Background: The addition of adipose-derived mesenchymal stromal cells to decellularized nerve allografts may improve outcomes of nerve reconstruction. Prior techniques used for cell seeding are traumatic to both the mesenchymal stromal cells and nerve graft. An adequate, reliable, and validated cell seeding technique is an essential step for evaluating the translational utility of mesenchymal stromal cell-enhanced decellularized nerve grafts. The purpose of this study was to develop a simple seeding strategy with an optimal seeding duration. Methods: A dynamic bioreactor was used to seed rat and human mesenchymal stromal cells separately onto rat and human decellularized nerve allografts. Cell viability was evaluated by MTS assays and cellular topology after seeding was determined by scanning electron microscopy. Cell density and distribution were determined by Live/Dead assays and Hoechst staining at four different time points (6, 12, 24, and 72 hours). The validity and reliability of the seeding method were calculated. Results: Cells remained viable at all time points, and mesenchymal stromal cells exhibited exponential growth in the frst 12 hours of seeding. Seeding efficiency increased significantly from 79.5 percent at 6 hours to 89.2 percent after 12 hours of seeding (p = 0.004). Both intrarater reliability (r = 0.97) and interrater reliability (r = 0.92) of the technique were high. Conclusions: This study describes and validates a new method of effectively seeding decellularized nerve allografts with mesenchymal stromal cells. This method is reproducible, distributes cells homogenously over the graft, and does not traumatize the intraneural architecture of the allograft. Use of this validated seeding technique will permit critical comparison of graft outcomes.
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