Traditional surgical and the employment of medical devices thought to restore the morphology, and functionality of the ureter do not allow to obtain satisfactory results, since they are limited by anatomical and biological issues. For this reason, the ureter regeneration continues to represent an important medical need to be satisfied. The tissue engineering approach could allow to overcome the challenges and limitations encountered in the urological surgical practice. Therefore, the present PhD thesis has the objective to develop and characterize antibacterial polymeric membranes obtained by Electrospinning (ELS), a widely used technique in the tissue engineering field which enables to synthetize nanofiber-based matrices in a simple and effective way. Among the various synthetic polymeric materials used to produce electrospun membranes, Polycaprolactone (PCL) was selected for its biocompatibility, slow biodegradability, and versatility. Moreover, to confer antibacterial properties to the final product, the PCL was combined with rifampicin (RIF), a broad-spectrum antibiotic, highly effective against Gram-positive bacteria. In detail, the first part of the present work describes the production and the characterization of planar PCL-based membranes loaded with RIF. By means of scanning electron microscope (SEM) analysis it was possible to analyze to evaluate the fibrous morphology, orientation, and the mean diameter. On Uniaxial tensile tests performed on both types of membranes showed optimal mechanical properties in terms of mechanical strength and elongation at break, making the membranes suitable candidates to be employed. The drug release from RIF-loaded membranes was then evaluated, displaying a burst release of the antibiotic agent in the first 24 hours and slowdown over time. This release guaranteed an inhibition of bacteria growth against bacterial strains, some belonging to the ESKAPE group. The biocompatibility was tested using Human urothelial bladder carcinoma cells (UCs) as cellular model revealing an optimal cell viability in the presence of the antibiotic drug. Considering the possible occurrence of bacterial infections caused by different pathogens, the development of a structure able to adequately combine different antibacterial strategies could be advantageous. For this reason, the second part of the thesis concerned the production of coaxial core/shell PCL-based electrospun fibers. In detail, the RIF was loaded within the core of the final structure, with the aim to slow its release and guarantee a prolonged antibacterial efficacy, while the shell of the fibers was functionalized with silver nanoparticles (AgNPs) to maintain an immediate effect upon the implantation in the surgical site. Their presence and homogeneous distribution were confirmed by Field-emission scanning electron microscopy (FE-SEM) and energy dispersive spectroscopy (EDS). A burst release of RIF from the core was registered in the first 24 hours, after that a plateau was reached. The rifampicin and silver released guaranteed an inhibitory activity against Staphylococcus aureus and Escherichia coli, respectively, while double functionalized membranes caused an inhibition of the bacterial growth on both tested bacterial strains. The produced membranes then showed good biocompatibility towards UCs, confirming that the release of both antibacterial agents does not affect cell viability. Lastly, the final part of thesis focused on the synthesis of the bi-layer PCL electrospun membranes and a tubular PCL electrospun scaffold with ureter-like architecture, in order to better mimic the native structure of the organ.

La chirurgia tradizionale e l'impiego di dispositivi medici per ripristinare la morfologia e la funzionalità dell'uretere non permettono di ottenere risultati soddisfacenti, in quanto limitati da problematiche anatomiche e biologiche. Per questo motivo, la rigenerazione dell'uretere continua a rappresentare un importante bisogno medico da soddisfare e attraverso l'ingegneria tissutale è possibile superare i problemi che si incontrano nella pratica chirurgica urologica. La presente tesi di dottorato ha l'obiettivo di sviluppare e caratterizzare membrane polimeriche antibatteriche ottenute mediante Electrospinning (ELS), una tecnica molto utilizzata nel campo dell'ingegneria tissutale consentendo di sintetizzare matrici a base di nanofibre in modo semplice ed efficace. Tra i vari materiali polimerici sintetici utilizzati per produrre membrane elettrofilate, il policaprolattone (PCL) è stato scelto per la sua biocompatibilità, la lenta biodegradabilità e la versatilità. Inoltre, per conferire proprietà antibatteriche al prodotto finale, il PCL è stato combinato con rifampicina (RIF), un antibiotico ad ampio spettro, molto efficace contro i batteri Gram-positivi. In dettaglio, la prima parte del presente lavoro descrive la produzione e la caratterizzazione di membrane planari a base di PCL caricate con RIF. Mediante l'analisi al microscopio elettronico a scansione (SEM) è stato possibile analizzare per valutare la morfologia fibrosa, l'orientamento e il diametro medio. Le prove di trazione uniassiale eseguite su entrambi i tipi di membrane hanno mostrato proprietà meccaniche ottimali in termini di resistenza meccanica e allungamento a rottura, rendendo le membrane idonee per l’applicazione. È stato valutato il rilascio del farmaco dalle membrane caricate con RIF, mostrando un rilascio a raffica dell'agente antibiotico nelle prime 24 ore e un rallentamento nel tempo. Questo rilascio ha garantito un'inibizione della crescita batterica nei confronti di ceppi batterici, alcuni appartenenti al gruppo ESKAPE. La biocompatibilità è stata testata utilizzando cellule di carcinoma della vescica uroteliale umana (UC) come modello cellulare, rivelando una vitalità cellulare ottimale in presenza del farmaco antibiotico. Considerando la possibile insorgenza di infezioni batteriche causate da diversi patogeni, potrebbe essere vantaggioso lo sviluppo di una struttura in grado di combinare adeguatamente diverse strategie antibatteriche. Per questo motivo, la seconda parte della tesi ha riguardato la produzione di fibre elettrofilate coassiali a base di PCL core/shell. Nel dettaglio, il RIF è stato caricato all'interno del nucleo della struttura finale, con l'obiettivo di rallentarne il rilascio e garantire un'efficacia antibatterica prolungata, mentre l’esterno delle fibre è stato funzionalizzato con nanoparticelle d'argento (AgNPs) per mantenere un effetto immediato all'impianto nel sito chirurgico. La loro presenza e distribuzione omogenea sono state confermate dalla microscopia elettronica a scansione a emissione di campo (FE-SEM) e dalla spettroscopia a dispersione di energia (EDS). Nelle prime 24 ore è stato registrato un rilascio massivo di RIF dal nucleo, dopodiché è stato raggiunto un plateau. La rifampicina e l'argento rilasciati hanno garantito un'attività inibitoria rispettivamente contro Staphylococcus aureus ed Escherichia coli, mentre le membrane a doppia funzionalizzazione hanno causato un'inibizione della crescita batterica su entrambi i ceppi batterici testati. Le membrane prodotte hanno poi mostrato una buona biocompatibilità nei confronti delle UC, confermando che il rilascio di entrambi gli agenti antibatterici non influisce sulla vitalità cellulare. Infine, la parte finale della tesi si è concentrata sulla sintesi delle membrane elettrofilate PCL a doppio strato e di uno scaffold tubolare elettrofilato PCL con architettura simile a un uretere, per mimare meglio la struttura nativa dell’organo.

SVILUPPO E CARATTERIZZAZIONE DI MEMBRANE POLIMERICHE ELETTROFILATE CON PROPRIETA' ANTIBATTERICHE PER LA RIGENERAZIONE URETERALE / Musciacchio, Luigi. - (2024 Mar 22).

SVILUPPO E CARATTERIZZAZIONE DI MEMBRANE POLIMERICHE ELETTROFILATE CON PROPRIETA' ANTIBATTERICHE PER LA RIGENERAZIONE URETERALE

MUSCIACCHIO, LUIGI
2024-03-22

Abstract

Traditional surgical and the employment of medical devices thought to restore the morphology, and functionality of the ureter do not allow to obtain satisfactory results, since they are limited by anatomical and biological issues. For this reason, the ureter regeneration continues to represent an important medical need to be satisfied. The tissue engineering approach could allow to overcome the challenges and limitations encountered in the urological surgical practice. Therefore, the present PhD thesis has the objective to develop and characterize antibacterial polymeric membranes obtained by Electrospinning (ELS), a widely used technique in the tissue engineering field which enables to synthetize nanofiber-based matrices in a simple and effective way. Among the various synthetic polymeric materials used to produce electrospun membranes, Polycaprolactone (PCL) was selected for its biocompatibility, slow biodegradability, and versatility. Moreover, to confer antibacterial properties to the final product, the PCL was combined with rifampicin (RIF), a broad-spectrum antibiotic, highly effective against Gram-positive bacteria. In detail, the first part of the present work describes the production and the characterization of planar PCL-based membranes loaded with RIF. By means of scanning electron microscope (SEM) analysis it was possible to analyze to evaluate the fibrous morphology, orientation, and the mean diameter. On Uniaxial tensile tests performed on both types of membranes showed optimal mechanical properties in terms of mechanical strength and elongation at break, making the membranes suitable candidates to be employed. The drug release from RIF-loaded membranes was then evaluated, displaying a burst release of the antibiotic agent in the first 24 hours and slowdown over time. This release guaranteed an inhibition of bacteria growth against bacterial strains, some belonging to the ESKAPE group. The biocompatibility was tested using Human urothelial bladder carcinoma cells (UCs) as cellular model revealing an optimal cell viability in the presence of the antibiotic drug. Considering the possible occurrence of bacterial infections caused by different pathogens, the development of a structure able to adequately combine different antibacterial strategies could be advantageous. For this reason, the second part of the thesis concerned the production of coaxial core/shell PCL-based electrospun fibers. In detail, the RIF was loaded within the core of the final structure, with the aim to slow its release and guarantee a prolonged antibacterial efficacy, while the shell of the fibers was functionalized with silver nanoparticles (AgNPs) to maintain an immediate effect upon the implantation in the surgical site. Their presence and homogeneous distribution were confirmed by Field-emission scanning electron microscopy (FE-SEM) and energy dispersive spectroscopy (EDS). A burst release of RIF from the core was registered in the first 24 hours, after that a plateau was reached. The rifampicin and silver released guaranteed an inhibitory activity against Staphylococcus aureus and Escherichia coli, respectively, while double functionalized membranes caused an inhibition of the bacterial growth on both tested bacterial strains. The produced membranes then showed good biocompatibility towards UCs, confirming that the release of both antibacterial agents does not affect cell viability. Lastly, the final part of thesis focused on the synthesis of the bi-layer PCL electrospun membranes and a tubular PCL electrospun scaffold with ureter-like architecture, in order to better mimic the native structure of the organ.
22-mar-2024
TURCO, GIANLUCA
36
2022/2023
Settore MED/50 - Scienze Tecniche Mediche Applicate
Università degli Studi di Trieste
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11368/3071830
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