The main topics of this doctoral thesis are bone regeneration and bone tissue analysis by means of nanotechnological strategies for both purposes. Briefly, the development of nanostructured membranes and scaffolds for bone regeneration procedures in oral surgery have been explored, together with the optimization of analysis protocols at the micro- and nano-scale. The first attempts were addressed to reproduce some of the recent results reported in the scientific literature. Thereafter the necessary components for the ELS process were acquired, assembled and tested. Different combinations of solvents for the preparation of polycaprolactone (PCL) based membranes were tested. Thereafter, an air-plasma cleaning process was applied in order to increase the membrane hydrophilicity. Qualitative characterization of membranes obtained by ELS, before and after plasma treatment has been performed together with the morphological analysis through Scanning Electronic Microscopy. The determination of surface wettability with contact angle measurements was performed. Lactose-modified chitosan (CTL) was added by chemical adsorption on PCL membranes in order to increase their bioactivity. Confocal microscopy analysis showed an improved adsorption of CTL for the membranes treated with air-plasma if compared with the untreated ones. Moreover, CTL was used to deliver, on the membranes, silver nanoparticles (nAg) synthesized within CTL solution. ETAAS analysis, performed to quantify nAg, showed a higher nAg content in membranes treated with a low energy air-plasma treatment and CTL-nAg at pH 7, thus confirming Raman findings. MG63 cells cultured on PCL membranes with or without CTL, showed a more sustained growth after 7 days on the CTL-coated membranes compared with untreated PCL membranes and PCL air-plasma treated membranes. Moreover, the presence of nAg did not hamper cell viability with respect to PCL membranes, as confirmed by lactate dehydrogenase (LDH) assay. The antibacterial activity of PCL-nAg membranes was tested in terms of biofilm inhibition on Pseudomonas aeruginosa (ATCC 27853) and Staphylococcus aureus (ATCC 25923), using the MTT test. The viability data and the SEM imaging clearly showed that the biofilm formation was strongly inhibited on the surface of PCL-CTL-nAg membranes. Mechanical resistance of the produced membranes, soaked and aged in Simulated Body Fluid (SBF) was tested by means of uniaxial tensile tests for the evaluation of elastic modulus and maximal deformation and stress. As second chapter of this thesis, the potentiality of another approach to produce nanostructured fibrin-based membranes was explored. These membranes were obtained by means of blood centrifugation and were tailored with bone-inducing molecule, such as nano-hydroxyapatite (nHAp). Thus, the durability of pristine membrane in SBF was tested. At each time point, one sample was analyzed with SEM with ImageJ processing tools revealing a mean fiber diameter of 0.103 ± 0.05µm without any statistically significant differences during time; degradation assay showed a two-folds increase of the weight related to the SBF absorption in the first 2 days. From the third day a constant degradation was observed. In the time frame of this experiment, the dimensional stability of the fibrin structure up to day 7 suggested that PRF membranes may also be used uncovered in the oral cavity. Subsequently, the effects of the nHAp addition during the forming process of PRF (thus during centrifugation) were investigated. As all the above-mentioned techniques were oriented to regenerate maxillofacial bone, a bone histomorphometric analysis protocol that, at the time of writing of the present thesis, was not already present in the University of Trieste) was optimized. This protocol was prepared and implemented with the ultrastructural SEM-EDS (Energy Dispersive Spectroscopy) analysis of hard tissues mounted on the histological slides).

Gli argomenti principali di questa tesi di dottorato sono la rigenerazione ossea e l'analisi del tessuto osseo mediante strategie nanotecnologiche per entrambi gli scopi. In breve, sono stati esplorati lo sviluppo di membrane e impalcature nanostrutturate per le procedure di rigenerazione ossea nella chirurgia orale, insieme all'ottimizzazione dei protocolli di analisi su micro e nanoscala. Sono stati acquisiti, assemblati e testati i componenti necessari per il processo ELS. Sono state testate diverse combinazioni di solventi per la preparazione di membrane a base di policaprolattone (PCL). Successivamente, è stato applicato un processo aria-plasma al fine di aumentare l'idrofilia della membrana. La caratterizzazione qualitativa delle membrane ottenute da ELS, prima e dopo il trattamento al plasma, è stata eseguita insieme all'analisi morfologica mediante microscopia elettronica a scansione. Il chitosano modificato con lattosio (CTL) è stato aggiunto mediante adsorbimento chimico sulle membrane del PCL al fine di aumentarne la bioattività. L'analisi microscopica confocale ha mostrato un miglior assorbimento del CTL per le membrane trattate con aria-plasma rispetto a quelle non trattate. Inoltre, CTL è stato utilizzato per fornire, sulle membrane, nanoparticelle d'argento (nAg) sintetizzate all'interno della soluzione CTL. L'analisi ETAAS, ha mostrato un maggiore contenuto di nAg nelle membrane trattate con un trattamento plasma-aria a bassa energia e CTL-nAg a pH 7, confermando così i risultati di Raman. Le cellule MG63 coltivate su membrane PCL con o senza CTL, hanno mostrato una crescita più sostenuta dopo 7 giorni sulle membrane rivestite con CTL rispetto alle membrane PCL non trattate e alle membrane trattate con aria-plasma PCL. Inoltre, la presenza di nAg non ha ostacolato la vitalità cellulare rispetto alle membrane PCL, come confermato dal test della lattato deidrogenasi (LDH). L'attività antibatterica delle membrane PCL-nAg è stata testata in termini di inibizione del biofilm su Pseudomonas aeruginosa e Staphylococcus aureus, utilizzando il test MTT. I dati di vitalità e l'imaging SEM hanno mostrato chiaramente che la formazione di biofilm era fortemente inibita sulla superficie delle membrane PCL-CTL-nAg. La resistenza meccanica delle membrane prodotte, imbevute e invecchiate nel fluido corporeo simulato (SBF) è stata testata mediante prove di trazione uniassiali per la valutazione del modulo elastico e la massima deformazione e stress. Come secondo capitolo di questa tesi, è stata esplorata la potenzialità di un altro approccio per produrre membrane a base di fibrina nanostrutturate. Queste membrane sono state ottenute mediante centrifugazione del sangue e sono state adattate con molecole che inducono l'osso, come la nano-idrossiapatite (nHAp). Pertanto, è stata testata la durabilità della membrana incontaminata in SBF. Ad ogni momento, un campione è stato analizzato con SEM con strumenti di elaborazione rivelando un diametro medio della fibra di 0,103 ± 0,05 µm senza differenze statisticamente significative nel tempo; il saggio di degradazione ha mostrato un aumento di due volte del peso correlato all'assorbimento di SBF nei primi 2 giorni. Dal terzo giorno è stato osservato un costante degrado. Successivamente, sono stati studiati gli effetti dell'aggiunta di nHAp durante il processo di formatura di PRF (quindi durante la centrifugazione). Poiché tutte le tecniche sopra menzionate erano orientate a rigenerare l'osso maxillo-facciale, è stato ottimizzato un protocollo di analisi istomorfometrica dell'osso che, al momento della stesura della presente tesi, non era ancora presente all'Università di Trieste). Questo protocollo è stato preparato e implementato con l'analisi ultrastrutturale SEM-EDS (Energy Dispersive Spectroscopy) dei tessuti duri montati sulle diapositive istologiche.

BIORESORBABLE ENGINEERED MEMBRANES FOR GUIDED BONE REGENERATION WITH ANTIMICROBIAL PROPERTIES / Berton, Federico. - (2020 Mar 06).

BIORESORBABLE ENGINEERED MEMBRANES FOR GUIDED BONE REGENERATION WITH ANTIMICROBIAL PROPERTIES

BERTON, FEDERICO
2020-03-06

Abstract

The main topics of this doctoral thesis are bone regeneration and bone tissue analysis by means of nanotechnological strategies for both purposes. Briefly, the development of nanostructured membranes and scaffolds for bone regeneration procedures in oral surgery have been explored, together with the optimization of analysis protocols at the micro- and nano-scale. The first attempts were addressed to reproduce some of the recent results reported in the scientific literature. Thereafter the necessary components for the ELS process were acquired, assembled and tested. Different combinations of solvents for the preparation of polycaprolactone (PCL) based membranes were tested. Thereafter, an air-plasma cleaning process was applied in order to increase the membrane hydrophilicity. Qualitative characterization of membranes obtained by ELS, before and after plasma treatment has been performed together with the morphological analysis through Scanning Electronic Microscopy. The determination of surface wettability with contact angle measurements was performed. Lactose-modified chitosan (CTL) was added by chemical adsorption on PCL membranes in order to increase their bioactivity. Confocal microscopy analysis showed an improved adsorption of CTL for the membranes treated with air-plasma if compared with the untreated ones. Moreover, CTL was used to deliver, on the membranes, silver nanoparticles (nAg) synthesized within CTL solution. ETAAS analysis, performed to quantify nAg, showed a higher nAg content in membranes treated with a low energy air-plasma treatment and CTL-nAg at pH 7, thus confirming Raman findings. MG63 cells cultured on PCL membranes with or without CTL, showed a more sustained growth after 7 days on the CTL-coated membranes compared with untreated PCL membranes and PCL air-plasma treated membranes. Moreover, the presence of nAg did not hamper cell viability with respect to PCL membranes, as confirmed by lactate dehydrogenase (LDH) assay. The antibacterial activity of PCL-nAg membranes was tested in terms of biofilm inhibition on Pseudomonas aeruginosa (ATCC 27853) and Staphylococcus aureus (ATCC 25923), using the MTT test. The viability data and the SEM imaging clearly showed that the biofilm formation was strongly inhibited on the surface of PCL-CTL-nAg membranes. Mechanical resistance of the produced membranes, soaked and aged in Simulated Body Fluid (SBF) was tested by means of uniaxial tensile tests for the evaluation of elastic modulus and maximal deformation and stress. As second chapter of this thesis, the potentiality of another approach to produce nanostructured fibrin-based membranes was explored. These membranes were obtained by means of blood centrifugation and were tailored with bone-inducing molecule, such as nano-hydroxyapatite (nHAp). Thus, the durability of pristine membrane in SBF was tested. At each time point, one sample was analyzed with SEM with ImageJ processing tools revealing a mean fiber diameter of 0.103 ± 0.05µm without any statistically significant differences during time; degradation assay showed a two-folds increase of the weight related to the SBF absorption in the first 2 days. From the third day a constant degradation was observed. In the time frame of this experiment, the dimensional stability of the fibrin structure up to day 7 suggested that PRF membranes may also be used uncovered in the oral cavity. Subsequently, the effects of the nHAp addition during the forming process of PRF (thus during centrifugation) were investigated. As all the above-mentioned techniques were oriented to regenerate maxillofacial bone, a bone histomorphometric analysis protocol that, at the time of writing of the present thesis, was not already present in the University of Trieste) was optimized. This protocol was prepared and implemented with the ultrastructural SEM-EDS (Energy Dispersive Spectroscopy) analysis of hard tissues mounted on the histological slides).
6-mar-2020
DI LENARDA, Roberto
31
2018/2019
Settore MED/28 - Malattie Odontostomatologiche
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/11368/2963760
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