The study of the different types of biocompatible scaffolds and their applications as surfaces for cell adhesion are among the main goals of regenerative medicine. In fact, tissue engineering involves the combination of scaffolds, cells and biologically active molecules in order to restore, maintain, or improve the functionality of damaged tissues or entire organs. In this regard, the analysis at molecular-level interactions between cells and biocompatible scaffolds becomes increasingly important in this field. Human Mesenchymal Stem Cells (MSCs) have proved to be particularly interesting for their unique biological properties, including self-renewal and multipotency. They are able to give in vivo and in vitro cells to various mesenchymal tissues such as bone, cartilage and adipose tissue, making them ideal for their applications in regenerative medicine. In this study, Human Mesenchymal Stem Cells derived from bone marrow (hBM-MSCs) were used for in vitro adhesion and interaction analysis with electrospun biocompatible nanofibers and functionalized with three different beta-lactam derivatives (GM18, LT25, SR610) for which it is it has been shown, in previous studies, to have an agonist action against different types of integrin receptors. The interaction and adhesion between cells and scaffolds was analyzed through vitality assays such as Alamar Blue and MTT, thus demonstrating increased cell adhesion on PLLA scaffolds in the presence of agonists versus their absence, and highlighting significant differences between the three various beta-lactam agonists. These differences are further examined by the morphological observation of the cells on the fibers in Electronic Scanning Microscopy (SEM). Secondly, the study was studied by analyzing specific proteins involved in the cell adhesion mechanism in Confocal Laser Scanning Microscopy (CLSM) and amplified by quantitative analysis by Western blot. The results obtained indicate that the presence of beta-lattamic integrin agonists in the electrospun PLLA structure significantly increases the adhesive capacity of the scaffold for MSCs. In fact, these innovative electrospun supports could function as "molecular glue" for cell adhesion by increasing the activation of specific integrins. The association of biologically active molecules such as beta-lattamic integrin agonists, therefore, could be particularly promising in regenerative medicine, ultimately making a beneficial approach to tissue repair and regeneration
Lo studio dei vari tipi di scaffold biocompatibili e la relativa applicazione come superfici per l'adesione cellulare è tra i principali obiettivi della medicina rigenerativa. L'ingegneria tissutale infatti prevede la combinazione di scaffold, cellule e molecole biologicamente attive al fine di ripristinare, mantenere o migliorare la funzionalità dei tessuti o di interi organi danneggiati. A tal proposito l’analisi delle interazioni a livello molecolare tra cellule e scaffold biocompatibili acquisisce sempre più importanza in questo ambito. Le Cellule Staminali Mesenchimali umane (hMSCs) si sono rivelate essere particolarmente interessanti per le loro proprietà biologiche uniche, tra cui la capacità di auto-rinnovamento e di multipotenza. Esse sono infatti in grado di dare origine, in vivo ed in vitro, a cellule di diversi tessuti mesenchimali come, ad esempio, quello osseo, cartilagineo e adiposo, rendendole ideali per una loro applicazione in medicina rigenerativa. In questo studio sono state dunque utilizzate Cellule Staminali Mesenchimali umane derivate dal midollo osseo (hBM-MSCs) per l’analisi in vitro dell’adesione e dell’interazione con nanofibre biocompatibili di PLLA elettrofilate e funzionalizzate con tre diversi derivati beta-lattamici (GM18, LT25, SR610), per i quali è stata dimostrata, in studi precedenti, un’azione agonista nei confronti di diversi tipi di recettori integrinici. L’interazione e l’adesione tra cellule e scaffold è stata analizzata attraverso test di vitalità come Alamar Blue ed MTT, dimostrando così una maggiore adesione cellulare su scaffold di PLLA in presenza di agonisti rispetto alla loro assenza, e evidenziando differenze rilevanti tra i tre diversi agonisti beta-lattamici. Tali differenze vengono ulteriormente esaminate mediante l’osservazione morfologica delle cellule sulle fibre in Microscopia Elettronica a Scansione (SEM). Lo studio è stato, in secondo luogo, approfondito mediante l’analisi di specifiche proteine coinvolte nel meccanismo di adesione cellulare in Microscopia Confocale a Scansione Laser (CLSM) e rafforzato da un’analisi quantitativa attraverso Western blot. In generale i risultati ottenuti indicano che la presenza di agonisti integrinici beta-lattamici nella struttura elettrofilata di PLLA aumenta in maniera significativa la capacità adesiva dello scaffold per le MSCs. Infatti questi innovativi supporti elettrofilati potrebbero funzionare da “collante molecolare” per l’adesione cellulare attraverso un aumento dell’attivazione di specifiche integrine. L’associazione di molecole biologicamente attive come agonisti integrinici beta-lattamici dunque, potrebbe essere particolarmente promettente in medicina rigenerativa, risultando in ultimo un approccio vantaggioso per la riparazione e rigenerazione cellulare tissutale
Studio in vitro dell’interazione tra Cellule Staminali Mesenchimali umane (hMSCs) con innovativi scaffold di nanofibre ellettrofilate di PLLA per la medicina rigenerativa.
CELLAMMARE, ALESSIA
2016/2017
Abstract
The study of the different types of biocompatible scaffolds and their applications as surfaces for cell adhesion are among the main goals of regenerative medicine. In fact, tissue engineering involves the combination of scaffolds, cells and biologically active molecules in order to restore, maintain, or improve the functionality of damaged tissues or entire organs. In this regard, the analysis at molecular-level interactions between cells and biocompatible scaffolds becomes increasingly important in this field. Human Mesenchymal Stem Cells (MSCs) have proved to be particularly interesting for their unique biological properties, including self-renewal and multipotency. They are able to give in vivo and in vitro cells to various mesenchymal tissues such as bone, cartilage and adipose tissue, making them ideal for their applications in regenerative medicine. In this study, Human Mesenchymal Stem Cells derived from bone marrow (hBM-MSCs) were used for in vitro adhesion and interaction analysis with electrospun biocompatible nanofibers and functionalized with three different beta-lactam derivatives (GM18, LT25, SR610) for which it is it has been shown, in previous studies, to have an agonist action against different types of integrin receptors. The interaction and adhesion between cells and scaffolds was analyzed through vitality assays such as Alamar Blue and MTT, thus demonstrating increased cell adhesion on PLLA scaffolds in the presence of agonists versus their absence, and highlighting significant differences between the three various beta-lactam agonists. These differences are further examined by the morphological observation of the cells on the fibers in Electronic Scanning Microscopy (SEM). Secondly, the study was studied by analyzing specific proteins involved in the cell adhesion mechanism in Confocal Laser Scanning Microscopy (CLSM) and amplified by quantitative analysis by Western blot. The results obtained indicate that the presence of beta-lattamic integrin agonists in the electrospun PLLA structure significantly increases the adhesive capacity of the scaffold for MSCs. In fact, these innovative electrospun supports could function as "molecular glue" for cell adhesion by increasing the activation of specific integrins. The association of biologically active molecules such as beta-lattamic integrin agonists, therefore, could be particularly promising in regenerative medicine, ultimately making a beneficial approach to tissue repair and regenerationÈ 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/20.500.14239/18147