James J. Hickman - US grants

Great strides have recently been made in using implanted electrodes to directly stimulate the cochlea, visual cortex, peripheral nerves, and other aspects of the human nervous system. Thus the electrodes interaction with the surrounding tissue is an important issue in its function. The surface of these electrode materials plays a key role in determining the body's response to the implanted electrode. Conventional materials can provoke an inflammation response that can be both painful and detrimental to the patient. Methods of altering this response include rendering the surface more inert, making it invisible to the immune system, or actively signaling the body to incorporate the implant. We will apply a novel method of changing the surfaces to make them more acceptable to the body with surface analytical methods to quantitate these changes. We will use self-assembled monolayers (SAMs) to modify the exposed surfaces of implanted electrodes in an attempt to make them more biocompatible. We will take advantage of the different chemical composites of a typical electrode, i.e., insulator and metal, to react two different monolayers to these surfaces. We will culture hippocampal and spinal cord neurons in serum-free and serum-containing media and determine the cells' response to the SAM-modified surface. We have previously shown that we can enhance or retard the growth of hippocampal and spinal cord neurons by modifying surfaces with different SAMs. We will quantitate the surface composition both before and after cell culture using X-ray Photoelectron Spectroscopy, Auger Electron Spectroscopy, ellipsometry, and contact angle measurements. The goal of this research is not lust to make the composite surface biocompatible, but to enhance its interaction with the surrounding tissue. We will also use monolayers of biological macromolecules attached to SAMs to recruit neurons for closer proximity to the electrodes. An understanding of basic cell-surface and tissue-surface interactions has a great many implications for the rehabilitation of impaired or disabled people. Issues we hope to address include chronic pain due to the development of inflammation and scar tissue around electrode implants, insights into the role of the surface in cell culture, as well as the incorporation of implanted electrodes in the body.

DESCRIPTION (provided by applicant): The reflex arc is a fundamental functional unit of the spinal cord. Disease or injury of any of the cellular elements in the unit can result in profound movement disorders. An engineering approach to this system would allow the evaluation of these problems and disorders in an in vitro system in a controlled, defined environment. This project proposes to develop a biologically integrated microfabricated silicon device to study synaptic communication development and reinnervation between motoneurons and myotubes. Our Hypothesis is that a combination of stem cells and growth factors will enable the reinnervation of muscle fibers and that human stem cells will innervate rat muscle in vitro to enable the in vivo evaluation of the above results. The motoneurons will be derived from embryonic rat as a control and compared/contrasted to adult rat motoneurons and motoneurons derived from human stem cells. The myotubes will be derived initially from embryonic rat, then adult rat and later adult human tissue. The primary goal of the project is to establish the ability of human stem cells to innervate adult rat muscle to determine the best conditions using the in vitro system to achieve in vivo reinnervation and functional recovery in a rat SCI model. Initially, in Aim 1 the motoneuron-to-muscle segment will be investigated at the single cell level to assess in vitro reinnervation in a system that is composed of patterned surfaces integrated with a MEMS construct. A chamber to mimic the PNS and CNS environment will be fabricated using MEMS fabrication methodology. Aim 1b examines the system created in Aim 1 where the target for the motoneurons is an adult myotube derived from human tissue. Aim 2 evaluates the system in the presence of growth factors. Aim 3 examines the effect of the presence of glial, Schwann and microglial cells on the functional capacity of the montoneuron to muscle segment. Aim 4 addresses the in vivo experiments that will evaluate the combination of growth factors and human stem cells optimized in Aims 1-3 to allow reinnervation of rat muscle and investigate functional recovery. An integration of fundamental neuroscience, cell biology, microsystem engineering, and surface chemistry will be implemented to build and test this hybrid device, leading eventually to designing schemes to prevent, diagnosis, and treat developmental abnormalities and chronic neurological/muscle disorders. New strategies for prosthetic and orthotic design and evaluation, and new approaches for spinal repair from the aspect of an activity dependent reinnervation of tissue are also envisioned. Finally, this technology if successful could be used to create functional assays to investigate human cells for maladies where animal models may not yet exist. The Nanoscience Technology Center at the University of Central Florida, the Neuroscience Institute at the Medical University of South Carolina, the Naval Research Laboratory (NRL), and Neuralstem (a biotech company devoted to human stem cell technology) have partnered to develop this technology.

[unreadable] DESCRIPTION (provided by applicant): This proposal focuses on the marriage of solid-state electronics and neuronal function to create a new high-throughput electrophysiological assay to determine a compound's or gene product's acute and chronic effect on a cell's function. We have integrated electronics, surface chemistry, biotechnology, genomics and fundamental neuroscience in a concept involving an assay where the reporter element is an array of electrically active cells. This innovative technology has potential to screen compounds from combinatorial chemistry, gene function analysis, and for basic neuroscience applications. Because the method is non-invasive, temporal analysis on a statistically relevant number of cells would now be possible. This would be a benefit over intracellular electrophysiology which perforates the membrane and kills the cell within 4-8 hours. Specific Aim 1 seeks to employ surface chemistry to establish a higher resistance seal between a NE108-15 cell and a metal microelectrode that recreates the interface that is present in patch-clamp electrophysiology utilizing glass micropipettes, so as to allow high fidelity extracellular electrophysiology on a microelectrode array. Specific Aim 2 relies on our previous research in which we used cultured neuronal cells as sensor elements for generic toxin detection. During the course of this study, we observed that most of the toxins tested stopped the action potential. However, how the action potential was interrupted showed differences that we postulate are due to individual toxins acting on different biochemical pathways, which in turn affect ion channels differentially, thereby changing the peak shape of the action potential in a unique manner for each toxin. We propose that algorithms developed jointly between UICU, UCF and CFDRC to analyze the action potential peak shape differences can be used to indicate the pathway(s) or cellular "functional categories" affected by the introduction of a compound to the system or from the activation of a gene. We believe this observation can ultimately be exploited to determine the functional category of biochemical action of unknown compounds or genes. Specific Aim 3 will combine these two advances to demonstrate the feasibility of using living cells as diagnostics for high throughput real-time assays of cell function. This would create a new diagnostic tool to enable the segregation of the effect of a compound or gene product on a cell's function into categories simply by monitoring changes in the action potential on a statistically relevant number of cells that is readily integrated with information obtained by other methodologies currently under development by CFDRC. This approach has the added capability to evaluate function temporally as well as under a variety of conditions, or at different times in a cell's lifecycle. The Nanoscience Technology Center at the University of Central Florida is lead for this proposal with collaborators from the University of Illinois and CFD Research Corporation, Huntsville, AL. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Our overall strategy is to utilize microphysiological systems in combination with functional readouts to establish systems capable of sophisticated analysis of drug candidates during pre-clinical testing. In the UH2 Phase, we will construct physiological systems that represent nervous, circulatory and gastrointestinal/liver. Hickman has published physiologically correct functional systems for cardiac, muscle, neuromuscular junction, myelination and neuronal networks. 3D liver models from RegeneMed and stem cell derived cardiomyocytes and hepatocytes from GE will be incorporated into these microphysiological systems. In the UH3 Phase our consortium will develop a low-cost in vitro predictive efficacy and toxicology system based on a novel pumpless microphysiological platform described in Sung et al 2010. The platform will contain electronic functional readouts and microanalytical systems for rapid high throughput biomarker sensing. Shuler's group has demonstrated microphysiological systems with up to 5 chambers that are a direct analog to a physiologically-based pharmacokinetic (PBPK) model. This system has been used to evaluate combination therapy for colon cancer and secondary toxicity from liver metabolites, response to endocrine disruptions, drug efficacy in multidrug resistant cancer systems, and model transport across a barrier tissue (e.g. GI tract) with systemic response (e.g. liver). Dr. Michael Shuler at Cornell University has pioneered the micro cell culture analog (¿CCA) or Body-on-a- Chip system, a realistic system using cell cultures to predict human response to drugs and biologics and will create a next-generation device. Dr. James Hickman at the University of Central Florida will develop functional in vitro human physiological systems and integrate them onto the microphysiological platform. RegeneMed will supply liver and skin constructs for the next-generation Body-on-a-Chip device. GE Global Research will develop analytical capabilities for integration onto the device, provide human stem cell-derived cardiomyocytes and hepatocytes and develop new human stem cell-derived cellular models, and will adapt their pioneering in silico prediction models for drug efficacy and validation. The Sanford-Burnham Institute has expertise in drug discovery and development and will compare the data generated with the in vitro system to known preclinical and clinical results, and provide regulatory guidance during the development process. LTC. Thomas at Walter Reed Army Institute of Research will develop immune system models for evaluating infectious disease on the device. In total, our consortium contains all of the skill sets required to construct, evaluate and commercialize the integrated system and associated components to achieve the goals outlined in the microphysiometer RFA. PUBLIC HEALTH RELEVANCE: We are developing microphsyiological modules to model the nervous system, circulatory system and gastrointestinal tract system utilizing our extensive experience in these areas in the UH2 Phase of this project. In the UH3 Phase, we will build a 10-organ system that will be low cost, yet highly functional for utilization in drug discovery, toxicity studies and eventually all aspects of pre clinical testing.

Project Summary Addiction to pain medications, especially opiates, has become a major health problem and systems to guide the understanding of repeat overdose treatments are needed. Our proposal seeks to build overdose models for four drugs (fentanyl, methadone, codeine, and morphine) in a multi-organ system and evaluate the acute and repeat dose, or chronic effects, of overdose treatments such as Naloxone on overdose recovery, efficacy as well as off-target toxicity for cardiac, muscle, kidney and liver. We have developed a low cost system using human cells in a pumpless multi-organ platform that allows continuous recirculation of a blood surrogate for up to 28 days. This system emulates the distribution of a parental compound and the formation of metabolites among all ?organ? compartments and predicts potential toxicity and efficacy of drugs better than in vitro single human organ or animal models. We will develop two different overdose models for both male and female phenotypes based on nociceptors and B?tzinger Complex (B?tC) neurons as they contain µ-opioid receptors but are thought to have different roles in response to overdose and treatment. We will also integrate functional immune components in the UH3 Phase that has been demonstrated to enable organ specific or systemic monocyte actuation. In addition, models for cardiomyopathy and an infection model will be utilized to more accurately represent the effects of therapeutics on comorbidities. We will establish a PKPD in vitro model of overdose and treatment to enable prediction in clinical environments for a range of variables including age and drug-drug interactions. Once established the system could be used to evaluate novel pain therapeutics for efficacy and off-target toxicity as well as additional overdose treatments in future studies. Interconnected systems with continuous recirculation of a blood surrogate allows both the parent compound and its metabolites to be evaluated in the same system since it is a low volume platform. This interconnected system is better suited for preclinical drug testing than single organ systems for the same reason that human and animal models are currently the gold standards for toxicity and efficacy determination as they allow communication between the organ systems in the body. To construct a well defined system we will use a common serum free medium with microelectrode arrays and cantilever systems that are integrated on chip that allow for noninvasive electronic and mechanical readouts of organ function. UCF and Hesperos in collaboration with clinicians seek to radically change established practice in drug discovery by bypassing animal experiments and extensive clinical trials to provide treatments for diseases and clinical conditions such as overdose. We have already been working with regulatory authorities to prepare for eventual acceptance of the systems for regular use in INDs. Since Hesperos is already offering multi-organ evaluations as a service to the pharmaceutical industry and clinicians there is a direct translational element in the proposal.