OBJECTIVES/GOALS: Load sharing across the arc of knee flexion of the medial knee ligaments (MKLs) is not well understood. The goal of this research is to characterize ligament engagement and in-situ force within the deep and superficial medial collateral ligament (dMCL, sMCL) and the posterior oblique ligament (POL) in response to externally applied multiplanar loads. METHODS/STUDY POPULATION: Ten human cadaveric knees, 5 male and 5 female, age 32±7 (25-42) [mean±SD (range min-max)] years, were mounted to a force sensor and a 6-degree-of-freedom robotic arm. Knee kinematics, before and after serial dissection of the sMCL, dMCL, and POL, were recorded from 0-30 degrees during applied isolated external rotation, valgus angulation, and anterior tibial moments, and the force (Newtons, N) borne by each structure was measured via the principle of superposition. Loads in the dMCL, sMCL, and POL will be compared across each knee and at each flexion angle with paired t-tests and repeated-measures analysis of variance with Tukey post hoc testing. Ten knees will provide >99% power to detect differences of 5N ± 3% at p=0.05, which is considered the threshold for clinically meaningful force differences. RESULTS/ANTICIPATED RESULTS: Our anticipated results include characterization of the means and standard deviations of the in-situ forces within the dMCL, sMCL, and POL in response to externally applied valgus angulation, tibial external rotation, and anterior-directed tibial loading at 0, 15, and 30 degrees of knee flexion. Our statistical analysis will determine if there are clinically meaningful differences (5N ± 3%) in the loads within each ligament at different knee flexion angles and will also provide data regarding differential relative ligament engagement for each applied force scenario, which is an indication of the percentage of contribution that each structure contributes to knee stability during application of forces and torques to the knee. DISCUSSION/SIGNIFICANCE: Data on ligament engagement and in-situ forces will help clinicians better diagnose potentially injured ligaments when they observe pathological knee laxity in an injured patient. Our results will also inform future computer modeling studies on injury mechanisms, individual anatomical variability, and surgical planning.