Despite the increasing incidence of knee osteoarthritis (OA), a world-leading cause of disability, its early diagnosis is still unattainable. The current diagnostic process is mainly based on the patients' clinical examination and the joint imaging. However, prescription for examination occur when the OA is already in an advanced stage. In addition, with this approach the biological processes activated during the OA, such as inflammation, are not considered. For these reasons the research is focusing on the finding of biological markers that can reflect the early biological alterations occurring in the joint. In this view, extracellular vesicles (EVs) isolated from biofluids with liquid biopsies are gaining importance as their content reflect the metabolic state of the origin cells. Contrarily to the classical view, it has been demonstrated that EVs include many subpopulations with different physicochemical features and biological roles. Thus, the first aim of this PhD project was to find the most effective technique to isolate and separate different size EV subpopulations from the synovial fluid (SF). To this end, I compared differential centrifugation, size exclusion chromatography, high performance liquid chromatography (HPLC) and asymmetrical flow field-flow fractionation (AF4). The AF4 resulted the most promising one, so I developed a new EV separation protocol at the Italian Institute of Technology in Genoa. Firstly, the flow rates in the AF4 were optimized until being able to isolate particles with a radius ranging from 20 up to more than 700 nm, that were gathered in 4 different subpopulations. I also obtained the EV profile and the relative percentage of each subpopulation. Then I characterized the EVs belonging to each subset by quantifying the Z potential, the protein and nucleic acid concentrations, by performing electron microscopy analysis to confirm the EV morphology and by evaluating the presence of EV-specific markers and the protein content, also with immune EM. The last part of the project was performed at the University of Gothenburg, Sweden. In addition to the lack of early biomarkers, no effective therapies able to revert the degeneration processes in the arthritic tissues are available, and the current approaches mainly aim at managing the pain. Different biological approaches have been proposed to fill this gap, but their clinical translation is not straightforward. In this scenario, the development of drug screening platforms can accelerate this translation, and microfluidics represents a promising approach. Hence, the second aim of my PhD project was focused on the development of a patient-specific microfluidic model to be used as drug screening platform for the evaluation of OA innovative treatments. The system consisted in a multi-channel microfluidic device that allowed the compartmentalized co-culture of primary and patient-matched synovial fibroblasts and chondrocytes in a 3D relevant hydrogel with synovial fluid interposed. The device was designed to allow the addition of biological treatments, mimicking an intra-articular injection, and the evaluation of their biological effects. To recreate a relevant cartilaginous compartment, I optimized commercially available hydrogels based on hyaluronic acid and/or type I collagen that were crosslinked enzymatically or via UV light. The chondrocytes cultured in these hydrogels showed higher expression of chondrocyte-specific markers. Then, I optimized the OA microenvironment within the model, evaluating the beneficial effect of the SF on the articular cells, that behaved differently when cultured with healthy or arthritic SF. Finally, the anti-inflammatory capabilities of adipose and bone-marrow mesenchymal stromal cells (MSCs) were assessed. The model effectively supported the injection of MSCs and the evaluation of their anti-inflammatory effects on the arthritic articular cells.

Nonostante l'incidenza dell'osteoartrosi (OA) del ginocchio, una delle maggiori cause di disabilità a livello mondiale, sia in crescita, la sua diagnosi precoce è ancora impossibile. L'attuale processo diagnostico è basato sull'esame clinico del paziente e sull'imaging dell'articolazione ma questi esami sono di norma prescritti quando l'OA è in uno stadio avanzato. In aggiunta, questo approccio non riesce a rilevare l'attivazione di processi biologici nell'instaurarsi dell'OA, come l'infiammazione. Per questi motivi la ricerca si è recentemente focalizzata sullo studio di biomarcatori che rilevino le alterazioni biologiche precoci nei tessuti articolari durante lo sviluppo dell'OA. In questa prospettiva, le vescicole extracellulari (EVs) isolate dai liquidi biologici tramite biopsie stanno acquisendo maggiore importanza in quanto esse rispecchiano lo stato metabolico della cellula di origine. Recentemente è stato dimostrato che le EVs consistono in numerose sottopopolazioni con caratteristiche fisico-chimiche ed effetti biologici diversi. Quindi, il primo obiettivo di questo progetto di dottorato è stato la ricerca della tecnica migliore per isolare e separare sottopopolazioni di EVs dal liquido sinoviale (LS) in base alla loro dimensione. Ho confrontato centrifugazione differenziale, cromatografia ad esclusione dimensionale, cromatografia liquida ad alta prestazione ed il frazionamento campo-flusso a flusso asimmetrico (AF4). Quest'ultima tecnica si è rivelata la più promettente, quindi ho sviluppato un nuovo protocollo di separazione delle EVs all'Istituto Italiano di Tecnologia di Genova. Prima di tutto ho ottimizzato i flussi nello strumento fino ad essere in grado di isolare particelle con un raggio compreso fra i 20 e i 700 nm, suddivise in 4 sottopopolazioni. Ho inoltre ottenuto il profilo dimensionale delle EVs e l'abbondanza relativa delle 4 sottopopolazioni. Successivamente ho caratterizzato le vescicole quantificando il potenziale Z e la concentrazione proteica e di acidi nucleici, effettuando un'analisi di microscopia elettronica per confermare la morfologia delle EVs e valutando la presenza di marcatori specifici ed il loro contenuto proteico, anche grazie all'immunomicroscopia elettronica. L'ultima parte del progetto è stata svolta presso l'Università di Göteborg, in Svezia. Alla mancanza di marcatori precoci dell'OA, si aggiunge l'indisponibilità di approcci terapeutici efficaci che invertano i processi degenerativi nei tessuti articolari artrosici. Sono stati proposti diversi trattamenti biologici per i quali però la traslazione in clinica non è semplice. In questo scenario, le piattaforme di screening per nuovi farmaci possono accelerare lo sviluppo di nuove terapie e la microfluidica è uno degli approcci utilizzati per creare queste piattaforme. Il secondo obiettivo del progetto di dottorato è stato quindi focalizzato sullo sviluppo di un modello microfluidico paziente specifico usato come piattaforma di screening per trattamenti innovativi per l'OA. Il device consiste in un chip microfluidico multi-compartimento che permette la coltura separata di fibroblasti sinoviali e condrociti primari in un ambiente 3D rilevante ed in presenza di LS, tutti isolati dallo stesso paziente. Il modello è stato disegnato per valutare l'effetto di trattamenti biologici, aggiunti al sistema simulando un'iniezione intra-articolare. Per ricreare un ambiente cartilagineo, ho ottimizzato idrogeli commerciali a base di acido ialuronico e/o collagene 1 che sono stati crosslinkati enzimaticamente o con luce UV; i condrociti coltivati in queste matrici hanno mostrato un'espressione più elevata di marcatori cartilaginei rispetto a matrici standard (es. fibrina). Successivamente ho ottimizzato l'ambiente del modello, valutando l'effetto di LS sano o artrosico sulle cellule. Infine, ho quantificato l'effetto antiinfiammatorio di cellule mesenchimali stromali da tessuto adiposo o midollo osseo.

(2022). Osteoarthritis theranostics: extracellular vesicles and drug microfluidic screening platforms as innovative tools. (Tesi di dottorato, Università degli Studi di Milano-Bicocca, 2022).

Osteoarthritis theranostics: extracellular vesicles and drug microfluidic screening platforms as innovative tools

D'ARRIGO, DANIELE
2022

Abstract

Despite the increasing incidence of knee osteoarthritis (OA), a world-leading cause of disability, its early diagnosis is still unattainable. The current diagnostic process is mainly based on the patients' clinical examination and the joint imaging. However, prescription for examination occur when the OA is already in an advanced stage. In addition, with this approach the biological processes activated during the OA, such as inflammation, are not considered. For these reasons the research is focusing on the finding of biological markers that can reflect the early biological alterations occurring in the joint. In this view, extracellular vesicles (EVs) isolated from biofluids with liquid biopsies are gaining importance as their content reflect the metabolic state of the origin cells. Contrarily to the classical view, it has been demonstrated that EVs include many subpopulations with different physicochemical features and biological roles. Thus, the first aim of this PhD project was to find the most effective technique to isolate and separate different size EV subpopulations from the synovial fluid (SF). To this end, I compared differential centrifugation, size exclusion chromatography, high performance liquid chromatography (HPLC) and asymmetrical flow field-flow fractionation (AF4). The AF4 resulted the most promising one, so I developed a new EV separation protocol at the Italian Institute of Technology in Genoa. Firstly, the flow rates in the AF4 were optimized until being able to isolate particles with a radius ranging from 20 up to more than 700 nm, that were gathered in 4 different subpopulations. I also obtained the EV profile and the relative percentage of each subpopulation. Then I characterized the EVs belonging to each subset by quantifying the Z potential, the protein and nucleic acid concentrations, by performing electron microscopy analysis to confirm the EV morphology and by evaluating the presence of EV-specific markers and the protein content, also with immune EM. The last part of the project was performed at the University of Gothenburg, Sweden. In addition to the lack of early biomarkers, no effective therapies able to revert the degeneration processes in the arthritic tissues are available, and the current approaches mainly aim at managing the pain. Different biological approaches have been proposed to fill this gap, but their clinical translation is not straightforward. In this scenario, the development of drug screening platforms can accelerate this translation, and microfluidics represents a promising approach. Hence, the second aim of my PhD project was focused on the development of a patient-specific microfluidic model to be used as drug screening platform for the evaluation of OA innovative treatments. The system consisted in a multi-channel microfluidic device that allowed the compartmentalized co-culture of primary and patient-matched synovial fibroblasts and chondrocytes in a 3D relevant hydrogel with synovial fluid interposed. The device was designed to allow the addition of biological treatments, mimicking an intra-articular injection, and the evaluation of their biological effects. To recreate a relevant cartilaginous compartment, I optimized commercially available hydrogels based on hyaluronic acid and/or type I collagen that were crosslinked enzymatically or via UV light. The chondrocytes cultured in these hydrogels showed higher expression of chondrocyte-specific markers. Then, I optimized the OA microenvironment within the model, evaluating the beneficial effect of the SF on the articular cells, that behaved differently when cultured with healthy or arthritic SF. Finally, the anti-inflammatory capabilities of adipose and bone-marrow mesenchymal stromal cells (MSCs) were assessed. The model effectively supported the injection of MSCs and the evaluation of their anti-inflammatory effects on the arthritic articular cells.
VANONI, MARCO ERCOLE
MORETTI, MATTEO
Osteoartrosi; EVs; Sottopopolazioni EVs; Microfluidica; Articolazione onchip
Osteoarthritis; EVs; EV subpopulations; Microfluidics; Articolazione onchip
BIO/10 - BIOCHIMICA
English
11-mag-2022
TECNOLOGIE CONVERGENTI PER I SISTEMI BIOMOLECOLARI (TeCSBi)
34
2020/2021
open
(2022). Osteoarthritis theranostics: extracellular vesicles and drug microfluidic screening platforms as innovative tools. (Tesi di dottorato, Università degli Studi di Milano-Bicocca, 2022).
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/10281/375387
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