Cellular repopulation of decellularized ACL allografts: Effects of bFGF, cell seeding approaches, and perfusion culture.

摘要

Injury to the anterior cruciate ligament (ACL) can lead to long-term joint instability and complications such as osteoarthritis. Currently, surgical intervention in the form of a patellar tendon autograft is favored as a method of ACL reconstruction. While this method has proved successful, complications such as joint stiffness and decreased range of motion may result in a patient being unable to return to previous activity levels.;The use of allograft ACL tissue as a method of ACL reconstruction allows for the original multiaxial structure of the ACL to be recaptured. Allograft tissue, however, presents the challenge of potentially immunogenic cellular components, which may result in an immune response in the host and poor graft performance. Through a series of washes and enzymatic digests, the cellular component can be removed, leaving the intact extracellular matrix, and maintaining the original structure and mechanical performance of the native ACL. By reseeding the allograft with autologous ACL fibroblast cells from the graft recipient, it is hypothesized that a living tissue graft can be created that will mimic the structure and function of the native ACL, and potentially serve as a new standard in ACL reconstruction. This study focuses on improving the cellular repopulation of decellularized ACL tissue, with a particular aim to determine the role of cell culture and seeding approaches steps in increasing cellular repopulation.;Porcine ACLs were decellularized using previously established protocols, and both whole and segments of decellularized ligaments were re-seeded with ACL fibroblasts obtained by explant from fresh tissue. Seeding and culture variables of tissue segments included (i) seeding time, (ii) seeding vessel, (iii) serum concentration in medium, (iv) exposure to basic fibroblast growth factor (bFGF,) and (v) location of seeding along length of ligament. Whole ligaments were reseeded and cultured under static and flow conditions. Cellular repopulation was assessed using cell quantification (Cyquant assay,) histology (hematoxylin and eosin staining,) and immunohistochemistry (MMP-1, procollagen III.);Seeding of tissue segments Was determined to be surface-area limited, and serum deprivation during culture was resulted in less cellular repopulation of tissue segments after 14 days in culture. Exposure of ACL fibroblasts to bFGF was found to increase proliferation in the absence of tissue, however, exposure did not result in increased repopulation of tissue after 28 days. Finally, the seeding and of whole ligaments under flow and static conditions demonstrated the possibility of whole ligament seeding, as well as the benefits of flow conditions, which resulted in significantly greater cellular repopulation after 14 days when compared to static culture.;This study provides a standard for seeding of tissue segments of decellularized porcine ACL tissue, and demonstrates methods for the seeding and culture of whole decellularized allografts. This concept provides proof of principle for the future study into the repopulation and performance of cellular repopulated decellularized ACL allografts in vivo.