Crimaldi, Luigi (2024) Controlled cell mechano-modulator for mechanomedicine applications. [Tesi di dottorato]

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Abstract

In contrast to traditional medicine, which relies primarily on the use of pharmaceuticals and chemical interventions to address diseases, mechanomedicine is a relatively newer paradigm that focuses on understanding and treating pathological processes through the administration of a new type of drug, namely mechanical stimulation. However, the applications of the mechanomedicine field are still limited to specific research areas, as the fundamental mechanisms regulating the cellular biological response to mechanical signals are not fully understood. As previously mentioned, cells possess the remarkable ability to integrate mechanical cues of the ECM which directly influence multiple cellular functions. For this reason, outlining the quantitative aspects of this relationship is crucial to how to actually modulate the mechanical stimuli to induce specific cellular responses. This knowledge not only advances our fundamental understanding of cell mechanobiology but also holds significant implications for various applications, including tissue engineering, regenerative medicine, and therapeutic interventions. In this PhD thesis, we presented the realization of an advanced tool to characterize in vitro cellular response to mechanical stimulation. This device, identified as a cell stretching system, is able to administer mechanical doses to the cells ensuring a coherent cell perception of spatio-temporal controlled stretching stimuli. In this configuration, we also provided a meticulous approach to calibrate the cell mechanical stimulation which can be generalized for any cell type and mechanomedicine application. Prior to delving into the core of the thesis, in the following chapter (Chapter II) it is provided an overview of the current state of art about the tools for in vitro cell mechanical stimulation. This section specifically concentrates on elucidating the working principles, design methodologies, biological relevance, as well as the inherent limitations of these devices. Subsequently, in Chapter III, we offer a detailed description of the steps followed for the design and realization of the custom-made cell stretching system. Methodologies employed to overcome the challenges related to the microscope integration and live-cell imaging compatibility are provided. Moreover, this chapter illustrates the large efforts spent in creating a synergistic environment that enhances the consistency and reliability of cellular responses to mechanical stimulation. By exploiting this powerful tool, in Chapter IV, we investigated the cell response to uniaxial sustained stretching. We focused our attention on quantitatively characterizing how the deformations applied to the substrate are dynamically perceived by the cells through the FAs and integrated at nuclear level by means of the actin cytoskeletal forces. Furthermore, morphological and biochemical changes of cell mechanosensory components were monitored over time to provide a better understanding of the mechanisms that regulate the cell mechanobiological response to mechanical stimulation. In conclusion the directions for future perspective will be summarized.

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