Skin-Derived Stem Cells Transplanted into Resorbable Guides Provide Functional Nerve Regeneration After Sciatic Nerve Resection

Marchesi C, Pluderi M, Colleoni F, Belicchi M, Meregalli M, Farini A, Parolini D, Draghi L, Fruguglietti ME, Gavina M, Porretti L, Cattaneo A, Battistelli M, Prelle A, Moggio M, Borsa S, Bello L, Spagnoli D, Gaini SM, Tanzi MC, Bresolin N, Grimoldi N, Torrente Y.

Summary: Severe lesions dealt to the body that result in the interruption of nerve tissue continuity most often lead to the loss of function in the peripheral nervous system (PNS). Several attempts have been made to help promote axonal nerve regeneration albeit to varying degrees of success. The first method was nerve grafts, but the lack of possible donor tissue made widespread usage almost impossible. In addition, artificial devices lacked the cellular elements of nerve stumps to properly initiate nerve regeneration. To combat this, researchers employed neural stem cells (NSC) and adult mesenchymal stem cells (MSC) because of their abilities to differentiate into Schwann-like cells to improve myelin recovery. However, NSC’s are hard to harvest and MSC’s failed to show significant evidence of promoting nerve regeneration in rat models, so a new method was needed.
The authors’ paper’s aim was to evaluate whether or not easily harvestable skin-derived stem cells (SDSC) could improve peripheral nerve regeneration. In order to test this, histological and functional methods were used to test the axonal regeneration of a rat with a 16 mm sciatic nerve gap repaired with varying degrees of SDSCs.
SDSC’s were isolated from 2 day-old rats, enzymatically disassociated and separated into to colonies, ones that adhered and ones that did not. Growth media was added and the cells were subjected to various mitogens to proliferate. The SDSC’s were then labeled with GFP and tested for the stem cell markers using cell cytometry. immunostaining, and the ELISA assay. Two nerve guides were prepared for the grafting procedure: the PLA-TMC guide and the Collagen guide.
Two-month old rats were surgically modified such that they had 16 mm gaps somewhere along the length of their nerve. Animals were then inserted with prepared SDSCs with one of the following ways: PLA-TMC guide filled with SDSCs, Type 1 collagen guide filled with SDSCs.
Progression of regeneration was observed with multiple methods. Functional methods included using a walking track analysis. Walking patterns of surgically repaired rats were tested to obtain a sciatic function index before surgery, 7 days after surgery, 30 days after surgery, and subsequently every 15 days until the end of the trial. Electrophysiological studies were also performed by stimulating the right sciatic nerve before and after surgery and measuring the compound muscle action potentials. The CMAPs were amplified and recorded to measure the latency times between action potentials. These results were compared to that of control rats without surgery both with and without sciatic nerve gaps to check for nerve regeneration.
Morphological Analysis was performed on the rats by fixing specimens in 4% glutaraldehyde in phosphate buffer and then dissecting them. Sections were stained using toluidine blue and using a Leica light microscope computer. Myelinated fibers were studied under an oil immersion lens and the numbers of axons were calculated using the publicc domain Image program. Tissue immunohistochemical analysis was achieved by performing immunofluorescent stainings at various time periods after the surgery. Schwann cell markers, basal lamina, myelin, endothelial sections, and nestin were all detected in the immunostaining process. Lastly, RT-PCR was performed using RNA isolated from rat sciatic nerve sections.
Presurgery SFI values were the same across all animal groups. Seven and 15 days after surgery, both cellular and SDSCs-filled groups showed improvement. However, 30 and 90 days after surgery, SDSCs-filled groups showed the most improvement, especially ones guided by collagen. Electrophysiological studies revealed that regeneration of the sciatic nerve was greater in rats that were treated with SDSCs at 15 days. The latency period of SDSC treated rats for the recorded CMAP levels were significantly lower than those that were treated with empty guides. However, 90 days after the surgery, conduction velocity of all rats showed no visible differences. Finally, immunohistochemical analysis was performed by checking for correlation of GFP positive cells and axonal regeneration. After 90 days, a significant increase in expression of neurofilaments and GFAP indicated significant recovery into the gap for rats treated with SDSCs.



Conclusions showed that SDSCs that were isolated and inserted into a 16mm gap of the sciatic nerve of rats showed significant evidence that they helped in regeneration of the peripheral nerve. The paper gives a scientific foundation for the further usage of SDSCs as a biomedical tool to promote full recovery in PNS patients dealing with oss of function.

Critique: This paper is very thorough in terms of its content and the way it carries out its experiments. Upon the insertion of SDSCs into the target site, it cross-compares results across the physiological, electrophysiological, and cellular levels of recovery. However, the set up of the experiment could use some tweaking. The cells were suspended in PBS, which isn’t necessarily beneficial for the cell. Rather than suspending the SDSCs in PBS, the author would probably have better results using a suspension with Matrigel, a gelatinous protein mixture that would provide the cells with a better suspension mechanism and nutrients. In addition, varying degrees of SDSC concentrations should have been mapped out to show the optimum concentration at which regeneration occurred the most.