UC Davis

Musculoskeletal injuries account for the highest percent of time away from work and affect over 100 million people in the United States [1,2]. Ligament and tendon injuries account for approximately 50% of all musculoskeletal injuries [3]. Despite the prevalence of these injuries, current tendon and ligament research is lacking, with little advancement in the treatment of tendinopathies for decades. Tendinopathy describes an injured or diseased tendon/ligament. Underneath this umbrella term, tendinitis is used to describe an injured tendon/ligament that has signs of inflammation. There are many molecular pathways which have been explored in great lengths in muscle and bone; however, the same signals in tendon/ligament have significantly less research devoted to them – including the pro-inflammatory pathways upregulated in tendinitis. In this dissertation, we characterize an engineered ligament model, determine the effects of pro-inflammatory cytokines on engineered ligaments, and explore possible interventions to treat tendinitis such as anti-inflammatories and isometric loading.

The in vitro engineered ligament model used throughout this dissertation was first developed by the Baar lab group [4]. In order to further validate the use of this model, we characterized the effect of passage number on cell gene expression and ligament function, as well as the ligament development over time. Despite previous studies suggesting multiple passages of fibroblasts led to decreased gene expression of typical tenoblast markers, our results found that there was no significant change in gene expression across multiple passages. Furthermore, from passage 4 to passage 11 there was no observed change in mechanical function or matrix composition of engineered ligaments. These results validate the use of this in vitro engineered ligament model using both early and later passage cells. We then characterized the development of engineered ligaments over the course of 5 weeks. The results showed that from Day 7 to Day 14 there was an exponential increase in both collagen content and percent collagen, while these measures began to plateau after Day 14. There also was a linear increase in mechanical function from Day 7 to Day 21. Taken together, this data suggests that Day 7 to Day 14 best represents a developing or regenerating tissue, while after Day 14 the plateau of collagen content best represents a more mature tissue. In total, the work in Chapter 2 demonstrates the efficacy of the model used throughout this dissertation and provides multiple timepoints for interventions based on desired modeling of a regenerating vs mature tissue.

To screen for potential therapeutics for tendinitis, we first sought to develop a model for tendinitis using engineered ligaments. Treating ligaments with pro-inflammatory cytokines TNF-, IL-1, and IL-6 individually led to decreased mechanical function and reduced collagen content. Using a Box-Behnken design of experiments, we developed an optimal combination of all three cytokines to decrease ligament mechanics by 50%. Treatment with this cytokine cocktail led to impaired mechanical function and decreased collagen, resulting in the first multi-cytokine in vitro model for tendinitis. Utilizing this reproducible model, we targeted the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-B) pathway since it had been identified as a potential target for treatment of chronic tendinopathies [5]. Surprisingly, treatment with a known NF-B inhibitor alone actually decreased the mechanical function of ligaments, while treatment alongside the cytokine cocktail had little to no positive effects. We then targeted another proinflammatory pathway – Janus kinase 1 (JAK1) and signal transducer and activator of transcription (STAT)3. Inhibition of JAK1, either alone and in the presences of the cytokine cocktail, resulted in increased mechanical function and increased collagen content in engineered ligaments. Furthermore, the quality of the tissue was improved by JAK1 inhibition, as demonstrated by an increase in enthalpy when undergoing differential scanning calorimetry. In total, the work presented in Chapter 3 resulted in the first multi-cytokine model for tendinitis in an in vitro engineered ligament. Additionally, inhibition of JAK1 has been identified as a possible new treatment for tendinitis.

Isometric loading has previously been reported to improve tendon function, reduce pain, and even reverse the appearance of a central core patellar tendinopathy on MRI [6–8]. Given the positive effect of isometric loading, we tested the combined effects of isometric loading and JAK1 inhibition on engineered ligaments in Chapter 4. Isometric load by itself tended to increase mechanical functional with no significant effect on collagen, resulting in the first successful reproduction of isometric loading effects in this model. Additionally, isometric load alone increased enthalpy which suggests that load improved mechanical function of the tissue through improved matrix organization. Inhibition of JAK1 again improved mechanical function and collagen content by itself. However, there was no interaction effect between load and JAK1 inhibition, instead there were independent positive effects that together led to an additive increase. Interestingly, the combination of isometric loading and JAK1 inhibition did not increase collagen content relative to load alone, suggesting that isometric load may actually prevent an increase in collagen while simultaneously improving collagen fibril organization, diameter, and/or cross-linking. Overall, the combined intervention of isometric load and JAK1 inhibition improve engineered ligament mechanical and material properties. This further supports the possibility of JAK1 inhibition as a novel therapeutic for tendinopathy and may improve upon the previously reported effective isometric loading protocols.

In sum, this dissertation better characterizes and validates the use of engineered human ligaments to make fundamental discoveries concerning tendon biology. Using this model, I developed a novel multi-cytokine treatment to reliably reproduce the negative effects of pro-inflammatory cytokines on ligaments and screen for therapeutics. This resulted in the identification of a new potential treatment for tendinitis – inhibition of JAK1 using a class of drugs that have FDA approval. Ultimately, this work furthers understanding within the field of tendon/ligament physiology and sets the foundation for future in vivo studies to improve treatment of tendinitis.