P44 | CROSSTALK AMONG NEURONS, MUSCLE AND BONE: HUMAN NEUROMUSCULAR JUNCTION ON A CHIP

M. Gatti^1,2, F. Beretti², M. Malenchini², M. Y. Follo¹, F. Mancarella³, F. Bonafè³, F. Gherardini⁴, T. Maraldi² | ¹Department of Biomedical and Neuromotor Science, Cellular Signalling Laboratory, University of Bologna, Italy; ²Department of Biomedical, Metabolic and Neural Sciences, University of Modena and Reggio Emilia, Modena, Italy; ³Istituto per lo Studio dei Materiali Nanostrutturati (ISMN), National Research Council (CNR), Bologna, Italy; ⁴Department of Engineering “Enzo Ferrari”, University of Modena and Reggio Emilia, Modena, Italy

Aging is associated with gradual degeneration, in mass and function, of the neuro-musculo-skeletal system. Neuromuscular junctions (NMJs) are specialized synapses, which are crucial for the communication between motor neurons and skeletal muscle and become vulnerable in muscle atrophy, where the impairment of NMJs results in muscle weakness. During aging this muscle wasting condition occurs and is called sarcopenia. Indeed, osteoporosis and sarcopenia - osteosarcopenia (OS) - are twinaging diseases. With the purpose to study the perturbation in NMJs occurring in OS, beside bone side defects, an ideal model would contain MNs, myotubes and osteoblasts to better recapitulate the human disease pathology. Therefore, we developed a personalized homemade microfluidic device to study compartmentalized human NMJs in an in vitro muscle model connected to the bone side. A selective pretreatment in bone compartment with dexamethasone (Dexa) was used to induce osteoporosis: in this way, we investigated if this pathological condition could be the driver to have an OS linked to NMJ perturbations. A soft master in SU-8 was fabricated through a photolithographic process tailored for high-aspect-ratio microstructures, enabling the creation of mold structures with micrometric resolution from which a replica in polydimethylsiloxane was obtained. This device is composed by three different compartments: a left-neuronal, a central-muscular and a right-bone one. The first two are connected by around 100 microgrooves. The 1µM diameter allows only neurites to pass through and take contact with the myotubes forming NMJs. The last compartment containing osteoblasts should be in an open or closed condition, to treat osteoblasts with Dexa separately from myotubes, but to allow afterwards the sharing of the medium. This innovative experimental model allowed us to perform immunofluorescence analysis on fixed samples or by live imaging. Collectively, this method will be useful to understand if the modifications induced in osteoblasts during bone disorders have a cascade in the muscle and neuron parts, such as in osteoporosis arising in females after menopause that leads to muscle weakness.