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1.  Key changes in denervated muscles and their impact on regeneration and reinnervation 
Neural Regeneration Research  2014;9(20):1796-1809.
The neuromuscular junction becomes progressively less receptive to regenerating axons if nerve repair is delayed for a long period of time. It is difficult to ascertain the denervated muscle's residual receptivity by time alone. Other sensitive markers that closely correlate with the extent of denervation should be found. After a denervated muscle develops a fibrillation potential, muscle fiber conduction velocity, muscle fiber diameter, muscle wet weight, and maximal isometric force all decrease; remodeling increases neuromuscular junction fragmentation and plantar area, and expression of myogenesis-related genes is initially up-regulated and then down-regulated. All these changes correlate with both the time course and degree of denervation. The nature and time course of these denervation changes in muscle are reviewed from the literature to explore their roles in assessing both the degree of detrimental changes and the potential success of a nerve repair. Fibrillation potential amplitude, muscle fiber conduction velocity, muscle fiber diameter, mRNA expression levels of myogenic regulatory factors and nicotinic acetylcholine receptor could all reflect the severity and length of denervation and the receptiveness of denervated muscle to regenerating axons, which could possibly offer an important clue for surgical choices and predict the outcomes of delayed nerve repair.
doi:10.4103/1673-5374.143424
PMCID: PMC4239769  PMID: 25422641
nerve regeneration; denervation; reinnervation; fibrillation potential; muscle fiber conduction velocity; muscle fiber diameter; maximal isometric force; neuromuscular junction; gene expression; neural regeneration
2.  Controlled release of vascular endothelial growth factor using poly-lactic-co-glycolic acid microspheres: In vitro characterization and application in polycaprolactone fumarate nerve conduits 
Acta biomaterialia  2011;8(2):511-518.
Vascular endothelial growth factor (VEGF) is a potent angiogenic stimulator. Controlled release of such stimulators may enhance and guide the vascularization process, and when applied in a nerve conduit may play a role in nerve regeneration. We report the fabrication and in vitro characterization of VEGF encapsulating poly-lactic-co-glycolic acid (PLGA) microspheres and the in vivo application of nerve conduits supplemented with VEGF-containing microspheres. PLGA microspheres containing VEGF were prepared by the double emulsion-solvent evaporation technique. This yielded 83.16% of the microspheres with a diameter < 53 µm. VEGF content measured by ELISA indicated 93.79 ±10.64% encapsulation efficiency. Release kinetics were characterized by an initial burst release of 67.6±8.25% within the first 24 hours, followed by consistent release of approximately 0.34% per day for 4 weeks. Bioactivity of the released VEGF was tested by human umbilical vein endothelial cell (HUVEC) proliferation assay. VEGF released at all time points enhanced HUVEC proliferation confirming that VEGF retained its bioactivity through the 4-week time period. When the microsphere delivery system was placed in a biosynthetic nerve scaffold, robust nerve regeneration was observed. This study established a novel system for controlled release of growth factors and enables in vivo studies of nerve conduits conditioned with this system.
doi:10.1016/j.actbio.2011.10.001
PMCID: PMC3972821  PMID: 22019759
microsphere; poly-lactic co-glycolic acid; vascular endothelial growth factor; bioactivity; biodegradation; nerve guide
3.  A systematic review of animal models used to study nerve regeneration in tissue-engineered scaffolds 
Biomaterials  2012;33(32):8034-8039.
Research on biomaterial nerve scaffolds has been carried out for 50 years. Only three materials (collagen, polycaprolactone and polyglycollic acid) have progressed to clinical use. Pre-clinical animal models are critical for testing nerve scaffolds prior to implementation in clinical practice. We have conducted a systematic review of 416 reports in which animal models were used for evaluation of nerve regeneration into synthetic conduits. A valid animal model of nerve regeneration requires it to reproduce the specific processes that take place in regeneration after human peripheral nerve injury. No distinct animal species meets all the requirements for an ideal animal model. Certain models are well suited for understanding regenerative neurobiology while others are better for pre-clinical evaluation of efficacy. The review identified that more than 70 synthetic materials were tested in eight species using 17 different nerves. Nerve gaps ranged from 1 to 90 mm. More than 20 types of assessment methodology were used with no standardization of methods between any of the publications. The review emphasizes the urgent need for standardization or rationalization of animal models and evaluation methods for studying nerve repair.
doi:10.1016/j.biomaterials.2012.07.056
PMCID: PMC3472515  PMID: 22889485
Peripheral nerve injury; peripheral nerve repair; nerve tube; nerve scaffold; biodegradable
4.  Comparison of polymer scaffolds in rat spinal cord: A step toward quantitative assessment of combinatorial approaches to spinal cord repair 
Biomaterials  2011;32(32):8077-8086.
The transected rat thoracic (T9/10) spinal cord model is a platform for quantitatively compa0ring biodegradable polymer scaffolds. Schwann cell-loaded scaffolds constructed from poly (lactic co-glycolic acid) (PLGA), poly(ε-caprolactone fumarate) (PCLF), oligo(polyethylene glycol) fumarate (OPF) hydrogel or positively charged OPF (OPF+) hydrogel were implanted into the model. We demonstrated that the mechanical properties (3-point bending and stiffness) of OPF and OPF+ hydrogels closely resembled rat spinal cord. After one month, tissues were harvested and analyzed by morphometry of neurofilament-stained sections at rostral, midlevel, and caudal scaffold. All polymers supported axonal growth. Significantly higher numbers of axons were found in PCLF (P < 0.01) and OPF+ (P < 0.05) groups, compared to that of the PLGA group. OPF+ polymers showed more centrally distributed axonal regeneration within the channels while other polymers (PLGA, PCLF and OPF) tended to show more evenly dispersed axons within the channels. The centralized distribution was associated with significantly more axons regenerating (P < 0.05). Volume of scar and cyst rostral and caudal to the implanted scaffold was measured and compared. There were significantly smaller cyst volumes in PLGA compared to PCLF groups. The model provides a quantitative basis for assessing individual and combined tissue engineering strategies.
doi:10.1016/j.biomaterials.2011.07.029
PMCID: PMC3163757  PMID: 21803415
OPF; PLGA; PCLF; axon regeneration; spinal cord injury; Schwann cell
5.  Accuracy of Motor Axon Regeneration Across Autograft, Single Lumen, and Multichannel Poly(lactic-co-glycolic Acid) (PLGA) Nerve Tubes 
Neurosurgery  2008;63(1):144-155.
Objective
Accuracy of motor axon regeneration becomes an important issue in the development of a nerve tube for motor nerve repair. Dispersion of regeneration across the nerve tube may lead to misdirection and polyinnervation. In this study, we present a series of methods to investigate the accuracy of regeneration, which we used to compare regeneration across autografts and single lumen poly(lactic-co-glycolic acid) (PLGA) nerve tubes. We also present the concept of the multichannel nerve tube that may limit dispersion by separately guiding groups of regenerating axons.
Methods
Simultaneous tracing of the tibial and peroneal nerves with fast blue (FB) and diamidino yellow (DY), 8 weeks after repair of a 1-cm nerve gap in the rat sciatic nerve, was performed to determine the percentage of double-projecting motoneurons. Sequential tracing of the peroneal nerve with DY 1 week before and FB 8 weeks after repair was performed to determine the percentage of correctly directed peroneal motoneurons.
Results
In the cases in which there was successful regeneration across single lumen nerve tubes, more motoneurons had double projections to both the tibial and peroneal nerve branches after single lumen nerve tube repair (21.4%) than after autograft repair (5.9%). After multichannel nerve tube repair, this percentage was slightly reduced (16.9%), although not significantly. The direction of regeneration was nonspecific after all types of repair.
Conclusion
Retrograde tracing techniques provide new insights into the process of regeneration across nerve tubes. The methods and data presented in this study can be used as a basis in the development of a nerve tube for motor nerve repair.
doi:10.1227/01.NEU.0000335081.47352.78
PMCID: PMC3463233  PMID: 18728579
misdirection; axon targeting; double labeling; peripheral nerve regeneration; rat sciatic nerve model; retrograde tracing
6.  In vitro and In vivo Release of Nerve Growth Factor from Biodegradable Poly-Lactic-Co-Glycolic-Acid Microspheres 
Regeneration of peripheral nerves after injury is suboptimal. We now report the long term delivery of nerve growth factor (NGF) by biodegradable poly-lactic-co-glycolic acid (PLGA) microspheres in vitro and in vivo. Lactic to glycolic acid ratios of 50:50 and 85:15 were fabricated using the double emulsion solvent, evaporation technique. Three different inherent viscosities (0.1dL/g: 1A, 0.4dL/g: 4A, 0.7dL/g: 7A) were analyzed. In vitro, release of NGF for 23 days was measured. Electron microscopy demonstrated intact spheres for at least 7 days (50:50 1A), 14 days (50:50 4A) or 35 days (50:50 7A and 85:15 7A). In vitro release kinetics were characterized by burst release, followed by release of NGF at a rate of 0.6%-1.6% a day. Release curves for 50:50 1A and 85:15 7A differed significantly from other compositions (p<0.01). In vivo, release was characterized by a novel radionuclide tracking assay. Release rates varied from 0.9%-2.2% per day with linear kinetics. All but the 85:15 type of spheres showed different release profiles in vivo compared to in vitro conditions. Based on the surface morphology and release profiles we found microspheres fabricated from 50:50 4A PLGA to be best suited for the use in a rat sciatic nerve injury model.
doi:10.1002/jbm.a.32900
PMCID: PMC2989534  PMID: 20878933
Nerve Growth Factor; Microspheres; Peripheral Nerve; Poly-lactic-co-glycolic-acid; Dorsal root ganglia
7.  Importance of the vasculature in cyst formation after spinal cord injury 
Journal of neurosurgery. Spine  2009;11(4):432-437.
Object
Glial scar and cystic formation greatly contribute to the inhibition of axonal regeneration after spinal cord injury (SCI). Attempts to promote axonal regeneration are extremely challenging in this type of hostile environment. The objective of this study was to examine the surgical methods that may be used to assess the factors that influence the level of scar and cystic formation in SCI.
Methods
In the first part of this study, a complete transection was performed at vertebral level T9–10 in adult female Sprague-Dawley rats. The dura mater was either left open (control group) or was closed using sutures or hyaluronic acid. In the second part of the study, complete or subpial transection was performed, with the same dural closure technique applied to both groups. Histological analysis of longitudinal sections of the spinal cord was performed, and the percentage of scar and cyst formation was determined.
Results
Dural closure using sutures resulted in significantly less glial scar formation (p = 0.0248), while incorporation of the subpial transection surgical technique was then shown to significantly decrease cyst formation (p < 0.0001).
Conclusions
In this study, the authors demonstrated the importance of the vasculature in cyst formation after spinal cord trauma and confirmed the importance of dural closure in reducing glial scar formation.
doi:10.3171/2009.4.SPINE08784
PMCID: PMC2981802  PMID: 19929340
traumatic spinal cord injury; vascular injury; glial cell response to injury
8.  Designing ideal conduits for peripheral nerve repair 
Neurosurgical focus  2009;26(2):E5.
Nerve tubes, guides, or conduits are a promising alternative for autologous nerve graft repair. The first biodegradable empty single lumen or hollow nerve tubes are currently available for clinical use and are being used mostly in the repair of small-diameter nerves with nerve defects of < 3 cm. These nerve tubes are made of different biomaterials using various fabrication techniques. As a result these tubes also differ in physical properties. In addition, several modifications to the common hollow nerve tube (for example, the addition of Schwann cells, growth factors, and internal frameworks) are being investigated that may increase the gap that can be bridged. This combination of chemical, physical, and biological factors has made the design of a nerve conduit into a complex process that demands close collaboration of bioengineers, neuroscientists, and peripheral nerve surgeons. In this article the authors discuss the different steps that are involved in the process of the design of an ideal nerve conduit for peripheral nerve repair.
doi:10.3171/FOC.2009.26.2.E5
PMCID: PMC2978041  PMID: 19435445
biomaterial; growth factor; nerve conduit; nerve guide; nerve tube; polymer; Schwann cell
9.  Axon Regeneration through Scaffold into Distal Spinal Cord after Transection 
Journal of Neurotrauma  2009;26(10):1759-1771.
Abstract
We employed Fast Blue (FB) axonal tracing to determine the origin of regenerating axons after thoracic spinal cord transection injury in rats. Schwann cell (SC)-loaded, biodegradable, poly(lactic-co-glycolic acid) (PLGA) scaffolds were implanted after transection. Scaffolds loaded with solubilized basement membrane preparation (without SCs) were used for negative controls, and nontransected cords were positive controls. One or 2 months after injury and scaffold implantation, FB was injected 0–15 mm caudal or about 5 mm rostral to the scaffold. One week later, tissue was harvested and the scaffold and cord sectioned longitudinally (30 μm) on a cryostat. Trans-scaffold labeling of neuron cell bodies was identified with confocal microscopy in all cell-transplanted groups. Large (30–50 μm diameter) neuron cell bodies were predominantly labeled in the ventral horn region. Most labeled neurons were seen 1–10 mm rostral to the scaffold, although some neurons were also labeled in the cervical cord. Axonal growth occurred bidirectionally after cord transection, and axons regenerated up to 14 mm beyond the PLGA scaffolds and into distal cord. The extent of FB labeling was negatively correlated with distance from the injection site to the scaffold. Electron microscopy showed myelinated axons in the transverse sections of the implanted scaffold 2 months after implantation. The pattern of myelination, with extracellular collagen and basal lamina, was characteristic of SC myelination. Our results show that FB labeling is an effective way to measure the origin of regenerating axons.
doi:10.1089/neu.2008.0610
PMCID: PMC2763055  PMID: 19413501
axonal tracing; biodegradable polymers; Fast Blue; Schwann cells; spinal cord injury
10.  Relationship between Scaffold Channel Diameter and Number of Regenerating Axons in the Transected Rat Spinal Cord 
Acta biomaterialia  2009;5(7):2551-2559.
Regeneration of endogenous axons through a Schwann cell (SC)-seeded scaffold implant has been demonstrated in the transected rat spinal cord. The formation of a cellular lining in the scaffold channel may limit the degree of axonal regeneration. Spinal cords of adult rats were transected and implanted with the SC-loaded polylactic co-glycollic acid (PLGA) scaffold implants containing seven parallel-aligned channels, either 450-μm (n=19) or 660-μm in diameter (n=14). Animals were sacrificed after 1, 2, and 3 months. Immunohistochemistry for neurofilament-expression was performed. The cross-sectional area of fibrous tissue and regenerative core was calculated. We found that the 450-μm scaffolds had significantly greater axon fibers per channel at the one month (186 ± 37) and three month (78 ± 11) endpoints than the 660-μm scaffolds (90 ± 19 and 40 ± 6, respectively) (P=0.0164 & 0.0149, respectively). The difference in the area of fibrous rim between the 450-μm and 660-μm channels was most pronounced at the one month endpoint, at 28,046 μm2 ± 6,551 and 58,633 μm2 ± 7,063, respectively (P=0.0105). Our study suggests that fabricating scaffolds with smaller diameter channels promotes greater regeneration over larger diameter channels. Axonal regeneration was reduced in the larger channels due to the generation of a large fibrous rim. Optimization of this scaffold environment establishes a platform for future studies of the effects of cell types, trophic factors or pharmacological agents on the regenerative capacity of the injured spinal cord.
doi:10.1016/j.actbio.2009.03.021
PMCID: PMC2731813  PMID: 19409869
Biomedical Engineering; Tissue Development and Growth; Central Nervous System; Polymeric Scaffolds
11.  Rigid Fixation of the Spinal Column Improves Scaffold Alignment and Prevents Scoliosis in the Transected Rat Spinal Cord 
Spine  2008;33(24):E914-E919.
Study Design
A controlled study to evaluate a new technique for spinal rod fixation after spinal cord injury in rats. Alignment of implanted tissue-engineered scaffolds was assessed radiographically and by magnetic resonance imaging.
Objective
To evaluate the stability of implanted scaffolds and the extent of kyphoscoliotic deformities after spinal fixation.
Summary of Background Data
Biodegradable scaffolds provide an excellent platform for the quantitative assessment of cellular and molecular factors that promote regeneration within the transected cord. Successful delivery of scaffolds to the damaged cord can be hampered by malalignment following transplantation, which in turn, hinders the assessment of neural regeneration.
Methods
Radio-opaque barium sulfate-impregnated poly-lactic-co-glycolic acid scaffolds were implanted into spinal transection injuries in adult rats. Spinal fixation was performed in one group of animals using a metal rod fixed to the spinous processes above and below the site of injury, while the control group received no fixation. Radiographic morphometry was performed after 2 and 4 weeks, and 3-dimensional magnetic resonance microscopy analysis 4 weeks after surgery.
Results
Over the course of 4 weeks, progressive scoliosis was evident in the unfixed group, where a Cobb angle of 8.13 ± 2.03° was measured. The fixed group demonstrated significantly less scoliosis, with a Cobb angle measurement of 1.89 ± 0.75° (P = 0.0004). Similarly, a trend for less kyphosis was evident in the fixed group (7.33 ± 1.68°) compared with the unfixed group (10.13 ± 1.46°). Quantitative measurements of the degree of malalignment of the scaffolds were also significantly less in the fixed group (5 ± 1.23°) compared with the unfixed group (11 ± 2.82°) (P = 0.0143).
Conclusion
Radio-opaque barium sulfate allows for visualization of scaffolds in vivo using radiographic analysis. Spinal fixation was shown to prevent scoliosis, reduce kyphosis, and reduce scaffold malalignment within the transected rat spinal cord. Using a highly optimized model will increase the potential for finding a therapy for restoring function to the injured cord.
doi:10.1097/BRS.0b013e318186b2b1
PMCID: PMC2773001  PMID: 19011531
spine fixation; transection spinal cord injury; scaffold; scoliosis
12.  Axon Regeneration through Scaffold into Distal Spinal Cord after Transection 
Journal of neurotrauma  2009;26(10):1759-1771.
We employed Fast Blue (FB) axonal tracing to determine the origin of regenerating axons after thoracic spinal cord transection injury in rats. Schwann cell (SC)-loaded, biodegradable, poly(lactic-co-glycolic acid) (PLGA) scaffolds were implanted after transection. Scaffolds loaded with solubilized basement membrane preparation (without SCs) were used for negative controls, and nontransected cords were positive controls. One or 2 months after injury and scaffold implantation, FB was injected 0–15 mm caudal or about 5 mm rostral to the scaffold. One week later, tissue was harvested and the scaffold and cord sectioned longitudinally (30 μm) on a cryostat. Trans-scaffold labeling of neuron cell bodies was identified with confocal microscopy in all cell-transplanted groups. Large (30–50 μm diameter) neuron cell bodies were predominantly labeled in the ventral horn region. Most labeled neurons were seen 1–10 mm rostral to the scaffold, although some neurons were also labeled in the cervical cord. Axonal growth occurred bidirectionally after cord transection, and axons regenerated up to 14 mm beyond the PLGA scaffolds and into distal cord. The extent of FB labeling was negatively correlated with distance from the injection site to the scaffold. Electron microscopy showed myelinated axons in the transverse sections of the implanted scaffold 2 months after implantation. The pattern of myelination, with extracellular collagen and basal lamina, was characteristic of SC myelination. Our results show that FB labeling is an effective way to measure the origin of regenerating axons.
doi:10.1089/neu.2008-0610
PMCID: PMC2763055  PMID: 19413501
axonal tracing; biodegradable polymers; Fast Blue; Schwann cells; spinal cord injury

Results 1-12 (12)