The fabrication of hydrogel microstructures based upon poly(ethylene glycol) diacrylates, dimethacrylates, and tetraacrylates patterned photolithographically on silicon or glass substrates is described. A silicon/silicon dioxide surface was treated with 3-(trichlorosilyl)propyl methacrylate to form a self-assembled monolayer (SAM) with pendant acrylate groups. The SAM presence on the surface was verified using ellipsometry and time-of-flight secondary ion mass spectrometry. A solution containing an acrylated or methacrylated poly(ethylene glycol) derivative and a photoinitiator (2,2-dimethoxy-2-phenylacetophenone) was spin-coated onto the treated substrate, exposed to 365 nm ultraviolet light through a photomask, and developed with either toluene, water, or supercritical CO2. As a result of this process, three-dimensional, cross-linked PEG hydrogel microstructures were immobilized on the surface. Diameters of cylindrical array members were varied from 600 to 7 micrometers by the use of different photomasks, while height varied from 3 to 12 micrometers, depending on the molecular weight of the PEG macromer. In the case of 7 micrometers diameter elements, as many as 400 elements were reproducibly generated in a 1 mm2 square pattern. The resultant hydrogel patterns were hydrated for as long as 3 weeks without delamination from the substrate. In addition, micropatterning of different molecular weights of PEG was demonstrated. Arrays of hydrogel disks containing an immobilized protein conjugated to a pH sensitive fluorophore were also prepared. The pH sensitivity of the gel-immobilized dye was similar to that in an aqueous buffer, and no leaching of the dye-labeled protein from the hydrogel microstructure was observed over a 1 week period. Changes in fluorescence were also observed for immobilized fluorophore labeled acetylcholine esterase upon the addition of acetyl acholine.

The long-term goal of our research is to engineer an acellular nerve graft for clinical nerve repair and for use as a model system with which to study nerve-extracellular matrix interactions during nerve regeneration. To develop this model acellular nerve graft we (1) examined the effects of detergents on peripheral nerve tissue, and (2) used that knowledge to create a nerve graft devoid of cells with a well-preserved extracellular matrix. Using histochemistry and Western analysis, the impact of each detergent on cellular and extracellular tissue components was determined. An optimized protocol was created with the detergents Triton X-200, sulfobetaine-16, and sulfobetaine-10. This study represents the most comprehensive examination to date of the effects of detergents on peripheral nerve tissue morphology and protein composition. Also presented is an improved chemical decellularization protocol that preserves the internal structure of native nerve more than the predominant current protocol.

Most neurotrophic factors are members of one of three families: the neurotrophins, the glial cell-line derived neurotrophic factor family ligands (GFLs) and the neuropoietic cytokines. Each family activates distinct but overlapping cellular pathways. Several studies have shown additive or synergistic interactions between neurotrophic factors from different families, though generally only a single combination has been studied. Because of possible interactions between the neurotrophic factors, the optimum concentration of a factor in a mixture may differ from the optimum when applied individually. Additionally, the effect of combinations of neurotrophic factors from each of the three families on neurite extension is unclear. This study examines the effects of several combinations of the neurotrophin nerve growth factor (NGF), the GFL glial cell-line derived neurotrophic factor (GDNF) and the neuropoietic cytokine ciliary neurotrophic factor (CNTF) on neurite outgrowth from young rat dorsal root ganglion (DRG) explants. The combination of 50 ng ml(-1) NGF and 10 ng ml(-1) of each GDNF and CNTF induced the highest level of neurite outgrowth at a 752 +/- 53% increase over untreated DRGs and increased the longest neurite length to 2031 +/- 97 microm compared to 916 +/- 64 microm for untreated DRGs. The optimum concentrations of the three factors applied in combination corresponded to the optimum concentration of each factor when applied individually. These results indicate that the efficacy of future therapies for nerve repair would be enhanced by the controlled release of a combination of neurotrophins, GFLs and neuropoietic cytokines at higher concentrations than used in previous conduit designs.

Synthetic polymers such as polypyrrole (PPy) are gaining significance in neural studies because of their conductive properties. We evaluated two novel biodegradable block co-polymers of PPy with poly(epsilon-caprolactone) (PCL) and poly(ethyl cyanoacrylate) (PECA) for nerve regeneration applications. PPy-PCL and PPy-PECA co-polymers can be processed from solvent-based colloidal dispersions and have essentially the same or greater conductivity (32 S/cm for PPy-PCL, 19 S/cm for PPy-PECA) compared to the PPy homo-polymer (22 S/cm). The PPy portions of the co-polymers permit electrical stimulation whereas the PCL or PECA blocks enable degradation by hydrolysis. For in vitro tests, films were prepared on polycarbonate sheets by air brushing layers of dispersions and pressing the films. We characterized the films for hydrolytic degradation, electrical conductivity, cell proliferation and neurite extension. The co-polymers were sufficient to carry out electrical stimulation of cells without the requirement of a metallic conductor underneath the co-polymer film. In vitro electrical stimulation of PPy-PCL significantly increased the number of PC12 cells bearing neurites compared to unstimulated PPy-PCL. For in vivo experiments, the PPy co-polymers were coated onto the inner walls of nerve guidance channels (NGCs) made of the commercially available non-conducting biodegradable polymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHB-HV). The NGCs were implanted in a 10 mm defect made in the sciatic nerve of rats, and harvested after 8 weeks. Histological staining showed axonal growth. The studies indicated that these new conducting degradable biomaterials have good biocompatibility and support proliferation and growth of PC12 cells in vitro (with and without electrical stimulation) and neurons in vivo (without electrical stimulation).

Recovery after peripheral nerve injury remains a significant challenge. Extracellular matrix proteins and hydrogels of extracellular matrix components have been shown to improve regeneration in peripheral nerve entubulation models, especially over long distances. The chemical properties, ligand identity and density, and mechanical properties of the hydrogel can affect neurite extension. However, the importance of combinatorial effects between different components in co-gels of several extracellular matrix components is unclear. In this study, we investigated neurite extension from explanted dorsal root ganglia cultured within co-gels made from laminin, fibronectin, collagen 1 and hyaluronic acid. Laminin had a strong, dose-dependent effect on both neurite length and outgrowth. Fibronectin was slightly, but generally not significantly, inhibitory to neurite extension. The concentration of collagen 1 and hyaluronic acid did not have significant effects on neurite extension. The combinatorial effects among the four components were additive rather than synergistic. A co-gel made with 1.5 mg/ml collagen 1 and 1.5 mg/ml laminin was optimum in this study, resulting in an average neurite length of 1532 +/- 91 microm versus 976 +/- 32 microm for controls, and an increase in overall volume outgrowth (reflecting neurite length and branching) of 85.9+/-9.3% over controls. This co-gel provides a mechanically stable scaffold with high ligand density and biochemical affinity. The results of this study support the use of co-gels of laminin and collagen 1 for promoting regeneration in peripheral nerve injuries and suggest that interactions among hydrogel components are not significant.

Peripheral nerve injury can result in debilitating outcomes including loss of function and neuropathic pain. Although nerve repair research and therapeutic development are widely studied, translation of these ideas into clinical interventions has not occurred at the same rate. At the turn of this century, approaches to peripheral nerve repair have included microsurgical techniques, hollow conduits, and autologous nerve grafts. These methods provide satisfactory results; however, they possess numerous limitations that can prevent effective surgical treatment. Commercialization of Avance, a processed nerve allograft, sought to address limitations of earlier approaches by providing an off‐the‐shelf alternative to hollow conduits while maintaining many proregenerative properties of autologous grafts. Since its launch in 2007, Avance has changed the landscape of the nerve repair market and is used to treat tens of thousands of patients. Although Avance has become an important addition to surgeon and patient clinical options, the product's journey from bench to bedside took over 20 years with many research and commercialization challenges. This article reviews the events that have brought a processed nerve allograft from the laboratory bench to the patient bedside. Additionally, this review provides a perspective on lessons and considerations that can assist in translation of future medical products.

Painful neuroma formation is a common and debilitating sequela of traumatic or oncologic nerve amputations. Studies suggest that isolating transected nerve stumps within protective caps during amputation surgery or revision procedures may assist in preventing symptomatic nerve-end neuroma formation. This study evaluated the local effects of two porcine small intestine submucosa (pSIS) nerve caps of differing configurations on a terminal nerve end in an animal model. The tibial nerves of 57 Sprague Dawley rats were transected and transposed to the lateral hind leg. The nerves were treated with one of three SIS materials, including (i) a nerve cap with spiraling chambering, termed spiral nerve cap (SNC), (ii) a nerve cap with bifurcated chambers termed chambered nerve cap (CNC), or (iii) an open tube. The surgical control consisted of nerve stumps that were not treated. Overall tissue response, axonal swirling, optical density of axons, and behavioral pain response were quantified at 8 and 12 weeks postoperatively. There were no notable differences between the performance of the SNC and CNC groups. The pSIS nerve caps mitigated aberrant axonal regeneration and decreased neuroma formation and associated pain response. These findings suggest that nerve caps with internal chambers for axonal outgrowth may improve axonal alignment, therefore reducing the likelihood of symptomatic neuroma formation.

BackgroundThe management of peripheral nerve injuries remains a large challenge for plastic surgeons. With the inability to fuse axonal endings, results after microsurgical nerve repair have been inconsistent. Our current nerve repair strategies rely upon the slow and lengthy process of axonal regeneration (~1 mm/d). Polyethylene glycol (PEG) has been investigated as a potential axonal fusion agent; however, the percentage of axonal fusion has been inconsistent. The purpose of this study was to identify a PEG delivery device to standardize outcomes after attempted axonal fusion with PEG.Materials and MethodsWe used a rat sciatic nerve injury model in which we completely transected and repaired the left sciatic nerve to evaluate the efficacy of PEG fusion over a span of 12 weeks. In addition, we evaluated the effectiveness of a delivery device's ability to optimize results after PEG fusion.ResultsWe found that PEG rapidly (within minutes) restores axonal continuity as assessed by electrophysiology, fluorescent retrograde tracer, and diffusion tensor imaging. Immunohistochemical analysis shows that motor axon counts are significantly increased at 1 week, 4 weeks, and 12 weeks postoperatively in PEG-treated animals. Furthermore, PEG restored behavioral functions up to 50% compared with animals that received the criterion standard epineurial repair (control animals).ConclusionsThe ability of PEG to rapidly restore nerve function after neurotmesis could have vast implications on the clinical management of traumatic injuries to peripheral nerves.