Anterior cruciate ligament (ACL) injuries lead to progressive degeneration of the knee joint and development of osteoarthritis in one-third of untreated patients. In the U.S., over 42,000 patients per year require surgical reconstruction of the ACL. Patellar tendon autografts are most frequently used, but acute graft necrosis delays neoligament formation. In theory, maintenance of graft viability should improve neoligament formation. New approaches are needed to test this theory. Acellular collagen scaffold implants can induce tissue ingrowth and perform mechanically similar to autografts after ACL reconstruction. The proposed research involves development of 'autograft analogues' (fibroblast-seeded collagen scaffolds) to test these hypotheses: Neoligament formation can be improved by implanting viable autogenous fibroblasts that proliferate and synthesize new matrix without undergoing acute necrosis. Fibroblast-seeded autograft analogues can remodel to approximate the structure and function of the ACL. The project has three specific aims: 1. Measure and optimize fibroblast attachment, proliferation, and matrix synthesis on fibroblast-seeded collagen scaffolds in vitro. Determine fibroblast viability and collagen scaffold strength as a function of time in vitro. 2. Evaluate neoligament formation, and neoligament remodeling, in ACLs surgically reconstructed using: a) patellar tendon autografts; b) unseeded collagen scaffolds; c) scaffolds seeded with intra-articular (ACL or synovial) fibroblasts; and d) scaffolds seeded with extraarticular (patellar tendon or skin) fibroblasts. 3. Evaluate the effects of mechanical loading on neoligament remodeling by using the same groups of implants as unloaded grafts in the contralateral knee. Neoligament tissue will be evaluated by biomechanical testing, histology, and analyses of the collagen network, for acute and chronic ACL reconstructions in rabbits. The ratio of viable seeded fibroblasts to newly-ingrown fibroblasts in the neoligament will be determined using fluorescent labelling and flow cytometry. Bone tunnel insertion sites will be examined using tetracycline labelling. These studies will elucidate the role of autogenous fibroblasts and mechanical loads in neoligament formation/remodeling. Autograft analogues are potentially useful for clinical ACL reconstruction: fibroblasts could be obtained from biopsy, cultured, seeded onto a collagen scaffold, and implanted as an ACL substitute into the same patient.
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