Macrophages and Schwann cells play a key role in the conducting the early events after peripheral nerve injury. Throughout this dissertation we have investigated one strategy for improved functional improvement by utilizing the PNM to manipulate the microenvironment at the site of nerve repair to promote modulation of the host inflammatory response and promote Schwann cell (SC) migration and axon extension across the repair site (Figure 46). We have shown that this is an effective approach. Here we determined the effects of the PNM hydrogel on macrophage gene expression, phenotype and migration using a combination of Nanostring technology and immunohistochemistry. We also evaluate effects on SC migration, numbers of motor neurons reaching their target and axon extension using rat models of sciatic and common peroneal nerve injuries. Finally, functional recovery was determined after both sciatic crush injury and common peroneal and sciatic nerve transection associated with a short gap. Control groups including uninjured animals, exposure of the nerve without injury, transection and ligation without repair and injection of hydrogel in uninjured nerves were also performed (n = 8/group). Animals were followed for 12 weeks and assessed longitudinally using multiple measures of sensory and functional recovery. Metrics included von Frey nociception assay, sciatic functional index, and kinematic analysis. At 12 weeks animals were subjected to electrophysiologic assessment of evoked compound motor action potential prior to euthanasia for tissue collection and subsequent
Figure 46: Graphical abstract of the use of PNM in augmenting peripheral nerve injury response
PNM promoted expression of Platelet Derived Growth Factor and M2 (regenerative) genes including Chi313, Retnla and IL4ra. Macrophage, particularly M2, recruitment to the injury site was increased in rats (p < 0.05). PMN promoted SC migration both in vitro and in vivo (p < 0.05). Axon extension determined by extension of axons in a crush model and across an 8mm gap was increased in the presence of PNM compared with an empty conduit (p < 0.05).
The amplitude of CMAP across the sciatic crush injury site, demonstrating axonal regrowth to the terminal muscle was increased by 50% compared to control following intraneural injection of PNM 12 weeks after nerve crush. Furthermore, CMAP amplitude was increased by 70% in an
These results demonstrate that an injectable, peripheral nerve matrix hydrogel derived from porcine sciatic nerve can modulate the response of two key cell types which conduct the early response to nerve injury. The present dissertation does not attempt to investigate the mechanisms by which the injectable ECM promote these phenomena; however, a recent study has described M2 macrophage mediated angiogenesis as a mechanism leading to Schwann cell chemotaxis and downstream regeneration [181]. However, though the exact mechanisms are not clear, these results link these phenomena to functional improvements downstream and suggest that PNM has the possibility to be used as a tool for improving nerve repair.
However, this dissertation was not without limitations, including the limited comparison to non-nerve specific ECM, several underpowered animal studies, and weakness of functional metrics assessed. In composition and in vitro assays, PNM was compared to UBM and SIS. Differences were observed in many of these experiments and these differences suggested that PNM would perform better in an animal model of peripheral nerve injury. However, this dissertation did not include either SIS or UBM in the majority of subsequent animal studies. UBM was only included with a large animal pilot study of recurrent laryngeal nerve transection and re- anastomosis repair. In this study, both PNM and UBM showed a similar trend towards improved recovery but little to no difference was observed between the two ECM sources. Future work could examine the use of non-nerve specific ECM hydrogels in nerve defect repair further. This work could elucidate the effect of nerve specific components from the general effect of having any extracellular matrix present. Furthermore, the inclusion of a simple collagen type I hydrogel as a
Chapter 4 of this dissertation focused on measuring return to function after peripheral nerve injuries. The studies incorporated several different measures of function including functional indexes, nociceptive withdrawal reflex testing, gait kinematics, and electrophysiology. This was meant to capture a full picture of nerve function while covering for limitations of single measurements. While we were able to make some conclusions from these experiments, we found that the indexes, nociceptive withdrawal reflex testing, gait kinematic measures of function were limited by a number of factors that limited the utility of these tests. We still found the motor function test valuable to understand the impact that the PNM treatment had on downstream function but do not believe that these tests were indicative of the full effect that PNM had on the regeneration after injury. The most accurate measures of regeneration are the histology and axon counts of the regenerating axons and secondly the electrical conduction of the nerve axons to the downstream muscles measured through CMAP electrophysiology.
To this point, PNM has yet to be combined with an FDA approved conduit and has only been investigated minimally in combination with other regeneration strategies. For the majority of nerve defect models, an inert nerve guidance conduit made from silicone was used. PNM was shown to improve outcomes when combined with this inert silicone conduit but did not exceed the nerve autograft in all studies. Current data suggests that PNM can improve the outcomes of all nerve guidance conduits when used as a lumen filler. Studies that compare the autograft repair to the use of PNM as a lumen filler for FDA approved conduits are needed. Furthermore, present studies showed an increased rate of regeneration during the first month after crush and transection injuries. Evidence suggests that this ECM hydrogel might only remain within the conduit for the first 1-2 weeks [97, 162, 269]. One possible future direction could be the additional supplementation of PNM to the injury site after the first month to boost regeneration. This process
would use already established techniques as well as developing new practices for using image- guided injection of the PNM hydrogel through the skin to refill the nerve guidance conduit. In addition, these techniques could be used to further investigate the injection of PNM directly into the nerve tissue to lead the nature regeneration as it regrows back through the distal end of the nerve.
Appendix A. Abbreviations Used
A B
BDNF (brain derived neurotrophic factor)
bFGF (basic fibroblast growth factor)
BM (Bone Marrow)
BMSC (Bone Marrow Stromal Cells)
BSA (Bovine serum albumin)
C
cm (Centimeter)
CNS (Central Nervous System)
C-NT (crush without treatment)
C-PNM (crush with PNM injection)
D
DNA (Deoxyribonucleic acid)
DNAse (Deoxyribonuclease)
DO-PNM (dissection down to nerve and
PNM injection into healthy nerve)
DRG (Dorsal Root Ganglion)
E
ECM (Extra Cellular Matrix)
EMG (Electromyography)
EVA (Ethylene-vinyl acetate)
FDA (Food and Drug Administration)
G
GAG (glycosaminoglycans)
GDNF (Glial derived neurotrophic factor)
GFAP (Glial Fibrillary Acidic Protein)
H
h (Hours)
HCT/P (Human Cellular and Tissue-based
Product) I IL-1 (Protein) K kg (Kilograms) L M mg (Milligram) min (Minute) mm (Millimeter) MMP (metalloproteinases)
MSC (Mesenchymal Stem Cells)
N
NI (No injury or surgery)
NIH (National Institutes of Health)
NT-3 (Neurotrophin-3)
NT-4 (Neurotrophin-4)
P
p (P-Value)
PCL (Poly(DL-Lactide-E-Caprolactone))
PDGF-β (Platelet derive growth factor β)
PGA (Polyglycolic Acid)
PHB (polyhydroxybutyrate)
PhD (Doctorate)
PHEMA-MMA
(Polyhydroxyethylmethacrylate)
PI (Principle Investigator)
PLGA (Polylactic-co-glycolic acid)
PNM (Peripheral nerve matrix)
PNM10 (PNM hydrogel at 10 mg/mL
concentration)
PNM20 (PNM hydrogel at 20 mg/mL
concentration)
PNM40 (PNM hydrogel at 40 mg/mL
PNS (Peripheral Nervous System)
R
R&D (Research and Development)
RNA (Ribonucleic acid)
S
s (Second)
S-100 (low molecular weight Protein)
SC (Schwann Cells)
SD (Sodium deoxycholate)
SDS (sodiumdodecyle sulfate)
Sham/DO (dissection down to nerve without
injury)
SIS (Porcine Small Intestinal Submucosa)
T
T-NT (transection and ligation)
TS (Toe-spread Test)
U
UBM (Urinary bladder matrix)
V
VEGF (Vascular Endothelial Growth Factor)
Appendix B. Programing Code