UC Davis

The ability to move without pain is essential to many occupations and sports. When chronic tendon pain becomes limiting, it is diagnosed as tendinopathy. At a minimum, this tendon pain limits quality of life. For athletes engaged in professional sport, tendinopathy can be devastating and potentially career ending. The exact etiology of tendinopathy is unknown. However, it is especially common in activities that involve repetitive movements and high jerk (typing, jumping, etc.). Since this describes many sports and professions, it should not be surprising that tendon sprains, strains, and pulls are the leading cause of time away from work. The prevalence of tendon injury is highest in professional basketball with ~75% of players having patellar tendon lesions (Benítez-Martínez et al., 2019). Despite the prevalence and high personal and economic costs, there has been no progress in treatments for chronic tendon injuries over the last 25 years. This is in part due to a lack of understanding about basic tendon biology and adaptations that occur with growth, injury, and repetitive loading. This dissertation investigated post-natal development of the Achilles and patellar tendons, developed a rat model of patellar tendinopathy, determined the molecular response of different types (isometric and dynamic) of loads on a tendinopathic tendon, and characterized spatial gene expression in a healthy adult rat patellar tendon. The molecular and mechanical changes that occur during rat post-natal Achilles and patellar tendon development were characterized at P7 (before walking), P14 (shortly after the onset of walking), and P28 (motor maturity). The results are presented in Chapter 2. From P7 to P28, The Achilles increased 3-fold in length, whereas the patellar tendon increased largely in cross-sectional area. Despite the different modes of growth there was a ~10-fold increase in ultimate tensile strength of both the Achilles and patellar tendons. The increase in Achilles length resulted in a greater increase in modulus, whereas the greater cross-sectional area of the patellar meant a larger increase in maximal tensile load. The tendons shared transcriptional similarity at P7 and P14 but diverged at P28. However, there were still many shared processes indicative of tendon post-natal development. These changes included increased extracellular matrix (ECM) gene expression and decreased cell cycle and mitochondrial gene expression. Ribosomal gene expression also significantly decreased in the Achilles tendon and tended to decrease in the patellar tendon. From the genes that were highly expressed at P28 in the patellar tendon, STAT signaling was identified as a downregulated pathway. We hypothesized that these genes contributed to tendon radial growth. STAT inhibition in engineered human ligaments caused an increase collagen content and maximal tensile load (MTL). These results suggest that our developmental transcriptomic data may provide insight into targets to improve tendon function both after injury and during development/training. There are many models of rodent tendon injury; however, no molecular comparison had been made to the human condition. We modeled tendinopathy in the rat using a biopsy punch to remove the central third of the patellar tendon, followed by two weeks of cage activity to allow scar formation. The scar tissue was sequenced with 3’ Tag RNA-seq. Differential gene expression analysis of the healthy versus scar tissues identified an increase in ECM gene expression, decrease in mitochondrial gene expression, and no change in inflammatory pathways. The increase in ECM and decrease in mitochondrial genes meant that the scar was not recapitulating the developmental pattern of gene expression observed in Chapter 2. There was also a visible increase in vascularity and cellularity in the scar tissue. These changes within the tendon are nearly identical to human tendinopathy (Jelinsky et al., 2011). Next, we studied the effect of two distinct types of mechanical load (dynamic and isometric) on gene expression in tendinopathy. The isometric load (4 x 30 s) caused an increased in scleraxis and collagen Ia1 expression relative to the contralateral control leg. In contrast, the time under tension-matched dynamic load (360 x 0.33 s) caused in increase in type II collagen expression relative to the isometric loaded group. These data suggest that isometric loads may be used to treat a tendon injury and that the type of load, rather than the time under tension, is an important consideration when exercising a tendinopathic tendon. The last chapter of the dissertation presents the first complete report of spatial gene expression in tendon. I used the 10X Visium Platform to profile cell type and gene expression location in two adult rat patellar tendon samples. The cell type classification was performed with an anchoring analysis to a single cell RNA sequencing data set from mouse Achilles tendon (de Micheli et al., 2020). There were four cell populations identified: tendon fibroblasts (two main subpopulations), red blood cells, immune cells, and pericytes. The tendon midsubstance contained one tendon fibroblast subpopulation, while the other tendon cell subpopulation was located to the periphery of the main population and within the strip of connective tissue to the side of the tendon. This loose connective tissue also contained red blood cells, immune cells, and pericytes. The top expressed spatially variable genes in both samples were genes with known function in tendon (Col1a1, Dcn, Fmod, Sparc, and Comp). Interestingly, the two most common collagens 1a1 and 3a1 were expressed in separate populations of fibroblasts. I also identified one gene (AABR07000398.1) without a known function that was highly expressed in a pattern opposite to Col1a1. These data are important because they describe the spatial separation of two distinct fibroblast populations and provide the basis for better understanding spatial gene expression following injury. This dissertation provides novel insight into tendon post-natal development and spatial gene expression. It also validates a rodent model of tendinopathy and identifies molecular changes that occur after two different types of exercise on a tendinopathic tendon. Together, these data support a model where force is passed through the center of a healthy tendon and this force is transmitted through a matrix that is maintained by a population of tendon fibroblasts. Following injury, the scarred portion of the tendon is shielded from stress/strain and this may allow the infiltration of the tendon by a second population of fibroblasts. In order to return normal function of the tendon after injury, long holds are necessary to overcome stress shielding. Further, my data suggest that JAK/STAT inhibitors may improve tendon regeneration after injury. These data fill important knowledge gaps in the field of tendon biology, provide evidence for the first drug to treat tendinopathy, and establish the basis for future studies to reverse tendinopathy.