Tendon pathologies, including both chronic injuries and acute tendon tears, are some of the most common musculoskeletal injuries. Chronic tendon injury, or tendinopathy, occurs both in athletes and in the general population, and can interfere with quality of life and ability to work [1, 2]. Overuse of the tendon during exercise plays a role in up to 50% of injuries in athletes, and affects multiple parts of the body including the supraspinatus tendon in the shoulder, and the Achilles tendon in the ankle [3, 4, 5]. Acute tears of tendons and ligaments, on the other hand, add substantially to the socioeconomic burden of tendon disease as a whole. These injuries also affect both upper and lower extremities, including the shoulder, ankle, hand, and wrist [6, 7, 8, 9]. While acute tendon and ligament injuries affect all ages, they typically have a higher prevalence among young adult patients engaging in intense physical activity [10, 11].

Historically, inflammatory processes have been thought to be of little importance in tendon pathology, due to the largely avascular nature of the healthy tissue. However, more recent literature has identified the presence of inflammation in both acute and chronic tendon injury. Because the literature on inflammation in tendon is in relatively nascent stages, there remain gaps in knowledge that hinder progress in the development of therapeutics to improve healing. Characterizing the inflammatory response in tendon, including the relative roles of different pathways and the role of mechanical loading, requires both cellular level and tissue level analysis.

To create a platform by which to investigate these objectives, an in vitro model was developed, wherein the complexity of the in vivo healing environment was simulated by M1 macrophage paracrine signaling, motivated by the well-established role of macrophages in driving tendon inflammatory responses [12]. This was achieved using conditioned media from M1 macrophages, which were applied to tendon fibroblasts after collection and filtration of the media. The inflammatory response in tendon fibroblasts was assessed via a combination of qPCR, multiplex protein assays for secreted cytokines, and bulk RNA sequencing. Characterization of the M1-CM and its effect on TFs revealed a robust inflammatory response in TFs, inducing the upregulation of over 500 genes and increased secretion of several cytokines. Additionally, the relative secretion of different cytokines within a pre-defined panel in M1-CM was distinct from that characterized in the TF supernatant upon stimulation with M1-CM. Thus, the macrophages were able to induce a distinct inflammatory profile in TFs through paracrine signaling that was specific to TFs.

Next, multiple immune-related pathways were manipulated in tendon fibroblasts in order to identify those necessary for inflammatory responses. Both the NF-kB pathway and the JAK/STAT signaling pathway were inhibited, to determine their respective roles in propagating inflammation. It was determined that both JAK/STAT and NF-kB were necessary for the response to M1-CM, and each pathway was responsible for different downstream responses to inflammation in TFs.

Additionally, the role of mechanical loading in tendon responses to inflammation was assessed, as mechanical stimulation is crucial in tendon function in homeostasis and in healing [13, 14, 15]. To determine how inflammatory activation affects tendon fibroblast responses to mechanical stimulation, M1 macrophage media was first applied to tendon fibroblasts in culture to induce inflammation. Then, the activated fibroblasts were subjected to tensile strain, and their responses to the loading regime were compared to that of untreated tendon fibroblasts. We found that the TF response to loading was altered by the presence of an inflammatory stimulus, with more genes being downregulated by loading than under control conditions. However, there were also responses to loading that were conserved regardless of the presence or absence of inflammation. Analysis of the genes that responded differently to loading under inflammatory conditions suggested changes in pathways involving extracellular matrix organization and G protein signaling.

Determining how responses to mechanical stimuli and inflammation interact in tendon is crucial to understanding healing processes in tendon overall. The results of this study indicate that the application of loading may serve to reduce ECM degradation processes, and calm the inflammatory response in tendon, without suppressing it entirely. Overall, it is clear that the role of mechanical stimulus is important to consider when studying inflammatory responses in tendon.