The aim of this PhD project is the engineering of hydrogel biomaterials in 2D and 3D to study how cell micro-environments instruct and guide cell behaviour. In particular, how the physicality of the extracellular matrix microenvironment influences the cell mechanoresponse, is of fundamental importance in disease development, as well as tissue engineering and regenerative medicine applications. 2D substrates are an ideal tool for an easier study of cell mechanotransduction. For this, we developed a fully defined biomaterial based on polyethyleneglycol chemically functionalized with cell-adhesive peptides, in which we were able to independently tune rigidities and adhesiveness, and so dissect the effects of these physical cues on mechanosignaling. Biological experiments on U2OS osteosarcoma cells seeded on these 2D substrates and the use of YAP/TAZ readout as cell mechanosignaling, demonstrated that the degree of YAP/TAZ nuclear localization on distinct substrates invariably correlates with the nuclear shape of individual cells, indicating the existence of a “nuclear ruler” for YAP/TAZ mechanosensing. In particular, we found that their nuclear accumulation occurs when the projected area of the nucleus surpasses a critical threshold of approximatively 150 μm2. Overall, this work suggests the existence of distinct checkpoints for cellular mechanosensing which can be placed under synthetic control. 3D cell culture systems might better mimic the extracellular matrix in which cells are embedded and recapitulating the natural cell microenvironment. For example, 3D hydrogels biomaterial may be used to developed more complex cell structure such as organoids. In this context, a second goal of this thesis was the engineering of a biomaterial with the aim to design a fully synthetic matrix for SH-SY5Y neuronal cell culture, and to study their behaviour in-vitro. We optimized the chemical and structural modification of hydrogels based on cellulose, a natural plant-derived polymer abundant in nature, non-animal derived and biocompatible, and with a fibrous structure. With the developed hydrogels, we achieved better results than the currently used 3D systems proposed in literature for this cellular system, in term of cell viability up to 14 days, number of long neurite (> 100 m) and number of cell cluster (< 2000 m2). As future development, this developed platform could be used to promote neuronal differentiation, facilitating the development of in-vitro models crucial for studying neurodegenerative diseases such as Parkinson’s disease (PD) or the growth of brain organoids in synthetic matrices. The third aim of this thesis was the development of a 3D immune niche for the delivering of cell-based immunotherapies, particularly in the field of cancer vaccines. The advantage of using an injectable biomaterial niche to deliver immune-therapies, is the generation of a depot from which vaccine components are released over prolonged, tunable and controlled timeframes, enabling a more precise targeting of therapeutic agents, thus reducing side effects and improving the therapeutic efficacy. In this context, we engineered a hydrogel biomaterial based on thiol-modified hyaluronic acid and studied its ability to recruit cells of the immune system in-vivo, allowing to study their phenotyping and consequently providing the possibility of formulating a more targeted therapy. We also found how, by tuning the gel crosslinking degree, it is possible to modulate and control the nanocarrier and drug release. Therefore, as a future perspective, this designed injectable hydrogel niche could be used as a “tool to study cell instruction” to understand and predict the best achievable immune response and the subsequent vaccine efficacy. Overall, in this thesis it was demonstrated how the targeted engineering of hydrogel biomaterials, can be exploited and crucial in diverse biomedical application.

Engineering of hydrogel biomaterials for biomedical applications / Torresan, Veronica. - (2024 May 06).

Engineering of hydrogel biomaterials for biomedical applications

TORRESAN, VERONICA
2024

Abstract

The aim of this PhD project is the engineering of hydrogel biomaterials in 2D and 3D to study how cell micro-environments instruct and guide cell behaviour. In particular, how the physicality of the extracellular matrix microenvironment influences the cell mechanoresponse, is of fundamental importance in disease development, as well as tissue engineering and regenerative medicine applications. 2D substrates are an ideal tool for an easier study of cell mechanotransduction. For this, we developed a fully defined biomaterial based on polyethyleneglycol chemically functionalized with cell-adhesive peptides, in which we were able to independently tune rigidities and adhesiveness, and so dissect the effects of these physical cues on mechanosignaling. Biological experiments on U2OS osteosarcoma cells seeded on these 2D substrates and the use of YAP/TAZ readout as cell mechanosignaling, demonstrated that the degree of YAP/TAZ nuclear localization on distinct substrates invariably correlates with the nuclear shape of individual cells, indicating the existence of a “nuclear ruler” for YAP/TAZ mechanosensing. In particular, we found that their nuclear accumulation occurs when the projected area of the nucleus surpasses a critical threshold of approximatively 150 μm2. Overall, this work suggests the existence of distinct checkpoints for cellular mechanosensing which can be placed under synthetic control. 3D cell culture systems might better mimic the extracellular matrix in which cells are embedded and recapitulating the natural cell microenvironment. For example, 3D hydrogels biomaterial may be used to developed more complex cell structure such as organoids. In this context, a second goal of this thesis was the engineering of a biomaterial with the aim to design a fully synthetic matrix for SH-SY5Y neuronal cell culture, and to study their behaviour in-vitro. We optimized the chemical and structural modification of hydrogels based on cellulose, a natural plant-derived polymer abundant in nature, non-animal derived and biocompatible, and with a fibrous structure. With the developed hydrogels, we achieved better results than the currently used 3D systems proposed in literature for this cellular system, in term of cell viability up to 14 days, number of long neurite (> 100 m) and number of cell cluster (< 2000 m2). As future development, this developed platform could be used to promote neuronal differentiation, facilitating the development of in-vitro models crucial for studying neurodegenerative diseases such as Parkinson’s disease (PD) or the growth of brain organoids in synthetic matrices. The third aim of this thesis was the development of a 3D immune niche for the delivering of cell-based immunotherapies, particularly in the field of cancer vaccines. The advantage of using an injectable biomaterial niche to deliver immune-therapies, is the generation of a depot from which vaccine components are released over prolonged, tunable and controlled timeframes, enabling a more precise targeting of therapeutic agents, thus reducing side effects and improving the therapeutic efficacy. In this context, we engineered a hydrogel biomaterial based on thiol-modified hyaluronic acid and studied its ability to recruit cells of the immune system in-vivo, allowing to study their phenotyping and consequently providing the possibility of formulating a more targeted therapy. We also found how, by tuning the gel crosslinking degree, it is possible to modulate and control the nanocarrier and drug release. Therefore, as a future perspective, this designed injectable hydrogel niche could be used as a “tool to study cell instruction” to understand and predict the best achievable immune response and the subsequent vaccine efficacy. Overall, in this thesis it was demonstrated how the targeted engineering of hydrogel biomaterials, can be exploited and crucial in diverse biomedical application.
Engineering of hydrogel biomaterials for biomedical applications
6-mag-2024
Engineering of hydrogel biomaterials for biomedical applications / Torresan, Veronica. - (2024 May 06).
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11577/3514442
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