Osteoarthritis and tendinopathy are common orthopaedic conditions with similar etiology, symptoms and clinical issues. They both are characterized by chronic inflammation and tissue degeneration, and the current conservative treatments do not effectively contrast the pathologic progression. The clinical need for innovative treatment has led to the development of regenerative medicine strategies, aimed to replace or regenerate cells and tissues. In this setting, mesenchymal stem cells (MSCs) represents the most promising tool, thanks to their ability to proliferate and differentiate towards specific cell lineages, as well as their paracrine action on the immune system and on the tissue resident cells. Although the most used sources of MSCs are bone marrow and adipose tissue, they are present in almost all body compartments, with a perivascular localization. Indeed, MSCs have been recently described as pericytes, deputed to respond to damage and restore tissue homeostasis. The interest in new sources of MSCs resides in the discovery of their limited in vivo differentiation ability, especially towards specific lineages such as tenocytes and chondrocytes. To face this, subpopulations of tendon and cartilage derived cells were found to possess clonogenic and differentiation abilities, and they were proposed as possible candidates for specific tissue regeneration. Contrary to their well-known ability to participate directly to tissue regeneration, the paracrine activity of these specific progenitors is still unclear, as well as their real nature as MSCs. Then, the first goal of this research was to identify the MSCs minimal criteria in these subpopulations, in terms of differentiation ability, immunophenotype and proliferation. We found that tendon stem/progenitor cells (TSPCs) possess these minimal criteria, and share the low immunogenicity and the immumodulatory ability with adipose derived stem cells (ASCs). On the contrary, while cartilage cells are the most effective in differentiate towards hyaline cartilage, they have low paracrine activity and thus a treatment based on the molecules released by ASCs could still result beneficial in case of joint inflammation or degeneration. The proof of concept for the application of ASCs conditioned medium, that is the complex of the molecules secreted during culture, was provided in an in vitro model of cartilage cell inflammation, resulting in a reduction of the catabolic response. After the characterization, we focused on the strategies to enhance the abilities of the progenitors with a therapeutic perspective. At first, we identified the role of different growth factors in the increase of TSPCs proportion within the tendon cell population during culture and in the induction of tenogenic differentiation. Then, we observed the in vitro effects of biophysical stimulations, such as extracorporeal shock waves (ESWs) and pulsed electromagnetic fields (PEMFs) on tendon cells, identifying a biological response with regenerative purport in terms of specific marker expression and paracrine activity. On these bases, we investigated the possibility to use PEMFs to enhance the potential of resident stem/progenitors cells to contrast the pathology progression, in a specially developed in vivo model of tendinopathy. The preliminary results from this study provided promising data, supportive of further investigations. Finally, to fill the gap between the experimental setting and the clinical practice, uncultured ASCs, in the form of stromal vascular fraction, were tested in the treatment of Achilles tendinopathy in a clinical trial, providing results supportive of their efficacy. In conclusion, the present work allowed increasing the knowledge in the field of MSCs applied to the most common orthopaedic conditions, with a panoramic view ranging from the in vivo to the clinical aspects. This research will contribute in the development of new therapeutic opportunities for these high impact pathologies.
L’artrosi e le tendinopatie sono patologie che presentano simili sintomi, quesiti clinici ed eziologia, entrambe caratterizzate da infiammazione e degenerazione tissutale. I trattamenti conservativi attualmente disponibili hanno dimostrato scarsa efficacia, portando interesse nello sviluppo di strategie di medicina rigenerativa, mirate a sostituire o rigenerare le cellule e i tessuti danneggiati. Le cellule staminali mesenchimali (MSC) rappresentano lo strumento più promettente per questi approcci, grazie alla loro capacità di proliferare e differenziarsi verso specifiche linee cellulari, oltre alle loro proprietà anti-infiammatorie e trofiche. Le principali fonti di MSC sono il midollo osseo e il tessuto adiposo, sebbene esse siano presenti in quasi tutti i distretti corporei, con localizzazione perivascolare. Infatti sono state recentemente descritte come periciti, cellule deputate a rispondere al danno tissutale ripristinando l'omeostasi. L'interesse verso fonti alternative di MSC è nato a causa della loro limitata capacità di differenziare in vivo, soprattutto verso la linea tenogenica e condrogenica. In questo contesto sono state identificate alcune sottopopolazioni di cellule tendinee e cartilaginee con abilità clonogeniche e differenziative, in grado di partecipare direttamente alla rigenerazione tissutale. Tuttavia, l'attività paracrina di questi progenitori, così come la loro reale natura di mesenchimali, è ancora dibattuta. Il primo obiettivo di questa ricerca è stato quindi quello di identificare i criteri minimi tipici delle MSC in queste sottopopolazioni in termini di capacità differenziative, proliferative e di immunofenotipo. Le cellule staminali/progenitrici del tendine (TSPC) sono risultate in possesso di questi criteri, mostrando anche una scarsa immunogenicità e una capacità immumodulatoria tipica delle ASC. Al contrario, le cellule cartilaginee possiedono una maggior capacità di differenziazione verso la cartilagine ialina, ma hanno bassa attività paracrina rispetto alle ASC. Perciò, aggiungendo mezzo condizionato da ASC in un modello in vitro di infiammazione su condrociti, è stato possibile ridurre la concentrazione dei marcatori del catabolismo cartilagineo. Una volta caratterizzate, ci siamo concentrati sulle strategie per aumentare le potenzialità terapeutiche di queste cellule. Inizialmente abbiamo identificato il ruolo di diversi fattori di crescita nell’aumento della proporzione di TSPC all'interno della popolazione di cellule tendinee, ma anche nella induzione della differenziamento tenogenico. Successivamente, abbiamo osservato gli effetti in vitro delle stimolazioni biofisiche, come le onde d'urto extracorporee (ESWs) e i campi elettromagnetici pulsati (PEMF) sulle cellule tendinee, individuando l’induzione di una risposta biologica in senso rigenerativo. Su queste basi, abbiamo studiato la possibilità di utilizzare i PEMF per aumentare il potenziale delle TSPC residenti nel contrastare la progressione patologica in un modello in vivo di tendinopatia appositamente sviluppato. I risultati preliminari di questo studio supportano l’efficacia di questo approccio. Per colmare il divario tra le condizioni sperimentali e la pratica clinica, le ASC, sotto forma di frazione stromale vascolare ovvero prima del passaggio in coltura, sono state applicate nel trattamento della tendinopatia d'Achille in uno studio clinico, ottenendo risultati a sostegno della loro efficacia. In conclusione, il presente lavoro ha contribuito ad aumentare le conoscenze nel campo delle MSC applicate alle patologie ortopediche più comuni, tenendo in considerazione tutte le problematiche presenti dagli scenari in vitro a quelli clinici. Partendo da questa ricerca sarà possibile in futuro sviluppare nuove strategie terapeutiche per queste patologie.
(2017). Mesenchymal Stem Cells for the treatment of osteoarthritis and tendinopathy. (Tesi di dottorato, Università degli Studi di Milano-Bicocca, 2017).
Mesenchymal Stem Cells for the treatment of osteoarthritis and tendinopathy
VIGANO', MARCO
2017
Abstract
Osteoarthritis and tendinopathy are common orthopaedic conditions with similar etiology, symptoms and clinical issues. They both are characterized by chronic inflammation and tissue degeneration, and the current conservative treatments do not effectively contrast the pathologic progression. The clinical need for innovative treatment has led to the development of regenerative medicine strategies, aimed to replace or regenerate cells and tissues. In this setting, mesenchymal stem cells (MSCs) represents the most promising tool, thanks to their ability to proliferate and differentiate towards specific cell lineages, as well as their paracrine action on the immune system and on the tissue resident cells. Although the most used sources of MSCs are bone marrow and adipose tissue, they are present in almost all body compartments, with a perivascular localization. Indeed, MSCs have been recently described as pericytes, deputed to respond to damage and restore tissue homeostasis. The interest in new sources of MSCs resides in the discovery of their limited in vivo differentiation ability, especially towards specific lineages such as tenocytes and chondrocytes. To face this, subpopulations of tendon and cartilage derived cells were found to possess clonogenic and differentiation abilities, and they were proposed as possible candidates for specific tissue regeneration. Contrary to their well-known ability to participate directly to tissue regeneration, the paracrine activity of these specific progenitors is still unclear, as well as their real nature as MSCs. Then, the first goal of this research was to identify the MSCs minimal criteria in these subpopulations, in terms of differentiation ability, immunophenotype and proliferation. We found that tendon stem/progenitor cells (TSPCs) possess these minimal criteria, and share the low immunogenicity and the immumodulatory ability with adipose derived stem cells (ASCs). On the contrary, while cartilage cells are the most effective in differentiate towards hyaline cartilage, they have low paracrine activity and thus a treatment based on the molecules released by ASCs could still result beneficial in case of joint inflammation or degeneration. The proof of concept for the application of ASCs conditioned medium, that is the complex of the molecules secreted during culture, was provided in an in vitro model of cartilage cell inflammation, resulting in a reduction of the catabolic response. After the characterization, we focused on the strategies to enhance the abilities of the progenitors with a therapeutic perspective. At first, we identified the role of different growth factors in the increase of TSPCs proportion within the tendon cell population during culture and in the induction of tenogenic differentiation. Then, we observed the in vitro effects of biophysical stimulations, such as extracorporeal shock waves (ESWs) and pulsed electromagnetic fields (PEMFs) on tendon cells, identifying a biological response with regenerative purport in terms of specific marker expression and paracrine activity. On these bases, we investigated the possibility to use PEMFs to enhance the potential of resident stem/progenitors cells to contrast the pathology progression, in a specially developed in vivo model of tendinopathy. The preliminary results from this study provided promising data, supportive of further investigations. Finally, to fill the gap between the experimental setting and the clinical practice, uncultured ASCs, in the form of stromal vascular fraction, were tested in the treatment of Achilles tendinopathy in a clinical trial, providing results supportive of their efficacy. In conclusion, the present work allowed increasing the knowledge in the field of MSCs applied to the most common orthopaedic conditions, with a panoramic view ranging from the in vivo to the clinical aspects. This research will contribute in the development of new therapeutic opportunities for these high impact pathologies.File | Dimensione | Formato | |
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Descrizione: tesi di dottorato
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