This thesis project explores the convergence of new 3D printing techniques (3D BioP) and bioreactors with the aim of recreating a model of skeletal muscle fiber using murine muscle cells, C2C12. The project was developed in the regenerative medicine laboratory (headed by Prof. Gabriele Ceccarelli, Dept. of 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. C2C12 murine muscle cells were encapsulated in fibrin hydrogel, a biomaterial promoting myogenic proliferation and differentiation. Once the suitable printing protocol for creating a 3D model of muscle fiber was identified, the bioink, i.e. the hydrogel, was extruded into three-dimensional, polycaprolactone (PCL) scaffolds. Since the 3D structure of the scaffold is essential for guiding proper cellular organization and extracellular matrix synthesis, initial experiments aimed to optimize the scaffold shape to limit cell leakage while ensuring fusion and differentiation possibilities. The scaffold had a serpentine-like structure containing a single track where the hydrogel was extruded. After printing, samples underwent morphological and molecular analysis demonstrating that PCL does not hinder cellular processes and promotes proliferation. However, from a differentiation perspective, only cells along the scaffold edges tended to elongate and mature, possibly perceiving the rigidity of PCL and the plastic of the culture plate. At this point, the next step was to replicate, as faithfully as possible, physiological growth conditions to recreate the structural and functional complexity of skeletal muscle. Since previous experiments indicated that static growth conditions alone are insufficient to induce the differentiation process, mechanical stimulation was added to cell culture through an engineered platform, a bioreactor. The culture chamber was initially 3D printed with nylon PA12, later coated with an impermeable material, polydimethylsiloxane, to ensure sterility maintenance. Furthermore, if testing the bioreactor requires a suitably flexible and stretchable three-dimensional support, the PCL scaffold was replaced with one made of polydimethylsiloxane (PDMS), a biocompatible and deformable material. The printing involves extruding the hydrogel into the tracks of three-dimensional scaffolds contained in boxes that allow subsequent connection to the bioreactor. Once the samples were obtained, mechanical stimulation was applied for 15 minutes followed by 15 minutes of rest, repeated for a total of 8 hours. At the end of the specified time, morphological and molecular analyses were conducted, showing good cellular density and viability. However, comparing the samples with the ones kept in static growth conditions, no significant improvements were identified, in terms of elongation and differentiation, while, regarding gene expression, the more myogenic genes had a significant induction with respect to static samples. This may be because C2C12 cells do not find a stable support for differentiation on PDMS, and the mechanical stimulation provided by the bioreactor may not be suitable for our sample type. Future studies will be aimed at investigating new growth and mechanical stimulation protocols, via bioreactor, to induce the myogenic differentiation process with the aim of recreating in vitro a skeletal muscle fiber as similar as possible to the physiological one, which can be used for both therapeutic and research purposes.
La presente tesi esplora la convergenza tra le nuove tecniche di stampa 3D (3D BioP) e i bioreattori con l’obiettivo di ricreare un modello di fibra muscolare scheletrica a partire da una linea di cellule muscolari murine, le C2C12. Le cellule C2C12 sono state incapsulate nell’idrogel di fibrina, biomateriale che promuove la proliferazione e il differenziamento miogenico. Una volta identificato il protocollo di stampa adatto per la creazione di un modello 3D di fibra muscolare, il bioinchiostro, cioè l’idrogel, è stato estruso all’interno di scaffold tridimensionali di policaprolattone (PCL). Poiché la struttura 3D dello scaffold è essenziale per guidare la corretta organizzazione cellulare e la sintesi della matrice extracellulare, i primi esperimenti hanno avuto come obiettivo l’ottimizzazione della forma dell’impalcatura per limitare la fuoriuscita cellulare, garantendo comunque la possibilità di fusione e di differenziamento. Lo scaffold presentava una struttura simile ad una serpentina, contenente un unico binario in cui è stato estruso l’idrogel. In seguito alla stampa, i campioni sono stati sottoposti ad analisi morfologiche e molecolari; queste hanno dimostrato che il PCL non ostacola i processi cellulari e favorisce la proliferazione, tuttavia, dal punto di vista differenziativo solamente le cellule disposte lungo i bordi dello scaffold tendono ad allungarsi e a maturare, probabilmente perché percepiscono la rigidità del PCL e della plastica della piastra di coltura. A questo punto il passo successivo è stato quello di replicare, il più fedelmente possibile, le condizioni di crescita fisiologiche per ricreare la complessità strutturale e funzionale del muscolo scheletrico. Poiché dagli esperimenti precedenti si è concluso che la sola crescita in condizioni statiche non è sufficiente ad indurre il processo di differenziamento miogenico, si è deciso di affiancare alla coltura cellulare la stimolazione meccanica. A questo scopo è stata sviluppata una piattaforma ingegnerizzata, cioè un bioreattore. La camera di coltura è stata inizialmente stampata in 3D con nylon PA12, successivamente rivestito con un materiale impermeabile, il polidimetilsilossano, per garantire il mantenimento della sterilità. Inoltre, se fino a questo momento per gli esperimenti era stato utilizzato uno scaffold di PCL, nel caso in cui si voglia testare l’efficacia del bioreattore, il supporto tridimensionale deve essere adeguatamente flessibile e allungabile, perciò, lo scaffold di PCL è stato sostituito con uno in polidimetilsilossano (PDMS), materiale biocompatibile e contemporaneamente deformabile. La stampa ha previsto l’estrusione dell’idrogel all’interno dei binari degli scaffold tridimensionali contenuti in scatolette che consentono il successivo collegamento al bioreattore. Una volta ottenuti i campioni si è proceduto con lo stimolo meccanico, 15 minuti di stimolazione e 15 minuti di riposo, per un totale di 5 ore al giorno per 2 giorni. Al termine del tempo stabilito per la stimolazione, si sono effettuate analisi morfologiche e molecolari, le quali hanno mostrato una buona densità e vitalità cellulare, ma, non miglioramenti significativi in termini di differenziamento, rispetto ai campioni mantenuti in condizioni statiche. Dal punto di vista, invece, dell’espressione genica, alcuni dei geni tipici del differenziamento miogenico risultano up-regolati nei costrutti stimolati rispetto ai campioni in condizioni statiche. Questi risultati, a volte discordanti, potrebbero essere dovuti al fatto che le cellule C2C12 non trovano sul PDMS un supporto stabile da garantire un adeguato processo di differenziamento, inoltre, la stimolazione meccanica fornita dal bioreattore potrebbe non essere adeguata al tipo di campione. Il processo, quindi, deve essere ottimizzato.
Sviluppo e ottimizzazione di un approccio integrato "Stampa 3D e Bioreattore" nell'ingegneria tissutale del muscolo scheletrico
SEGADELLI, LETIZIA
2022/2023
Abstract
This thesis project explores the convergence of new 3D printing techniques (3D BioP) and bioreactors with the aim of recreating a model of skeletal muscle fiber using murine muscle cells, C2C12. The project was developed in the regenerative medicine laboratory (headed by Prof. Gabriele Ceccarelli, Dept. of 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. C2C12 murine muscle cells were encapsulated in fibrin hydrogel, a biomaterial promoting myogenic proliferation and differentiation. Once the suitable printing protocol for creating a 3D model of muscle fiber was identified, the bioink, i.e. the hydrogel, was extruded into three-dimensional, polycaprolactone (PCL) scaffolds. Since the 3D structure of the scaffold is essential for guiding proper cellular organization and extracellular matrix synthesis, initial experiments aimed to optimize the scaffold shape to limit cell leakage while ensuring fusion and differentiation possibilities. The scaffold had a serpentine-like structure containing a single track where the hydrogel was extruded. After printing, samples underwent morphological and molecular analysis demonstrating that PCL does not hinder cellular processes and promotes proliferation. However, from a differentiation perspective, only cells along the scaffold edges tended to elongate and mature, possibly perceiving the rigidity of PCL and the plastic of the culture plate. At this point, the next step was to replicate, as faithfully as possible, physiological growth conditions to recreate the structural and functional complexity of skeletal muscle. Since previous experiments indicated that static growth conditions alone are insufficient to induce the differentiation process, mechanical stimulation was added to cell culture through an engineered platform, a bioreactor. The culture chamber was initially 3D printed with nylon PA12, later coated with an impermeable material, polydimethylsiloxane, to ensure sterility maintenance. Furthermore, if testing the bioreactor requires a suitably flexible and stretchable three-dimensional support, the PCL scaffold was replaced with one made of polydimethylsiloxane (PDMS), a biocompatible and deformable material. The printing involves extruding the hydrogel into the tracks of three-dimensional scaffolds contained in boxes that allow subsequent connection to the bioreactor. Once the samples were obtained, mechanical stimulation was applied for 15 minutes followed by 15 minutes of rest, repeated for a total of 8 hours. At the end of the specified time, morphological and molecular analyses were conducted, showing good cellular density and viability. However, comparing the samples with the ones kept in static growth conditions, no significant improvements were identified, in terms of elongation and differentiation, while, regarding gene expression, the more myogenic genes had a significant induction with respect to static samples. This may be because C2C12 cells do not find a stable support for differentiation on PDMS, and the mechanical stimulation provided by the bioreactor may not be suitable for our sample type. Future studies will be aimed at investigating new growth and mechanical stimulation protocols, via bioreactor, to induce the myogenic differentiation process with the aim of recreating in vitro a skeletal muscle fiber as similar as possible to the physiological one, which can be used for both therapeutic and research purposes.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/20.500.14239/17267