A simple dynamic strategy to deliver stem cells to decellularized nerve allografts

Background: The addition of adipose-derived Mesenchymal Stromal Cells (MSCs) to decellularized nerve allografts may improve outcomes of nerve reconstruction. Prior techniques used for cell seeding are traumatic to both the MSCs and nerve graft. An adequate, reliable and validated cell seeding technique is an essential step for evaluating the translational utility of MSC-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 MSCs separately onto rat and human decellularized nerve allografts. Cell viability was evaluated by MTS assays and cellular topology after seeding was determined by SEM microscopy. Cell density and distribution were determined by LIVE/DEAD assays and Hoechst staining at 4 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 MSCs exhibited exponential growth in the first 12 hours of seeding. Seeding efficiency increased significantly from 79.5% at 6 hours to 89.2% after 12 hours of seeding (p = 0.004). Both intra-rater (r = 0.97) and inter-rater reliability (r = 0.92) of the technique were high. Conclusions: This study describes and validates a new method to effectively seed decellularized nerve allografts with MSCs. This method is reproducible, distributes cells homogenously over the graft and does not traumatize the intra-neural architecture of the allograft. Utilization of this validated seeding technique will permit critical comparison of graft outcomes. Financial Disclosure Statement: Funding for this study was received from the Mayo Clinic Center for Regenerative Medicine Acknowledgements: We thank the American Donor Services for providing fresh human cadaveric motor nerves. We also thank Drs. Anthony Windebank, Allan Dietz, Amel Dudakovic and Roman Thaler for their expertise and guidance. Presentations: Part of this work was presented at the Annual Meeting of the American Society for Peripheral Nerve, Hawaii, United States, January 13th, 2017. Author Contributions: Nadia Rbia: contributed to the conception, analysis, interpretation of the data, as well as drafting of the manuscript for submission.Liselotte F. Bulstra: contributed to the conception, analysis, interpretation of the data, as well as the drafting of the manuscript for submission. Allen T. Bishop: contributed to the conception and interpretation of data, as well as edited the manuscript. Andre J. van Wijnen: contributed to the conception and interpretation of data, as well as edited the manuscript. Alexander Y. Shin: contributed to the conception and interpretation of data, as well as the acquisition of funding and editing of this manuscript. Corresponding Author: Alexander Y. Shin, M.D., Professor of Orthopedic Surgery, Professor of Neurosurgery, Mayo Clinic, 200 1st St. SW, Rochester, MN, 55905, Phone: 507-284-0475, Email: shin.alexander@mayo.edu. ©2018American Society of Plastic Surgeons