Influence of Magnesium Degradation on Schwannoma Cell Responses to Nerve Injury Using an In Vitro Injury Model.

Nerve guidance conduits for peripheral nerve injuries can be improved using bioactive materials such as magnesium (Mg) and its alloys, which could provide both structural and trophic support. Therefore, we investigated whether exposure to Mg and Mg-1.6wt%Li thin films (Mg/Mg-1.6Li) would alter acute Schwann cell responses to injury. Using the RT4-D6P2T Schwannoma cell line (SCs), we tested extracts from freeze-killed cells (FKC) and nerves (FKN) as in vitro injury stimulants. Both FKC and FKN induced SC release of the macrophage chemoattractant protein 1 (MCP-1), a marker of the repair SC phenotype after injury. Next, FKC-stimulated cells exposed to Mg/Mg-1.6Li reduced MCP-1 release by 30%, suggesting that these materials could have anti-inflammatory effects. Exposing FKC-treated cells to Mg/Mg-1.6Li reduced the gene expression of the nerve growth factor (NGF), glial cell line-derived neurotrophic factor (GDNF), and myelin protein zero (MPZ), but not the p75 neurotrophin receptor. In the absence of FKC, Mg/Mg-1.6Li treatment increased the expression of NGF, p75, and MPZ, which can be beneficial to nerve regeneration. Thus, the presence of Mg can differentially alter SCs, depending on the microenvironment. These results demonstrate the applicability of this in vitro nerve injury model, and that Mg has wide-ranging effects on the repair SC phenotype.

Nerve Regeneration with a Scaffold Incorporating an Absorbable Zinc-2% Iron Alloy Filament to Improve Axonal Guidance.

Peripheral nerve damage that results in lost segments requires surgery, but currently available hollow scaffolds have limitations that could be overcome by adding internal guidance support. A novel solution is to use filaments of absorbable metals to supply physical support and guidance for nerve regeneration that then safely disappear from the body. Previously, we showed that thin filaments of magnesium metal (Mg) would support nerve regeneration. Here, we tested another absorbable metal, zinc (Zn), using a proprietary zinc alloy with 2% iron (Zn-2%Fe) that was designed to overcome the limitations of both Mg and pure Zn metal. Non-critical-sized gaps in adult rat sciatic nerves were repaired with silicone conduits plus single filaments of Zn-2%Fe, Mg, or no metal, with autografts as controls. After seventeen weeks, all groups showed equal recovery of function and axonal density at the distal end of the conduit. The Zn alloy group showed some improvements in early rat health and recovery of function. The alloy had a greater local accumulation of degradation products and inflammatory cells than Mg; however, both metals had an equally thin capsule (no difference in tissue irritation) and no toxicity or inflammation in neighboring nerve tissues. Therefore, Zn-2%Fe, like Mg, is biocompatible and has great potential for use in nervous tissue regeneration and repair.

Effects of Altering Magnesium Metal Surfaces on Degradation In Vitro and In Vivo during Peripheral Nerve Regeneration.

In vivo use of biodegradable magnesium (Mg) metal can be plagued by too rapid a degradation rate that removes metal support before physiological function is repaired. To advance the use of Mg biomedical implants, the degradation rate may need to be adjusted. We previously demonstrated that pure Mg filaments used in a nerve repair scaffold were compatible with regenerating peripheral nerve tissues, reduced inflammation, and improved axonal numbers across a short-but not long-gap in sciatic nerves in rats. To determine if the repair of longer gaps would be improved by a slower Mg degradation rate, we tested, in vitro and in vivo, the effects of Mg filament polishing followed by anodization using plasma electrolytic oxidation (PEO) with non-toxic electrolytes. Polishing removed oxidation products from the surface of as-received (unpolished) filaments, exposed more Mg on the surface, produced a smoother surface, slowed in vitro Mg degradation over four weeks after immersion in a physiological solution, and improved attachment of cultured epithelial cells. In vivo, treated Mg filaments were used to repair longer (15 mm) injury gaps in adult rat sciatic nerves after placement inside hollow poly (caprolactone) nerve conduits. The addition of single Mg or control titanium filaments was compared to empty conduits (negative control) and isografts (nerves from donor rats, positive control). After six weeks in vivo, live animal imaging with micro computed tomography (micro-CT) showed that Mg metal degradation rates were slowed by polishing vs. as-received Mg, but not by anodization, which introduced greater variability. After 14 weeks in vivo, functional return was seen only with isograft controls. However, within Mg filament groups, the amount of axonal growth across the injury site was improved with slower Mg degradation rates. Thus, anodization slowed degradation in vitro but not in vivo, and degradation rates do affect nerve regeneration.

Embedding magnesium metallic particles in polycaprolactone nanofiber mesh improves applicability for biomedical applications.

Magnesium (Mg) metal is of great interest in biomedical applications, especially in tissue engineering. Mg exhibits excellent in vivo biocompatibility, biodegradability and, during degradation, releases Mg ions (Mg2+) with the potential to improve tissue repair. We used electrospinning technology to incorporate Mg particles into nanofibers. Various ratios of Mg metal microparticles (<44 µm diameter) were incorporated into nanofiber polycaprolactone (PCL) meshes. Physicochemical properties of the meshes were analyzed by scanning electron microscopy (SEM), Fourier-transform infrared spectroscopy (FTIR), mechanical tensile testing, X-ray diffractometry and UV-VIS spectrophotometry. Biological properties of meshes were evaluated in vitro and in vivo. Under mammalian cell culture conditions, Mg-containing meshes released hydrogen gas and relative amounts of free Mg2+ that reflected the Mg/PCL ratios. All meshes were non-cytotoxic for 3T3 fibroblasts and PC-12 pheochromocytoma cells. In vivo implantation under the skin of mice for 3, 8 and 28 days showed that Mg-containing meshes were well vascularized, with improved measures of inflammation and healing compared to meshes without Mg. Evidence included an earlier appearance and infiltration of tissue repairing macrophages and, after 28 days, evidence of more mature tissue remodeling. Thus, these new composite nanofiber meshes have promising material properties that mitigated inflammatory tissue responses to PCL alone and improved tissue healing, thus providing a suitable matrix for use in clinically relevant tissue engineering applications. STATEMENT OF SIGNIFICANCE: The biodegradable metal, magnesium, safely biodegrades in the body, releasing beneficial byproducts. To improve tissue delivery, magnesium metal particles were incorporated into electrospun nanofiber meshes composed of a biodegradable, biocompatible polymer, polycaprolactone (PCL). Magnesium addition, at several concentrations, did not alter PCL chemistry, but did alter physical properties. Under cell culture conditions, meshes released magnesium ions and hydrogen gas and were not cytotoxic for two cell types. After implantation in mice, the mesh with magnesium resulted in earlier appearance of M2-like, reparative macrophages and improved tissue healing versus mesh alone. This is in agreement with other studies showing beneficial effects of magnesium metal and provides a new type of scaffold material that will be useful in clinically relevant tissue engineering applications.

An in vitro and in vivo characterization of fine WE43B magnesium wire with varied thermomechanical processing conditions.

Absorbable implants made of magnesium alloys may revolutionize surgical intervention, and fine magnesium wire will be critical to many applications. Functionally, the wires must have sufficient mechanical properties to withstand implantation and in-service loading, have excellent tissue tolerance, and exhibit an appropriate degradation rate for the application. Alloy chemistry and thermomechanical processing conditions will significantly impact the material's functional performance, but the exact translation of these parameters to implant performance is unclear. With this in mind, fine (127 µm) WE43B magnesium alloy wires in five thermomechanical process (TMP) conditions (90% cold work [CW], and 250, 375, 400, and 450°C heat treatments) were investigated for their effect on mechanical and corrosion behavior. The TMP conditions gave clear metallurgical differences: transverse grain dimensions ranged from 200 nm (CW) to 3 µm (450°C), UTS varied from 324 MPa (450°C) to 608 MPa (250°C), and surgical knotting showed some were suitable (CW, 400°C, 450°C) while others were not (250°C, 350°C). In vitro and in vivo corrosion testing yielded interesting and in some cases conflicting results. After 1 month immersion in cell culture medium, wire corrosion was extensive, and TMP conditions altered the macrocorrosion morphology but not the rate or total release of magnesium ions. After 1 month subdermal implantation in mice, all wires were well tolerated and showed very little corrosion (per µCT and histology), but differences in localized corrosion were detected between conditions. This study indicates that WE43B wires treated at 450°C may be most suitable for surgical knotting procedures. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 1987-1997, 2018.

Short and long gap peripheral nerve repair with magnesium metal filaments.

A current clinical challenge is to replace autografts for repair of injury gaps in peripheral nerves, which can occur due to trauma or surgical interruption. Biodegradable metallic magnesium filaments, placed inside hollow nerve conduits, could support nerve repair by providing contact guidance support for axonal regeneration. This was tested by repairing sciatic nerves of adult rats with single magnesium filaments placed inside poly(caprolactone) nerve conduits. Controls were empty conduits, conduits containing titanium filaments and/or isografts from donor rats. With a nerve gap of 6 mm and 6 weeks post-repair, magnesium filaments had partially resorbed. Regenerating cells had attached to the filaments and axons were observed in distal stumps in all animals. Axon parameters were improved with magnesium compared to conduits alone or conduits with single titanium filaments. With a longer gap of 15 mm and 16 weeks post-repair, functional parameters were improved with isografts, but not with magnesium filaments or empty conduits. Magnesium filaments were completely resorbed and no evidence of scarring was seen. While axon outgrowth was not improved with the longer gap, histological measures of the tissues were improved with magnesium compared to empty conduits. Therefore, the use of magnesium filaments is promising because they are biocompatible and improve aspects of nerve regeneration. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 3148-3158, 2017.

Cytotoxicity and apoptotic gene expression in an in vitro model of the blood-brain barrier following exposure to poly(butylcyanoacrylate) nanoparticles.

Background Poly(butylcyanoacrylate) (PBCA) nanoparticles (NPs) loaded with doxorubicin (DOX) and coated with polysorbate 80 (PS80) have shown efficacy in the treatment of rat glioblastoma. However, cytotoxicity of this treatment remains unclear. Purpose The purpose of this study was to investigate cytotoxicity and apoptotic gene expression using a proven in vitro co-culture model of the blood-brain barrier. Methods The co-cultures were exposed to uncoated PBCA NPs, PBCA-PS80 NPs or PBCA-PS80-DOX NPs at varying concentrations and evaluated using a resazurin-based cytotoxicity assay and an 84-gene apoptosis RT-PCR array. Results The cytotoxicity assays showed PBCA-PS80-DOX NPs exhibited a decrease in metabolic function at lower concentrations than uncoated PBCA NPs and PBCA-PS80 NPs. The apoptosis arrays showed differential expression of 18 genes in PBCA-PS80-DOX treated cells compared to the untreated control. Discussion As expected, the cytotoxicity assays demonstrated enhanced dose-dependent toxicity in the DOX loaded NPs. The differentially expressed apoptotic genes participate in both the tumor necrosis factor receptor-1 and mitochondria-associated apoptotic pathways implicated in current DOX chemotherapeutic toxicity. Conclusion The following data suggest that the cytotoxic effect may be attributed to DOX and not the NPs themselves, further supporting the use of PBCA-PS80 NPs as an effective drug delivery vehicle for treating central nervous system conditions.

Evaluation of peripheral nerve regeneration through biomaterial conduits via micro-CT imaging.

OBJECTIVE: Hollow nerve conduits made of natural or synthetic biomaterials are used clinically to aid regeneration of peripheral nerves damaged by trauma or disease. To support healing, conduit lumen patency must be maintained until recovery occurs. New methods to study conduit structural integrity would provide an important means to optimize conduits in preclinical studies. We explored a novel combined technique to examine structural integrity of two types of nerve conduits after in vivo healing. STUDY DESIGN: Micro-CT imaging with iodine contrast was combined with histological analysis to examine two different nerve conduits after in vivo nerve reconstruction in rats. MATERIALS AND METHODS: Sciatic nerve gaps in adult Lewis rats were reconstructed with poly(caprolactone) (PCL, 1.6 cm gap, 14-week survival) or silicone (1 cm gap, 6-week survival) conduits (N = 12 total). Conduits with regenerating tissues were imaged by micro-CT with iodine contrast and compared to the histology (hematoxylin and eosin, immunostaining for axons) of regenerated tissues after iodine removal. RESULTS: PCL nerve conduits showed extensive breakage throughout their length, but all showed successful nerve growth through the conduits. The silicone conduits remained intact, although significant constriction was uniquely detected by micro-CT, with 1 of 6 animals showing incomplete tissue regeneration. CONCLUSIONS: Micro-CT with iodine contrast offers a unique and valuable means to determine 3D structural integrity of nerve conduits and nerve healing following reconstruction. Furthermore, this paper shows that even if conduit compression and degradation occur, nerve regeneration can still take place.

Combining micro-computed tomography with histology to analyze biomedical implants for peripheral nerve repair.

BACKGROUND: Biomedical implants used in tissue engineering repairs, such as scaffolds to repair peripheral nerves, can be too large to examine completely with histological analyses. Micro-computed tomography (micro-CT) with contrast agents allows ex vivo visualization of entire biomaterial implants and their interactions with tissues, but contrast agents can interfere with histological analyses of the tissues or cause shrinkage or loss of antigenicity. NEW METHOD: Soft tissue, ex vivo micro-CT imaging using Lugol's iodine was compatible with histology after using a rapid (48 h) method of removing iodine. RESULTS: Adult normal and repaired rat sciatic nerves were infiltrated ex vivo with iodine, imaged with micro-CT and then the iodine was removed by incubating tissues in sodium thiosulfate. Subsequent paraffin sections of normal nerve tissues showed no differences in staining with hematoxylin and eosin or immunostaining with multiple antibodies. Iodine treatment and removal did not alter axonal diameter, nuclear size or relative area covered by immunostained axons (p>0.05). Combining imaging modalities allowed comparisons of macroscopic and microscopic features of nerve tissues regenerating through simple nerve conduits or nerve conduits containing a titanium wire for guidance. COMPARISON WITH EXISTING METHODS: Quantification showed that treatment with iodine and sodium thiosulfate did not result in tissue shrinkage or loss of antigenicity. CONCLUSIONS: Because this combination of treatments is rapid and does not alter tissue morphology, this expands the ex vivo methods available to examine the success of biomaterial implants used for tissue engineering repairs.

Poly(ε-caprolactone)/keratin-based composite nanofibers for biomedical applications.

Keratin-based composite nanofibers have been fabricated by an electrospinning technique. Aqueous soluble keratin extracted from human hair was successfully blended with poly(ε-caprolactone) (PCL) in different ratios and transformed into nanofibrous membranes. Toward the potential use of this nanofibrous membrane in tissue engineering, its physicochemical properties, such as morphology, mechanical strength, crystallinity, chemical structure, and integrity in aqueous medium were studied and its cellular compatibility was determined. Nanofibrous membranes with PCL/keratin ratios from 100/00 to 70/30 showed good uniformity in fiber morphology and suitable mechanical properties, and retained the integrity of their fibrous structure in buffered solutions. Experimental results, using cell viability assays and scanning electron microscopy imaging, showed that the nanofibrous membranes supported 3T3 cell viability. The ability to produce blended nanofibers from protein and synthetic polymers represents a significant advancement in development of composite materials with structural and material properties that will support biomedical applications. This provides new nanofibrous materials for applications in tissue engineering and regenerative medicine.

Effects of elevated magnesium and substrate on neuronal numbers and neurite outgrowth of neural stem/progenitor cells in vitro.

Because a potential treatment for brain injuries could be elevating magnesium ions (Mg(2+)) intracerebrally, we characterized the effects of elevating external Mg(2+) in cultures of neonatal murine brain-derived neural stem/progenitor cells (NSCs). Using a crystal violet assay, which avoids interference of Mg(2+) in the assay, it was determined that substrate influenced Mg(2+) effects on cell numbers. On uncoated plastic, elevating Mg(2+) levels to between 2.5 and 10mM above basal increased NSC numbers, and at higher concentrations numbers decreased to control or lower levels. Similar biphasic curves were observed with different plating densities, treatment durations and length of time in culture. When cells were plated on laminin-coated plastic, NSC numbers were higher even in basal medium and no further effects were observed with Mg(2+). NSC differentiation into neurons was not altered by either substrate or Mg(2+) supplementation. Some parameters of neurite outgrowth were increased by elevated Mg(2+) when NSCs differentiated into neurons on uncoated plastic. Differentiation on laminin resulted in increased neurites even in basal medium and no further effects were seen when Mg(2+) was elevated. This system can now be used to study the multiple mechanisms by which Mg(2+) influences neuronal biology.

Polymer Coating of Carbon Nanotube Fibers for Electric Microcables.

Carbon nanotubes (CNTs) are considered the most promising candidates to replace Cu and Al in a large number of electrical, mechanical and thermal applications. Although most CNT industrial applications require macro and micro size CNT fiber assemblies, several techniques to make conducting CNT fibers, threads, yarns and ropes have been reported to this day, and improvement of their electrical and mechanical conductivity continues. Some electrical applications of these CNT conducting fibers require an insulating layer for electrical insulation and protection against mechanical tearing. Ideally, a flexible insulator such as hydrogenated nitrile butadiene rubber (HNBR) on the CNT fiber can allow fabrication of CNT coils that can be assembled into lightweight, corrosion resistant electrical motors and transformers. HNBR is a largely used commercial polymer that unlike other cable-coating polymers such as polyvinyl chloride (PVC), it provides unique continuous and uniform coating on the CNT fibers. The polymer coated/insulated CNT fibers have a 26.54 µm average diameter-which is approximately four times the diameter of a red blood cell-is produced by a simple dip-coating process. Our results confirm that HNBR in solution creates a few microns uniform insulation and mechanical protection over a CNT fiber that is used as the electrically conducting core.

Ghrelin enhances olfactory sensitivity and exploratory sniffing in rodents and humans.

Olfaction is an integral part of feeding providing predictive cues that anticipate ingestion. Although olfactory function is modulated by factors such as prolonged fasting, the underlying neural mechanisms remain poorly understood. We recently identified ghrelin receptors in olfactory circuits in the brain. We therefore investigated the role of the appetite-stimulating hormone ghrelin in olfactory processing in rodents and humans, testing the hypothesis that ghrelin lowers olfactory detection thresholds and enhances exploratory sniffing, both being related to food seeking. In rats, intracerebroventricular ghrelin decreased odor detection thresholds and increased sniffing frequency. In humans, systemic ghrelin infusions significantly enhanced sniff magnitudes in response to both food and nonfood odorants and air in comparison to control saline infusions but did not affect the pleasantness ratings of odors. This is consistent with a specific effect on odor detection and not the hedonic value of odors. Collectively, our findings indicate that ghrelin stimulates exploratory sniffing and increases olfactory sensitivity, presumably enhancing the ability to locate, identify, and select foods. This novel role is consistent with ghrelin's overall function as a signal amplifier at the molecular interface between environmental and nutritional cues and neuroendocrine circuits controlling energy homeostasis.

Differential effects of antipsychotic and antidepressant drugs on neurogenic regions in rats.

Increased neurogenesis in the hippocampus and subventricular zone (SVZ) of the brain of animals has been demonstrated following administration of several psychotropic medications. Such changes are thought to regenerate tissues and contribute to the beneficial effects of the medications. This study sought to determine if another neurogenic tissue, the peripheral olfactory epithelium (OE), might also exhibit changes after treatment with psychotropic medications. Young adult male rats were treated with risperidone and paliperidone, atypical antipsychotic medications; fluoxetine, a selective serotonin reuptake inhibitor (SSRI) antidepressant; and diluent control for 28days via drinking water. Bromodeoxyuridine (BrdU) was injected to label dividing cells and positive cells were quantified in the OE, cortical SVZ, and dentate gyrus (DG) of the hippocampus. In the first of two studies, paliperidone and risperidone treatment (at 1mg/kg/day) resulted in increased numbers over controls of BrdU positive cells in the OE. In the second study, examining OE, SVZ and DG in the same animal, paliperidone, but not risperidone or fluoxetine (0.6 mg/kg/day) resulted in increased cells in the OE and posterior SVZ. However, fluoxetine, but not paliperidone or risperidone treatment increased BrdU positive cells in the DG. These results show that psychotropic drug-induced cell proliferation occurs in the OE and parallels changes in the SVZ but not DG. Thus, the peripheral OE can serve as a proxy for certain psychotropic drug-induced actions on SVZ brain cell proliferation. This olfactory model can be employed in human research as a method to explore the neurogenesis effects of various pharmacologic treatments of neuropsychiatric disorders.

A carbon nanotube needle biosensor.

A carbon nanotube needle biosensor was developed to provide fast, cost effective and highly sensitive electrochemical detection of biomolecules. The sensor was fabricated based on an array of aligned multi-wall carbon nanotubes synthesized by chemical vapor deposition. A bundle of nanotubes in the array was welded onto the tip of a tungsten needle under a microscope. The needle was then encased in glass and a polymer coating leaving only the tip of the needle exposed. Cyclic voltammetry was performed to examine the redox behavior of the nanotube needle. The cyclic voltammetry results showed a steady-state response attributable to radial diffusion with a high steady-state current density. An amperometric sensor was then developed for glucose detection by physically attaching glucose oxidase on the nanotube needle. The amperometric response of these nanotube needles showed a high sensitivity with a low detection limit. It is expected that the nanotube needle can be sharpened to increase the sensitivity to the point where the current is almost too small to measure. The simple manufacturing method should allow commodity level production of highly sensitive electronic biosensors.

Spatiotemporal distribution of the insulin-like growth factor receptor in the rat olfactory bulb.

Insulin-like growth factor I (IGF-I) and its receptor (IGF-IR) are involved in growth of neurons. In the rat olfactory epithelium, we previously showed IGF-IR immunostaining in subsets of olfactory receptor neurons. We now report that IGF-IR staining was heaviest in the olfactory nerve layer of the rat olfactory bulb at embryonic days 18, and 19 and postnatal day 1, with labeling of protoglomeruli. In the adult, only a few glomeruli were IGF-IR-positive, some of which were unusually small and strongly labeled. Some IGF-IR-positive fibers penetrated deeper into the external plexiform layer, even in adults. In developing tissues, IGF-IR staining co-localized with that for olfactory marker protein and growth associated protein GAP-43, but to a lesser extent with synaptophysin. In the adult, IGF-IR-positive fibers were compartmentalized within glomeruli. IGF-I may play a role in glomerular synaptogenesis and/or plasticity, possibly contributing to development of coding patterns for odor detection or identification.