, elongation was greater in the IFM compared to the FM. Stress deprivation also induced a loss of biomechanical integrity, primarily in the IFM. Static loading reduced both the biomechanical degradation and cilia elongation. The different responses to stress deprivation in the two tendon regions are likely to be important for the aetiology of tendinopathy. Furthermore, these data suggest that primary cilia elongate in response to biomechanical degradation rather than simply the removal of load. This response to degradation is likely to have important consequences for cilia signalling in tendon and as well as in other connective tissues. © 2016 The Authors. Journal of Orthopaedic Research Published by Wiley Periodicals, Inc. on behalf of Orthopaedic Research Society. J Orthop Res 34:2146-2153, 2016., (© 2016 The Authors. Journal of Orthopaedic Research Published by Wiley Periodicals, Inc. on behalf of Orthopaedic Research Society.)

MRI biomarkers such as T 2 , T 2 * and T 1rho have been widely used, but are confounded by the magic angle effect. The purpose of this study is to investigate the use of the two-dimensional ultrashort echo time magnetization transfer (UTE-MT) sequence for potential magic angle independent MR biomarkers. Magnetization transfer was investigated in cadaveric Achilles tendon samples using the UTE-MT sequence at five MT powers and five frequency offsets ranging from 2 to 50 kHz. The protocol was applied at five sample orientations ranging from 0 to 90° relative to the B 0 field. The results were analyzed with a two-pool quantitative MT model. Multiple T E data were also acquired and mono-exponential T 2 * was calculated for each orientation. Macromolecular proton fractions and exchange rates derived from UTE-MT modeling did not appreciably change between the various orientations, whereas the T 2 * relaxation time demonstrated up to a sixfold increase from 0° to 55°. The UTE-MT technique with two-pool modeling shows promise as a clinically compatible technique that is resistant to the magic angle effect. This method provides information on the macromolecular proton pool that cannot be directly obtained by other methods, including regular UTE techniques., (Copyright © 2016 John Wiley & Sons, Ltd.)

Carpal tunnel syndrome is characterized by non-inflammatory fibrosis of the subsynovial connective tissue (SSCT), a paratenon-like structure inside the carpal tunnel. This pathology suggests repetitive and/or excessive shear forces are involved in injury development. We assessed relative motion between the flexor digitorum superficialis (FDS) tendon and adjacent paratenon in Zone V using colour Doppler imaging as 16 healthy participants completed three long finger movements (metacarpophalangeal joint flexion, proximal and distal interphalangeal joint flexion, full finger flexion) in three wrist postures (30° extension, 0°, 30° flexion). While the type of finger movement did not affect tendon-paratenon relative motion, we found a significant main effect of wrist posture (p < 0.001). Relative displacement between the FDS tendon and paratenon (as a percentage of tendon displacement) increased from 27.2% (95%CI = 24.8-29.5%) in 30° wrist extension to 39.9% (95%CI = 37.3-42.4%) in 30° wrist flexion. Optical motion capture confirmed that wrist posture did not affect metacarpophalangeal joint range of motion (p = 0.265) or proximal interphalangeal joint range of motion (p = 0.582). These results indicate that relative motion increased due to paratenon strain when the wrist was flexed. While our findings agree with previous cadaveric research in wrist flexion, we found that relative displacement decreased in 30° wrist extension (compared to 0°). These results differ from cadaveric research, possibly due to challenges maintaining anatomic fidelity of the viscoelastic paratenon tissue in vitro. Overall, our study suggests a greater susceptibility to shear injury during repetitive finger movements, particularly when the wrist is flexed. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:1248-1255, 2016., (© 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.)

The optimal mechanical loading regimen for the healing of a tendon graft in a bone tunnel is unknown. We developed a rat model that directly tensions a healing tendon graft, without the use of confounding joint motion. Fifty cycles of either 0, 3, or 6 N of tension were applied to groups daily for 3 or 6 weeks. At 3 weeks the low load (3 N) group had the highest failure load (p = 0.009), but by 6 weeks there were no differences in failure load among groups. At 3 weeks the high load (6 N) group had greater osteoclast activity compared to the immobilized (0 N) group (p < 0.05), and by 6 weeks there were significantly more osteoclasts in the high load group compared to the low load group (p = 0.01). Bone volume fraction was higher in the immobilized group compared to the 3 N load group at 3 weeks (p = 0.014) and 6 weeks (p = 0.007). At 6 weeks, the immobilized group had greater trabecular number compared to both loading groups (p < 0.05). In conclusion, low magnitude loading had a beneficial early effect but continued loading led to poorer new bone formation over time and no beneficial effect at 6 weeks, perhaps due to delayed maturation from cumulative loads. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:852-859, 2016., (© 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.)

The outcomes of flexor tendon repair are highly variable. As recent efforts to improve healing have demonstrated promise for growth factor- and cell-based therapies, the objective of the current study was to enhance repair via application of autologous adipose derived stromal cells (ASCs) and the tenogenic growth factor bone morphogenetic protein (BMP) 12. Controlled delivery of cells and growth factor was achieved in a clinically relevant canine model using a nanofiber/fibrin-based scaffold. Control groups consisted of repair-only (no scaffold) and acellular scaffold. Repairs were evaluated after 28 days of healing using biomechanical, biochemical, and proteomics analyses. Range of motion was reduced in the groups that received scaffolds compared to normal. There was no effect of ASC + BMP12 treatment for range of motion or tensile properties outcomes versus repair-only. Biochemical assays demonstrated increased DNA, glycosaminoglycans, and crosslink concentration in all repair groups compared to normal, but no effect of ASC + BMP12. Total collagen was significantly decreased in the acellular scaffold group compared to normal and significantly increased in the ASC + BMP12 group compared to the acellular scaffold group. Proteomics analysis comparing healing tendons to uninjured tendons revealed significant increases in proteins associated with inflammation, stress response, and matrix degradation. Treatment with ASC + BMP12 amplified these unfavorable changes. In summary, the treatment approach used in this study induced a negative inflammatory reaction at the repair site leading to poor healing. Future approaches should consider cell and growth factor delivery methods that do not incite negative local reactions., (© 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.)

The manner in which strains are passed down the hierarchical length scales of tendons dictates how cells within the collagen network regulate the tissue response to loading. How tendons deform in different hierarchical levels under shear and compression is unknown. The aims of this study were: (i) to evaluate whether specific regions of bovine deep digital flexor tendons exhibited different strain attenuation from macro to micro length scales, and (ii) to elucidate mechanisms responsible for tendon deformation under shear and compression. Samples from distal and proximal regions of flexor tendons were subjected to three-step incremental stress-relaxation tests. Images of tissue markers, photobleached lines on collagen fibers, and nuclei locations were collected before and after loading. Results showed that strain transfer was attenuated from tissue to local matrix under both shear and compression. Nuclear aspect ratios exhibited smaller changes for distal samples, suggesting that cells are more shielded from deformation in the distal region. Collagen fiber sliding was observed to contribute significantly in response to shear, while uncrimping and fiber reorganization were the predominant mechanisms under compression. This study provides insight into microscale mechanisms responsible for multiscale strain attenuation of tendons under non-tensile macroscale loading., (© 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.)

The long head of the biceps tendon (LHBT) occupies a unique proximal intra-articular and distal extra-articular position within the human shoulder. In the presence of a rotator cuff (RC) tear, the LHBT is recruited into an accelerated role undergoing potential mechanical and biochemical degeneration. Intra-articular sections of the LHBT were harvested during primary shoulder arthroplasty from patients with an intact or deficient RC. LHBTs were stained (H&E, Alcian Blue) and subjected to histologic analysis using the semiquantitative Bonar scale and measurement of collagen orientation. LHBTs (n = 12 per group) were also subjected to gene-expression analyses via an RT(2) -PCR Profiler Array quantifying 84 genes associated with cell-cell and cell-matrix interactions. LHBTs (n = 18 per group) were biomechanically tested with both stress-relaxation and load-to-failure protocols and subsequently modeled with the Quasilinear Viscoelastic (QLV) and Structural-Based Elastic (SBE) models. While no histologic differences were observed, significant differences in mechanical testing, and viscoelastic modeling parameters were found. PCR arrays identified five genes that were differentially expressed between RC-intact and RC-deficient LHBT groups. LHBTs display signs of pathology regardless of RC status in the arthroplasty population, which may be secondary to both glenohumeral joint arthritis and the additional mechanical role of the LHBT in this population., (© 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.)