The common treatments of cartilage diseases include both pharmacological and surgical therapies. Thanks to the ability of Mesenchymal Stem Cells (MSCs) to directly participate to tissue regeneration as well as to restore tissue homeostasis throughout their paracrine activity, the interest on MSCs applications for the treatment of a wide range of musculoskeletal pathologies is progressively increasing. However, the precise and effective local delivery of cells at the injury site still represent a crucial point of this kind of therapy. Hence, the present study, in line with the cell therapy field, has aimed to optimize the use of lyophilized silk/fibroin alginate microcarriers (L-FAMs) for the local articular delivery of adipose-derived mesenchymal stem cell (ASCs), with the final goal to make the groundwork for the development of a one-step autologous therapy for the treatment of patients affected by osteoarthritis. Basing on a protocol for cell adhesion onto L-FAMs previously developed in the frame of the StemDelivery project lead by the Cell Delivery System Lab of the University of Pavia in collaboration with the Galeazzi Orthopaedic Institute, Milan, the influence of different combinations of parameters (seeding volume, seeding time, presence or absence of a pause during the agitation time and seeding speed) on cell adhesion rate has been evaluated in order to obtain a rapid and effect process. The experimental conditions have been identified with the use of the Design of Experiment (DoE), a statistical approach which permits to define the minimal experimental conditions to test in order to have information with statistical significant meaning. The final aim of study was to identify the best cell seeding condition to obtain the higher cell adhesion rate on L-FAMs in the shortest time, but at the same time allowing for the maintenance of the physiological MSCs morphology and a good rate of homogeneity in term of distribution onto microcarriers. Cell seeding was performed in dynamic conditions with the use of a bioreactor, testing both the continuous and the intermittent rotatory stirring. A set of the 13 experimental conditions were tested with ASCs isolated from 3 different donors. In all the cases, a maximum of two hours was allowed to obtain cell adhesion, in order to develop a final protocol which could be suitable for a future clinical use of one-step MSCs therapy where the surgical time is limited. Immediately after the stirring time, cell adhesion rate has been quantitatively evaluated by Alamar Blue and CyQuant assay, whereas the qualitative analysis of cell viability has been performed by Live&Dead. After collecting the whole results, the statistical analysis allowed to interpret the different combinations of parameters initially suggested by the DoE approach, providing the identification of the best experimental condition. The results suggested that the combination of an intermittent stirring seeding (with a pause of 10 minutes), a rotation speed of 12 rpm, 400 µl of cell suspension and is able to optimize cell adhesion on these microcarriers in 85 minutes. This final optimal condition was then tested on 3 further ASCs populations deriving from different donors, confirming the results and thus and the suitability of L-FAMs as a delivery system for one-step local procedures. Even if in these experiments the ASCs were previously expanded in vitro, which guarantee for the method reproducibility, this research represents the fundament for future applications of minimally manipulated cells, such as the adipose tissue stromal vascular fraction (SVF). This study this represent the starting point of a following more extensive study that will aim to introduce this cell delivery system in the clinical practice for the one-step regeneration of articular cartilage lesions.
Il trattamento chirurgico rappresenta l’unica soluzione definitiva per il trattamento di patologie a carico della cartilagine articolare. La necessità di sviluppare terapie alternative, che agiscano sulle cause della patologia e che non siano invasive, ha creato un forte interesse da parte della ricerca scientifica verso l’utilizzo di cellule staminali mesenchimali (MSC). Grazie al loro potenziale multi-differenziativo e alla loro capacità di rilasciare molecole con proprietà anti-infiammatorie ed immunomodulatorie, le MSC svolgono un importante ruolo nei processi rigenerativi, agendo in talune situazioni come farmaci. Per svolgere al meglio la propria funzione è importante che le MSC riescano a raggiungere efficacemente il sito di lesione. Questo progetto di tesi ha avuto come obiettivo quello di ottimizzare le prestazioni di microcarrier liofilizzati costituiti da alginato di sodio e rivestiti di fibroina della seta (L-FAM) nel veicolare MSC derivate da tessuto adiposo (ASC) in sede articolare, al fine di ottimizzare lo sviluppo di una terapia autologa one-step per il trattamento della patologia artrosica. Partendo dal protocollo di adesione cellulare messo a punto precedentemente, in questo progetto di tesi è stata valutata l’influenza di diversi parametri (volume di semina cellulare, velocità di rotazione, tempo di agitazione e presenza/assenza di pause durante l’agitazione) sulla rapidità ed efficacia di adesione delle ASC sui L-FAM. Le condizioni sperimentali sono state individuate grazie al metodo del Design of Experiment (DoE), approccio statistico che individua il numero minimo di condizioni da testare per ottenere un’informazione che sia statisticamente significativa. Questa tecnica permette di identificare le migliori condizioni sperimentali che favoriscano l’adesione delle cellule ai L-FAM nel minor tempo possibile, permettendo il mantenimento della normale morfologia allungata di queste cellule e un’omogenea distribuzione sui L-FAM, condizione necessaria affinché svolgano la normale attività terapeutica. L’adesione cellulare è stata ottenuta in condizioni di semina dinamica mediante agitazione continua o intermittente, in bioreattore. Su 3 diverse popolazioni di ASC sono state testate 13 differenti condizioni di semina, con una durata massima di due ore, in modo tale da ottenere un protocollo finale idoneo a future procedure chirurgiche di trapianto cellulare autologo one-step. Al termine del periodo di agitazione, l’adesione cellulare è stata valutata mediante i saggi quantitativi Alamar Blue e CyQuant e il saggio qualitativo Live&Dead. L’analisi statistica dei dati ha permesso di interpretare le diverse combinazioni di parametri inizialmente fornite dal metodo DoE, portando ad individuare la condizione sperimentale ottimale. Dai risultati ottenuti si evince che la combinazione di una velocità di agitazione di 12 rpm, la presenza di una pausa di 10 minuti e un volume di semina cellulare di 400 µl è in grado di massimizzare l’adesione cellulare sui L-FAM in un tempo di 85 minuti, che risulta congruo alle richieste cliniche. Tale condizione è stata quindi testata su ulteriori 3 popolazioni di ASC, confermando l’idoneità dei L-FAM come sistemi di delivery cellulare. Sebbene nel presente studio siano state utilizzate cellule precedentemente espanse in coltura che hanno il vantaggio di garantire la riproducibilità del metodo, tale progetto si propone di porre le basi per future applicazioni in cui verranno utilizzate cellule minimamente manipolate come la frazione vascolare stromale (SVF) autologa derivante da tessuto adiposo. Tale progetto perciò rappresenta il primo passo di un futuro studio più ampio che avrà come scopo principale l’introduzione di questo sistema di veicolazione cellulare nella pratica clinica per la rigenerazione one-step di lesioni della cartilagine articolare.
Adesione di cellule staminali mesenchimali su microcarrier di alginato e fibroina: cell delivery system per la rigenerazione cartilaginea
FABRO FONTANA, FRANCESCA
2015/2016
Abstract
The common treatments of cartilage diseases include both pharmacological and surgical therapies. Thanks to the ability of Mesenchymal Stem Cells (MSCs) to directly participate to tissue regeneration as well as to restore tissue homeostasis throughout their paracrine activity, the interest on MSCs applications for the treatment of a wide range of musculoskeletal pathologies is progressively increasing. However, the precise and effective local delivery of cells at the injury site still represent a crucial point of this kind of therapy. Hence, the present study, in line with the cell therapy field, has aimed to optimize the use of lyophilized silk/fibroin alginate microcarriers (L-FAMs) for the local articular delivery of adipose-derived mesenchymal stem cell (ASCs), with the final goal to make the groundwork for the development of a one-step autologous therapy for the treatment of patients affected by osteoarthritis. Basing on a protocol for cell adhesion onto L-FAMs previously developed in the frame of the StemDelivery project lead by the Cell Delivery System Lab of the University of Pavia in collaboration with the Galeazzi Orthopaedic Institute, Milan, the influence of different combinations of parameters (seeding volume, seeding time, presence or absence of a pause during the agitation time and seeding speed) on cell adhesion rate has been evaluated in order to obtain a rapid and effect process. The experimental conditions have been identified with the use of the Design of Experiment (DoE), a statistical approach which permits to define the minimal experimental conditions to test in order to have information with statistical significant meaning. The final aim of study was to identify the best cell seeding condition to obtain the higher cell adhesion rate on L-FAMs in the shortest time, but at the same time allowing for the maintenance of the physiological MSCs morphology and a good rate of homogeneity in term of distribution onto microcarriers. Cell seeding was performed in dynamic conditions with the use of a bioreactor, testing both the continuous and the intermittent rotatory stirring. A set of the 13 experimental conditions were tested with ASCs isolated from 3 different donors. In all the cases, a maximum of two hours was allowed to obtain cell adhesion, in order to develop a final protocol which could be suitable for a future clinical use of one-step MSCs therapy where the surgical time is limited. Immediately after the stirring time, cell adhesion rate has been quantitatively evaluated by Alamar Blue and CyQuant assay, whereas the qualitative analysis of cell viability has been performed by Live&Dead. After collecting the whole results, the statistical analysis allowed to interpret the different combinations of parameters initially suggested by the DoE approach, providing the identification of the best experimental condition. The results suggested that the combination of an intermittent stirring seeding (with a pause of 10 minutes), a rotation speed of 12 rpm, 400 µl of cell suspension and is able to optimize cell adhesion on these microcarriers in 85 minutes. This final optimal condition was then tested on 3 further ASCs populations deriving from different donors, confirming the results and thus and the suitability of L-FAMs as a delivery system for one-step local procedures. Even if in these experiments the ASCs were previously expanded in vitro, which guarantee for the method reproducibility, this research represents the fundament for future applications of minimally manipulated cells, such as the adipose tissue stromal vascular fraction (SVF). This study this represent the starting point of a following more extensive study that will aim to introduce this cell delivery system in the clinical practice for the one-step regeneration of articular cartilage lesions.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/20.500.14239/25735