The AAPS 101st Annual Meeting

Background:

Local modulation of the nerve reconstruction micro-environment has been an area of interest to enhance peripheral nerve regeneration. Neuromodulatorsinclude:

•Adipose-derived mesenchymal stem cells (MSCs):

-Act through paracrine properties to enhance regeneration

-Limitations: cell culture, cel instability, tumorigenesis

•Tacrolimus (FK506): FDA approved immunosuppressant

-Accelerates nerve regeneration, reduces fibrosis at nerve reconstruction site.

-Local delivery to avoid unwanted side effects of drug toxicity.

•Exosomes (EX) are nanosized cell-free membrane extracellular vesicle

•Purified exosome product (PEP)

•Blood-derived, lypholizedpowder, Off-the-shelf available

Aim:

To evaluate the neurite extension after local delivery of MSCs, PEP or FK506 alone or combined in vitro

Methods:

Construction 3D compartmented cell culture model

1.Autografts are harvested from Lewis rats. Allografts are harvested from Sprague-Dawley rats and decellularized according to our decellularization protocol.

2.MSC seeding using undifferentiated Lewis rat adipose derived MSCs, Passage 5 according to our dynamic seeding protocol.

3.Dorsal root ganglions (DRG) serve as the neuronal cells: harvested from Neonatal Lewis rat pups postnatal day 3-7.

4.DRGs are attached to the nerve graft using Tisseel fibrin glue to create DRG-nerve graft constructs.

5.Constructs are placed in the model, isolating the DRG (central nerve) from the nerve graft (peripheral nerve: treatment side).

Outcome measures

•Confirming stem cell presence and characterization

-Immunohistochemical staining against CD90/DAPI

•Neurite extension (µm) as a measure of nerve regeneration

-Immunohistochemical staining against Neurofilament-160

Results:

•Stem cell viability was confirmed in all MSC-treated nerve grafts.

•Autograft superior to untreated Allograft (p<0.002)

•Allograft + PEP, Allograft + FK506 + PEP and Allograft + FK506 + MSC superior to untreated Allograft (all p<0.001)

•Allograft + PEP, Allograft + FK506 + PEP and Allograft + FK506 + MSC comparable to untreated Autograft (all p>0.99)

•Allograft + FK506 + MSCs superior to Allograft + MSCs (p=0.0012).

•Allograft + FK506 + PEP (longest neurite extension) comparable to Allograft + FK506 + MSCs (p>0.99)

Discussion & Conclusion:

•The treatment of PEP and FK506 combined resulted in the longest neurite extension, superior to untreated allografts and comparable to autografts, the current gold standard, isolated PEP treatment, and MSCs and FK506 combined.

•This cell-free exosome product is a novel and promising candidate to enhance nerve allograft regeneration, that can overcome limitations of current cell-based therapies, suggesting possible avenues for clinical translation.

•In vivo animal models are needed to further investigate the effect of PEP alone and combined with FK506 on nerve regeneration.

The AAPS 101st Annual Meeting

The Effect Of Local Exosomes, Stem Cells And Tacrolimus On Neurite Extension

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