We have recently discovered that adolescent animals have a functionally successful repair response to ACL injury when a tissue engineering repair strategy is used. This strategy utilizes a collagen scaffold with an optimized platelet solution (a collagen-platelet composite, CPC) to enhance healing in the ligament wound site. When using the CPC, we discovered that the strength of the ACL after three months of healing was equivalent to an ACL reconstruction graft, the current gold standard of treatment. Further study demonstrated that the strength of the bio-enhanced repairs doubled between 3 and 6 months, while the strength of ACL reconstruction has been previously reported to plateau during that period in similar animal models. These results are extremely promising for this new technique; however, all of these studies have had the injury created and treated at the same setting (immediate repair). In the clinical setting, there is often a delay of 2 weeks or more between the time of injury and surgery which is different from our prior studies. We recently found that delays of even two weeks between injury and repair can be deleterious for ACL healing. In this grant, we will define the mechanisms responsible for the loss of healing efficacy with a two week delay. We hypothesize that by understanding the key cellular and molecular changes which occur following ACL injury, we will be able to intervene in this process and further improve the functional outcome of ACL repair. The following Specific Aims will be performed: Aim 1: Determine the changes in cytokine production and gene expression in serum, synovial fluid, and tissue over the 14 days following an acute ACL injury. Aim 2: Define the cellular mechanisms affected by changes in cytokine levels and their effects on cruciate fibroblasts obtained from porcine and human ACL tissue. Aim 3: Improve the functional healing of the ACL through delivery of a targeted intervention after injury. The proposed studies will identify early changes at the molecular, cellular and tissue levels which may affect the success of the repaired ligament using a variety of techniques. Changes in gene expression will be identified via RNASeq, in situ hybridization, and laser capture microdissection while protein expression will be quantified using ELISA and proteomics approaches. Characterization of these catabolic pathways which impair healing will provide the foundation for identifying a means of mitigating the negative effects which occur after injury and improve the functional performance of ACL repair. PUBLIC HEALTH RELEVANCE: Anterior cruciate ligament (ACL) rupture affects over 400,000 patients each year in the US. Unfortunately, even with the best surgical reconstruction techniques available today, as many as 75% of patients will develop premature osteoarthritis. Thus, there is great interest in developing improved treatments for this injury. One promising technique is bio-enhanced ACL repair, where instead of replacing the torn ACL with a tendon graft, the ACL can be sutured back together with a bioactive scaffold that stimulates healing. This technique is working well in preclinical studies when performed immediately after an ACL injury, but is not as successful when the repair is delayed for several weeks. Overcoming this problem is critical, as human patients typically have at least a two-week delay between injury and surgery. This proposal aims to define changes occurring at the molecular, cellular and tissue levels during that two week delay which may contribute to the loss in the ability to attain successful bio-enhanced repair of the ACL. Once we define the timing and pattern of the catabolic changes, a targeted pharmaceutical intervention can be used to block these changes and improve the repair and regeneration of the torn ACL.