Mechanical cues of the cell surrounding affect cells' behaviour via mechanotransduction. Indeed, cells are able to perceive and respond to external mechanical forces from growing microenvironment, as well as to the stiffness of the underlying extracellular matrix. However, the interrelated effects of mechanical stimuli on cellular response are poorly understood yet. In this project, I will propose a novel experimental approach to couple the substrate stiffness with an external applied stretch to elucidate and compare the physiologic and altered cellular mechano-signalling. On this basis, I will optimize an innovative cell culture system, developed in our laboratory, to provide uniaxial longitudinal stretch by a linear actuator on the silicone chamber surface where cells will be seeded. An additive linear motor will be devised to automatically compensate the longitudinal displacement imposed by the main actuator, thus allowing a continuous cells' observation during the stretch. Moreover, optical software will be employed to elaborate post-processed cellular images for biological analysis. In this view, I will first develop a real-time program in LabVIEW for the cellular imaging acquisition and for the actuators' synchronization. In parallel, I will design and realize the silicone stretch chamber for cell culture through a 3D printed mold. Subsequently, I will incorporate to the silicone chamber an ad-hoc customized substrates, realized to simulate the native matrices stiffness. A strict characterization of each custom-made substrate in terms of the matrix stiffness and strain field distribution on the bottom area will be performed before cell testing. Finally, a series of tests will be carried on studying the cellular response to different substrates rigidities in static/dynamic stretch, testing healthy cells, like osteoblasts and muscle cells, and pathologic ones, such as osteosarcoma and transfected SOD cells, respectively.
Mechanical cues of the surrounding environment affect cells¿ behaviour via mechanotransduction.
Indeed, cells are able to perceive and respond to external mechanical forces from the physiologic
microenvironment, as well as to the stiffness of the extracellular matrix. Moreover, this complex
process is also involved in various diseases. Even though it is clear that the cells are able to respond
separately to an external force or to the underlying matrix stiffness, which mechanotransduction
mechanisms are involved and the combined effects of these two mechanical stimuli on cellular
behaviour are poorly understood yet. To date, several commercial systems are employed to simulate
in-vivo forces by applying external mechanical loads on cells adherent to substrates. However, most
of these commercial layers are stiffer than the cellular native matrices.
In this context, I will develop a new experimental method to couple substrate stiffness with a
static/dynamic stretch.
The innovation of this approach is based on the development of a stretch system combined with
homemade substrates-based stretch chambers of different stiffness. The optimized system will be
low-cost, non-invasive and versatile. First, I will realize a customized a program for imaging
acquisition and motors¿ synchronization for an accurate cell monitoring. Indeed, since the
measurement techniques for cellular analysis are optical, the presence of two actuators will
guarantee the centring of cells on the substrate region of interest by stretch compensation, therefore
allowing the imaging acquisition for the entire stretching. The proposed device will be also
designed with low-cost and lightweight materials to be easily moved and placed on any platform or
microscope. Moreover, customized stretch chambers will be developed to test various cells type
(e.g. bone and muscle cells), incorporating into them substrates with different rigidities into the
cellular matrix range. Therefore, the proposed project will be useful to investigate the coupling
effect of substrate rigidity and mechanical stretch on the cellular physiological and altered
behaviours, thus providing crucial outcomes about the mechanotransduction role in different
pathologies.