The Introduction Of Human Mesenchymal Stem Cells To Clinically Available Nerve Substitutes
Femke Mathot, MD1,2; Nadia Rbia, MD1,2; Mana Saffari, MD1,2; Allen T. Bishop, MD3; Andre van Wijnen, PhD4; Alexander Y. Shin, MD3
1Erasmus MC, Rotterdam, Netherlands, 2Mayo Clinic, Rochester, MN, 3Department of Orthopedic Surgery, Microvascular Research Laboratory, Mayo Clinic, Rochester, MN, 4Orthopedic Surgery, Mayo Clinic, Rochester, MN
Hypothesis
We hypothesized that seeding human adipose derived Mesenchymal Stem Cells (MSCs) on the surface of clinically available nerve replacement treatments (Avance Nerve graft and NeuraGen Nerve guide) could provide extra biological support and potentially enhances the outcomes of these nerve substitutes.
Methods: An MTS assay examined the viability of human MSCs at different time intervals when a 2mm segment of Avance Nerve Graft (group I) or NeuraGen Nerve Guide (group II) was added to their environment (n=3 per group per time point). MSCs were seeded by using a non-traumatic dynamic seeding strategy, preserving the inner ultrastructure of the nerve substitutes. 1 million MSCs per nerve substitute were transferred to conical tubes containing either a 10mm Avance nerve graft or a 10mm NeuraGen nerve guide. The conical tubes were rotated in a bioreactor for 6, 12 and 24 hours after which cell counts were performed to obtain seeding efficiencies. The distribution of the MSCs was mapped with live/dead and Hoechst stains. Fixed cross-sectional sections of the seeded nerve substitutes were Hoechst stained to observe the migration of cells inside the graft.
Results: The viability of MSCs was not influenced by the presence of both nerve substitutes. For group I, a seeding efficiency of 18.23% was obtained after 6 hours, increasing to 66.46% after 12 hours (p<0.001) after which the efficiency decreased to 59.90% after 24 hours (p=1.00). For group II, the seeding efficiency increased from 52.08% after 6 hours to 94.17% after 12 hours (p=0.009) and decreased to 52.50% after 24 hours (p=0.009). Seeding efficiencies were significantly higher for group 2 after 6 and 12 hours of seeding (p=0.007 and p=0.025). Live/dead stains and Hoechst stains on all time points showed a uniform distribution of viable MSCs over the entire surface of both nerve substitutes. Cross-sectional sections revealed that the MSCs were absent on the inside of group I, but were present on the inside of group II.
Conclusion
Viable human MSCs can be seeded on both nerve substitutes without harming the inner ultrastructure; 12 hours is the optimal seeding duration. Human MSCs were seeded on the surface of both nerve substitutes in a uniform matter. MSCs only migrated into the NeuraGen nerve guide during dynamic seeding. Our method showed to have great clinical potential to improve and individualize peripheral nerve repair.
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