Effect of extraction protocols and epidermal growth factor on the cellular repopulation of decellularized anterior...

We are developing a decellularized bone-anterior cruciate ligament (ACL)-bone allograft for treatment of ACL disruption in young or active patients. This study demonstrates the feasibility of seeding decellularized ACL tissue with primary ligament fibroblasts. Porcine ACLs were decellularized by one of three protocols, each differing only by the detergent/solvent used during the second wash (SDS, Triton-X, or TnBP). Porcine ACL fibroblasts were obtained by explant and seeded onto tissue samples of decellularized ACL. Culture conditions were varied to compare the relative effect of three different decellularization protocols on cellular repopulation. Culture condition variables included (1) the number of cells used for seeding, (2) the addition of epidermal growth factor (EGF), and (3) culture duration. Cellular ingrowth was assessed by metabolic activity (MTT assay), DNA quantification (Hoescht dye), and histology (H&E staining). Cell counting on histological sections demonstrated that Triton-X-and TnBP-treated ligaments were more receptive to cellular ingrowth than SDS-treated samples. The addition of EGF to culture medium did not significantly increase cellular ingrowth. Both the Triton-X and TnBP decellularization treatments provide suitable, naturally derived scaffolds for the ingrowth of primary ACL fibroblasts, and should be further investigated in the development of an allograft-derived bone-ACL-bone graft.