Olfactory Derived Stem Cells Delivered in a Biphasic Conduit Promote Peripheral Nerve Repair In Vivo

Abstract

Peripheral nerve injury presents significant therapeutic challenges for recovery of motor and sensory function in patients. Different clinical approaches exist but to date there has been no consensus on the most effective method of treatment. Here, we investigate a novel approach to peripheral nerve repair using olfactory derived stem (ONS) cells delivered in a biphasic collagen and laminin functionalized hyaluronic acid based nerve guidance conduit (NGC). Nerve regeneration was studied across a 10-mm sciatic nerve gap in Sprague Dawley rats. The effect of ONS cell loading of NGCs with or without nerve growth factor (NGF) supplementation on nerve repair was compared to a cell-free NGC across a variety of clinical, functional, electrophysiological, and morphologic parameters. Animals implanted with ONS cell loaded NGCs demonstrated improved clinical and electrophysiological outcomes compared to cell free NGC controls. The nerves regenerated across ONS cell loaded NGCs contained significantly more axons than cell-free NGCs. A return of the nocioceptive withdrawal reflex in ONS cell treated animals indicated an advanced repair stage at a relatively early time point of 8 weeks post implantation. The addition of NGF further improved the outcomes of the repair indicating the potential beneficial effect of a combined stem cell/growth factor treatment strategy delivered on NGCs. Stem Cells Translational Medicine 2017;6:1894-1904.

Keywords: Biomaterials; Nerve guidance conduit; Olfactory derived stem cells; Peripheral nerve injury.

Figure 1

Nerve guidance conduit (NGC) in situ. Cuff sutured to perineurium. Treatment groups were implanted with either NGC alone, NGC and ONS cells, or NGC, ONS, and nerve growth factor.

Figure 2

Olfactory neuroepithelial derived stem (ONS) cells in culture on HA hydrogels. (A–C): Spheroidal configuration of ONS cells on unmodified HA hydrogel. (D–F): ONS cells on HA‐Lam hydrogel. In comparison with unmodified HA hydrogels, ONS cells demonstrated more phenotypic extension (blue arrows). (A, D) single staining for nestin, (B, E) single staining for β‐ tubulin (C, F) composite images with nestin (red) nuclear stain (blue) and β‐tubulin (green). The scale bar for all images is 20 µm. Abbreviations: HA, hyaluronic acid; HA‐Lam, HA‐Laminin.

Figure 3

Olfactory neuroepithelial derived stem (ONS) cells in culture on HA hydrogels with media supplemented with nerve growth factor (NGF). (A–C): Spheroidal configuration of ONS cells on unmodified HA hydrogel supplemented with NGF. Matrix invasion and cell extension were not observed. (D–F): ONS cells on HA‐Lam hydrogel supplemented with NGF. In comparison with unmodified HA hydrogels, ONS cells cultured on HA‐Lam with NGF demonstrated marked phenotypic extension (F Insert) and matrix invasion. (A, D) single staining for nestin, (B, E) single staining for β‐ tubulin (C, F) composite images with nestin (red) nuclear stain (blue) and β‐tubulin (green). The scale bar for all images is 20 µm. Abbreviations: HA, hyaluronic acid; HA‐Lam, HA‐Laminin.

Figure 4

Biphasic NGC. (A): SEM photomicrograph demonstrating the cross‐sectional appearance of the biphasic nerve guidance conduit (NGC). The dotted yellow line demonstrates the tubular collagen conduit—HA‐Laminin luminal filler interface (Image Courtesy of Austyn Matheson and Alan Ryan TERG). (B): High magnification image of longitudinal section of the insert is shown with phalloidan stained cytoskeleton by cells distributed through the bi‐layered tissue engineered NGC. Cell nuclei were counter stained with Hoechst nuclear stain.

Figure 5

Improved clinical outcomes were noted in all experimental treatment groups. Table (top) demonstrating clinical outcomes per group. Images (bottom) showing recovery of the nerve morphology following treatment (A–C) compared to the nonoperated positive control (Left hind‐limb). Contractures (reduced angle shown within the black squares), ulcers (yellow circles) and autophagy of the lateral toes (blue arrows) are highlighted. (A): Animal treated with the NGC alone. (B): Animal treated with NGC and ONS. (C): Animal treated with NGC, ONS, and NGF. Abbreviations: NGC, nerve guidance conduit; NGF, nerve growth factor; ONS, olfactory neuroepithelial derived stem.

Figure 6

Electrophysiological recovery was detected in all treatment groups. Electromyographical testing (A) demonstrated that the addition of ONS cells resulted in 2.79‐fold increase in compound muscle action potential versus the NGC alone (p < .05), while the further addition of NGF resulted in a similar increase of 2.76‐fold (p > .05). (B) Peak compression force was increased by 2.6‐fold with the addition of ONS cells + NGF, although this was nonsignificant (p = .092). (C) Peak tension was improved by 1.9‐fold with the addition of ONS cells (p > .05) and by 2.9‐fold with the further addition of NGF (p < .05). Asterisk indicates statistically significant difference (<0.05%) versus NGC control group. Abbreviations: NGC, nerve guidance conduit; NGF, nerve growth factor; ONS, olfactory neuroepithelial derived stem.

Figure 7

Recovery of nerve morphology was detected in all treatment groups. Representative immunofluorescent images showing recovery of the nerve morphology versus the control (A) following treatment with NGC (B), NGF + ONS (C), and NGF + ONS +NGF (D). Mid‐conduit sections in all treatment showed signs of morphological recovery comparable to the positive control. Nuclei = blue. Extracellular matrix laminin = red and fibronectin = green. Scale bar in all images – 50 µm. The addition of ONS cells resulted in (E) a 44.5% increase in axon count (p > .05,) while the combination of ONS cells + NGF resulted in a 212.5% increase in axon count versus the NGC alone (p < .05). Axon diameter (F) was improved by 11.7% (p > .05) with ONS cell addition and 26% by the combination of ONS cells + NGF (p > .05). Axon alignment (G) was not significantly altered with the addition of either ONS cells alone or in combination with NGF (2.2% and 4.4% increases, respectively, p > .05). Asterisk indicates statistically significant difference (<0.05%) versus NGC control group. Abbreviations: NGC, nerve guidance conduit; NGF, nerve growth factor; ONS, olfactory neuroepithelial derived stem.