Device Design and Diagnostic Imaging of Radiopaque 3D Printed Tissue Engineering Scaffolds.

3D printed biomaterial implants are revolutionizing personalized medicine for tissue repair, especially in orthopedics. In this study, a radiopaque Bi 2 O 3 doped polycaprolactone ( PCL ) composite is developed and implemented to enable the use of diagnostic X-ray technologies, especially photon counting X-ray computed tomography ( PCCT ), for comprehensive in vivo device monitoring. PCL filament with homogeneous Bi 2 O 3 nanoparticle ( NP ) dispersion (0.8 to 11.7 wt%) are first fabricated. Tissue engineered scaffolds ( TES ) are then 3D printed with the composite filament, optimizing printing parameters for small feature size and severely overhung geometries. These composite TES are characterized via micro-computed tomography ( µ CT ), tensile testing, and a cytocompatibility study, with Bi 2 O 3 mass fractions as low as 2 wt% providing excellent radiographic distinguishability, improved tensile properties, and equivalent cytocompatibility of neat PCL. The excellent radiographic distinguishability is validated in situ by imaging 4 and 7 wt% TES in a mouse model with µCT, showing excellent agreement with in vitro measurements. Subsequently, CT image-derived swine menisci are 3D printed with composite filament and re-implanted in their corresponding swine legs ex vivo . Re-imaging the swine legs via clinical CT allows facile identification of device location and alignment. Finally, the emergent technology of PCCT unambiguously distinguishes implanted menisci in situ.

Antagonism of kappa opioid receptors accelerates the development of L-DOPA-induced dyskinesia in a preclinical model of moderate dopamine depletion.

Levels of the opioid peptide dynorphin, an endogenous ligand selective for kappa-opioid receptors (KORs), its mRNA and pro-peptide precursors are differentially dysregulated in Parkinson's disease (PD) and following the development of l-DOPA-induced dyskinesia (LID). It remains unclear whether these alterations contribute to the pathophysiological mechanisms underlying PD motor impairment and the subsequent development of LID, or whether they are part of compensatory mechanisms. We sought to investigate nor-BNI, a KOR antagonist, 1) in the dopamine (DA)-depleted PD state, 2) during the development phase of LID, and 3) via measuring of tonic levels of striatal DA. While nor-BNI (3 mg/kg; s.c.) did not lead to functional restoration in the DA-depleted state, it affected the dose-dependent development of abnormal voluntary movements (AIMs) in response to escalating doses of l-DOPA in a rat PD model with a moderate striatal 6-hydroxdopamine (6-OHDA) lesion. We tested five escalating doses of l-DOPA (6, 12, 24, 48, 72 mg/kg; i.p.), and nor-BNI significantly increased the development of AIMs at the 12 and 24 mg/kg l-DOPA doses. However, after reaching the 72 mg/kg l-DOPA, AIMs were not significantly different between control and nor-BNI groups. In summary, while blocking KORs significantly increased the rate of development of LID induced by chronic, escalating doses of l-DOPA in a moderate-lesioned rat PD model, it did not contribute further once the overall severity of LID was established. While we observed an increase of tonic DA levels in the moderately lesioned dorsolateral striatum, there was no tonic DA change following administration of nor-BNI.

Antagonism of kappa opioid receptors accelerates the development of L-DOPA-induced dyskinesia in a preclinical model of moderate dopamine depletion.

Levels of the opioid peptide dynorphin, an endogenous ligand selective for kappa-opioid receptors (KORs), its mRNA and pro-peptide precursors are differentially dysregulated in Parkinson disease (PD) and following the development of L-DOPA-induced dyskinesia (LID). It remains unclear, whether these alterations contribute to the pathophysiological mechanisms underlying PD motor impairment and the subsequent development of LID, or whether they are part of compensatory mechanisms. We sought to investigate nor-BNI, a KOR antagonist, 1) in the dopamine (DA)-depleted PD state, 2) during the development phase of LID, and 3) with measuring tonic levels of striatal DA. Nor-BNI (3 mg/kg; s.c.) did not lead to functional restoration in the DA-depleted state, but a change in the dose-dependent development of abnormal voluntary movements (AIMs) in response to escalating doses of L-DOPA in a rat PD model with a moderate striatal 6-hydroxydopamine (6-OHDA) lesion. We tested five escalating doses of L-DOPA (6, 12, 24, 48, 72 mg/kg; i.p.), and nor-BNI significantly increased the development of AIMs at the 12 and 24 mg/kg L-DOPA doses. However, after dosing with 72 mg/kg L-DOPA, AIMs were not significantly different between control and nor-BNI groups. In summary, while blocking KORs significantly increased the rate of development of LID induced by chronic, escalating doses of L-DOPA in a moderate-lesioned rat PD model, it did not contribute further once the overall severity of LID was established. While we saw an increase of tonic DA levels in the moderately lesioned dorsolateral striatum, there was no tonic DA change following administration of nor-BNI.

In Vitro Biofouling Performance of Boron-Doped Diamond Microelectrodes for Serotonin Detection Using Fast-Scan Cyclic Voltammetry.

Neurotransmitter release is important to study in order to better understand neurological diseases and treatment approaches. Serotonin is a neurotransmitter known to play key roles in the etiology of neuropsychiatric disorders. Fast-scan cyclic voltammetry (FSCV) has enabled the detection of neurochemicals, including serotonin, on a sub-second timescale via the well-established carbon fiber microelectrode (CFME). However, poor chronic stability and biofouling, i.e., the adsorption of interferent proteins to the electrode surface upon implantation, pose challenges in the natural physiological environment. We have recently developed a uniquely designed, freestanding, all-diamond boron-doped diamond microelectrode (BDDME) for electrochemical measurements. Key potential advantages of the device include customizable electrode site layouts, a wider working potential window, improved stability, and resistance to biofouling. Here, we present a first report on the electrochemical behavior of the BDDME in comparison with CFME by investigating in vitro serotonin (5-HT) responses with varying FSCV waveform parameters and biofouling conditions. While the CFME delivered lower limits of detection, we also found that BDDMEs showed more sustained 5-HT responses to increasing or changing FSCV waveform-switching potential and frequency, as well as to higher analyte concentrations. Biofouling-induced current reductions were significantly less pronounced at the BDDME when using a "Jackson" waveform compared to CFMEs. These findings are important steps towards the development and optimization of the BDDME as a chronically implanted biosensor for in vivo neurotransmitter detection.

Next-Generation Diamond Electrodes for Neurochemical Sensing: Challenges and Opportunities.

Carbon-based electrodes combined with fast-scan cyclic voltammetry (FSCV) enable neurochemical sensing with high spatiotemporal resolution and sensitivity. While their attractive electrochemical and conductive properties have established a long history of use in the detection of neurotransmitters both in vitro and in vivo, carbon fiber microelectrodes (CFMEs) also have limitations in their fabrication, flexibility, and chronic stability. Diamond is a form of carbon with a more rigid bonding structure (sp3-hybridized) which can become conductive when boron-doped. Boron-doped diamond (BDD) is characterized by an extremely wide potential window, low background current, and good biocompatibility. Additionally, methods for processing and patterning diamond allow for high-throughput batch fabrication and customization of electrode arrays with unique architectures. While tradeoffs in sensitivity can undermine the advantages of BDD as a neurochemical sensor, there are numerous untapped opportunities to further improve performance, including anodic pretreatment, or optimization of the FSCV waveform, instrumentation, sp2/sp3 character, doping, surface characteristics, and signal processing. Here, we review the state-of-the-art in diamond electrodes for neurochemical sensing and discuss potential opportunities for future advancements of the technology. We highlight our team's progress with the development of an all-diamond fiber ultramicroelectrode as a novel approach to advance the performance and applications of diamond-based neurochemical sensors.

Moving Fast-Scan Cyclic Voltammetry toward FDA Compliance with Capacitive Decoupling Patient Protection.

Carbon-fiber microelectrodes allow for high spatial and temporal measurements of electroactive neurotransmitter measurements in vivo using fast-scan cyclic voltammetry (FSCV). However, common instrumentation for such measurements systems lack patient safety precautions. To add safety precautions as well as to overcome chemical and electrical noise, a two-electrode FSCV headstage was modified to introduce an active bandpass filter on the electrode side of the measurement amplifier. This modification reduced the measured noise and ac-coupled the voltammetric measurement and moved it from a classical direct current response measurement. ac-coupling not only reduces the measured noise, but also moves FSCV toward compliance with IEC-60601-1, enabling future human trials. Here, we develop a novel ac-coupled voltammetric measurement method of electroactive neurotransmitters. Our method allows for the modeling of a system to then calculate a waveform to compensate for added impedance and capacitance for the system. We describe how first by measuring the frequency response of the system and modeling the analogue response as a digital filter we can then calculate a predicted waveform. The predicted waveform, when applied to the bandpass filter, is modulated to create a desired voltage sweep at the electrode interface. Further, we describe how this modified FSCV waveform is stable, allowing for the measurement of electroactive neurotransmitters. We later describe a 32.7% sensitivity enhancement for dopamine with this new measurement as well as maintaining a calibration curve for dopamine, 3,4-dihydroxyphenylacetic acid, ascorbic acid, and serotonin in vitro. We then validate dopamine in vivo with stimulated release. Our developed measurement method overcame the added capacitance that would traditionally make a voltammetric measurement impossible, and it has wider applications in electrode sensor development, allowing for measurement with capacitive systems, which previously would not have been possible.