Project Summary: Anterior cruciate ligament (ACL) injuries are highly common especially in individuals with active lifestyles. Based on a recent report from American Orthopedic Society for Sports Medicine, more than 150,000 Americans suffer from ACL injuries each year costing an estimated 500 million US dollars. Current treatment strategies such as autografts and allografts are highly limited due to donor site morbidity and risk of immune rejection, respectively. Synthetic grafts are a promising alternative but suffer from major drawbacks that include poor graft integration and mechanical mismatch at the graft-tissue interface resulting in suboptimal healing, repetitive graft failure and poor clinical outcome. Tissue engineering of the graft-tissue interface has significant potential to improve the clinical outcome of ACL reconstruction surgeries using synthetic grafts. The central hypothesis of the proposed study is that incorporation of a biomimetic bioglass gradient that compositionally, mechanically and biologically mimics the native ACL enthesis will improve synthetic graft integration by promoting cell migration, composition-directed cell differentiation and de novo matrix formation. Studies in Aim 1 of the proposal will employ a 3D Raman spectral mapping based approach to discern the collagen:mineral compositional gradient at the native bone-fibrocartilage-ligament interface of the rabbit ACL. The Raman spectral data will be converted into STL files and used to 3D print collagen-based biomimetic bioglass gradient incorporated matrices (BioGIMs) that replicate the compositional gradient of the native ACL. Studies in Aim 2 of the proposal will focus upon achieving material-directed differentiation of MSCs by mimicking the compositional and mechanical properties of the BioGIMs to that of native ACL enthesis. First, bioglass particle size will be modulated (35 nm and 10 m) and the most optimal bioglass particle size that yields composition- directed differentiation will be identified. Second, genipin crosslinking will be employed and modulated for BioGIMs with the most optimal particle size to converge upon the mechanical properties of native enthesis and thus further enhance material-directed differentiation. Cellular differentiation studies will be performed in different culture medium conditions (normal growth medium, and osteogenic medium) to assess the bioactivity of the BioGIMs without the addition of external factors. Studies in Aim 3 of the proposal will investigate matrix reorganization on BioGIMs that yield material-directed differentiation as per Aim 2. BioGIM functionality will be assessed via confirmation and typification of cell-synthesized tissue-specific matrix, evaluation of biomechanical properties of BioGIMs after culture and assessment of cellular distribution and de novo matrix components by Raman spectroscopy and conventional biological assay methods. Overall, the expected outcomes of the proposed study is to deliver a mechanically competent Bio-GIM with material-directed MSC differentiation and matrix reorganization which can then be integrated onto synthetic grafts to improve the outcome of ACL surgeries in future small-animal models studies. Finally, this combined approach of Raman spectroscopy and 3- D printing for the development of biomimetic gradients is not limited to ACL and is easily applicable for the regeneration of other vital joints such as the rotator cuff tendon and articular cartilage.