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The Effect of Local Exosomes, Stem Cells and Tacrolimus on Neurite Extension
Tara Sara Saffari, MD, MSc1,2; Daan J Rademakers, BSc1,2; Tiam M Saffari, MD, PhD, MSc1,2,3; Nicholas Pulos, MD1; Shin Y Alexander, MD4
1Mayo Clinic, Rochester, MN; 2Radboudumc, Nijmegen, Netherlands; 3The Ohio State University Wexner Medical Center, Columbus, OH; 4Department of Orthopedic Surgery, Mayo Clinic, Rochester, MN

Background: The effect of Purified Exosome Product (PEP) on enhancing nerve regeneration has been an undiscovered topic of interest. PEP is an off-the-shelve product and may overcome the limitations of mesenchymal stem cells (MSC). Tacrolimus (FK506) is a FDA approved immunosuppressant with neuroregenerative properties. The aim of this study was to investigate the combined effect of local delivery of PEP, MSCs and FK506 alone and combined on nerve regeneration.
Materials & Methods: A three-dimensional in vitro compartmented cell culture system was used to evaluate regenerating neurites from a rat neonatal dorsal root ganglion (DRG) into the adjacent nerve graft. Decellularized allografts were augmented with local application of (i) dynamic undifferentiated MSC seeding, (ii) PEP (5%), (iii) FK506 (100 ng/mL), (iv) combined PEP+FK506, and (v) combined MSC+FK506 (N=9/group). Outcomes were compared to untreated autografts and allografts. After 48 hours of incubation, constructs were collected and stained against Neurofilament-160 to measure neurite extension. CD90-DAPI staining was used to confirm stem cell characterization.
Results: Stem cell viability was confirmed in all MSC-treated nerve grafts using CD90. Combined PEP+FK506 treatment to allografts resulted in the longest neurite extension and significantly improved outcomes of untreated allografts (p<0.05), reaching comparability to autografts (p=0.66). In allografts, combined PEP+FK506 treatment was comparable to PEP alone and MSC+FK506 (respectively, p=0.97 and p=0.80). PEP alone and MSC+FK506 treatment were found superior to negative controls (P<0.05).
Conclusions: PEP combined with FK506 in allografts resulted in the longest neurite extension and was superior to untreated allografts, and comparable to autograft (gold standard), PEP alone and MSC+FK506 treatment. These results suggest that the neuroregenerative effect of PEP is similar to MSCs. PEP could overcome the limitations of harvesting, culturing and seeding of stem cells, making this more translatable to clinical care. In vivo animal models are needed to further investigate the effect of PEP and PEP combined with FK506 in nerve regeneration.
Figure 1 Longitudinal sections of dorsal root ganglion (DRG)-nerve graft constructs stained with Neurofilament-160 (in red). Growing neurites are visualized in (A) autografts and (B) untreated allografts. Allografts are treated with (C) MSCs, (D) 5% PEP, (E) 100 ng/ml tacrolimus, (F) combined PEP+FK506 combined, and (G) combined MSC+ FK506 combined. Neurites grew from the DRG into the nerve graft; from the circular top to bottom.


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