Anterior Cruciate Ligament Injury Stimulates Cytokine Activation and Morphological Maladaptions in Patellar Tendon

Over 250,000 individuals suffer an ACL tear annually in the United States alone, with a lifetime treatment cost over $10 billion. Research has predominately focused on the recovery of quadriceps muscle strength and knee biomechanics after ACL injury; however, other knee structures that contribute to functional strength and performance have garnered less attention. After ACL injury and reconstruction, patellar tendon shortening is observed with and without harvesting of a portion of patellar tendon for grafting, indicating morphological alterations to the tendon even in the absence of a direct insult. 30% of patients report patellofemoral pain 12 months after hamstring-tendon ACL reconstruction, and 7.6% show clear radiographic evidence of patellar tendinopathy after ACL rupture. Furthermore, any previous knee injury is an established risk factor for development of patellar tendinopathy. Patellar tendinopathy is a common cause of knee pain in athletes, resulting in reduced playing time and impaired performance. Most cases are treated with non-operative interventions, such as physical therapy. Healthy tendons are avascular and primarily composed of tightly organized collagen 1 fibrils with resident tenocytes supporting tissue homeostasis. Tendinopathy is associated with development of fibrovascular tissue within the tendon with disorganized extracellular matrix proteins, disproportionate deposition of collagen 3, and altered interstitial cell abundance. In direct tendon injury models, cytokine signaling, particularly TGFâ, mediates proliferation of fibrogenic cells, contributing to development of pathogenic fibrosis that is not fully resolved; however, the contribution of resident tenocytes compared to infiltrating fibrogenic cell types is not well understood. Our lab has identified upregulated cytokine signaling after ACL injury that mediates cellular and morphological alterations in vastus lateralis in the absence of a direct muscular injury; however, diffuse ACL injury-induced cytokine upregulation and associated cellular and morphological maladaptions in patellar tendon have not been investigated. The objective of this proposal is to determine the time course of cytokine activation and deleterious cellular and morphological changes to patellar tendon resultant from a torn ACL using a novel murine model, which may identify future therapeutic targets to mitigate tendon pathology after ACL injury. Aim 1. Define the time course of cytokine signaling within the patellar tendon in a mouse ACL transection model. We will perform ACL transection surgery unilaterally on C57BL/6 mice. The alternate limb will undergo sham surgery to serve as an internal control. Patellar tendons will be harvested from each limb at 1, 3, 7, 10, 14, and 21 days after ACL transection and sham surgeries, and expression of a panel of cytokines as well as downstream signaling will be measured. Hypothesis: ACL injury will induce upregulated cytokine activation and downstream signaling in the patellar tendon in a progressive manner. Aim 2. Determine collagen isoform frequency, capillarization, and fibrogenic cell abundance and activity in the patellar tendon in a mouse ACL transection model. ACL transection and sham surgeries will be performed on mice followed by daily intraperitoneal (IP) injections with EdU to track cell proliferation, and patellar tendons will be harvested at 1, 3, 7, 10, 14, and 21 days after ACL transection and sham surgeries. Patellar tendon area, proportion of collagen 1 relative to collagen 3, capillarization, and fibrogenic cell abundance (tenocytes, myofibroblasts, fibrogenic adipogenic progenitors/FAPs) and proliferation will be measured using immunohistochemical techniques with fluorescent microscopy. Incorporation of EdU will be used to measure proliferation of each fibrogenic cell type. Sports Medicine Impact: These experiments will show that cytokine expression and subsequent downstream signaling are progressively upregulated in the patellar tendon after ACL transection, supporting deleterious cellular and morphological adaptations representative of patellar tendinopathy. The results will support future human studies and identify potential therapeutic targets to mitigate tendon maladaption and associated pain after ligamentous injury, likely promoting improved participation in physical therapy to restore quadriceps strength and subsequently enhanced return to sport and performance outcomes.