Abstract 1. Introduction Cardiovascular diseases are one of the main causes of death in developed countries. Among them, one of the most common is coronary atherosclerosis, characterized by arterial thickening and the development of atherosclerotic plaques that that reduce arterial lumen size. Nowadays the gold standard atherosclerosis treatment is percutaneous transluminal coronary angioplasty (PTCA) with or without stent implantation [5]. In particular, the most widely implanted stents release an antiproliferative drug (DES) to prevent the re-narrowing of the treated vessel which is known as in-stent restenosis (ISR). Although the introduction of DES has decreased the incidence of ISR, this disease still hampers the long-term efficacy of PTCA. An alternative approach to the loading of anti-proliferative drugs is the treatment of the stent with biomimetic techniques, to improve the tissue integration of the device. In the last years, in particular, significant results in this field have been achieved in the development of biomimetic techniques on titanium like the Anodic Spark Deposition. This treatment, allows the creation of a thin titanium oxide layer on the surface of the treated device, promoting an excellent tissue integration. For its great effectiveness, nowadays the ASD is already widely used in the orthodontic field and, potentially, its use could also be expanded to other areas such as stents. So, the potential improvement of ASD about tissue integration reopens the study of titanium as an alternative material in the production of stents. In order that this biomimetic technique can be used on a stenti s necessary to previously improve surface characteristics. The surface finish, for example, should be as smooth and uniform as possible to promote a good hemodynamics and avoid thrombus formation [66]. To optimize surface characteristics, therefore, it was decided to use electropolishing, a technique that allows to polish devices characterized by complex and smaller shapes. It is preferable to traditional mechanical methods, that often scratch and deform their surfaces. So, the main purpose of this thesis is the definition of the best titanium electropolishing process parameters and verify the effectiveness of this treatment for a possible future use in the biomedical cardiovascular field. Other goals of this work are verification of the possibility of using on titanium the ASD treatment in synergy with electropolishing and, finally, the comparison of the latter with the treatments used for the materials currently on the market for stents production. 2. Materials and methods 2.1 Samples In addition to titanium specimens, electropolishing tests were carried out also on of stainless steel and cobalt-chromium, to have a initial comparison also with the materials currently used for stents production. Titanium All the samples used for the electropolishing treatment, with different geometries and surface finishes, are of pure titanium grade 2. Before and after each electrochemical process (hence also before each analysis technique), each sample was treated with the following steps: pickling solution with 5% hydrofluoric acid (Fluka, 47600) and 30% nitric acid (Sigma-Alorich, 438073) for 10 seconds, ultrasonic washing bath in Acetone (Sigma-Aldrich, 32201) for 5 min, ultrasonic washing bath in Millipore for 5 min, ultrasonic washing bath in Ethanol (Sigma-Aldrich, 02870) for 5 min and drying in a stove at 37 ° C for 45 min. To verify the effectiveness of the treatment of different geometries, different types of samples have been used: disks, small bars, networks with different meshes and stents. Stainless steel and cobalt-chromium All samples for the two materials used for the electropolishing treatments were obtained through punching of plates, respectively, of AISI 316L stainless steel and CoCr L605. Before and after each electrochemical process each sample was treated with the same procedure previously described for the titanium samples. 2.2 Electropolishing The treatment of electropolishing of all the titanium samples was performed with the following electrolytic solution [59]: 1.4 l of Ethanol (Sigma-Aldrich, 02870), 0.6 l of Propanol (Sigma-Aldrich, 33539), 120 g of Aluminium Chloride (Sigma-Aldrich, 206911) and 500 g of Zinc Chloride (Sigma-Aldrich, 229997). The process parameters are as follow [59]: temperature of 30 ° C, current density of 2 kA / m², maximum voltage of 70 V and variable exposure times ( range 5 - 30 min). For stainless steel and CoCr samples, the electrolyte solutions and the process parameters are taken from literature [60-62]. 2.3 Anodic Spark Deposition This biomimetic surface modification technique has been performed only on titanium samples. The electrolytic solution used was obtained by mixing the following reagents [63]: Sodium Silicate (Carlo Erba Reagents, 373 908), Calcium Acetate (Fluka, 21052), Sodium Hydroxide (Fluka, 71690) and Beta-glycerophosphate salt disodium pentahydrate (Fluka, 50020). The parameters used for this treatment are: temperature 0 ° C, current of 30 mA and maximum voltage 175 V. 2.4 Observation of samples and evaluation of morphological characteristics The evaluation of the surface morphology of all the treated samples, both through electropolishing and ASD, was carried out before and after the corresponding techniques. During the execution of the experiments, a first observation of the surface of the samples was carried out through a stereoscopic microscope (DFC290 model, Leica Microsystems). The use of a scanning electron microscope (Stereoscan 360, Cambridge Instruments), allowed the evaluation of the effectiveness and the uniformity of the treatment by the observation of surface morphology. An atomic force microscope (NT-MDT SOLVER PRO model), finally, has been used to obtain 3D images of the surface morphology of network samples treated. 2.5 Roughness measurements The parameters related to the roughness of the electropolishing techniques and the ASD were measured both before and after the respective treatments. The measures relating to the disc-shaped samples were made through laser profilometry (UBM Microfocus 5600). An atomic force microscope (NT-MDT SOLVER PRO model) was also used to value networks roughness. 2.6 Mass loss measurements All samples, about all geometry and material, were weighed both before and after the electropolishing and ASD process using a precision microbalance Gibertini E 50 / S3, in order to calculate the mass loss. 2.7 Static contact angle The relative measurements of static contact angle were carried out for the comparison of the wettability between various treatments and different types of material. In this study the static contact angle was evalueted using the Drop Shape Analysis software (DSA) version 1.92.1.1 [HS] for Windows, developed by Kruess. Each drop, about 2μl of size, has been placed on the material surface manually using a syringe. 2.8 Elemental ASD analysis An EDS (model Inca Energy200, Oxford Instruments) was used for the elemental analysis of the titanium oxide layer after the ASD treatments, both on non-modified and electropolished samples. 2.9 Statistical analysis For all the comparisons between roughness, mass loss and static contact angle values, a One Way Anova test with subsequent post-hoc Tukey test method was used. 3. Results 3.1 Observation of samples and evaluation of morphological characteristics SEM images relating to all electropolished titanium samples show that the treatment removed all the burrs along the edge without deforming profile geometry (Fig 1). Electropolishing also allows the elimination of all imperfections in the central area of the untreated sample, giving a smooth and uniform morphology (Fig 2). The effectiveness related to the roughness reduction and the uniformity of treatment is also shown in the AFM images, regarding 5 minutes electropolished networks (Fig. 3). About ASD, however, this image shows the uniformity of the titanium oxide layer formed on disks surface, both non-modified and previously electropolished for 5 minutes (Fig 4). Morphologically, there are no differences. 3.2 Roughness measurements Table 1° shows the comparison between average roughness datas for different types of treatment used on titanium disks. As we can see from the table, after a 5 minute electropolishing treatment, the roughness of the sample decreases significantly compared to the initial value (p <0.001). The process of ASD, instead, does not cause a considerable decrease in roughness. Their composition, EP+ASD, returns a middle roughness value between those; this data is not considerably different from the electropolishing one. Same considerations can be extended to the results obtained using the AFM analysis, shown in Table 1b. In table 2 are compared data related to the percentage roughness decrease after electropolishing treatments performed on different material samples. Among their roughness variations there is no significant difference. 3.3 Mass loss measurements Table 3 represents the comparison between percentage mass loss values for various type of treatments The electropolishing treatment, from now, will refer to a time process of 5 minutes. Electropolishing causes an high mass loss. Even the ASD, despite being a treatment that involves material deposition on the sample, causes a minimum dissolution. The two processes composition (EP + ASD), therefore, results in a higher material loss, due to the sum of both their dissolution actions; this value, however, is not significantly different from that relating to only electropolishing process. Table 4, however, represents the comparison between percentage mass loss values, related to electropolishing treatments performed on different material samples. As we can see from the table, stainless steel mass loss values and CoCr are similar, while titanium loss is significantly lower (p <0.001). 3.4 Static contact angle Table 5 represents the comparison between static contact angle values of various types of treatments used on titanium disks. As we can see from the data, the treatment type carried out influences material wettability. In particular, while electropolishing samples give an high static contact angle value (and so a more hydrophobic surface), ASD give lower values. The particular morphology of the porous titanium oxide layer covering the samples, in fact, allows a greater interaction with water particles. Table 6, on the other hand, represents the comparison between static contact angle values for electropolishing treatments performed on different material samples. Stainless steel is the most congenial to water, followed, in order of increasing in hydrophobicity, by CoCr and titanium. While between steel and CoCr there is a substantial difference, between titanium and steel instead exists (p < 0.05). 3.5 Elemental ASD analysis As demonstrated by EDS analysis, the different combination of electropolishing and ASD treatments has little effect on the titanium oxide layer chemical composition. Calcium and phosphoruos peaks, in fact, are almost similar. 4 Conclusions After this study it can be concluded that, in order to obtain repeatable and consistent results in terms of uniformity of treatment that roughness, are enough adequate agitation, contact symmetry for the passage of the current in the sample and a duration of the treatment time of 5 minutes. As regards the electrical parameters, instead, it is possible to confirm the effectiveness of the values obtained by Tajima et al. [59]. Thinking about a potential use at an industrial level, it is therefore possible to conclude that this treatment constitutes a surface finishing method of the simple and inexpensive titanium, both in terms of waste of economic resources of that time. Subsequently, tests were carried out on different electropolishing treatment times and evaluated the results for roughness and average mass loss of each. As regards the verification of the treatment effectiveness for a possible future use into the cardiovascular field of stents, is possible to state that 5 minutes electropolishing guarantee good premises both in terms of roughness and mass loss. The latter, in fact, is sufficiently low that it can be assumed negligible and ensure a good maintenance of the mechanical and geometrical characteristics of the material. The roughnesses are also comparable with those mentioned in recent articles about surface morphology of titanium stents, that show how surfaces characterized by a submicron roughness ensure less platelet adhesion [67] and a decrease thrombus formation, as well as a better cellular integration [68]. In general terms, it is possible therefore to assert that with the experimental conditions identified in this study it is possible to obtain a significant decrease of the roughness of the titanium samples, as well as a uniform treatment on their profile and a low material mass loss. Among the objectives of this work it has been verified the possibility to use Anodic Spark Deposition, previously studied for stents application, after the developed electropolishing process. The study leads to the conclusion that the combination of the two treatments, in particular a sequential process of electropolishing and ASD, allows to obtain a sample characterized by a porous surface oxide layer with low roughness, without any geometric deformation or considerable mass losses. In conclusion, then, we can adfirm the possibility of a future sequential use of electropolishing and Anodic Spark Deposition. From the initial comparison with the materials currently on the market and their associated electrochemical industrial treatments, good characteristics of titanium have been emerged. This material, in fact, ensured the best compromise between reduction of average roughness and mass loss during electropolishing tests, as well as the higher static contact angle value. To better investigate electropolishing treatment for cardiovascular devices, future may be carried out on stents manufactured with femtosecond laser cutting. Moreover, the study of its possible synergic use with ASD should be expanded by performing mechanical tests to verify superficial adhesion of the oxide layer. Ad hoc dopants, that ensures better tissue integration with vessels, for ASD stents treatments should be also thought and ensured by clinical trials in order to assess platelet aggregation and cell adhesion.
Sommario 1. Introduzione Le malattie cardiovascolari sono una delle principali cause di morte nei paesi sviluppati. Tra di esse, una delle più comuni è l’aterosclerosi coronarica, caratterizzata da un ispessimento dell’arteria e dallo sviluppo di placche aterosclerotiche che diminuiscono il diametro del lume del vaso. Oggigiorno il principale intervento per curare questa malattia è l’angioplastica percutanea con palloncino (PTCA), con o senza impianto di stent [5]. In particolare, gli stent più impiantati rilasciano un farmaco anti-proliferativo (DES) per prevenire il fenomeno di ristenosi (ISR), caratterizzato da un nuovo restringimento del lume del vaso. Nonostante l’introduzione dei DES abbia diminuito l’incidenza di ISR, questa malattia ancora oggi riduce l’efficacia a lungo termine di PTCA. Un approccio alternativo al caricamento di farmaci anti-proliferativi è il trattamento dello stent con tecniche biomimetiche, in modo da migliorare l’integrazione tessutale del dispositivo. Negli ultimi decenni, in particolare, considerevoli risultati in questo ambito sono stati raggiunti nello sviluppo di tecniche biomimetiche sul titanio come, ad esempio, l’Anodic Spark Deposition. Questo trattamento, permette la creazione di un sottile strato di ossido di titanio sulla superficie del dispositivo trattato, favorendone un’eccellente integrazione tessutale. Per la sua grande efficacia, oggigiorno l’ASD trova già largo impiego nel campo ortodontico e, potenzialmente, il suo utilizzo potrebbe anche essere ampliato ad altri ambiti come, ad esempio, quello degli stents. Il potenziale miglioramento riguardo alla diminuzione delle probabilità di restenosi portato dall’ASD, quindi, riapre lo studio del titanio come materiale alternativo nella produzione di stents, inizialmente messo in disparte a favore delle leghe CoCr. Affinchè questa tecnica biomimetica possa essere utilizzata su di uno stent, tuttavia, occorre preventivamente ottimizzarne le caratteristiche superficiali. La finitura superficiale, ad esempio, deve essere il più possibile liscia ed uniforme per favorire una buona emodinamica ed evitare la formazione di trombi [66]. Per ottimizzare le caratteristiche superficiali, quindi, si è deciso di utilizzare l’elettrolucidatura, una tecnica che permette la finitura di dispositivi caratterizzati da geometrie complesse e di dimensioni ridotte, preferibile ai metodi meccanici tradizionali che tendono a graffiare e deformarene le superfici. Lo scopo principale di questa tesi è, quindi, la definizione dei parametri di processo migliori per operare efficacemente la lucidatura del titanio per via elettrochimica, con particolare attenzione a componenti di dimensioni e geometrie di interesse per il settore cardiovascolare, anche in confronto ai materiali attualmente utilizzati (acciai inossidabili e leghe di cobalto-cromo). Tra gli obiettivi di questo lavoro è stata verificata la possibilità di far seguire al trattamento di elettrolucidatura sviluppato un trattamento elettrochimico di Anodic Spark Deposition, precedentemente messo a punto per l’applicazione in questo specifico ambito. 2. Materiali e metodi 2.1 Campioni Oltre ad i campioni in titanio, ovvero il materiale relativo a questo studio, sono state effettuate prove di elettrolucidatura anche su acciaio inossidabile e Cobalto-Cromo, per avere un termine di paragone anche con i materiali attualmente utilizzati in commercio per la produzione di stents. Titanio Tutti i campioni utilizzati per i trattamenti di elettrolucidatura, con diverse geometrie e finiture superficiali, sono di titanio puro grado 2. Prima e dopo di ogni processo elettrochimico (quindi anche prima di ogni tecnica di analisi), ogni campione è stato trattato con la seguente procedura: decapaggio con soluzione 5% Acido Fluoridrico (Fluka, 47600) e 30 % Acido Nitrico (Sigma-Alorich, 438073) per 10 secondi, lavaggio in bagno ad ultrasuoni in Acetone (Sigma-Aldrich, 32201) per 5 min, lavaggio in bagno ad ultrasuoni in acqua Millipore per 5 min, lavaggio in bagno ad ultrasuoni in Etanolo (Sigma-Aldrich, 02870) per 5 min ed asciugatura in stufa a 37°C per 45 min. Per verificare l’efficacia del trattamento su geometrie differenti, sono state utilizzate diverse tipologie di campioni: dischetti, sbarrette, reti con maglie differenti e stents. Acciaio inossidabile e Cobalto-Cromo Tutti i campioni relativi ai due materiali utilizzati per i trattamenti di elettrolucidatura sono stati ottenuti tramite punzonatura di lastre, rispettivamente, di acciaio inossidabile AISI 316L e CoCr L605. Prima e dopo ogni processo elettrochimico ogni campione è stato trattato con la medesima procedura, precedentemente descritta per i campioni di titanio. 2.2 Elettrolucidatura Il trattamento di elettrolucidatura di tutti i campioni in titanio è stato effettuato con la seguente soluzione elettrolitica [59]: 1.4 l di Etanolo (Sigma-Aldrich, 02870), 0.6 l di Propanolo (Sigma-Aldrich, 33539), 120 g di Cloruro di Alluminio (Sigma-Aldrich, 206911) e 500 g di Cloruro di Zinco (Sigma-Aldrich, 229997). I parametri del processo sono i seguenti [59]: temperatura di 30°C, densità di corrente di 2 kA/m² , tensione massima imposta di 70 V e tempo di esposizione variabile (range 5 – 30 min). Anche per quanto riguarda i campioni in acciaio inossidabile e CoCr, le soluzioni elettrolitiche ed i parametri di processo derivano da letteratura [60-62]. 2.3 Anodic Spark Deposition Questa tecnica biomimetica di modifica superficiale è stata effettuata solo sui campioni in titanio. La soluzione elettrolitica utilizzata è stata ottenuta tramite la miscelazione dei seguenti reagenti [63]: silicato di sodio (Carlo Erba Reagenti, 373908), acetato di calcio (Fluka, 21052), idrossido di sodio (Fluka, 71690) e sale beta-glicerofosfato disodico pentaidratato (Fluka, 50020). I parametri utilizzati per il trattamento ASD sono: temperatura 0°C, corrente di 30 mA e tensione massima imposta 175 V. 2.4 Osservazione dei campioni e valutazione delle caratteristiche morfologiche La valutazione della morfologia superficiale di tutti i campioni trattati, sia attraverso elettrolucidatura che ASD, è stata effettuata sia prima che dopo le relative tecniche. Durante l’esecuzione degli esperimenti, una prima osservazione della superficie dei campioni è stata effettuata attraverso un microscopio stereoscopico (modello DFC290, Leica Microsystems). L’utilizzo di un microscopio elettronico a scansione (Stereoscan 360, Cambridge Instruments), invece, ha permesso la valutazione dell’efficacia e dell’uniformità dei trattamenti attraverso l’osservazione della morfologia superficiale. Un microscopio a forza atomica (modello NT-MDT SOLVER PRO), infine, è stato utilizzato per avere immagini tridimensionali della morfologia superficiale dei campioni di rete trattati con le due tecniche. 2.5 Misure di rugosità I parametri relativi alla rugosità delle tecniche di elettrolucidatura ed l’ASD sono stati misurati sia prima che dopo i rispettivi trattamenti. Le misure relative ai campioni discoidali sono state effettuate attraverso profilometria laser (Microfocus UBM 5600). Un microscopio a forza atomica (modello NT-MDT SOLVER PRO), inoltre, ha permesso di ottenere i valori di rugosità di campioni dalla geometria più complessa, quali sono le reti. 2.6 Misure di perdita di massa Tutti i campioni, di ogni geometria e materiale, sono stati pesati sia prima che dopo i processi di elettrolucidatura ed ASD utilizzando una microbilancia di precisione Gibertini E 50/S3, in modo da poter calcolarne la perdita di massa. 2.7 Angolo di contatto statico Le misurazioni relative all’angolo di contatto statico sono state effettuate per il confronto della bagnabilità tra i vari trattamenti effettuati, oltre che per quello tra i diversi tipi di materiale. In questo studio è stato valutato l’angolo di contatto statico tramite il software Drop Shape Analysis (DSA) versione 1.92.1.1 [HS] per Windows, sviluppato dalla Kruess. Ciascuna goccia, delle dimensioni di circa 2µl, è stata messa manualmente a contatto con il materiale tramite una siringa. 2.8 Analisi elementale dell’ASD Uno spettroscopio a dispersione di energia (modello Inca Energy200,Oxford Instruments), è stato utilizzato per l’analisi elementale dello strato di ossido di titanio dopo i trattamenti di ASD su campioni non modificati ed elettrolucidati. 2.9 Analisi statistica Per tutti i confronti relativi ai valori di rugosità, perdita di massa e angolo di stato utilizzato un test Anova ad una via con successivo test post-hoc con metodo Tukey. 3. Risultati 3.1 Osservazione dei campioni e valutazione delle caratteristiche morfologiche Le immagini SEM relative a tutti i campioni in titanio elettrolucidati mostrano come il trattamento abbia eliminato le bave di lavorazione lungo il bordo senza andare, tuttavia, a deformare la geometria del profilo (Fig 1). L’ elettrolucidatura consente inoltre l’eliminazione di tutte le asperità e le creste superficiali che caratterizzavano la zona centrale del campione non trattato, restituendo una morfologia levigata ed uniforme(Fig 2). L’efficacia relativa alla diminuzione di rugosità ed all’uniformità di trattamento è mostrata anche nelle immagini AFM, relative a reti elettrolucidate per 5 minuti (Fig. 3). L’immagine dell’ASD, invece, mostra l’uniformità dello strato di ossido di titanio formatosi sulla superficie di dischetti in titanio non modificati e preventivamente elettrolucidati per 5 minuti (Fig 4). Morfologicamente, non sussistono differenze sostanziali. 3.2 Misure di rugosità Nella tabella 1a sono confrontati i dati relativi ai valori di rugosità medi per i diversi tipi di trattamento utilizzati su campioni discoidali di titanio grado 2. Come si può vedere dalla tabella, dopo un trattamento di elettrolucidatura di 5 minuti, la rugosità del campione diminuisce notevolmente rispetto a quella iniziale (p < 0.001). Il processo di ASD, al contrario, non ne provoca una considerevole diminuzione di rugosità. La loro composizione, EP+ASD, restituisce un valore dirugosità non significativamente differente da quello della sola elettrolucidatura. La tabella 1b mostra i risultati ottenuti tramite l’analisi AFM. Nella tabella 2 sono confrontati i dati relativi alla diminuzione di rugosità percentuale dopo trattamenti di elettrolucidatura eseguiti su campioni di materiali diversi. Tra le loro variazioni di rugosità non sussiste nessuna differenza significativa. 3.3 Misure di perdita di massa La tabella 3 rappresenta i valori di perdita di massa percentuale, messi a confronto, delle varie tipologie di trattamenti utilizzati su campioni discoidali di titanio grado 2. I trattamenti di elettrolucidatura, d’ora in poi, si riferiscono a processi della durata di 5 minuti. L’elettrolucidatura provoca la maggiore perdita di massa. Anche l’ASD, tuttavia, pur essendo un trattamento che comporta la deposizione di materiale sul campione, ne provoca una minima dissoluzione. La composizione dei due processi (EP+ASD), quindi, risulta in una maggiore perdita di materiale dovuta alla somma di entrambe le loro azioni di dissoluzione; valore, tuttavia, non significativamente differente da quello relativo al solo processo di elettrolucidatura. La tabella 4, invece, rappresenta i valori di perdita di massa percentuale, messi a confronto, relativi ai trattamenti di elettrolucidatura eseguiti su campioni discoidali di materiali diversi. Come si può notare dalla tabella, i valori relativi alla perdita di massa dei campioni in acciaio inossidabile e CoCr sono analoghi, mentre la perdita di materiale relativa al titanio risulta essere significativamente inferiore (p < 0.001). 3.4 Angolo di contatto statico La tabella 5 rappresenta i valori di angolo di contatto statico, messi a confronto, delle varie tipologie di trattamenti utilizzati su campioni di titanio grado 2. Come si può notare dai dati, diversi tipi di trattamenti risultano in differenti valori dell’angolo di contatto statico. In particolare, mentre i valori dei campioni elettrolucidati sono più alti (quindi la superficie è maggiormente idrofobica), quelli dell’ASD sono minori. La particolare morfologia porosa dello strato di ossido di titanio che ricopre i campioni, infatti, permette una maggiore interazione con le particelle di acqua. La tabella 6, invece, rappresenta i valori di angolo di contatto statico, messi a confronto, relativi ai trattamenti di elettrolucidatura eseguiti su campioni di materiali diversi. Considerando la media delle facce, l’acciaio inossidabile risulta il più affine all’acqua e, a seguire in ordine di idrofobicità crescente, vengono CoCr e titanio. 3.5 Analisi elementale dell’ASD Come dimostrato dall’analisi EDS, la diversa combinazione dei trattamenti di elettrolucidatura ed ASD influisce poco sulla composizione chimica finale dello strato di ossido di titanio che andrà a ricoprire il campione. I picchi relativi sia al calcio che al fosforo, infatti, sono quasi analoghi. 4 Conclusioni Dopo questo studio è possibile concludere che, per poter ottenere risultati ripetibili ed omogenei sia in termini di uniformità di trattamento che di rugosità, occorrono una adeguata agitazione, simmetria di contatto per il passaggio della corrente nel campione ed un tempo di trattamento della durata di 5 minuti. Per quanto riguarda i parametri elettrici, invece, è possibile confermare l’efficacia dei valori ottenuti da Tajima et al. [59]. Pensando ad un potenziale utilizzo a livello industriale, è quindi possibile concludere che questo trattamento costituisca un metodo di finitura superficiale del titanio semplice e poco oneroso. Successivamente sono state svolte prove di elettrolucidatura su diversi tempi di trattamento e valutati i risultati relativi a rugosità e perdita di massa media di ognuno. Per quanto riguarda la verifica dell’efficacia di tale trattamento per un possibile futuro impiego nel settore biomedicale cardiovascolare degli stents è possibile affermare che un’ elettrolucidatura di 5 minuti garantisca delle buone premesse sia in termini di rugosità che di perdita di massa. Quest’ultima, infatti, è sufficientemente bassa da poter essere ipotizzata trascurabile e garantire un buon mantenimento delle caratteristiche meccaniche e geometriche del materiale. I valori di rugosità di questi ultimi, inoltre, risultano comparabili con quelli citati in recenti articoli sulla morfologia della superficie di stents in titanio, che dimostrano come superfici caratterizzate da una rugosità submicrometrica garantiscano una minore adesione piastrinica [67] ed una diminuzione della formazione di trombi, oltre che una migliore integrazione cellulare [68]. In termini generali, è possibile quindi asserire che con le condizioni sperimentali individuate in questo studio è possibile ottenere una diminuzione significativa della rugosità dei campioni in titanio, oltre che un trattamento uniforme lungo tutto il loro profilo ed una perdita di massa del materiale ridotta. Tra gli obiettivi di questo lavoro è stata verificata la possibilità di far seguire al trattamento di elettrolucidatura sviluppato un trattamento elettrochimico di Anodic Spark Deposition, precedentemente studiato per l’applicazione in questo specifico ambito. Lo studio condotto permette di concludere che la combinazione dei due trattamenti, in particolare un processo sequenziale di elettrolucidatura ed ASD, consente di ottenere un campione caratterizzato da uno strato superficiale poroso di ossido dalla rugosità ridotta, senza deformazioni nella geometria iniziale o considerevoli perdite di massa. In conclusione, quindi, si può affermare la possibilità di un futuro utilizzo sequenziale delle tecniche di elettrolucidatura ed Anodic Spark Deposition. Dall’iniziale confronto con i materiali attualmente sul mercato ed i loro relativi trattamenti di elettrolucidatura già utilizzati a livello industriale, sono emerse le buone caratteristiche del titanio. Questo materiale, infatti, è quello che ha garantito il miglior compromesso tra diminuzione di rugosità media e perdita di massa durante le prove di elettrolucidatura, oltre che il maggior valore di angolo di contatto. Per approfondire lo studio del trattamento di elettrolucidatura per dispositivi biomedicali cardiovascolari in titanio, in futuro si potrebbero effettuare prove su stents fabbricati con taglio laser a femtosecondi. Per quanto riguarda studio del suo possibile utilizzo sinergico con l’ASD sarebbe utile che questo venisse ampliato effettuando dei test meccanici per verificare l’adesione superficiale dello strato di ossido. Si potrebbe valutare inoltre un dopaggio ad hoc per gli stents, che garantisca la migliore integrazione tissutale con le pareti dei vasi di impianto, verificandolo poi con dei trials in vitro in modo da valutare i fenomeni di aggregazione piastrinica ed adesione cellulare e, quindi, il processo di endotelizzazione.
Sviluppo di trattamenti di elettrolucidatura su titanio per il settore biomedicale cardiovascolare
COLOMBO, GABRIELE
2015/2016
Abstract
Abstract 1. Introduction Cardiovascular diseases are one of the main causes of death in developed countries. Among them, one of the most common is coronary atherosclerosis, characterized by arterial thickening and the development of atherosclerotic plaques that that reduce arterial lumen size. Nowadays the gold standard atherosclerosis treatment is percutaneous transluminal coronary angioplasty (PTCA) with or without stent implantation [5]. In particular, the most widely implanted stents release an antiproliferative drug (DES) to prevent the re-narrowing of the treated vessel which is known as in-stent restenosis (ISR). Although the introduction of DES has decreased the incidence of ISR, this disease still hampers the long-term efficacy of PTCA. An alternative approach to the loading of anti-proliferative drugs is the treatment of the stent with biomimetic techniques, to improve the tissue integration of the device. In the last years, in particular, significant results in this field have been achieved in the development of biomimetic techniques on titanium like the Anodic Spark Deposition. This treatment, allows the creation of a thin titanium oxide layer on the surface of the treated device, promoting an excellent tissue integration. For its great effectiveness, nowadays the ASD is already widely used in the orthodontic field and, potentially, its use could also be expanded to other areas such as stents. So, the potential improvement of ASD about tissue integration reopens the study of titanium as an alternative material in the production of stents. In order that this biomimetic technique can be used on a stenti s necessary to previously improve surface characteristics. The surface finish, for example, should be as smooth and uniform as possible to promote a good hemodynamics and avoid thrombus formation [66]. To optimize surface characteristics, therefore, it was decided to use electropolishing, a technique that allows to polish devices characterized by complex and smaller shapes. It is preferable to traditional mechanical methods, that often scratch and deform their surfaces. So, the main purpose of this thesis is the definition of the best titanium electropolishing process parameters and verify the effectiveness of this treatment for a possible future use in the biomedical cardiovascular field. Other goals of this work are verification of the possibility of using on titanium the ASD treatment in synergy with electropolishing and, finally, the comparison of the latter with the treatments used for the materials currently on the market for stents production. 2. Materials and methods 2.1 Samples In addition to titanium specimens, electropolishing tests were carried out also on of stainless steel and cobalt-chromium, to have a initial comparison also with the materials currently used for stents production. Titanium All the samples used for the electropolishing treatment, with different geometries and surface finishes, are of pure titanium grade 2. Before and after each electrochemical process (hence also before each analysis technique), each sample was treated with the following steps: pickling solution with 5% hydrofluoric acid (Fluka, 47600) and 30% nitric acid (Sigma-Alorich, 438073) for 10 seconds, ultrasonic washing bath in Acetone (Sigma-Aldrich, 32201) for 5 min, ultrasonic washing bath in Millipore for 5 min, ultrasonic washing bath in Ethanol (Sigma-Aldrich, 02870) for 5 min and drying in a stove at 37 ° C for 45 min. To verify the effectiveness of the treatment of different geometries, different types of samples have been used: disks, small bars, networks with different meshes and stents. Stainless steel and cobalt-chromium All samples for the two materials used for the electropolishing treatments were obtained through punching of plates, respectively, of AISI 316L stainless steel and CoCr L605. Before and after each electrochemical process each sample was treated with the same procedure previously described for the titanium samples. 2.2 Electropolishing The treatment of electropolishing of all the titanium samples was performed with the following electrolytic solution [59]: 1.4 l of Ethanol (Sigma-Aldrich, 02870), 0.6 l of Propanol (Sigma-Aldrich, 33539), 120 g of Aluminium Chloride (Sigma-Aldrich, 206911) and 500 g of Zinc Chloride (Sigma-Aldrich, 229997). The process parameters are as follow [59]: temperature of 30 ° C, current density of 2 kA / m², maximum voltage of 70 V and variable exposure times ( range 5 - 30 min). For stainless steel and CoCr samples, the electrolyte solutions and the process parameters are taken from literature [60-62]. 2.3 Anodic Spark Deposition This biomimetic surface modification technique has been performed only on titanium samples. The electrolytic solution used was obtained by mixing the following reagents [63]: Sodium Silicate (Carlo Erba Reagents, 373 908), Calcium Acetate (Fluka, 21052), Sodium Hydroxide (Fluka, 71690) and Beta-glycerophosphate salt disodium pentahydrate (Fluka, 50020). The parameters used for this treatment are: temperature 0 ° C, current of 30 mA and maximum voltage 175 V. 2.4 Observation of samples and evaluation of morphological characteristics The evaluation of the surface morphology of all the treated samples, both through electropolishing and ASD, was carried out before and after the corresponding techniques. During the execution of the experiments, a first observation of the surface of the samples was carried out through a stereoscopic microscope (DFC290 model, Leica Microsystems). The use of a scanning electron microscope (Stereoscan 360, Cambridge Instruments), allowed the evaluation of the effectiveness and the uniformity of the treatment by the observation of surface morphology. An atomic force microscope (NT-MDT SOLVER PRO model), finally, has been used to obtain 3D images of the surface morphology of network samples treated. 2.5 Roughness measurements The parameters related to the roughness of the electropolishing techniques and the ASD were measured both before and after the respective treatments. The measures relating to the disc-shaped samples were made through laser profilometry (UBM Microfocus 5600). An atomic force microscope (NT-MDT SOLVER PRO model) was also used to value networks roughness. 2.6 Mass loss measurements All samples, about all geometry and material, were weighed both before and after the electropolishing and ASD process using a precision microbalance Gibertini E 50 / S3, in order to calculate the mass loss. 2.7 Static contact angle The relative measurements of static contact angle were carried out for the comparison of the wettability between various treatments and different types of material. In this study the static contact angle was evalueted using the Drop Shape Analysis software (DSA) version 1.92.1.1 [HS] for Windows, developed by Kruess. Each drop, about 2μl of size, has been placed on the material surface manually using a syringe. 2.8 Elemental ASD analysis An EDS (model Inca Energy200, Oxford Instruments) was used for the elemental analysis of the titanium oxide layer after the ASD treatments, both on non-modified and electropolished samples. 2.9 Statistical analysis For all the comparisons between roughness, mass loss and static contact angle values, a One Way Anova test with subsequent post-hoc Tukey test method was used. 3. Results 3.1 Observation of samples and evaluation of morphological characteristics SEM images relating to all electropolished titanium samples show that the treatment removed all the burrs along the edge without deforming profile geometry (Fig 1). Electropolishing also allows the elimination of all imperfections in the central area of the untreated sample, giving a smooth and uniform morphology (Fig 2). The effectiveness related to the roughness reduction and the uniformity of treatment is also shown in the AFM images, regarding 5 minutes electropolished networks (Fig. 3). About ASD, however, this image shows the uniformity of the titanium oxide layer formed on disks surface, both non-modified and previously electropolished for 5 minutes (Fig 4). Morphologically, there are no differences. 3.2 Roughness measurements Table 1° shows the comparison between average roughness datas for different types of treatment used on titanium disks. As we can see from the table, after a 5 minute electropolishing treatment, the roughness of the sample decreases significantly compared to the initial value (p <0.001). The process of ASD, instead, does not cause a considerable decrease in roughness. Their composition, EP+ASD, returns a middle roughness value between those; this data is not considerably different from the electropolishing one. Same considerations can be extended to the results obtained using the AFM analysis, shown in Table 1b. In table 2 are compared data related to the percentage roughness decrease after electropolishing treatments performed on different material samples. Among their roughness variations there is no significant difference. 3.3 Mass loss measurements Table 3 represents the comparison between percentage mass loss values for various type of treatments The electropolishing treatment, from now, will refer to a time process of 5 minutes. Electropolishing causes an high mass loss. Even the ASD, despite being a treatment that involves material deposition on the sample, causes a minimum dissolution. The two processes composition (EP + ASD), therefore, results in a higher material loss, due to the sum of both their dissolution actions; this value, however, is not significantly different from that relating to only electropolishing process. Table 4, however, represents the comparison between percentage mass loss values, related to electropolishing treatments performed on different material samples. As we can see from the table, stainless steel mass loss values and CoCr are similar, while titanium loss is significantly lower (p <0.001). 3.4 Static contact angle Table 5 represents the comparison between static contact angle values of various types of treatments used on titanium disks. As we can see from the data, the treatment type carried out influences material wettability. In particular, while electropolishing samples give an high static contact angle value (and so a more hydrophobic surface), ASD give lower values. The particular morphology of the porous titanium oxide layer covering the samples, in fact, allows a greater interaction with water particles. Table 6, on the other hand, represents the comparison between static contact angle values for electropolishing treatments performed on different material samples. Stainless steel is the most congenial to water, followed, in order of increasing in hydrophobicity, by CoCr and titanium. While between steel and CoCr there is a substantial difference, between titanium and steel instead exists (p < 0.05). 3.5 Elemental ASD analysis As demonstrated by EDS analysis, the different combination of electropolishing and ASD treatments has little effect on the titanium oxide layer chemical composition. Calcium and phosphoruos peaks, in fact, are almost similar. 4 Conclusions After this study it can be concluded that, in order to obtain repeatable and consistent results in terms of uniformity of treatment that roughness, are enough adequate agitation, contact symmetry for the passage of the current in the sample and a duration of the treatment time of 5 minutes. As regards the electrical parameters, instead, it is possible to confirm the effectiveness of the values obtained by Tajima et al. [59]. Thinking about a potential use at an industrial level, it is therefore possible to conclude that this treatment constitutes a surface finishing method of the simple and inexpensive titanium, both in terms of waste of economic resources of that time. Subsequently, tests were carried out on different electropolishing treatment times and evaluated the results for roughness and average mass loss of each. As regards the verification of the treatment effectiveness for a possible future use into the cardiovascular field of stents, is possible to state that 5 minutes electropolishing guarantee good premises both in terms of roughness and mass loss. The latter, in fact, is sufficiently low that it can be assumed negligible and ensure a good maintenance of the mechanical and geometrical characteristics of the material. The roughnesses are also comparable with those mentioned in recent articles about surface morphology of titanium stents, that show how surfaces characterized by a submicron roughness ensure less platelet adhesion [67] and a decrease thrombus formation, as well as a better cellular integration [68]. In general terms, it is possible therefore to assert that with the experimental conditions identified in this study it is possible to obtain a significant decrease of the roughness of the titanium samples, as well as a uniform treatment on their profile and a low material mass loss. Among the objectives of this work it has been verified the possibility to use Anodic Spark Deposition, previously studied for stents application, after the developed electropolishing process. The study leads to the conclusion that the combination of the two treatments, in particular a sequential process of electropolishing and ASD, allows to obtain a sample characterized by a porous surface oxide layer with low roughness, without any geometric deformation or considerable mass losses. In conclusion, then, we can adfirm the possibility of a future sequential use of electropolishing and Anodic Spark Deposition. From the initial comparison with the materials currently on the market and their associated electrochemical industrial treatments, good characteristics of titanium have been emerged. This material, in fact, ensured the best compromise between reduction of average roughness and mass loss during electropolishing tests, as well as the higher static contact angle value. To better investigate electropolishing treatment for cardiovascular devices, future may be carried out on stents manufactured with femtosecond laser cutting. Moreover, the study of its possible synergic use with ASD should be expanded by performing mechanical tests to verify superficial adhesion of the oxide layer. Ad hoc dopants, that ensures better tissue integration with vessels, for ASD stents treatments should be also thought and ensured by clinical trials in order to assess platelet aggregation and cell adhesion.| File | Dimensione | Formato | |
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https://hdl.handle.net/10589/121468