The research proposed in this thesis applied three-dimensional bioprinting technique to create in vitro a reliable model of skeletal muscle fibres (3D BioP). The project was performed in the Regenerative Medicine laboratory (headed by Prof. Gabriele Ceccarelli, Dept. Public Health, Experimental and Forensic Medicine) in collaboration with the research team of Prof. Michele Conti from the Department of Civil Engineering and Architecture of the University of Pavia. For this purpose, a murine muscle cell line (C2C12 cells) and various types of hydrogels combined or not with different scaffolds were used. The best ink for the proliferation and differentiation of muscle cells was first determined through several experimental rounds that were carried out with the goal of finding a suitable 3D BioP protocol to aid in the development of an in vitro 3D muscle fiber model. According to the morphological and molecular biology findings, the fibrin hydrogel was the most effective at promoting the differentiation of the C2C12 murine myoblast line out of all the hydrogels tested. Subsequently, the biomaterial polycaprolactone (PCL) was tested to build a scaffold that could hold C2C12 muscle cells and fibrin hydrogel promoting cell elongation. Therefore, several 3D printing experiments were carried out using two PCL substrates with various shapes and structures. Additionally, it was assessed whether fibrinogen alone or in combination with a PCL structure could promote cell differentiation and proliferation. The structures tested were: (i) a grid with seven "tracks" into which C2C12 cell-containing hydrogel made of fibrinogen was extruded; (ii) a Serpentine-like PCL structure into which the hydrogel was extruded. On these different structures, cell viability and myogenic differentiation using molecular biology analysis and morphological tests were evaluated. Results showed that PCL scaffolds do not interfere with cellular processes and that fibrinogen hydrogel acts as a good extracellular matrix (ECM) substitute, promoting C2C12 cell proliferation and differentiation, making it a promising material for skeletal muscle tissue regeneration. During the third phase of our study, we co-printed C2C12 cells into two spherical PCL structures, one with a large radius and one with a small radius. Morphological analysis results obtained during the several time points emphasized the progression of good cell differentiation especially at the edges of the circular structures where probably the cells “feel” better the stiffness made by PCL and the plastic of the petri dish. The next step of this thesis project was to create an engineered platform to replicate the structural and functional complexity of skeletal muscle. The first experiment utilized a 3D-printed culture chamber made of nylon PA12 for the bioreactor's setup. Unfortunately, because the culture chamber was created using a 3D printing technology that processes materials in the form of powder, causing high porosity of the printed parts, this condition did not meet the waterproofing requirement required for the bioreactor design. By coating the cell chambers with polydimethylsiloxane (PMDS), the experiment was repeated. We were able to complete the necessary waterproofing and pass the sterility test thanks to this treatment. In this regard, up to 21 days, no contamination of the cell culture was observed. Future research will focus into how dynamic cultures (with bioreactors) might be used to stretch these bio complexes and further promote the process of muscle differentiation and elongation to obtain an in vitro muscle fiber to study skeletal muscle tissue and spatial organization in large number of physiological and pathological contexts.
L’obiettivo di questo elaborato di tesi è stato quello di generare un modello di fibra muscolare scheletrica utilizzando la tecnica della manifattura additiva o stampa 3D (3D BioP). Il progetto è stato condotto utilizzando una linea di cellule muscolari murine (C2C12) e diversi tipi di idrogel combinati o meno con scaffold in grado di mimare l’impalcatura della matrice extracellulare muscolare. Il progetto è stato sviluppato nel laboratorio di Medicina Rigenerativa (guidato dal prof. Gabriele Ceccarelli, Dip.to di Sanità Pubblica, Med. Sperimentale e Forense) in collaborazione con il gruppo di ricerca del Prof. Michele Conti. Inizialmente sono state svolte ricerche ed analisi per identificare l’inchiostro (quindi l’idrogel) più adatto alla proliferazione e al differenziamento delle cellule muscolari. Pertanto, sono stati condotti diversi esperimenti con l'obiettivo di identificare un protocollo di stampa adatto alla formazione di un modello 3D di fibra muscolare in vitro. I risultati morfologici e di biologia molecolare hanno dimostrato che l'idrogel di fibrina promuove maggiormente il differenziamento miogenico delle cellule rispetto a tutti gli altri idrogel testati. Successivamente è stato introdotto il biomateriale policaprolattone (PCL) per creare uno scaffold più rigido che potesse contenere l'idrogel di fibrina combinato con le cellule muscolari C2C12. A tal proposito, sono stati eseguiti due cicli di stampa, utilizzando due substrati di PCL di diversa forma e struttura. Inoltre, è stata valutata la capacità del fibrinogeno di promuovere il differenziamento e la proliferazione cellulare da solo o accoppiato con la struttura diversa di PCL. Su queste due strutture sono state eseguite analisi morfologiche e di biologia molecolare. I risultati hanno determinato che l'idrogel di fibrinogeno in combinazione con il PCL fornisce un buon sostituto della matrice extracellulare (ECM) promuovendo la proliferazione e il differenziamento miogenico e che le impalcature del PCL non ostacolano i processi cellulari, rendendo quest’ultimo un materiale promettente per la rigenerazione del tessuto muscolare scheletrico. Nella terza fase della nostra ricerca, siamo passati al co-printing 3D di due strutture di PCL circolari, una con raggio grande e una con raggio piccolo, in cui sono state stampate le cellule muscolari C2C12. I risultati hanno evidenziato che il differenziamento miogenico avviene soprattutto ai bordi delle strutture circolari dove le cellule "percepiscono" meglio la rigidità data dal PCL e dalla plastica della piastra di coltura. L’ulteriore obiettivo del progetto di tesi è stato quello di creare una piattaforma ingegnerizzata per replicare la complessità strutturale e funzionale del muscolo scheletrico. Tale piattaforma si è basata sulla progettazione di un bioreattore. Per la messa a punto del bioreattore il primo esperimento ha previsto la progettazione di una camera di coltura stampata in 3D con nylon PA12. Purtroppo, questa struttura non andava a soddisfare il requisito di impermeabilità richiesto per il design del bioreattore, in quanto, la camera di coltura è stata realizzata mediante una tecnologia di stampa 3D che processa materiali sotto forma di polvere, causando quindi un’elevata porosità delle parti stampate. Abbiamo, di conseguenza, ripetuto l’esperimento andando ad impermeabilizzare le camere cellulari tramite un coating di polidimetilsilossano (PDMS). Questo trattamento ci ha permesso di soddisfare il requisito richiesto di impermeabilità e di superare la prova di sterilità. Studi futuri cercheranno quindi di indagare come le colture dinamiche (con bioreattori) potrebbero essere utilizzate per migliorare il processo di differenziamento miogenico al fine di creare in vitro un modello il più vicino possibile a quello fisiologico della fibra muscolare scheletrica, da impiegare per studi di medicina rigenerativa sia in contesti fisiologici che patologici.
Design e biofabbricazione di scaffold 3D per applicazioni nella medicina rigenerativa muscolare
BARILLARI, ALESSIO
2021/2022
Abstract
The research proposed in this thesis applied three-dimensional bioprinting technique to create in vitro a reliable model of skeletal muscle fibres (3D BioP). The project was performed in the Regenerative Medicine laboratory (headed by Prof. Gabriele Ceccarelli, Dept. Public Health, Experimental and Forensic Medicine) in collaboration with the research team of Prof. Michele Conti from the Department of Civil Engineering and Architecture of the University of Pavia. For this purpose, a murine muscle cell line (C2C12 cells) and various types of hydrogels combined or not with different scaffolds were used. The best ink for the proliferation and differentiation of muscle cells was first determined through several experimental rounds that were carried out with the goal of finding a suitable 3D BioP protocol to aid in the development of an in vitro 3D muscle fiber model. According to the morphological and molecular biology findings, the fibrin hydrogel was the most effective at promoting the differentiation of the C2C12 murine myoblast line out of all the hydrogels tested. Subsequently, the biomaterial polycaprolactone (PCL) was tested to build a scaffold that could hold C2C12 muscle cells and fibrin hydrogel promoting cell elongation. Therefore, several 3D printing experiments were carried out using two PCL substrates with various shapes and structures. Additionally, it was assessed whether fibrinogen alone or in combination with a PCL structure could promote cell differentiation and proliferation. The structures tested were: (i) a grid with seven "tracks" into which C2C12 cell-containing hydrogel made of fibrinogen was extruded; (ii) a Serpentine-like PCL structure into which the hydrogel was extruded. On these different structures, cell viability and myogenic differentiation using molecular biology analysis and morphological tests were evaluated. Results showed that PCL scaffolds do not interfere with cellular processes and that fibrinogen hydrogel acts as a good extracellular matrix (ECM) substitute, promoting C2C12 cell proliferation and differentiation, making it a promising material for skeletal muscle tissue regeneration. During the third phase of our study, we co-printed C2C12 cells into two spherical PCL structures, one with a large radius and one with a small radius. Morphological analysis results obtained during the several time points emphasized the progression of good cell differentiation especially at the edges of the circular structures where probably the cells “feel” better the stiffness made by PCL and the plastic of the petri dish. The next step of this thesis project was to create an engineered platform to replicate the structural and functional complexity of skeletal muscle. The first experiment utilized a 3D-printed culture chamber made of nylon PA12 for the bioreactor's setup. Unfortunately, because the culture chamber was created using a 3D printing technology that processes materials in the form of powder, causing high porosity of the printed parts, this condition did not meet the waterproofing requirement required for the bioreactor design. By coating the cell chambers with polydimethylsiloxane (PMDS), the experiment was repeated. We were able to complete the necessary waterproofing and pass the sterility test thanks to this treatment. In this regard, up to 21 days, no contamination of the cell culture was observed. Future research will focus into how dynamic cultures (with bioreactors) might be used to stretch these bio complexes and further promote the process of muscle differentiation and elongation to obtain an in vitro muscle fiber to study skeletal muscle tissue and spatial organization in large number of physiological and pathological contexts.È consentito all'utente scaricare e condividere i documenti disponibili a testo pieno in UNITESI UNIPV nel rispetto della licenza Creative Commons del tipo CC BY NC ND.
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https://hdl.handle.net/20.500.14239/15852