Bioadhesion and strength of attachment of neurons to biomaterials, pyrolytic carbon and commercially pure titanium, subjected to radio frequency glow discharge and autoclave sterilization
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Improved electrically conductive biomaterials, that minimize or eliminate encapsulation by fibrous tissue from the generally seen "foreign body reaction", would be beneficial for longer-term reliability of neural electrode stimulation and signal recording functions. Improved soft tissue adhesion and integration with electrode surfaces could favor better electronic signal transfer while also preventing micromotion of the electrodes near and beyond target tissue sites. This investigation selected two conductive biomaterials with good prior records of long-term implant biocompatibility. These included pyrolytic carbon (PyC) as used in synthetic heart valves, and commercially pure titanium (cpTi) as used in osseointegrating dental implants. Surface energy modification by low-temperature sterilizing Radio Frequency Glow Discharge treatment (RFGD) was utilized to obtain better neural cell adhesion strengths. A dense cell-to-substratum implant/microtissue adhesion model was developed by 48-hour plating and culture of approximately 300,000 freshly harvested P3-P4 postnatal rat spiral ganglion neurons on approximately 300 mm 2 areas of standard autoclaved PyC and cpTi specimens, and on identical specimens prepared by 2.5 minutes exposure to RFGD to elevate their surface energies. Material surface properties were documented by Comprehensive Contact Angle measurements for Critical Surface Tension (CST) determination, by stylus profilometry for surface roughness, and supporting characterization by Scanning Electron Microscopy(SEM), Energy Dispersive X-ray analysis (EDS), and Electron Spectroscopy for Chemical Analysis (ESCA). These methods verified that the main effects of the mild RFGDT were reduction of superficial organic contaminants and increased surface oxidation, leading to increased CST values from less than 40 mN/m to greater than 70 mN/m for the autoclaved and RFGDT specimens, respectively. Initially attached cell numbers and neurofilament-antibody-stainable areas were greater for the autoclaved than RFGD specimens, as documented by fluorescence and confocal microscopy followed by quantitative image analysis. A water-jet impingement technique was utilized to determine differential retention strengths of the initially attached cells, revealing neuron-to-substratum attachment strengths ranging from a low value of 66 dynes/cm 2 on conventionally autoclaved PyC to more than 114 dynes/cm 2 on RFGD cpTi. Stereomicroscopic inspection of the water-jet impacted cell carpets revealed more effective detachment of cells and sharp, built-up edges of the detachment area margins for autoclaved specimens. In contrast, graded cellular detachment areas and margins in the jet-impacted carpets were observed for the RFGD treated specimens, correlating with findings of greater strengths of cell adhesion to RFGD treated substrata. Future work will focus on determining whether this increased neuronal cell attachment strength can limit or prevent fibrous encapsulation of implanted electrodes to better maintain design values for signal recording and tissue stimulation.