Cartilage disorders due to traumas and aging, particularly arthritic conditions, represent a major cause of morbidity globally and an enormous cost for health and social care systems. Unfortunately, it is predicted that the increase in life expectancy will further exacerbate this burden in future years. As available therapies either lack of efficacy or exhibit severe side effects, there is an urgent need for novel approaches that can effectively treat cartilage damage when the progressive lesioning process is initiated. Remarkably, thanks to the recent findings in tissue engineering, cellular and molecular biology and biomaterials, it appears that progress in this field might have finally turned a corner. Specifically, experimental therapies using human mesenchymal stromal cells (hMSCs) are receiving an increasing amount of interest, due to encouraging preclinical and clinical results, and the extraordinary potential offered for cartilage reconstruction. In this regards, one of the main challenges that research has to deal with is the control of cell differentiation. Indeed, while hMSCs have proven able to reform cartilage, the conditions needed to induce a stable mature chondrocytic phenotype in vivo, as well as a sufficient production of cartilage matrix, are not defined. The research described in this thesis was mainly aimed at the design of novel strategies based on the use of hMSCs depleted of negative regulators of differentiation to enhance the repair of cartilage defects. In doing so, the employment of molecular and functional analysis also allowed us to unveil regulatory interplays involved in chondrogenesis, the knowledge of which is the prerequisite for the development of targeted and more efficient tools to induce cartilage reconstruction. Our work was mainly focused on two cell regulators that have been recently proposed as anti-chondrogenic factors, miR-221 and Slug. We first demonstrated that the depletion of miR-221 and Slug by gene silencing is sufficient to address hMSCs from different sources towards chondrogenesis in vitro, without requiring supplementation with conventional chondrogenic inducers. To improve the validity and relevance of our experimental evidence, we then established and employed specific 3D-culture systems, i.e. combination of hMSCs with biocompatible scaffolds (scaffold-based tissue engineering), pellet culture and co-culture aggregate techniques (scaffold-less aggregate tissue engineering), and in vitro osteochondral constructs. Notably, these culture models will serve as novel advanced platforms to test the differentiation/therapeutic potential of manipulated hMSCs, being one step closer to the in vivo osteochondral microenvironment. With the aim to confer a translational value to our work, we eventually assessed and proved that the silencing of miR-221 is indeed an efficient strategy to induce hMSCs to rapidly differentiate into chondrocytes in vivo and produce cartilage matrix in the context of an osteochondral defect. Taken as a whole, our data were useful to define yet uncharacterized regulatory interplays in chondrogenesis, and to identify novel molecular targets to be properly manipulated in the effort to control and stabilize the chondrogenic phenotype of differentiated hMSCs. We speculate that approaches based on the modulation of endogenous molecular cues, such as ours, will soon allow to obtain hMSCs populations displaying a higher differentiation and therapeutic potential, laying the basis for important applications in basic research and regenerative medicine.

Le lesioni della cartilagine dovute a traumi o patologie legate all’invecchiamento, in particolare artrosiche, rappresentano una delle principali cause di morbilità a livello mondiale, nonchè un gravoso onere economico per i sistemi sanitari nazionali. È ragionevole ipotizzare che l’aumento dell’aspettativa di vita determinerà un ulteriore incremento dell’incidenza di tali problematiche nel corso dei prossimi anni. Sfortunatamente, le poche terapie attualmente disponibili sono caratterizzate da un’efficacia limitata e/o un elevato tasso di fallimento. Da qui deriva la necessità e l’urgenza di sviluppare approcci innovativi in grado di arrestare e possibilmente revertire la degenerazione del tessuto cartilagineo, un processo che, una volta innescato, procede inesorabilmente verso uno stato infiammatorio cronico. Grazie alle recenti scoperte nel campo dell’ingegneria tissutale, della biologia cellulare e molecolare e dei biomateriali, la ricerca in questo campo sembra essere arrivata ad un punto di svolta. In particolare, le terapie basate sulle cellule stromali mesenchimali (hMSCs) suscitano attualmente forte interesse, in seguito ai notevoli risultati ottenuti dagli studi preclinici e dai trials clinici, e per l’innegabile potenziale terapeutico per la rigenerazione dei tessuti. Prima che questo obiettivo possa essere raggiunto, l’ostacolo principale che rimane da superare è probabilmente il controllo del differenziamento cellulare. Mentre infatti le hMSCs sono in grado di differenziare in senso condrogenico e produrre matrice cartilaginea, le condizioni ottimali in grado di determinare un fenotipo condrocitario stabile in vivo, come pure una produzione sufficiente di matrice, non sono note. Il progetto di ricerca in oggetto è stato principalmente rivolto all’ideazione di strategie innovative basate sull’utilizzo di hMSCs ingegnerizzate, in cui viene bloccata l’espressione di specifici regolatori anti-condrogenici, per stimolare il riparo della cartilagine. In tale contesto, l’utilizzo di tecnologie molecolari e analisi funzionali ha inoltre permesso di delucidare network cellulari che intervengono nella regolazione della condrogenesi, la conoscenza dei quali è il pre-requisito per lo sviluppo di approcci terapeutici più efficaci e mirati. Il lavoro si è principalmente focalizzato su due regolatori cellulari che solo recentemente sono stati proposti come fattori anti-condrogenici, il microRNA miR-221 e il fattore trascrizionale Slug. In una prima fase, gli esperimenti hanno dimostrato come l’inibizione di miR-221 o Slug mediante silenziamento genico sia sufficiente per differenziare in vitro hMSCs derivanti da diverse fonti in senso condrocitario, senza richiedere l’aggiunta di induttori convenzionali. Al fine di rafforzare la validità delle evidenze ottenute, sono stati quindi messi a punto ed impiegati specifici sistemi di coltura in 3D, come la combinazione delle hMSCs con scaffold biocompatibili (“scaffold-based tissue engineering”), sistemi di coltura in pellet o mediante aggregati cellulari (“scaffold-less aggregate tissue engineering”), e modelli osteocondrali in vitro. Va sottolineato come tali sistemi di coltura potranno essere sfruttati come piattaforme avanzate per testare il potentiale differenziativo e terapeutico di hMSCs ingegnerizzate, essendo più rappresentativi del microambiente osteocondrale in vivo. Al fine di conferire un valore traslazionale al progetto di tesi, è stato infine valutato e dimostrato come il silenziamento di miR221 possa effettivamente rappresentare una strategia perseguibile per differenziare le hMSCs in condrociti in vivo, e per incrementare la produzione di matrice cartilaginea nel contesto di un difetto osteocondrale. In conclusione, il lavoro di tesi ha permesso non solo di definire aspetti della regolazione della condrogenesi non ancora caratterizzati, ma anche di identificare nuovi target molecolari che possono essere opportunamente manipolati al fine di stabilizzare il fenotipo condrocitario delle hMSCs differenziate. Questo ci porta ad ipotizzare che approcci basati sulla manipolazione di fattori endogeni chiave, come quelli qui proposti, potranno essere impiegati per ottenere hMSCs dotate di un elevato potenziale differenziativo e terapeutico, gettando le basi per importanti applicazioni nel campo della medicina rigenerativa.

Validation of the anti-chondrogenic properties of the microRNA-221 and Slug transcription factor by different in vitro and in vivo models: new perspectives for cartilage regeneration

LOLLI, Andrea
2016

Abstract

Cartilage disorders due to traumas and aging, particularly arthritic conditions, represent a major cause of morbidity globally and an enormous cost for health and social care systems. Unfortunately, it is predicted that the increase in life expectancy will further exacerbate this burden in future years. As available therapies either lack of efficacy or exhibit severe side effects, there is an urgent need for novel approaches that can effectively treat cartilage damage when the progressive lesioning process is initiated. Remarkably, thanks to the recent findings in tissue engineering, cellular and molecular biology and biomaterials, it appears that progress in this field might have finally turned a corner. Specifically, experimental therapies using human mesenchymal stromal cells (hMSCs) are receiving an increasing amount of interest, due to encouraging preclinical and clinical results, and the extraordinary potential offered for cartilage reconstruction. In this regards, one of the main challenges that research has to deal with is the control of cell differentiation. Indeed, while hMSCs have proven able to reform cartilage, the conditions needed to induce a stable mature chondrocytic phenotype in vivo, as well as a sufficient production of cartilage matrix, are not defined. The research described in this thesis was mainly aimed at the design of novel strategies based on the use of hMSCs depleted of negative regulators of differentiation to enhance the repair of cartilage defects. In doing so, the employment of molecular and functional analysis also allowed us to unveil regulatory interplays involved in chondrogenesis, the knowledge of which is the prerequisite for the development of targeted and more efficient tools to induce cartilage reconstruction. Our work was mainly focused on two cell regulators that have been recently proposed as anti-chondrogenic factors, miR-221 and Slug. We first demonstrated that the depletion of miR-221 and Slug by gene silencing is sufficient to address hMSCs from different sources towards chondrogenesis in vitro, without requiring supplementation with conventional chondrogenic inducers. To improve the validity and relevance of our experimental evidence, we then established and employed specific 3D-culture systems, i.e. combination of hMSCs with biocompatible scaffolds (scaffold-based tissue engineering), pellet culture and co-culture aggregate techniques (scaffold-less aggregate tissue engineering), and in vitro osteochondral constructs. Notably, these culture models will serve as novel advanced platforms to test the differentiation/therapeutic potential of manipulated hMSCs, being one step closer to the in vivo osteochondral microenvironment. With the aim to confer a translational value to our work, we eventually assessed and proved that the silencing of miR-221 is indeed an efficient strategy to induce hMSCs to rapidly differentiate into chondrocytes in vivo and produce cartilage matrix in the context of an osteochondral defect. Taken as a whole, our data were useful to define yet uncharacterized regulatory interplays in chondrogenesis, and to identify novel molecular targets to be properly manipulated in the effort to control and stabilize the chondrogenic phenotype of differentiated hMSCs. We speculate that approaches based on the modulation of endogenous molecular cues, such as ours, will soon allow to obtain hMSCs populations displaying a higher differentiation and therapeutic potential, laying the basis for important applications in basic research and regenerative medicine.
PIVA, Maria Roberta
CUNEO, Antonio
File in questo prodotto:
File Dimensione Formato  
Tesi.pdf

accesso aperto

Tipologia: Tesi di dottorato
Licenza: PUBBLICO - Pubblico senza Copyright
Dimensione 13.05 MB
Formato Adobe PDF
13.05 MB Adobe PDF Visualizza/Apri

I documenti in SFERA sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.

Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11392/2403461
 Attenzione

Attenzione! I dati visualizzati non sono stati sottoposti a validazione da parte dell'ateneo

Citazioni
  • ???jsp.display-item.citation.pmc??? ND
  • Scopus ND
  • ???jsp.display-item.citation.isi??? ND
social impact