Osso e cartilagine costituiscono i principali componenti del sistema scheletrico. Diverse patologie possono inficiare tali tessuti, causando danni rilevanti ed ampie ripercussioni sulla qualità della vita, provocando dolore e disabilità. Varie terapie sono state proposte al fine di promuoverne la rigenerazione, ma un ripristino completo è stato ottenuto in un ridotto numero di pazienti. L’ingegneria tissutale (IT) è emersa come un approccio promettente, sebbene la combinazione ideale di cellule, biomateriali e molecole bioattive ancora non esista. In questo contesto una migliore caratterizzazione dei biomateriali e delle cellule utilizzabili in IT, insieme ad una maggiore comprensione dei meccanismi molecolari implicati nel differenziamento cellulare e nella rigenerazione tissutale, è altamente desiderabile. La ricerca presentata in questa tesi si inserisce in questo ambito, ed è focalizzata su due punti principali: 1) la realizzazione di costrutti cellulari in vitro per l’IT ossea e cartilaginea, in grado di riprodurre le interazioni cellula-cellula e cellula-matrice extracellulare (ECM) tipiche del microambiente in vivo, e 2) la caratterizzazione di nuove molecole coinvolte nell’osteogenesi/condrogenesi di cellule staminali mesenchimali umane (hMSC) con implicazioni in IT. Per quanto riguarda il primo aspetto, il lavoro è stato sviluppato in quattro diverse direzioni: a) la produzione di microfibre composite di alginato e biomateriali derivati dalla ECM, come la gelatina o la matrice decellularizzata della vescica urinaria (UBM). Un approccio di microfluidica è stato sviluppato per ottenere un controllo rigoroso sulle caratteristiche strutturali e morfologiche delle microfibre, che potrebbero in seguito inficiare le loro prestazioni biologiche; b) l’uso delle microfibre composite per la coltura in vitro di condrociti de-differenziati, per permettere a tali cellule di riacquisire le loro proprietà condrogeniche senza l’uso di induttori. Abbiamo dimostrato come la presenza di UBM sia rilevante per il mantenimento del fenotipo re-differenziato, indicando come i segnali della ECM siano cruciali per la funzionalità cellulare e supportando l’uso di questo tipo di materiali nell’IT della cartilagine; c) la realizzazione di costrutti miniaturizzati in vitro in grado di mimare il microambiente osseo in vivo, attraverso la coltura di osteoblasti (hOB) e osteoclasti (hOC) umani nel bioreattore “Rotary Cell Culture System” senza l’utilizzo di biomateriali. La formazione di aggregati cellulari ben strutturati è stata ottenuta grazie ad un adeguato bilanciamento tra l’attività osteoblastica e osteoclastica, anche nel caso di OB da osso necrotico; d) la caratterizzazione di molecole collageniche scarsamente studiate, come il collageno di tipo 15 (ColXV), riconosciuto come fondamentale nell’osteogenesi delle hMSC e nel processo di mineralizzazione: l’espressione del ColXV è mantenuta ad alti livelli nelle fasi iniziali, mentre va progressivamente diminuendo, fino a scomparire, quando una fase avanzata di mineralizzazione è stata raggiunta. Relativamente alle indagini molecolari, sono state principalmente focalizzate su: a) la dimostrazione del ruolo di NFATc1 come regolatore trascrizionale del DNA mitocondriale in hMSC osteo-differenziate. È stato dimostrato il reclutamento in vivo di NFATc1 nella regione regolatoria D-loop del DNA mitocondriale in concomitanza con una diminuzione di espressione di geni cruciali del metabolismo durante la fase di mineralizzazione; b) l’identificazione di geni target del fattore anti-condrogenico microRNA-221. Attraverso il sequenziamento del trascrittoma e analisi bioinformatiche, sono stati identificati 110 geni. Nella maggior parte dei casi una loro implicazione nella condrogenesi è sconosciuta, e per questo motivo è stato ipotizzato lo sviluppo di un sistema di screening condrogenico nelle hMSCs, attualmente in corso.

Bone and cartilage constitute the main components of the skeletal system, sustaining body movements and protecting soft tissues. Several pathological conditions could affect these tissues, with relevant damages which could highly impact on life quality, causing pain and disabilities. Various therapeutic strategies have been proposed to enhance the regenerative ability of bone and cartilage, but fully restoration has been achieved only in a small subset of patients. Tissue engineering (TE) has emerged as a promising solution for unresolved clinical issues, although the ideal combination of cells, biomaterials and bioactive molecules does not still exist. In this perspective a better characterization of biomaterials and cells which could be used in TE, together with a deeper comprehension of the molecular mechanisms guiding cell differentiation and tissue repair, is highly desirable. The research presented in this thesis addresses this context, and is focused on two main points: 1) the realization of in vitro cell-based constructs for bone and cartilage TE, resembling the in vivo microenvironment with cell-cell and cell-extracellular matrix (ECM) interactions and 2) the characterization of new molecular factors involved in the osteogenesis/chondrogenesis of human mesenchymal stem cells (hMSCs) with important impact on TE approaches. Regarding the first aspect the experimental work has been developed in four different directions: a) the production of composite microfibers made of alginate and biomaterials derived from ECM, such as gelatin or decellularized urinary bladder matrix (UBM). A microfluidic approach was developed to obtain a strict control over the morphological and structural characteristics of the microfibers, which could subsequently affect biological performances of the devices; b) the use of composite microfibers for the in vitro culture of de-differentiated chondrocytes, to allow the re-acquisition by the cells of the chondrogenic properties, without the need to add chondrogenic inducers. For the first time we demonstrated how the presence of the UBM was relevant for the maintenance of re-differentiated phenotype, indicating how signaling from ECM is crucial for cell functionality and supporting the employment of this kind of materials in cartilage TE; c) the realization of a miniaturized in vitro construct able to mimic the in vivo bone microenvironment, through the co-culture of human osteoblasts (hOBs) and osteoclasts (hOCs) in the Rotary Cell Culture System (RCCS) bioreactor in a scaffold-free approach. The formation of well-organized cell aggregates as a result of an adequate balance between OB and OC activity was obtained, also when OBs from necrotic bone was employed; d) the characterization of poorly studied collagenic molecules, such as the collagen type 15 (ColXV), which was recognized as fundamental to ensure an adequate hMSCs osteogenic differentiation and mineralization process: ColXV expression is maintained at high level in the early phases of osteogenesis, while it progressively decreases, up to disappear, when a great amount of ECM mineralization is achieved. Concerning the molecular investigations, they were mainly focused on: a) the demonstration of the role of NFATc1 as transcriptional regulator of mitochondrial genome in osteo-differentiated hMSCs. For the first time, the in vivo recruitment of NFATc1 at the regulatory D-loop region of mitochondrial DNA correlated with a decrease of the expression of crucial metabolic genes has been demonstrated during the calcification phase; b) the identification of target genes of the anti-chondrogenic factor microRNA-221 (miR-221). Through RNA-sequencing technique and bioinformatic analysis, 110 genes were identified as possible targets. In most of the cases their implication in the chondrogenic process is still unknown and for this reason the development of a chondrogenic screening system in hMSCs has been hypothesized and is currently in progress.

Cell-based models for bone and cartilage tissue engineering: in vitro investigations from a biomaterialistic, cellular and molecular perspective.

ANGELOZZI, MARCO
2017-04-20T00:00:00+02:00

Abstract

Bone and cartilage constitute the main components of the skeletal system, sustaining body movements and protecting soft tissues. Several pathological conditions could affect these tissues, with relevant damages which could highly impact on life quality, causing pain and disabilities. Various therapeutic strategies have been proposed to enhance the regenerative ability of bone and cartilage, but fully restoration has been achieved only in a small subset of patients. Tissue engineering (TE) has emerged as a promising solution for unresolved clinical issues, although the ideal combination of cells, biomaterials and bioactive molecules does not still exist. In this perspective a better characterization of biomaterials and cells which could be used in TE, together with a deeper comprehension of the molecular mechanisms guiding cell differentiation and tissue repair, is highly desirable. The research presented in this thesis addresses this context, and is focused on two main points: 1) the realization of in vitro cell-based constructs for bone and cartilage TE, resembling the in vivo microenvironment with cell-cell and cell-extracellular matrix (ECM) interactions and 2) the characterization of new molecular factors involved in the osteogenesis/chondrogenesis of human mesenchymal stem cells (hMSCs) with important impact on TE approaches. Regarding the first aspect the experimental work has been developed in four different directions: a) the production of composite microfibers made of alginate and biomaterials derived from ECM, such as gelatin or decellularized urinary bladder matrix (UBM). A microfluidic approach was developed to obtain a strict control over the morphological and structural characteristics of the microfibers, which could subsequently affect biological performances of the devices; b) the use of composite microfibers for the in vitro culture of de-differentiated chondrocytes, to allow the re-acquisition by the cells of the chondrogenic properties, without the need to add chondrogenic inducers. For the first time we demonstrated how the presence of the UBM was relevant for the maintenance of re-differentiated phenotype, indicating how signaling from ECM is crucial for cell functionality and supporting the employment of this kind of materials in cartilage TE; c) the realization of a miniaturized in vitro construct able to mimic the in vivo bone microenvironment, through the co-culture of human osteoblasts (hOBs) and osteoclasts (hOCs) in the Rotary Cell Culture System (RCCS) bioreactor in a scaffold-free approach. The formation of well-organized cell aggregates as a result of an adequate balance between OB and OC activity was obtained, also when OBs from necrotic bone was employed; d) the characterization of poorly studied collagenic molecules, such as the collagen type 15 (ColXV), which was recognized as fundamental to ensure an adequate hMSCs osteogenic differentiation and mineralization process: ColXV expression is maintained at high level in the early phases of osteogenesis, while it progressively decreases, up to disappear, when a great amount of ECM mineralization is achieved. Concerning the molecular investigations, they were mainly focused on: a) the demonstration of the role of NFATc1 as transcriptional regulator of mitochondrial genome in osteo-differentiated hMSCs. For the first time, the in vivo recruitment of NFATc1 at the regulatory D-loop region of mitochondrial DNA correlated with a decrease of the expression of crucial metabolic genes has been demonstrated during the calcification phase; b) the identification of target genes of the anti-chondrogenic factor microRNA-221 (miR-221). Through RNA-sequencing technique and bioinformatic analysis, 110 genes were identified as possible targets. In most of the cases their implication in the chondrogenic process is still unknown and for this reason the development of a chondrogenic screening system in hMSCs has been hypothesized and is currently in progress.
BORGATTI, Monica
PIVA, Maria Roberta
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11392/2487900
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