Surface treatment on AZ31 magnesium alloy to improve stability and biocompatibility

Introduction Evolution of biomaterials employed as essential substances for fixation of fractures has a significant role in orthopedic surgery. Biomaterials contribute significantly to the improvement of the health and well-being of humankind. The human bodies are prone to painful and disabling injuries such as strains, sprains, dislocation and fractures. Fractures are simply a break in bone which is caused by the forces that exceed the strength of osseous tissue in the bone (1) (2). One of challenging area in orthopedic and oral surgery would refer to the reconstruction of damaged or lost bone in the maxillofacial region which is a major clinical issue. Maxillofacial and skull bones play significant role as are vital for the physiological form and function of many other organs in the head area. The undeniable impacts of maxillofacial structures on the oral and nasal cavity, orbits, and adjacent cranial structures make it a functionally and cosmetically important structure. Orthopedic biomaterials usually are implanted into or near a bone fracture to facilitate healing or to compensate for a loss of bone tissue. (2) Craniomaxillofacial (CMF) implants are used in surgeries of the maxillofacial region such as head, face, neck, oral, and jaw surgeries. These can be done by either placing the implant permanently or temporarily in the body which can be removed as it is no longer needed. Global Craniomaxillofacial (CMF) Implants market Size is anticipated to grow at 6.9% compound annual growth rate from 2018 to 2023 and it is estimated that the global market was valued at USD 2.05 billion in 2018 and is expected to reaches USD 2.86 billion by 2023. In terms of materials, CMF implants were divided into ceramics, non-degradable metals and bio-absorbable substances. Resorbable internal fixation avoids the disadvantages of permanent metal internal fixation devices such as palpability, visibility, stress shielding, dysesthesia, temperature sensitivity, and interactions with diagnostic or therapeutic radiation. The traditional and currently used material for degradable CMF implants are polymer-based ones. However, number of drawbacks have been reported by using these materials, such as: capable to be used only in non-load-bearing regions, due to their brittleness and low elastic modulus and low degradation rate which can lead to activation of inflammatory cascades. (3) Explorations in the biodegradable materials are required to enhance device performance, to improve function, deliver bioactive compounds and achieve the goal of tissue regeneration. The development of metallic biomaterials has gained interest. However, the biggest drawback is the non-degradability of these materials in the body environment which demands the secondary surgical procedure for the removal of implants after the bone heals. Therefore, at present, great amount of research is focused on developing biodegradable, low density and highly bioactive implants without compromising on strength. In particular, magnesium is a metal characterized by good biocompatibility that makes it interesting for biomedical applications. However, magnesium is characterized by a very low structural strength and by an inadequate degradation rate. Because of those reasons, magnesium is usually used in the form of alloy (4). Over the years, different kind of magnesium alloys have been evaluated for different fields of application, such as AZ91, AM60 and AZ31, that can be selected like good candidates for CMF. In particular, the AZ31 alloy guarantees a particularly good compromise between the mechanical properties and the biocompatibility as well as being able to be extruded. Despite of using alloying elements to enhance the pure magnesium mechanical strength, the rapid corrosion rate remains the fundamental critical issue: the detachment of corrosion products causes the local embrittlement of the material with probable formation of cracks (4) (5) (6). A promising method to tackle this issue is conducting surface treatments; the two main categories are the conversion coatings, like electrophoretic deposition and micro arc oxidation (MAO), and coatings for deposition such as dipping and deposition by air spray. Micro arc oxidation (MAO) is a surface modification that converts the outer layer of samples by applying an electric field; the final result is a microporous surface with physical characteristics similar to that of a ceramic material, made of magnesium oxide, inside which chemical species, present inside the electrolytic bath, can be incorporated (7) (8). This technique exhibited numerous advantages as it improves the corrosion resistance of the material, it has low cost, it does not cause pollution, it’s simple to be performed and doesn’t require a particular preparation of the sample (9) (10). In order to obtain a protective coating by MAO process, it’s necessary to consider different process parameters such as: electrolytic solution, current density, voltage and processing time. These parameters, according to literature, are not uniquely defined and they can be selected from a wide range (4). Another technique as a surface treatment which recently have been employed to generate a protective layer on magnesium is the hydrothermal treatment. This kind of treatment has number of advantages like, the low temperatures of process (100-200°C), high purity, high coating thickness, simplicity, eco-compatibility, good adhesion to bellow substrate and can be done on 3D structures with complex geometries (11) (12). The principle of the hydrothermal treatment involves the insertion of a sample in a solution in static condition. The procedure is conducted with a fixed temperature in a determined time (hours) in order to obtain the desired surface modification. This treatment, as seen for MAO, leads to different results depending on the process parameters such as working fluid, temperature ramp, working temperature and treatment time. The aim of this work is to analyze and to optimize the principal parameters of the hydrothermal treatment as a secondary surface treatment on optimized MAO coated sample to obtain a thin, compact and protective coating on AZ31 magnesium alloy samples suitable for use as constituent substance in CMF implants. The specimens will be evaluated in term of morphology, thickness and corrosion. Finally, a preliminary biological characterization will be done to evaluate the possible release of toxic products for the cellular growth by performing an indirect cytotoxicity test in vitro. In this test, samples obtained will be compared to the bare magnesium alloy (AZ31). Materials and methods Strips (20 * 0.5 * 0.05 cm) were cut from the AZ31 foil and were used as substrate materials. Each strip was grinded using the sandpaper P1200 and polished with a 0,05 μm grain alumina suspension. Before MAO treatment, samples were etched using a NITAL solution (pure ethanol and nitric acid with a 95:5) for 30 seconds followed by the same amount of time of immersion in pure ethanol. MAO treatment was carried out using optimized solution according to the previous work in Chiesa et al. group; the MAO bath solution composed of: 10 g/L Na2SiO3, 3M KOH and 4ml/L glycerol was used in all MAO treatments. MAO process parameters were optimized with initial values from proposed values according to the literature. In particular, maximum working voltage (70, 80, 90 and 100 V), treatment time (7, 10, 15 and 20 min) and current density (15, 20, 40 and 60 mA/cm-2) were evaluated. After MAO treatment, each foil strip was rinsed with Millipore water, air dried and rest to become completely dry, then cut for 1.5 cm in order to handle the coated area, which was 1 cm long, without damaging it. Hydrothermal treatment was performed on specimen treated with optimized MAO process parameters. All hydrothermal process were conducted at constant temperature in de-ionized water as bath solution filled 75% of the autoclave. To evaluate and optimize the process parameters of hydrothermal process, a wide range of test values were selected both for temperature and time of treatment; all combination of temperature as 100, 130 and 160 ˚ C and duration of treatment as 4, 8, 16 and 24 hours were performed. In order to evaluate the corrosion resistance, an immersion degradation test performed by using the Simulated Body Fluid (SBF) according to the Kokubo report (13). During this test (the portion of the sample without coating was covered by several Teflon wraps to protect the untreated part from the corrosion environment) each sample was immersed in 50 ml of SBF. The experiment was carried out for 7, 10, 14 and 21 days at 37°C. For each time point, 3 specimens for each selected process parameters were used as well as 3 samples of bare AZ31 magnesium alloy as control. pH of degradation bath solution was measured every day, in order to track the variation induced by sample’s corrosion; after this measurement, 50% volume of SBF solution was changed with fresh one in order to apply an approximation of the physiological fluid’s replenish (14). In order to remove and clean the surface of the specimens from the degradation by-products a post-degradation treatment was conducted. At each time point, samples were immersed in a 180 g/l chromic acid solution for 20 minutes at room temperature, then rinsed with Millipore water and dried. To evaluate the effects of degradation, mass variation was measured using a balance with a resolution of 0,001 g. The phase composition of the coating was examined with X-ray Diffraction (XRD). The surface morphology of the prepared coating before and after degradation was studied with stereomicroscope and scanning electron microscopy (SEM). The thickness of each coating was measured through the evaluation of prepared metallographic section of samples by optical microscope. In order to quantify the variation in concentration of elements as a consequence of degradation of specimens in SBF, optical emission spectroscopy was performed. An indirect cytotoxicity test was performed to assess the cell viability and cytotoxicity induced from degradation of different coated samples along with bare magnesium alloy (AZ31). The Alamar Blue® assay was employed based on ISO10993; L929 cell line (murine fibroblast NCTC clone 929 [L cell, L-929, derivative of Strain L] (ATCC® CCL-1™)) was used in Alamar Blue® assay. The test was performed for 7, 14 and 21 days of degradation in non-refreshed sterilized 15 ml of SBF to obtain a concentrated solution of corrosion products in SBF. Each sample used for the indirect cytotoxicity test was used also to evaluate the corrosion resistance in 15 ml of SBF. In order to quantify and correlate the variation in concentration of elements as a consequence of degradation of specimens in SBF compared to induced cytotoxicity, optical emission spectroscopy was performed. Results and discussion OPTIMIZATION OF MAO PROCESS PARAMETERS Regarding the maximum set voltage on the generator, the 70 (V) seemed to be not sufficient to form a protective layer on the specimen and led to a very thin coating. Increasing voltage resulted in formation of thicker oxide layer. The maximum reached voltage during the treatment, even by setting 140 (V) on the generator was 107 (V) also by letting the time extended to 20 min. The fig. i shows the evolution of voltage over time for different set voltage values. Figure i. Voltage evaluation during MAO treatment according to different maximum reached voltage According to the fig. i after 7 min of MAO treatment regardless of set voltage on the generator, the instantaneous voltage during process only fluctuated around 100 (V). According to the SEM images, by increasing the voltage; the number of pores, micro-cracks and thickness of coating increased. Considering the morphology of the formed layer, 100 (V) selected as the optimized value for the tension limit of MAO treatment. The increase of treatment time from 7 to 15 min caused the formation of thicker oxide layer followed by a slightly decrease of thickness by increasing the time from 15 to 20 min, which is similar to results in literature. In addition, more cracks and inhomogeneity were observed in the SEM images by increasing the time of treatment. According to the SEM images and optical images from metallographic sections, 10 min was selected as the optimum duration of MAO treatment to form a protective oxide layer on surface of samples. To optimize the current density, increasing from 20 mA*cm-2 to 60 mA/cm-2 caused formation of large, powerful and less dynamic sparks which led to increase of roughness and inhomogeneity of formed oxide layer. MAO treatment performed at 15 mA/cm-2 revealed to be slow and exhibited low value of instantaneous voltage for the first 4 minutes of treatment. According to the observations during treatment and evaluation of obtained surface morphology by stereomicroscope and SEM, 20 mA*cm-2 was selected as optimum value for current density. OPTIMIZATION OF HYDROTHERMAL TREATMENT Performing Hydrothermal treatment, regardless of value of the process parameters, led to formation of a layer composing of nanostructures which covered the micro-pores of MAO coating (fig. ii). The SEM images of samples after conducting hydrothermal treatment with different process parameters outlined the formation of two major nano-sized particles: rod- and flower-shaped particles. Figure ii. SEM images of MAO coated sample, M-100-20-10 (left) and hydrothermally treated sample, HM-130-8(right) Increasing the temperature of hydrothermal process had different effects which depended on duration of treatment; while treating MAO coated samples for 4 hours, increasing the temperature from 100 to 160 ˚C, enhanced the number, size and compactness of formed Nano-particles. However, increasing the temperature in case of 16 and 24 hours treatments resulted to less number of Nano rod-shapes particles and smaller size of flower-shaped particles. Considering the treatment time of hydrothermal process, by treating the specimens at 100 ˚C, increasing the duration of process led to enhancing the number and compactness of formed nanostructures; treating the samples at 130 ˚C, increasing the time of treatment from 4 to 8 hours increased the thickness of coating by enhancing the tightness of rod-shaped nano-particles. However, increasing the treatment time while processing at 160 ˚C more than 4 hours resulted to detachment of MAO coatings. IMMERSION DEGRADATION TEST Mass variation during immersion degradation test was shown in fig. to illustrate the kinetics of degradation of samples in 50 ml of refreshing SBF. Figure iii. Mass loss (% initial mass) during immersion degradation test According to the fig. iii, MAO treatment (M-100-20-10) could slightly protect the substrate but not more than 14 days. However, the optimized hydrothermally treated samples which had a thin layer composed of nanostructures of magnesium hydroxide had significant potential to decrease the mass loss of samples. The fig. iv exhibits the pH of SBF as degradation solution each day before refreshing 50% volume of solution. Figure iv. pH variation during immersion degradation test According to the graph, the MAO coated sample and the bare AZ31 magnesium alloy sample which held the highest corrosion rates illustrated the highest pH values. The phenomena would be justified considering the higher amounts of hydrogen would be produced which results to higher pH in case of higher corrosion rates according to bellow reactions during degradation of magnesium physiological medium: The mass variation of samples in 15 ml of non-refreshed sterilized SBF from which the extracts were taken for Alamar Blue® assay was shown in fig. v. Figure v. Mass loss (% initial mass) for indirect cytotoxic test According to the graph, the trends of degradation rates were similar to immersion degradation test as was expected. INDIRECT CYTOTOXICITY TEST Fig.vi illustrates the variation of cell viability during 3 weeks of sample’s degradation. Figure vi. Cell viability (% vs CTR) during indirect cytotoxicity test (* p <0.05, ** p <0.01, *** P < 0.001) According to the graph, the hydrothermal treatment showed better results in term of cell viability compared to MAO coated sample and compared to bare magnesium alloy samples after 3 weeks of degradation. The elevation of pH and the concentration of corrosion products during the indirect cytotoxicity test were measured at each time step of 7, 14 and 21 days by pH meter and optical emission spectrometer which the results were shown in fig and respectively. Figure vii. pH variation during indirect cytotoxic test (left), magnesium concentration variation during indirect cytotoxic test (right) According to the graphs, the higher pH values as a result of alkanization of solution and higher concentration of corrosion products both because of higher corrosion rates of bare magnesium alloys and single step MAO treatment on specimens would be two major causes of higher induced toxicity according to these two types of samples compared to hydrothermally treated one. Conclusions The goal of this research work was to evaluate and analyze the potential of hydrothermal treatment as a second surface treatment on MAO coated samples to enhance the corrosion resistance of AZ31 magnesium alloys. Considering the MAO coated samples, the surface illustrated the formation of non-uniform and porous layer which contained micro-cracks as well. The immersion degradation test in SBF revealed that the MAO treatment could decrease the corrosion rate of samples slightly for a short duration of 2 weeks. Interestingly in this work, the hydrothermally treated specimens proved the significant potential of this treatment to cover the porous MAO coating by a layer composed of magnesium hydroxide nanostructures and in general improve the corrosion resistance of magnesium alloys for longer period of time. The immersion degradation test in SBF illustrated the decrease in corrosion rate of samples where the mass loss of optimized hydrothermally treated MAO coated samples decrease 15% and 12% compared to the MAO coated sample and bare magnesium alloys respectively. The results of Alamar blue® assay signified that neither MAO nor hydrothermal treatment would induce cytotoxicity as the sample degrades in SBF. In addition, the hydrothermal treatment illustrated better result in term of cell viability. It exhibited 14% and 12% increase of cell viability as incubated in extracts from SBF solution mixed with corrosion products of hydrothermally treated sample compared to MAO coated specimen and uncoated AZ31 magnesium alloy respectively, after 3 weeks of sample’s corrosion.

Lo scopo di un impianto cranio-maxillo-facciale è fornire un adeguato supporto meccanico nel sito implantare per i primi mesi successivi all'operazione; inoltre, nel caso di impianti biodegradabili, deve avere un tasso di degradazione compatibile con la rigenerazione dei tessuti. Il magnesio e in particolare le sue leghe sembrano materiali in grado di soddisfare questi requisiti, considerando le loro proprietà meccaniche simili a quelle del tessuto osseo e le loro caratteristiche di biocompatibilità. Il principale svantaggio di queste leghe è la bassa resistenza alla corrosione in ambiente fisiologico. Di conseguenza, in questa ricerca si è deciso di valutare gli effetti del trattamento idrotermico su lega di magnesio AZ31 precedentemente rivestita mediante ossidazione con microarco, ipotizzando che possa creare un rivestimento protettivo che presenti un buon compromesso tra proprietà meccaniche e biocompatibilità, con l'obiettivo di ottenere un materiale con caratteristiche superficiali adatte per una futura applicazione nel campo della chirurgia cranio-maxillo-facciale in termini di stabilità e biocompatibilità. Inizialmente sarà studiato l'effetto protettivo di un rivestimento ottenuto mediante ossidazione con microarco (MAO), un processo che forma uno strato di ossido in grado di aumentare la resistenza alla corrosione del materiale. Successivamente, sul campione rivestito con MAO verrà eseguito un trattamento idrotermico e valutata la sua efficacia nel migliorare la stabilità e la biocompatibilità del materiale. Per ottenere un rivestimento sottile, compatto e altamente protettivo, dopo un’analisi della letteratura, verranno ottimizzati i parametri elettrici del processo MAO (densità di corrente, tensione massima e tempo di trattamento). Questo perché, secondo la letteratura, non sono ancora stati individuati parametri definiti per ottimizzare tale processo, sebbene si tratti di un noto trattamento di anodizzazione già studiato da tempo. I campioni così ottenuti saranno valutati esaminando la morfologia superficiale mediante osservazione allo stereomicroscopio ed analisi SEM; inoltre sarà analizzata la composizione chimica della superficie mediante analisi XRD e lo spessore del rivestimento sarà misurato analizzando le sezioni metallografiche mediante microscopio ottico. I campioni rivestiti tramite MAO e ottimizzati in termini di morfologia superficiale e spessore del rivestimento saranno sottoposti a trattamento idrotermico in acqua deionizzata, con diversi valori di test per due dei principali parametri di processo: temperatura e tempo di trattamento. I campioni trattati idrotermicamente ottimizzati in termini di morfologia superficiale del rivestimento saranno sottoposti a un test di degradazione in SBF per studiare la cinetica di corrosione dei diversi rivestimenti rispetto al campione rivestito MAO ottimizzato e alla lega di magnesio AZ31 non trattata. Il comportamento dei diversi rivestimenti sarà valutato attraverso l'analisi morfologica e la variazione ponderale; inoltre verranno effettuate quotidiane misurazioni del pH per valutare eventuali variazioni. Infine, verrà condotta una caratterizzazione biologica preliminare per valutare il possibile rilascio di prodotti tossici a seguito della degradazione e la loro eventuale influenza sulla crescita cellulare mediante test di citotossicità indiretta in vitro. La lega AZ31 non trattata sarà confrontata con i campioni rivestiti con un singolo trattamento MAO e con campioni sottoposti a trattamento MAO seguito da trattamento idrotermico. I campioni verranno messi a contatto con SBF per 1, 2 e 3 settimane, al termine di ogni intervallo temporale verrà valutata la variazione di massa causata dalla degradazione. La linea cellulare L929 (clone 929 del fibroblasto murino NCTC) verrà posta a contatto con gli estratti in SBF contenenti prodotti di corrosione ottenuti dopo ogni fase temporale e la vitalità cellulare sarà valutata con il saggio Alamar Blue®. Inoltre saranno condotte analisi ICP-OES per quantificare l'aumento della concentrazione di elementi relativi ai prodotti di corrosione.