Repairing Peripheral Nerves: Is there a Role for Carbon Nanotubes?

Peripheral nerve injury continues to be a major global health problem that can result in debilitating neurological deficits and neuropathic pain. Current state-of-the-art treatment involves reforming the damaged nerve pathway using a nerve autograft. Engineered nerve repair conduits can provide an alternative to the nerve autograft avoiding the inevitable tissue damage caused at the graft donor site. Commercially available nerve repair conduits are currently only considered suitable for repairing small nerve lesions; the design and performance of engineered conduits requires significant improvements to enable their use for repairing larger nerve defects. Carbon nanotubes (CNTs) are an emerging novel material for biomedical applications currently being developed for a range of therapeutic technologies including scaffolds for engineering and interfacing with neurological tissues. CNTs possess a unique set of physicochemical properties that could be useful within nerve repair conduits. This progress report aims to evaluate and consolidate the current literature pertinent to CNTs as a biomaterial for supporting peripheral nerve regeneration. The report is presented in the context of the state-of-the-art in nerve repair conduit design; outlining how CNTs may enhance the performance of next generation peripheral nerve repair conduits.

Influence of different types of carbon nanotubes on muscle cell response.

The aim of this study was to evaluate the impact of multi-walled carbon nanotubes (MWCNTs), before and after chemical surface functionalization on muscle cell response in vitro and in vivo conditions. Prior to biological tests the surface physicochemical properties of the carbon nanotubes (CNTs) deposited on a polymer membrane were investigated. To 'evaluate microstructure and structure of CNTs scanning electron microscopy (SEM) and Fourier transformation infrared spectroscopy (FTIR) were used. During in vitro study CNTs deposited on polymer membrane were contacted directly with myoblast cells, and after 7 days of culture cytotoxicity of samples was analyzed. Moreover, cell morphology in contact with CNTs was observed using SEM and fluorescence microscopy. The cytotoxicity of CNTs modified in a different way was comparable and significantly lower in comparison with pure polymer membrane. Microscopy analysis of cultured myoblasts confirms intense cell proliferation of all investigated samples with CNTs while for two kinds of CNTs myoblasts' differentiation into myotubes was observed. Histochemical reactions for the activity of enzymes such as acid phosphatase, cytochrome C oxidase, and non-specific esterase allowed the analysis of the extent of inflammation, degree of regeneration process of the muscle fibers resulting from the presence of the satellite cells and the neuromuscular junction on muscle fibers in contact with CNTs after implantation of CNTs into gluteal muscle of rat.

In vitro biocompatibility of multiwalled carbon nanotubes with sensory neurons.

Multiwalled carbon nanotubes (MWCNTs) possess unique properties rendering them a potentially useful biomaterial for neurobiological applications such as providing nanoscale contact-guidance cues for directing axon growth within peripheral nerve repair scaffolds. The in vitro biocompatibility of MWCNTs with postnatal mouse spinal sensory neurons was assessed for this application. Cell culture medium conditioned with MWCNTs was not significantly toxic to dissociated cultures of postnatal mouse dorsal root ganglia (DRG) neurons. However, exposure of DRG neurons to MWCNTs dispersed in culture medium resulted in a time- and dose-dependent reduction in neuronal viability. At 250 µg mL⁻¹, dispersed MWCNTs caused significant neuronal death and unusual neurite morphologies illustrated by immunofluorescent labelling of the cytoskeletal protein beta (III) tubulin, however, at a dose of 5 µg mL⁻¹ MWCNTs were nontoxic over a 14-day period. DRG neurons grown on fabricated MWCNT substrates produced neurite outgrowths with abnormal morphologies that were significantly inferior in length to neurons grown on the control substrate laminin. This evidence demonstrates that to be utilized as a biomaterial in tissue scaffolds for nerve repair, MWCNTs will require robust surface modification to enhance biocompatibility and growth promoting properties.

Effect of MWCNT surface and chemical modification on in vitro cellular response.

The aim of this study was to evaluate the impact of multi-walled carbon nanotubes (MWCNTs with diameter in the range of 10-30 nm) before and after chemical surface functionalisation on macrophages response. The study has shown that the detailed analysis of the physicochemical properties of this particular form of carbon nanomaterial is a crucial issue to interpret properly its impact on the cellular response. Effects of carbon nanotubes (CNTs) characteristics, including purity, dispersity, chemistry and dimension upon the nature of the cell environment-material interaction were investigated. Various techniques involving electron microscopy (SEM, TEM), infrared spectroscopy (FTIR), inductively coupled plasma optical emission spectrometry, X-ray photoelectron spectroscopy have been employed to evaluate the physicochemical properties of the materials. The results demonstrate that the way of CNT preparation prior to biological tests has a fundamental impact on their behavior, cell viability and the nature of cell-nanotube interaction. Chemical functionalisation of CNTs in an acidic ambient (MWCNT-Fs) facilitates interaction with cells by two possible mechanisms, namely, endocytosis/phagocytosis and by energy-independent passive process. The results indicate that MWCNT-F in macrophages may decrease the cell proliferation process by interfering with the mitotic apparatus without negative consequences on cell viability. On the contrary, the as-prepared MWCNTs, without any surface treatment produce the least reduction in cell proliferation with reference to control, and the viability of cells exposed to this sample was substantially reduced with respect to control. A possible explanation of such a phenomenon is the presence of MWCNT's agglomerates surrounded by numerous cells releasing toxic substances.

Identification of microcephalin, a protein implicated in determining the size of the human brain.

Primary microcephaly (MIM 251200) is an autosomal recessive neurodevelopmental condition in which there is a global reduction in cerebral cortex volume, to a size comparable with that of early hominids. We previously mapped the MCPH1 locus, for primary microcephaly, to chromosome 8p23, and here we report that a gene within this interval, encoding a BRCA1 C-terminal domain-containing protein, is mutated in MCPH1 families sharing an ancestral 8p23 haplotype. This gene, microcephalin, is expressed in the developing cerebral cortex of the fetal brain. Further study of this and related genes may provide important new insights into neocortical development and evolution.

Expression of mOb1, a novel atypical 73 amino acid K50-homeodomain protein, during mouse development.

We report the initial characterization of mOb1 (Odd homeoBox 1), which encodes an atypical 73 amino acid K50-homeodomain protein localised in the cytoplasm and absent from nuclei during mouse development. Conserved orthologues were present in man, rat, cow, pig and chicken, but not in fish, amphibians or invertebrates. Temporo-spatial patterns of mOb1 transcript and mOb1 protein expression were coincident in developing mouse embryos. Cardiac expression was first observed at E8.25 in linear heart tube myocardium and briefly in both horns of the sinus venosus. Myocardial expression peaked at E13.5, where after it diminished and was not detectable above background by adulthood. At no stage was expression observed in endocardium, endocardial cushion tissue, the coronary arteries or great vessels. At E13.5 and E15.5, mOb1 expression broadened to include skeletal muscle, stratified epithelium (upper aerodigestive tract and skin), epithelium of developing airways, vibrissae, midbrain/hindbrain junction, meninges, mesenchymal cellular condensations that preceded cartilage formation and chondrocytes.

Expression of mOb1, a novel atypical 73 amino acid K50-homeodomain protein, during mouse development.

We report the initial characterization of mOb1 (Odd homeoBox 1), which encodes an atypical 73 amino acid K50-homeodomain protein localised in the cytoplasm and absent from nuclei during mouse development. Conserved orthologues were present in man, rat, cow, pig and chicken, but not in fish, amphibians or invertebrates. Temporo-spatial patterns of mOb1 transcript and mOb1 protein expression were coincident in developing mouse embryos. Cardiac expression was first observed at E8.25 in linear heart tube myocardium and briefly in both horns of the sinus venosus. Myocardial expression peaked at E13.5, where after it diminished and was not detectable above background by adulthood. At no stage was expression observed in endocardium, endocardial cushion tissue, the coronary arteries or great vessels. At E13.5 and E15.5, mOb1 expression broadened to include skeletal muscle, stratified epithelium (upper aerodigestive tract and skin), epithelium of developing airways, vibrissae, midbrain/hindbrain junction, meninges, mesenchymal cellular condensations that preceded cartilage formation and chondrocytes.