Optical and biological properties of transparent nanocrystalline hydroxyapatite obtained through spark plasma sintering.

Transparent bioceramics have attracted a large amount of research interest as they facilitate direct observation of biointerfacial reactions. Thus far, attempts to achieve transparent hydroxyapatite have been focused on augmenting the sintering pressure and/or extending the sintering duration. This study aims at fabricating transparent HA using a direct and fast spark plasma sintering process with appropriate starting powder and moderate sintering pressure. Three types of raw powder, namely micro-spheres, nano-rods and nano-spheres, were sintered to investigate the optical and biological properties of the compacted pellets. It was found that in terms of transparency, the micro-sphere pellet sintered at 1000°C stood out with an in-line transmittance as high as 84% achieved at 1300nm for a 2mm thick sample. In addition, pellets fabricated from micro-spheres demonstrated the highest cell viability in in vitro biological tests with L929 cells. Living cells cultured on a transparent micro-sphere pellet could be directly and clearly observed by light microscopy. It is thus concluded that the micro-sphere powder is the most desirable raw material to manufacture transparent hydroxyapatite because it could enable dense pellets with notably high transparency and outstanding in vitro biocompatibility to be readily obtained.

Cell compatible encapsulation of filaments into 3D hydrogels.

Tissue engineering scaffolds for nerve regeneration, or artificial nerve conduits, are particularly challenging due to the high level of complexity the structure of the nerve presents. The list of requirements for artificial nerve conduits is long and includes the ability to physically guide nerve growth using physical and chemical cues as well as electrical stimulation. Combining these characteristics into a conduit, while maintaining biocompatibility and biodegradability, has not been satisfactorily achieved by currently employed fabrication techniques. Here we present a method combining pultrusion and wet-spinning techniques facilitating incorporation of pre-formed filaments into ionically crosslinkable hydrogels. This new biofabrication technique allows the incorporation of conducting or drug-laden filaments, controlled guidance channels and living cells into hydrogels, creating new improved conduit designs.

Effect of the dopant anion in polypyrrole on nerve growth and release of a neurotrophic protein.

The dopant anion in polypyrrole plays a critical role in determining the physical and chemical properties of these conducting polymers. Here we demonstrate an additional effect on the ability to incorporate and release a neurotrophic protein - neurotrophin-3. The multi-faceted role of the dopant is critical in ensuring optimal performance of polypyrroles in their use as platforms for nerve growth. In this paper, the effect of changing the co-dopant used in electrochemical polypyrrole synthesis on the compatibility with primary auditory nerve tissue is considered and compared to some of the physical properties of the films. Significant differences in the controlled-release properties of the films were also observed. The ability of the polymers to enhance nerve growth and survival in vitro with neurotrophin-3 release was also studied, which is a function of both compatibility with the neural tissue and the ability of the polymer to release sufficient neurotrophic protein to affect cell growth. A small synthetic dopant, para-toluene sulphonate, was found to perform favourably in both aspects and ultimately proved to be the most suitable material for the application at hand, which is the delivery of neurotrophins for inner-ear therapies.

Nanostructured aligned CNT platforms enhance the controlled release of a neurotrophic protein from polypyrrole.

An aligned CNT array membrane electrode has been used as a nanostructured supporting platform for polypyrrole (PPy) films, exhibiting significant improvement in the controlled release of neurotrophin. In terms of linearity of release, stimulated to unstimulated control of NT-3 release and increased mass and % release of incorporated NT-3, the nanostructured material performed more favourably than the flat PPy film.

Conducting polymers, dual neurotrophins and pulsed electrical stimulation--dramatic effects on neurite outgrowth.

In this study the synergistic effect of delivering two neurotrophins simultaneously to encourage neuron survival and neurite elongation was explored. Neurotrophin-3 (NT-3) and brain-derived neurotrophic factor (BDNF) were incorporated into polypyrrole (PPy) during electrosynthesis and the amounts incorporated and released were determined using iodine-125 ((125)I) radio-labelled neurotrophins. Neurite outgrowth from cochlear neural explants grown on the conducting polymer was equivalent to that on tissue culture plastic but significantly improved with the incorporation of NT-3 and BDNF. Neurite outgrowth from explants grown on polymers containing both NT-3 and BDNF showed significant improvement over PPy doped only with NT-3, due to the synergistic effect of both neurotrophins. Neurite outgrowth was significantly improved when the polymer containing both neurotrophins was electrically stimulated. It is envisaged that when applied to the cochlear implant, these conducting and novel polymer films will provide a biocompatible substrate for storage and release of neurotrophins to help protect auditory neurons from degradation after sensorineural hearing loss and encourage neurite outgrowth towards the electrodes.

Polypyrrole-coated electrodes for the delivery of charge and neurotrophins to cochlear neurons.

Sensorineural hearing loss is associated with gradual degeneration of spiral ganglion neurons (SGNs), compromising hearing outcomes with cochlear implant use. Combination of neurotrophin delivery to the cochlea and electrical stimulation from a cochlear implant protects SGNs, prompting research into neurotrophin-eluting polymer electrode coatings. The electrically conducting polypyrrole/para-toluene sulfonate containing neurotrophin-3 (Ppy/pTS/NT3) was applied to 1.7 mm2 cochlear implant electrodes. Ppy/pTS/NT3-coated electrode arrays stored 2 ng NT3 and released 0.1 ng/day with electrical stimulation. Guinea pigs were implanted with Ppy/pTS or Ppy/pTS/NT3 electrode arrays two weeks after deafening via aminoglycosides. The electrodes of a subgroup of these guinea pigs were electrically stimulated for 8 h/day for 2 weeks. There was a loss of SGNs in the implanted cochleae of guinea pigs with Ppy/pTS-coated electrodes indicative of electrode insertion damage. However, guinea pigs implanted with electrically stimulated Ppy/pTS/NT3-coated electrodes had lower electrically-evoked auditory brainstem response thresholds and greater SGN densities in implanted cochleae compared to non-implanted cochleae and compared to animals implanted with Ppy/pTS-coated electrodes (p<0.05). Ppy/pTS/NT3 did not exacerbate fibrous tissue formation and did not affect electrode impedance. Drug-eluting conducting polymer coatings on cochlear implant electrodes present a clinically viable method to promote preservation of SGNs without adversely affecting the function of the cochlear implant.

A conducting-polymer platform with biodegradable fibers for stimulation and guidance of axonal growth.

A biosynthetic platform composed of a conducting polypyrrole sheet embedded with unidirectional biodegradable polymer fibers is described (see image; scale bar = 50 µm). Such hybrid systems can promote rapid directional nerve growth for neuro-regenerative scaffolds and act as interfaces between the electronic circuitry of medical bionic devices and the nervous system.

Promoting neurite outgrowth from spiral ganglion neuron explants using polypyrrole/BDNF-coated electrodes.

Release of neurotrophin-3 (NT3) and brain-derived neurotrophic factor (BDNF) from hair cells in the cochlea is essential for the survival of spiral ganglion neurons (SGNs). Loss of hair cells associated with a sensorineural hearing loss therefore results in degeneration of SGNs, potentially reducing the performance of a cochlear implant. Exogenous replacement of either or both neurotrophins protects SGNs from degeneration after deafness. We previously incorporated NT3 into the conducting polymer polypyrrole (Ppy) synthesized with para-toluene sulfonate (pTS) to investigate whether Ppy/pTS/NT3-coated cochlear implant electrodes could provide both neurotrophic support and electrical stimulation for SGNs. Enhanced and controlled release of NT3 was achieved when Ppy/pTS/NT3-coated electrodes were subjected to electrical stimulation. Here we describe the release dynamics and biological properties of Ppy/pTS with incorporated BDNF. Release studies demonstrated slow passive diffusion of BDNF from Ppy/pTS/BDNF, with electrical stimulation significantly enhancing BDNF release over 7 days. A 3-day SGN explant assay found that neurite outgrowth from explants was 12.3-fold greater when polymers contained BDNF (p < 0.001), although electrical stimulation did not increase neurite outgrowth further. The versatility of Ppy to store and release neurotrophins, conduct electrical charge, and act as a substrate for nerve-electrode interactions is discussed for specialized applications such as cochlear implants.

Optimising the incorporation and release of a neurotrophic factor using conducting polypyrrole.

In this study, a neurotrophin delivery system based on an inherently conducting polymer (ICP) has been developed. Direct incorporation of neurotrophin-3 (NT-3) was investigated and controlled release was tested under various electrochemical conditions. The loading capacity and amount of NT-3 released from the polymer was determined using (125)I-labelled NT-3. Electrochemical stimulation of polypyrrole by pulsed voltage, pulsed current or cyclic voltammetry promoted the release of NT-3 at a greater rate than natural diffusion of NT-3. NT-3 was released from polypyrrole as an initial burst in the first 24 h followed by prolonged release over a subsequent 6 days of sampling. The amount of NT-3 incorporated into the polymer could be controlled by varying the polymerisation time, with longer growth periods incorporating more NT-3. The NT-3 release results indicated that the polymers grown for longer released a lower percentage of the incorporated NT-3 compared to the polymers grown for shorter times. Polymer-based neurotrophin delivery systems have the potential to be incorporated into future treatments for nerve injuries to prevent nerve degradation and promote nerve protection.