Project Grant F31DE035400

MULTIMODAL MULTI-ELECTRODE ARRAY (MEA) FOR PROBING TOOTH ROOT ORGANOID MATURATION AND SENSORY INNERVATION - PROJECT SUMMARY IN THE UNITED STATES, THE AVERAGE NUMBER OF TEETH IN ADULTS DECREASES BY 35% BY THE AGE OF 65. MISSING TEETH ARE REPLACED BY ARTIFICIAL DENTAL IMPLANTS, WHICH, AMONG OTHER DRAWBACKS, DO NOT PROVIDE SENSORY CUES DURING MASTICATION DUE TO THE ABSENCE OF NERVE INNERVATION. THE SYED-PICARD LAB AT THE UNIVERSITY OF PITTSBURGH HAS DEVELOPED A SELF-ASSEMBLING TOOTH ROOT ORGANOID WITH REMARKABLE DIFFERENTIATION INTO DISTINCT SPATIAL LAYERS USING POSTNATAL STEM CELLS DERIVED FROM HUMAN PATIENTS DURING WISDOM TOOTH EXTRACTION. NERVE INNERVATION OF THE TOOTH ROOT OCCURS IN A PRECISE SPATIOTEMPORAL MANNER. HERE, WE AIM TO EVALUATE PROPER INNERVATION OF THE TOOTH ROOT ORGANOID BY INVESTIGATING THE SPATIOTEMPORAL DYNAMICS OF NEUROTROPHIC FACTORS IN THE ORGANOID AND THE NERVE INNERVATION ITSELF OVER THE COURSE OF THE ORGANOID'S 14-DAY MATURATION PERIOD. MOREOVER, WE EVALUATE THE FUNCTIONALITY OF THE INNERVATED SENSORY NERVES THROUGH ELECTROPHYSIOLOGY. MICROELECTRODE ARRAYS (MEAS) ARE USED EXTENSIVELY IN VITRO AND IN VIVO TO RECORD AND MODULATE NEURAL ACTIVITIES AND ARE BEING EXPLORED AS A POWERFUL TOOL FOR BIOCHEMICAL SENSING WITH HIGH SPATIAL AND TEMPORAL RESOLUTION. APTAMERS HAVE BEEN APPLIED AS BIO-RECOGNITION ELEMENTS IN CUSTOMIZABLE BIOSENSOR MEAS, BUT THEIR POOR STABILITY IN BIOLOGICAL ENVIRONMENTS CAN RESULT IN A VERY SHORT SENSOR LIFETIME. MOREOVER, THE STANDARD METHOD FOR ATTACHING APTAMERS ON MEAS DOES NOT ALLOW FOR SITE- SPECIFICITY, PREVENTING THE DESIGN OF MULTI-MODAL SENSORS THAT CAN SENSE MULTIPLE ANALYTES AND PERFORM ELECTROPHYSIOLOGY. IN THIS RESEARCH, WE AIM TO DEVELOP A HIGHLY STABLE APTAMER FUNCTIONALIZED MEA FOR MULTIMODAL INTERROGATION OF THE ORGANOID. IN AIM 1, I WILL DEVELOP A HIGHLY CUSTOMIZABLE AND STABLE ELECTROCHEMICAL BIOSENSOR THROUGH ELECTRODEPOSITION OF POROUS NANOPARTICLES FUNCTIONALIZED WITH THE APTAMERS OF CHOICE. THE MEA WILL BE CUSTOM-DESIGNED AND FABRICATED TO INTERFACE WITH THE MULTIPLE LAYERS OF THE ORGANOID, SENSE MULTIPLE ANALYTES, AND STIMULATE NEURONS. IN AIM 2, I WILL IMPLANT MY APTAMER FUNCTIONALIZED MEAS FOR BDNF AND NGF DETECTION AND TRACK THEIR SPATIOTEMPORAL EXPRESSION OVER THE 14-DAY MATURATION PERIOD. IN AIM 3, I WILL CO-CULTURE SENSORY NEURONS WITH THE ORGANOIDS TO STIMULATE AND TRACK NEURAL FIRING AND CORRELATE IT SPATIOTEMPORALLY WITH BDNF AND NGF EXPRESSION AS SENSORY INNERVATION MATURES. NOCICEPTIVE STIMULI WILL BE APPLIED TO THE MATURE ORGANOIDS IN THE FORM OF CAPSAICIN, ACID, AND HEAT, AND THE NEURONAL RESPONSES WILL BE MEASURED. I WILL RECEIVE TRAINING FROM DR. TRACY CUI ON NEUROCHEMICAL SENSORS AND NEURAL RECORDING AND DR. FATIMA SYED-PICARD ON STEM-CELL BIOLOGY, ORGANOIDS, AND NEUROREGULATORY PROCESSES DIRECTING INNERVATION DURING TOOTH DEVELOPMENT. THE UNIVERSITY OF PITTSBURGH IS A WORLD-CLASS NEURAL ENGINEERING RESEARCH CENTER AND HIGHLY RANKED DENTAL AND CRANIOFACIAL RESEARCH INSTITUTION. DRS. CUI AND SYED-PICARD HAVE AN ESTABLISHED COLLABORATIVE RELATIONSHIP AND WILL PROVIDE THE OPTIMAL ENVIRONMENT FOR THIS PROJECT.