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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.

Patients with type 2 diabetes (T2D) have a lower risk of Mycobacterium tuberculosis infection, progression from infection to tuberculosis (TB) disease, TB morality and TB recurrence, when being treated with metformin. However, a detailed mechanistic understanding of these protective effects is lacking. Here, we use mass cytometry to show that metformin treatment expands a population of memory-like antigen-inexperienced CD8+CXCR3+ T cells in naive mice, and in healthy individuals and patients with T2D. Metformin-educated CD8+ T cells have increased (i) mitochondrial mass, oxidative phosphorylation, and fatty acid oxidation; (ii) survival capacity; and (iii) anti-mycobacterial properties. CD8+ T cells from Cxcr3−/− mice do not exhibit this metformin-mediated metabolic programming. In BCG-vaccinated mice and guinea pigs, metformin enhances immunogenicity and protective efficacy against M. tuberculosis challenge. Collectively, these results demonstrate an important function of CD8+ T cells in metformin-derived host metabolic-fitness towards M. tuberculosis infection., Metformin is an anti-diabetic drug that has shown promise to reduce M. tuberculosis susceptibility. Here the authors show that this effect is a result of metformin-mediated activation of anti-mycobacterial memory-like antigen-inexperienced CD8+CXCR3+ T cells, an effect that also boosts response to BCG vaccination.

Methionine biosynthetic pathway, essential for the growth of Mycobacterium tuberculosis (Mtb) in the host, represents an attractive target for the development of novel anti-tuberculars. Here, we have biochemically characterized homoserine acetyl transferase (HSAT viz. MetA) of Mtb, which catalyses the first committed step of methionine and S-adenosylmethionine (SAM) biosynthesis. High-throughput screening of a 2300 compound library resulted in identification of thiram, an anti-fungal organosulfur compound, as the most potent MetA inhibitor. Further analysis of thiram analogs led to the identification of orally bioavailable disulfiram (DIS, an anti-alcoholism FDA approved drug) as a novel inhibitor of MetA. Both thiram and DIS restricted the growth of drug-sensitive and drug-resistant Mtb strains in a bactericidal manner. ThermoFlour assay demonstrated direct binding of DIS with MetA. Metabolomic and transcriptomic studies showed DIS mediated perturbation of methionine and redox homeostasis, respectively, in Mtb. In concordance, the effect of DIS on Mtb growth was partially rescued by supplementation with either L-methionine as well as N-acetyl cysteine, suggesting a multi-target killing mechanism. In Mtb-infected mice, DIS administration restricted bacterial growth, increased efficacy of isoniazid, ameliorated lung pathology, modulated lung immune cell landscape and protective immune response. Taken together, our results demonstrate that DIS can be repurposed for designing an effective anti-tubercular therapy.

Diabetic patients taking metformin have lower risk for Mycobacterium tuberculosis (Mtb) infection, progression from infection to tuberculosis (TB) disease, TB morality and TB recurrence. However, a detailed mechanistic understanding of metformin’s protective immunological benefits on host resistance to TB is lacking. In this study, using mass cytometry we show that metformin treatment expands memory-like antigen-inexperienced CD8+CXCR3+ T cells in naïve mice, and in healthy and diabetic humans. Metformin-educated CD8+ T cells have increased (i) mitochondrial mass, oxidative phosphorylation, and fatty acid oxidation; (ii) survival capacity; and (iii) anti-mycobacterial properties. CD8+ T cells from CXCR3−/− mice did not exhibit metformin-mediated metabolic programming. In BCG-vaccinated mice and guinea pigs, metformin enhanced immunogenicity and protective efficacy against Mtb challenge. Collectively, our results demonstrate an important role of CD8+ T cells in metformin-derived host metabolic-fitness towards Mtb infection.

One of the most common bath solutions used in musculoskeletal mechanical testing is phosphate buffered saline (PBS). In tendon, swelling induced by physiological PBS results in decreased tendon modulus and induces microstructural changes. It is critical to evaluate the multiscale mechanical behavior of tendon under swelling to interpret prior work and provide information to design future studies. We compared the effects of physiological PBS and 8% polyethylene glycol and saline bathing solutions on tendon multiscale tendon mechanics and damage as well as microstructure with TEM in order to understand the effect of swelling on tendon. At the tissue level, tendons in PBS had a lower modulus than SPEG samples. PBS samples also showed an increased amount of non-recoverable sliding, which is an analog for microscale damage. SPEG had a higher microscale to tissue-scale strain ratio, showing the fibrils experienced less strain attenuation. From the TEM data, we showed the fibril spacing of SPEG samples was more similar to fresh control than PBS. We concluded that swelling alters multiscale mechanics and damage in addition to tendon microstructure. Future mechanical testing should consider using SPEG as a bath solution with an osmotic pressure which preserves fresh tissue water content.

Methionine biosynthetic pathway, essential for the growth of Mycobacterium tuberculosis (Mtb) in the host, represents an attractive target for the development of novel anti-tuberculars. Here, we have biochemically characterized homoserine acetyl transferase (HSAT viz. MetA) of Mtb, which catalyses the first committed step of methionine and S-adenosylmethionine (SAM) biosynthesis. High-throughput screening of a 2300 compound library resulted in identification of thiram, an anti-fungal organosulfur compound, as the most potent MetA inhibitor. Further analysis of thiram analogs led to the identification of orally bioavailable disulfiram (DIS, an anti-alcoholism FDA approved drug) as a novel inhibitor of MetA. Both thiram and DIS restricted the growth of drug-sensitive and drug-resistant Mtb strains in a bactericidal manner. ThermoFlour assay demonstrated direct binding of DIS with MetA. Metabolomic and transcriptomic studies showed DIS mediated perturbation of methionine and redox homeostasis, respectively, in Mtb. In concordance, the effect of DIS on Mtb growth was partially rescued by supplementation with either L-methionine as well as N-acetyl cysteine, suggesting a multi-target killing mechanism. In Mtb-infected mice, DIS administration restricted bacterial growth, increased efficacy of isoniazid, ameliorated lung pathology, modulated lung immune cell landscape and protective immune response. Taken together, our results demonstrate that DIS can be repurposed for designing an effective anti-tubercular therapy. Funding Statement: The research fellowship to D.C. and T.P.G. from Department of Biotechnology, Govt. of India is acknowledged. Ava.S. fellowship was supported by the University of Grants commission. R.B. acknowledges Department of Science and Technology India and FICCI for providing C.V. Raman International fellowship. R.S. acknowledge THSTI, and Department of Biotechnology, Govt. of India (BT/PR29075/BRB/10/1699/2018) for funding. This research was supported by SIgN A*STAR, A*STAR JCO-CDA grant (#1518251030), and Singapore-India Joint grant (#1518224018) to A.S. Declaration of Interests: None. Ethics Approval Statement: The proposed animal experiments were approved by the institutional animal ethics committee of Defence Science Organization (DSO), Singapore.

Tendon’s viscoelastic behaviors are important to the mechanical function and mechanobiology. When loaded in longitudinal tension, tendons often have a very large Poisson’s ratio (ν > 2) that exceeds the limit of incompressibility for isotropic material (ν = 0.5), indicating that tendon experiences volume loss, inducing poroelastic fluid exudation in the transverse direction. Therefore, transverse poroelasticity is an important contributor to tendon material behavior. Tendon hydraulic permeability which is required to evaluate the fluid flow contribution to viscoelasticity, is mostly unavailable, and where available, varies by several orders of magnitude. In this manuscript, we quantified the transverse poroelastic material parameters of rat tail tendon fascicles by conducting transverse osmotic loading experiments, in both tension and compression. We used a multi-start optimization method to evaluate the parameters based on biphasic finite element modeling. Our tendon samples had a transverse hydraulic permeability of 10(−4) to 10(−5) mm(4) (Ns)(−1) and showed a significant tension-compression nonlinearity in the transverse direction. Further, using these results, we predict hydraulic permeability during longitudinal (fiber-aligned) tensile loading, and the spatial distribution of fluid flow during osmotic loading. These results reveal novel aspects of tendon mechanics and can be used to study the physiomechanical response of tendon in response to mechanical loading.

Rodent tendons are widely used to study human pathologies such as tendinopathy and repair, and to address fundamental physiological questions about development, growth, and remodeling. However, how the gross morphology and multi-scale hierarchical structure of rat tendons, such as the tail, plantaris, and Achilles tendons, compare with that of human tendons are unknown. In addition, there remains disagreement about terminology and definitions. Specifically, the definitions of fascicle and fiber are often dependent on diameter sizes, not their characteristic features, and these definitions impair the ability to compare hierarchical structure across species, where the sizes of the fiber and fascicle may change with animal size and tendon function. Thus, the objective of the study was to select a single species that is commonly used for tendon research (rat) and tendons with varying mechanical functions (tail, plantaris, Achilles) to evaluate the hierarchical structure at multiple length scales using histology, SEM, and confocal imaging. With the exception of the specialized rat tail tendon, we confirmed that in rat tendons there are no fascicles and the fiber is the largest subunit. In addition, we provided a structurally based definition of a fiber as a bundle of collagen fibrils that is surrounded by elongated cells, and this definition was supported by both histologically processed and unprocessed samples. In all rat tendons studied, the fiber diameters were consistently between 10 and 50 μm, and this diameter range appears to be conserved across larger species. Specific recommendations were made highlighting the strengths and limitations of each rat tendon as a research model. Understanding the hierarchical structure of tendon can advance the design and interpretation of experiments and development of tissue-engineered constructs.

Inelastic behaviors, such as softening, a progressive decrease in modulus before failure, occur in tendon andare important aspect in degeneration and tendinopathy. These in elastic behaviors are generally attributed to two potential mechanisms: plastic deformation and damage. However, it is not clear which is primarily responsible.In this study, we evaluated these potential mechanisms of tendon in elasticity by using a recently developed reactive in elasticity model (RIE), which is a structurally-inspired continuum mechanics frame work that models tissue in elasticity based on the molecular bond kinetics. Using RIE, we formulated two material models, one specific toplastic deformation and the other to damage. The models were independently fit to published experimental tensiletests of rat tail tendons. We quantified the inelastic effects and compared the performance of the two models infitting the mechanical response during loading, relaxation, unloading, and reloading phases. Additionally, we validated the models by using the resulting fit parameters to predict an independent set of experimental stress-straincurves from ramp-to-failure tests. Overall, the models were both successful in fitting the experiments and predicting the validation data. However, the results did not strongly favor one mechanism over the other. As a result, to distinguish between plastic deformation and damage, different experimental protocols will be needed. Nevertheless, these findings suggest the potential of RIE as a comprehensive framework for studying tendon inelastic behaviors.

It is common in biomechanics to use previously frozen tissues, where it is assumed that the freeze-thaw process does not cause consequential mechanical or structural changes. We have recently quantified multiscale tendon mechanics and damage mechanisms using previously frozen tissue, where damage was defined as an irreversible change in the microstructure that alters the macroscopic mechanical parameters. Because freezing has been shown to alter tendon microstructures, the objective of this study was to determine if freezing alters tendon multiscale mechanics and damage mechanisms. Multiscale testing using a protocol that was designed to evaluate tendon damage (tensile stress-relaxation followed by unloaded recovery) was performed on fresh and previously frozen rat tail tendon fascicles. At both the fascicle and fibril levels, there was no difference between the fresh and frozen groups for any of the parameters, suggesting that there is no effect of freezing on tendon mechanics. After unloading, the microscale fibril strain fully recovered, and interfibrillar sliding only partially recovered, suggesting that the tendon damage is localized to the interfibrillar structures and that mechanisms of damage are the same in both fresh and previously frozen tendons.

Tendon pathology is associated with damage. While tendon damage is likely initiated by mechanical loading, little is known about the specific etiology. Damage is defined as an irreversible change in the microstructure that alters the macroscopic mechanical parameters. In tendon, the link between mechanical loading and microstructural damage, resulting in macroscopic changes, is not fully elucidated. In addition, tendon damage at the macroscale has been proposed to initiate when tendon is loaded beyond a strain threshold, yet the metrics to define the damage threshold are not determined. We conducted multi-scale mechanical testing to investigate the mechanism of tendon damage by simultaneously quantifying macroscale mechanical and microstructural changes. At the microscale, we observe full recovery of the fibril strain and only partial recovery of the interfibrillar sliding, indicating that the damage initiates at the interfibrillar structures. We show that non-recoverable sliding is a mechanism for tendon damage and is responsible for the macroscale decreased linear modulus and elongated toe-region observed at the fascicle-level, and these macroscale properties are appropriate metrics that reflect tendon damage. We concluded that the inflection point of the stress-strain curve represents the damage threshold and, therefore, may be a useful parameter for future studies. Establishing the mechanism of damage at multiple length scales can improve prevention and rehabilitation strategies for tendon pathology. Statement of Significance Tendon pathology is associated with mechanically induced damage. Damage, as defined in engineering, is an irreversible change in microstructure that alters the macroscopic mechanical properties. Although microstructural damage and changes to macroscale mechanics are likely, this link to microstructural change was not yet established. We conducted multiscale mechanical testing to investigate the mechanism of tendon damage by simultaneously quantifying macroscale mechanical and microstructural changes. We showed that non-recoverable sliding between collagen fibrils is a mechanism for tendon damage. Establishing the mechanism of damage at multiple length scales can improve prevention and rehabilitation strategies for tendon pathology.