Characterization of an in vitro engineered ligament model.

In vivo tendon and ligament research can be limited by the difficultly of obtaining tissue samples that can be biochemically analyzed. In this study, we characterize the most widely used in vitro engineered ligament model. Despite previous works suggesting multiple passages change gene expression in 2D primary tenocytes, we found no relationship between passage number and expression of classical tendon fibroblast markers across different biological donors. When engineered into 3D ligaments, there was an increase in maximal tensile load between 7 and 14 days in culture, that corresponded with an increase in collagen content. By contrast, percent collagen increased logarithmically from Day 7 to Day 14, and this was similar to the increase in the modulus of the tissue. Importantly, there was no relationship between passage number and mechanical function or collagen content in the two independent donors tested. These results suggest that the model develops quickly and is reliable across differing passage numbers. This provides the field with the ability to 1) consistently determine functional changes of interventions out to passage number 10; and 2) to time interventions to the appropriate developmental stage: developing/regenerating (Day 7) or mature (Day 14) tissue.

Inhibiting JAK1, not NF-κB, reverses the effect of pro-inflammatory cytokines on engineered human ligament function.

The role of inflammation in chronic tendon/ligament injury is hotly debated. There is less debate about inflammation following acute injury. To better understand the effect of acute inflammation, in this study we developed a multi-cytokine model of inflammatory tendinitis. The combined treatment with TNF-α, IL-1ß, and IL-6, at dosages well below what are routinely used in vitro, decreased the mechanical properties and collagen content of engineered human ligaments. Treatment with this cytokine mixture resulted in an increase in phospho-NF-κB and MMP-1, did not affect procollagen production, and decreased STAT3 phosphorylation relative to controls. Using this more physiologically relevant model of acute inflammation, we inhibited NF-κB or JAK1 signaling in an attempt to reverse the negative effects of the cytokine mixture. Surprisingly, NF-κB inhibition led to an even greater decrease in mechanical function and collagen content. By contrast, inhibiting JAK1 led to an increase in mechanical properties, collagen content and thermal stability concomitant with a decrease in MMP-1. Our results suggest that inhibition of JAK1, not NF-κB, reverses the negative effects of pro-inflammatory cytokines on collagen content and mechanics in engineered human ligaments.

The rat Achilles and patellar tendons have similar increases in mechanical properties but become transcriptionally divergent during postnatal development.

The molecular events that drive post-natal tendon development are poorly characterized. In this study, we profiled morphological, mechanical, and transcriptional changes in the rat Achilles and patellar tendon before walking (P7), shortly after onset of walking (P14), and at motor maturity (P28). The Achilles and patellar tendons increased collagen content and mechanical strength similarly throughout post-natal development. However, at P28 the patellar tendon tended to display a higher maximal tensile load (MTL) (P = 0.0524) than the Achilles tendon, but a similar ultimate tensile strength (UTS; load relative to cross-sectional area) probably due to its increased cross-sectional area during development. The tendons started transcriptionally similar, with overlapping PCA clusters at P7 and P14, before becoming transcriptionally distinct at P28. In both tendons, there was an increase in extracellular matrix (ECM) gene expression and a concomitant decrease in cell cycle and mitochondrial gene expression. The transcriptional divergence at P28 suggested that STAT signalling was lower in the patellar tendon where MTL increased the most. Treating engineered human ligaments with the STAT inhibitor itacitinib increased collagen content and MTL. Our results suggest that during post-natal development, cellular resources are initially allocated towards cell proliferation before shifting towards extracellular matrix development following the onset of mechanical load and provide potential targets for improving tendon function. KEY POINTS: Little is known about mechanisms of post-natal tendon growth. We characterized morphological, mechanical, and transcriptional changes that occur before (P7), and early (P14) and late after (P28) rats begin to walk. From P7 to P28, the Achilles tendon increased in length, whereas the patellar tendon increased in cross-sectional area. Mechanical and material properties of the Achilles and patellar tendon increased from P7 to P28. From P7 to P28, the Achilles and patellar tendons increased expression of ECM genes and decreased mitochondrial and cell cycle gene expression. Ribosomal gene expression also significantly decreased in the Achilles and tended to decrease in the patellar tendon. At P28, STAT1 signalling tended to be lower in the patellar tendon which had grown by increasing cross-sectional area and inhibiting STAT activation in vitro improved mechanical properties in engineered human ligaments.

Cannabidiol Does Not Impact Acute Anabolic or Inflammatory Signaling in Skeletal Muscle In Vitro.

Background: Cannabidiol (CBD) is becoming increasingly popular for the treatment of clinical conditions including as an aid for muscle recovery. Previous work demonstrated that CBD exhibited mild effects on skeletal muscle, with a tendency to increase anabolic signaling and decrease inflammatory signaling. Methods: To gain mechanistic insight and extend these findings, we conducted a set of experiments using C2C12 myotubes. Results: Increasing the dose of CBD (1-5 µM) provided with insulin-like growth factor 1 (IGF-1) showed no effect on anabolic signaling through mTORC1 (S6K1 [Thr389], p=0.27; rpS6 [Ser240/244], p=0.81; or 4E-BP1 [Thr37/46], p=0.87). Similarly, inflammatory signaling through nuclear factor kappa B (NF-κB) (p105, p=0.88; p50, p=0.93; or phosphorylated p65 [Ser536], p=0.84) in response to tumor necrosis factor α (TNFα) was unaffected by CBD (2.5 µM), whereas dioscin, a natural product that blocks NF-κB signaling, reduced p105 and phosphorylated p65 (Ser536) compared with the TNFα and the TNFα + CBD condition (p<0.01 and p<0.05, respectively). Finally, cannabinoid receptor type 1 (CB1) receptor levels were measured in C2C12 cells, murine skeletal muscle, cortex, and hippocampus. Although CB1 was not detectable in muscle cells or muscle tissue, high levels were observed in brain tissue. Conclusion: In conclusion, CBD does not directly modulate anabolic or inflammatory signaling in myotubes in vitro, which can likely be explained by the lack of functional receptors.

A mutation in desmin makes skeletal muscle less vulnerable to acute muscle damage after eccentric loading in rats.

Desminopathy is the most common intermediate filament disease in humans. The most frequent mutation causing desminopathy in patients is a R350P DES missense mutation. We have developed a rat model with an analogous mutation in R349P Des. To investigate the role of R349P Des in mechanical loading, we stimulated the sciatic nerve of wild-type littermates (WT) (n = 6) and animals carrying the mutation (MUT) (n = 6) causing a lengthening contraction of the dorsi flexor muscles. MUT animals showed signs of ongoing regeneration at baseline as indicated by a higher number of central nuclei (genotype: P < .0001). While stimulation did not impact central nuclei, we found an increased number of IgG positive fibers (membrane damage indicator) after eccentric contractions with both genotypes (stimulation: P < .01). Interestingly, WT animals displayed a more pronounced increase in IgG positive fibers with stimulation compared to MUT (interaction: P < .05). In addition to altered histology, molecular signaling on the protein level differed between WT and MUT. The membrane repair protein dysferlin decreased with eccentric loading in WT but increased in MUT (interaction: P < .05). The autophagic substrate p62 was increased in both genotypes with loading (stimulation: P < .05) but tended to be more elevated in WT (interaction: P = .05). Caspase 3 levels, a central regulator of apoptotic cell death, was increased with stimulation in both genotypes (stimulation: P < .01) but more so in WT animals (interaction: P < .0001). Overall, our data indicate that R349P Des rats have a lower susceptibility to structural muscle damage of the cytoskeleton and sarcolemma with acute eccentric loading.

Muscle-tendon cross talk during muscle wasting.

In organisms from flies to mammals, the initial formation of a functional tendon is completely dependent on chemical signals from muscles (myokines). However, how myokines affect the maturation, maintenance, and regeneration of tendons as a function of age is completely unstudied. Here we discuss the role of four myokines-fibroblast growth factors (FGF), myostatin, the secreted protein acidic and rich in cysteine (SPARC) miR-29-in tendon development and hypothesize a role for these factors in the progressive changes in tendon structure and function as a result of muscle wasting (disuse, aging, and disease). Because of the close relationship between mechanical loading and muscle and tendon regulation, disentangling muscle-tendon cross talk from simple mechanical loading is experimentally quite difficult. Therefore, we propose an experimental framework that hopefully will be useful in demonstrating muscle-tendon cross talk in vivo. Though understudied, the promise of a better understanding of muscle-tendon cross talk is the development of new interventions that will improve tendon development, regeneration, and function throughout the lifespan.