Effect of extraction protocols and epidermal growth factor on the cellular repopulation of decellularized anterior cruciate ligament allografts

Abstract

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.

Freeze-Thaw Cycles Enhance Decellularization of Large Tendons

“…19 Sodium dodecyl sulfate (SDS) was reported to be effective but to reduce cytocompatibility due to matrix alterations; however, the protocol used in these studies did not only include incubation in SDS, but also in Triton X-100. 11,17,22,23 Further, several groups performed detergent-based decellularization with samples that had previously been frozen or freezedried 15,16,24 ; it should be acknowledged that this may have had an additional effect on decellularization effectiveness.…”

Comparison of Three Methods for the Derivation of a Biologic Scaffold Composed of Adipose Tissue Extracellular Matrix

“…11,[14][15][16][17][18][19][20][21][22][23][24] When tissues have been efficiently decellularized, the ECM-specific structural and functional components that remain (collagen, laminin, fibronectin, growth factors, etc.) are relatively well conserved across mammalian species and, therefore, do not evoke adverse immune reaction.…”

Whole-organ engineering with natural extracellular materials

“…While verification of cell removal is typically quantified by DNA content post-decellularization by means of spectrophotometry [29][30][31][32], further characterization of the remaining major histocompatibility complexes (MHCs) is essential for clinical human allograft studies. For the decellularized human whole-trachea graft, MHC class I and II removal was monitored.…”