Chitosan/Graphene Oxide Nanocomposite Coatings on Mg alloy: Corrosion and Biocompatibility Properties

doi:10.21203/rs.3.rs-2222903/v1

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Chitosan/Graphene Oxide Nanocomposite Coatings on Mg alloy: Corrosion and Biocompatibility Properties

https://doi.org/10.21203/rs.3.rs-2222903/v1

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In the present study, optimal conditions for the fabrication of chitosan (CS)/graphene oxide (GO) nanocomposite coatings were evaluated by the pulse electrodeposition process (PED) on Mg–2wt%Zn scaffolds. The size distribution of CS, GO, and nanocomposite coatings were evaluated using dynamic light scattering (DLS). The coatings microstructure and morphologies were investigated by the Fourier transform infrared spectroscopy (FT-IR), ultraviolet-visible spectroscopy (UV- visible), Raman spectroscopy, thermogravimetric analysis (TGA), derivative thermal gravimetric (DTG), differential thermal analysis (DTA), and scanning electron microscopy (SEM). Taguchi statistical method was used to optimize the effect of PED parameters, including peak current density (CD) and duty cycle (DC) as well as GO nanosheets content (1,2, and 3wt%). Results showed that optimal coatings were produced under the conditions of 2 wt% GO, CD = 20 mA/cm ², DC = 0.5 and pH = 5. The process's time, temperature, and frequency were 20 min, 37^oC, and 1000 Hz. The biocompatibility of coatings was assessed by in vitro test. The results of cell viability and adhesion of MG63 cells on optimal coating are promising for application in bone tissue engineering.

Nanocomposite

Biocompatibility

Graphene Oxide

Corrosion

Taguchi Method

Mg alloys have attracted exceptional attention in medical applications [1]. The Mg alloys have specific density, Young's modulus, and yield strength closer to human bones. Thus, stress shielding can be avoided [2, 3]. However, the application of the Mg scaffold is not generally used because the Mg degradation rate is higher at the pH level of body fluid [4]. So, alloying and surface coatings are used for improving the corrosion resistance of Mg alloys to expand Mg alloy scaffold and comfort their usage in clinical applications [5]. Generally, the surface morphology, microstructure, and composition, which play critical roles in the influence of artificial scaffolds, can make protein absorption, thus mediating favorable and unfavorable cells [6]. Meanwhile, applying a protective coating consisting of biocompatible materials such as CS is the most successful method offering the benefit of exceptional biocompatibility and inhibiting the corrosion rate of Mg. CS is a natural biopolymer indicating different biological activities containing excellent biocompatibility, osteoconductivity, and antimicrobial properties. Hence, it can be used in biomedical applications [7]. CS-based composites can help regenerate and repair damaged tissues [8]. One of the most lately used composite compounds with CS in medical matters is GO, which improves its properties [9]. Nanocomposites of CS/GO have provided encouraging results for tissue engineering applications. According to previous research, the CS/GO nanocomposites were used to regenerate diverse tissues such as bone, [10, 11] skin,[12] heart,[13] and nerve [14]. Compared to other tissues, research in bone tissue engineering has taken up a large part. CS/GO nanocomposites are most commonly used for bone tissue engineering because CS's molecular structure is close to glycosaminoglycans[15]. When GO is composed of CS, it indicates the ability to increase osteogenic differentiation[16]. Currently, the potential of GO has captured incredible attention as a two-dimensional planar coating of three-dimensional porous scaffolds for the differentiation of diverse styles of stem cells towards osteogenic families [17, 18]. The fabricated CS/GO nanocomposites are commonly 3D scaffolds that stimulate new bone tissue growth by imitating bone microarchitecture [19]. The excellent porosity, chemical and physical aspects and mineralization of CS/GO nanocomposite caused efficient applications for bone tissue [20]. Many studies have demonstrated that surface modification through depositing a nanocomposite coating layer improves biocompatibility. However, it encourages bone ingrowth, osseointegration induction, and mesenchymal stem cell proliferation on Mg alloys [3, 21]. Among specific methods, sol-gel,[22] electrophoretic depositions (EPD),[23] and plasma electrolyte oxidation (PEO)[24] have found significant sorption in recent years. EPD is a current method used in developing nanocomposite coatings among these technologies. It has unique characteristics resulting from forming a uniform coating on a porous scaffold, controlling the coating's thickness, low deposition temperature, and chemical composition [25]. However, the usual electrodeposition process may develop loose, porous, and low adhesive coatings due to the non-optimal effect of concentration polarization and hydrogen production. This non-stick coating is scaled during biodegradation. It prevents the fixation of implanted material to the host’s tissue. Therefore, to solve these problems, it is suggested to use the PED method to deposit uniform and adhesive coating and optimal morphology [26].Several studies have recently focused on fabricating CS/GO nanocomposite coatings to promote the coating's biological properties and mechanical strength using electrochemical or electrophoretic deposition [27–29]. A few studies have been conducted on improving the PED process's corrosion resistance and biocompatibility of nanocomposite coatings on Mg-2Zn scaffolds. This method is often used to prepare coatings on implants to control the chemical composition and suppress the reduction of H₂ and coating thickness [30–32].

It was required to keep all the parameters constant and evaluate each parameter's effects to investigate the effects of the parameters on usual experimental methods. Experimental design science originated from the abundant number of experiments, high cost, and incompetence in explaining the relationship between chemical and physical variables. Therefore, an assessment method is required to analyze the limited number of available information. A developed statistical route to enhance the quality of products, which has been recently applied to engineering, has been introduced by Genichi Taguchi. [33] and biotechnology [34, 35]. Taguchi’s design of experiments has attracted the attention of statisticians as well as researchers as a promising method to achieve the goals [36]. The Taguchi method was applied in this research to identify and find the optimum samples to save time and costs.

The present study aimed to fabricate CS/GO nanocomposite coatings and evaluate corrosion behavior and biocompatibility activity. CS/GO coatings were deposited on a scaffold of Mg alloy containing 2wt%Zn using the PED process. Four factors of GO content, DC, CD, and pH were optimized using the Taguchi method. Finally, the optimal coating's corrosion resistance, thermal stability, morphology, and biocompatibility were tested.

2.1. Materials

To Fabricate the Mg-2Zn scaffolds, Mg and Zn powders (manufactured by Merck), with a purity of 97% and 99%, respectively, were used. Carbamide (CO (NH₂)₂) (supplied by Merck) was applied as the space-holder agent. The chitosan (supplied by Aldrich Chemical Co. with MW = 161,000 g/mol, degree of deacetylation (DD) of 75.6%, and viscosity of 1406 m. Pas) and graphene oxide (produced by Hammer’s method with concentration and length of 1g/lit and 2.7–6.8 µm) were employed to fabricate the nanocomposites.

2.2. Fabrication Of Scaffolds

The Carbamide was added at various volume contents of 5, 10, 15 and 20% to the mixture of Mg-2Zn powders. Then, using the powder metallurgy method, the porous scaffolds were produced with the formerly described mixtures. The scaffold samples were manufactured in a cylindrical mold of 1 cm in height as well as inner diameter under a press pressure of 200 MPa. Then, they were heat treated at two cycles: 1) heating to 250°C at an amount of 1°C/min and holding this temperature for two hours, and 2) heating to 550°C at an amount of 10°C/min and kept at this temperature for two hours.

2.3. Coating Process

The chitosan (~ 1g) was dissolved in an acetic acid solution (1 wt%) using a mixer at 1100 rpm for 20h. Then, the GO was dispersed in the obtained CS mixture by sonicating using HD 3200 series, Landline sonicator at the maximum power of 75W for 1h at room temperature. It is noteworthy that the GO was added gradually to CS at various contents of 1, 2 and 3 wt% to enhance the dispersion of nanosheets which were then coated on the prepared scaffolds. To deposit the nanocomposite n the scaffolds, the uncoated ones were connected to the cathode pole while the anode pole contained the graphite electrodes parallelly. After the deposition of the coatings, the prepared samples were washed with deionized water a. second dried. The conditions of the coating process were pulse-peak current density of 10 & 20 mA/cm² in the medium pH of 5 at the temperature of 37°C at the pulse duty cycles of 0.1, 0.2 and 0.5 for 20min. The duty cycle (DC) is defined as t_on/ (t_on + t_off) [31] where t_on is the duration of applied pulse potential and t_off is the time of no current/potential. For example, DC = 0.4 means that t_off is 1.5 times t_on.

2.4. Characterization

This study's structural analysis of obtained coatings determined the JASCO 4700 FT-IR in 4000 − 400 cm^-1 wave number. The UV-Vis spectra were measured using a Lambda 850 spectrometer, Perkin Elmer Co. The Raman Takram N1-541Teksan microscope model was utilized along with a Broker-based D8-Advance device detector for the XRD measurements. The XRD test was performed at all angles of 2θ (from 5° to 45°) with an increasing step of 0.1° and a stop time of 0.1 seconds per step at room temperature. Thermal TGA, DTG, and DTA studied the thermal properties of the coatings. The Shimadzu TGA 209 F₁thermogravimetric instrument acquired TGA results in the range was 25–450 ^oC using a ramp rate of 10 ^oC/min. TA instrument was utilized for DTA measurements by heating at the rate of 10°C/min under N₂ at a flow rate of 100 mL/min. The particle size and zeta potentials of the suspensions were evaluated by Zeta sizer Nano ZS90 (Malvern Instruments, UK). The corrosion tests were carried out by a linear potentiodynamic experiment using a conventional potentiostat of the three electrodes, containing working, reference (Ag/AgCl), and counter electrodes (Pt). The experiment was performed in an SBF environment at -2.2-0.2 V with a constant scan rate of 100 mV/s at 37°C for 45 seconds in the solution body fluids (SBF). In electrochemical impedance spectroscopy (EIS) measurements, the AC frequency range extended from 100 kHz to 1 Hz. In this investigation, an SEM (the TeScan-Mira III model) equipped with an energy dispersive x-ray spectrometer (EDS) and atomic force microscope (ENTEGRA AFM NT-MDT model) were used to assess the levels and quantitatively investigate the chemical surface structure of the material. After three days, the quantitative toxicity MG63 cell culture tests were performed on the scaffolds to study biocompatibility. The human osteosarcoma cell line (MG-63) was obtained from the national cell bank (The Iran Pasteur Institute, Tehran, Iran).

After defrosting the cells, they were transferred to a flask containing DMEM (Dulbecco's Modified Eagle Medium) culture medium with 10% FBS. Then, the flask was incubated at the humidity of 37°C and 90% and 5% carbon dioxide concentration. It should be noted that the culture medium was changed every 3–4 days. The extraction process was performed according to ISO 10993-5 standards to investigate the toxicity of the samples and their effect on cell growth and proliferation [37]. The amount of 1 mm of the culture medium was added to each sterilized sample for every 3 cm² of the surface. Then, after 1 and 3 days, the medium was removed and added to the cells. A specific amount of culture medium (DMEM) was also considered a control.

For evaluating the cell proliferation, 1×10⁴ cells with 100 µl of culture medium were poured into each well of the 96-well culture plate and incubated for 24 hours at 37°C to permit the cells to stick to the bottom of the plate. Then, a possible amount of the culture medium was removed from the cells, and 90 µl of each sample extract, the extracts diluted with the culture medium, and ten µl of FBS were added up to each culture well. Then culture medium was removed, and 100 µl of MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) with a concentration of 0.5 mg/ml and poured into each well and incubated for four h. After four h, the solution was removed from the cells, and isopropanol was added to dissolve the dark blue crystals.

To better dissolve the MTT precipitate, the plate was set on a shaker for 15 minutes. Then, for calculating the concentration of the substance dissolved in isopropanol, an ELISA device (BioTek ELx808, USA) was used at a wavelength of 570 nm. The wells with more cells show higher optical density (OD) than those with fewer cells. Accordingly, it is possible to determine the amount of more cells from the well below and compare it with the control sample. The viability of cells was obtained as follows:

$$Viability\%=\left(\frac{mean\text{ OD of a sample}}{ mean\text{ OD of control}}\right)\times 100$$

2.5. Taguchi Method

The Taguchi method investigated the PED parameters' effect in this study and determined the optimal conditions. The Taguchi method was selected based on four variables at 2–3 levels L₆ (2¹×3³) to optimize the experimental conditions. The effect of PED parameters on the coating properties, including CD, DC, GO content (%), and pH, were investigated. Table 1 indicates the design of the Taguchi Mixed level design.

Here, L₆ (2¹×3³) was selected, representing six experiments with three levels and four factors. However, 54 experiments were required to consider all possible combinations of input parameters in the traditional or classical approach.

Table 1

The design of Taguchi Mixed level (L₆ with 4 factors and 2–3 levels)
Parameters	Levels
Parameters	1	2	3
DC	0.1	0.2	0.5
CD (mA cm^− 2)	10	20	-
GO content (%)	1	2	3
pH	4	5	6

Note: The time, temperature, and frequency of the process were 20 min, 37^oC, and 1000 Hz, respectively.

3.1. Particle size distribution and zeta potential

Size distribution of GO nanosheets dispersed in water (pH = 4.5), CS and CS/GO nanocomposites investigated by DLS analyses. The standard spherical particle models were used in DLS. Results reported in Fig. 1(a) show two types of peaks corresponding to 800 and 3200 nm size populations (poly dispersion index; PdI = 6.75). It is worth noting that the heights of the two peaks shown in Fig. 1(a) are related to the scattering intensities and not the amount of small and big GO particles. The low value of PDI indicates that the GO sample is constituted of only two populations, but we cannot say which is the most abundant. Our outcomes are similar to the results obtained by Liu et al. regarding the size of GO and rGO. As described earlier, the model-derived diameters are not their actual sizes because most graphene-based materials are not spherical particles. DLS results only to illustrate the size contrasts between the two materials. The size, thickness and corresponding height profile of information for GO nanosheets were observed by AFM (Fig. 1(b)). The AFM images of the GO reveal the presence of irregularly shaped sheets with uniform thickness and different lateral dimensions. Nanosheets can be observed with different sizes ranging from a few tens of nanometers to hundreds of nanometers. As shown from the height profiles recorded at different locations, the measured thickness of the GO sheets was uniform (∼1 nm), while lateral dimensions ranged from 1.6 to 6.6 µm.

The calculated average size of CS and CS/GO nanocomposite particles was about 4.5 and 6.6 µm, respectively (Fig. 2 (a-c)). The size distribution of CS/GO nanocomposite particles is more significant than that of CS polymer and GO nanosheets. It seems that GO nanosheets were isolated by CS polymer.

After mixing GO with CS solution by ultrasonication, the suspension indicated good stability for several weeks. The zeta potential values of CS and CS/GO dispersion indicate a significant effect of the negative charge of GO on CS polymer. The Zeta potential of the CS was + 42 ± 0.8 mV, and this value changed as a result of GO addition to + 40.6 ± 0.5 mV and + 36.6 ± 0.6 mV for the CS/1wt%GO and CS/2wt%GO nanocomposites respectively. Moreover, when the zeta potential index in the colloidal systems is above ± 30 mV, particles are stable in electrostatic repulsion forces [29]. Good dispersion of GO nanosheets in CS solution may be the consequence of the hydrophilic groups in GO and electrostatic interaction between the cationic CS and the GO surface's negative charge.

3.2. Characteristics Of The Nanocomposite Coatings

The GO, CS, and CS/2wt% GO coating were first evaluated by FT-IR (Fig. 3(a)). The GO spectrum indicated two adsorption peaks at 1721 cm^− 1 and 1625 cm^− 1, which were assigned to the C = O stretches of the carboxylic group and the distortion of the O–H bond in water, respectively. The peak appearing at 3240 cm^− 1 was ascribed to the hydroxyl group on GO [38]. The FT-IR spectrum of CS shows a broad absorption band between 3600 cm^− 1 and 2964 cm^− 1, centered at 3260 cm^− 1, due to O–H stretching vibration, N–H extension vibration, and the intermolecular H-bonds of the polysaccharide moieties [39]. A spectral band at 2870 cm^− 1 is observed, corresponding to the axial stretching of C–H bonds. A peak at 1642 cm^− 1 represents the acetamide group's axial stretching of C = O bonds. Another spectral band at 1540 cm^− 1 is associated with the amino group's angular deformation of N–H bonds. A band at 1388 cm^− 1 due to symmetrical angular deformation of CH₃ and the amide III band at 1331cm^− 1 are also observed. The spectral band corresponding to the polysaccharide skeleton, including vibrations of the glycoside bonds and C–O and C–O–C stretching, are also observed in the FT-IR spectrum of chitosan within 1156–655 cm^− 1 [38]. In the spectra of CS/GO, the dominant peaks at 1020 cm^− 1 and 1550 cm^− 1 correspond to the absorbance of glucosidic bond, stretching vibration from C = O of -NHCO- and the N-H bending NH₂, respectively, compared to the pure CS and GO [40].

Figure 3(b) shows the UV spectra of CS/GO nanocomposite coatings with different GO percentages. The UV spectra of CS/1wt % GO coating exhibit a specific peak at 219 nm, related to the electron transfer of π–π* from the aromatic rings of C = C bonds [41]. This absorption peak red-shifted to 223 nm by increasing the GO contents to 3wt%, resulting from overlapping π-π* GO bonds transmission and CS polymerization of n-π* bonds [41]. This result shows a successful chemical relocation of CS polymer with GO.^33,34 The observed peak as a shoulder at about 297 nm is related to the transfer of n-π* from the C = O bond for CS/1wt% GO nanocomposite coating. By contrast, increasing the GO contents to 3wt%, the shoulder peak exhibits a small red shift to 305 nm, indicating an increase in the GO layers in the nanocomposite coating [42].

To further identify the existence of the GO in nanocomposite coating, Raman analysis was applied. Figure 3(c) shows the Raman spectrum of GO and the CS/2wt% GO nanocomposite. As expected, both the conspicuous and characteristic peaks of GO were observed at 1350 cm^− 1 (D band, associated with the disorder in GO structure) and 1575 cm^− 1 (G band, caused by the vibration of sp² carbon atom). However, the G band for the CS/2wt% GO was shifted upward to 1596 cm^− 1. The spectral shifts could be ascribed to disturbing the GO structure caused by the physical or chemical interactions between the carbon/oxide atoms of GO and the reactive sites of CS [43].

The XRD patterns of the coatings with GO different values are indicated in Fig. 3(d). The characteristic XRD peak of GO appeared at 2θ = 10.5° [44]. All nanocomposite coatings illustrate two peaks, one at 15.3º corresponding to the hydrated crystalline structure, whereas the broadened peak at about 20.3º exhibits an amorphous structure [45].

The diffraction angles of the nanocomposite coatings were similar to the CS, and the diffraction peaks corresponding to GO were not seen in the XRD peak of nanocomposite coatings. It is indicated that the addition of GO did not change the amorphous structure of CS, and GO was well exfoliated with CS in nanocomposite coatings. The diffraction intensity of CS was a little stronger than that of nanocomposite coatings at 2θ = 15.3°, implying that the crystalline degree of chitosan decreased after adding GO into CS. Also, the chemical structure of the CS in the nanocomposite coatings was changed with the increasing GO values, indicating that there was mainly physical interaction between CS and GO.

3.3. Determination Of The Optimal Coating By Taguchi Method

Table 2 shows nanocomposite coatings suggested by Taguchi's design. Using Minitab software, the Taguchi method offers the best levels for each parameter to prepare the optimal nanocomposite coating with the highest properties. Figure 4 represents the best PED parameters and levels (2:2:3:2) for nanocomposite coatings suggested by the Taguchi method (CD = 20 mAcm^− 2, GO wt %=2, DC = 0.5, and pH = 5).

Table 2

The results of PED parameters suggested by Taguchi design for CS/GO nanocomposite coatings
Coating	CD (mA cm^− 2)	GO content (%)	DC	pH
1	10	1	0.1	4
2	10	2	0.2	5
3	10	3	0.2	6
4	20	2	0.5	4
5	20	2	0.1	5
6	20	3	0.5	6

3.4. Determination Of The Highest Corrosion Resistance Of Cs/go Coatings By The Taguchi Method

EIS measurements were also carried out in SBF to elucidate the corrosion protection mechanism of coatings. Figure 5 presents the Bode curves of nanocomposite coatings. The sample obtained by Taguchi factors is the optimal coating. The curves in the high-frequency range correspond to the solution resistance, while the curves in the middle-frequency range are related to the properties of passive layers. Moreover, the curves in the low-frequency range correspond to the interface between the passive layers and the Mg-2Zn surface. The impedance magnitude of the coating suggested by the Taguchi method is about 1.47 ×10¹⁰ Ω, which is the highest among all the coatings. This result shows that the coating suggested by the Taguchi method can significantly improve the corrosion resistance compared to the other coatings. Also, the slope of the curve in the middle of this coating is more significant than other coatings, indicating the capacitive properties. It is noteworthy that the Bode curve of the nanocomposite coating suggested by the Taguchi method is closer to Taguchi 5 and 6 (Table 2).

3.5. Evaluation Of Corrosion Resistance Of The Optimal Coating

The potentiodynamic polarization curves are used for comparing the corrosion resistance of optimal coating with Mg-2Zn scaffold and CS coating. Figure 6(a) indicates the polarization curves of coatings at DC = 0.5, CD = 20 mA/cm², pH = 5, t = 20 min, T = 37 ^oC, and f = 1000 Hz in SBF. Table 3 lists the parameters extracted from the polarization curves using Corr. View Software. The optimal coating's corrosion potential (E_corr) was increased. However, the corrosion current density was decreased compared to the Mg-2Zn and CS coating. Also, the corrosion rate of the optimal coating was 0.1442 mm/year, which was very low and appropriate compared to the CS coating and Mg-2Zn, with corrosion rates of 0.4058 and 2.1065 mm/year, respectively.

Figure 6(b) shows the relationship between the frequency and impedance magnitude of Mg-2Zn coatings. The bode plot denotes the higher impedance modulus at low frequency. The impedance magnitude of Mg-2Zn, CS, GO, and optimal coatings at low frequencies are about 395, 9395, 3268, and 1.47 ×10¹⁰ Ω, respectively. The impedance magnitude shows that coating with optimal conditions improves corrosion resistance. In addition, the slope of the curves in the middle-frequency range for the optimal coating is more significant than other coatings, indicating the capacitive properties.

Table 3

The parameters extracted from the polarization curves
Scaffold	Corrosion potential (V)	Corrosion current density (µA/cm²)	Corrosion rate (mm /year)
Mg-2Zn	-1.64 ± 0.02	97.60 ± 1.22	2.1065 ± 0.9245
CS coating	-1.59 ± 0.02	18.88 ± 0.63	0.4058 ± 0.0051
Optimal coating	-1.52 ± 0.02	6.71 ± 0.34	0.1442 ± 0.0027

3.6. Thermal Stability Analysis

TGA, DTG, and DTA were used to characterize the thermal properties of GO, CS, and optimal coating (Fig. 7 (a and b)). TGA is considered the most important method for studying the thermal stability of polymers. The peaks represent the weight loss at the specific temperature range. According to the TGA curve, there are three stages in the thermal decomposition of CS. The first stage centered at about 70°C to 100°C due to the moisture content. The second stage begins at 284°C and ends at 330°C, which shows the degradation of leading polymer chains, [46] and three-stage is related to the degradation of the lateral chains. The TGA curve indicates that the significant weight loss is about 17% which is attributed to the degradation of the polymer backbone [46]. Two more peaks centered at 284°C and 333°C for CS coating are related to the initial and final decomposition stages in correlation to DTG and DTA curves. The GO had a weight loss of almost 15% below 200°C from water evaporation and about 40% at 318°C due to the elimination of oxygen-containing functional groups [47]. Here, it can be noted that the DTA maximum temperature order is the same as DTG. In the case of the optimal coating, the second peak at 294°C is related to the decomposition of epoxy and carboxylic groups, which is 10°C higher than the second peak of CS (284°C). Since the optimal coating mainly comprises CS, its thermal decomposition diagram is similar to the CS coating.

Moreover, nanocomposite coating exhibits better thermal stability than GO and CS coatings. The weight loss at the temperature range of 284°C-333°C for CS and 94°C-333°C for CS/2wt% GO is 22% and 17%, respectively. At around 450°C, the remaining weight of GO, CS, and optimal coatings was 54%, 56%, and 61%, respectively. The reason for the low weight loss of the optimal coating is the existence of functional groups on the surface of GO that interacts with the matrix of CS. Also, proper dispersion of GO sheets in the matrix of pure CS plays an essential role in this field. These factors prevent the slip of CS polymer chains during heat treatment in the weight loss test.

3.7. Surface Morphology Of The Optimal Coating

SEM and AFM techniques characterized the morphology of CS and the optimal coating. The SEM micrographs of the CS polymer and the optimal coating are shown in Fig. 8. Porosity is seen in the SEM image of the CS biopolymer (Fig. 8(a)), whereas the CS-2wt% GO optimal coating exhibits a somewhat rough surface and less porosity (Fig. 8(b)). The dispersion and the exfoliation of GO nanosheets in the CS matrix can affect porosity. The pore size of the CS coating is approximately 100–400 µm (Fig. 8(c)). However, the optimal coating is between 50–250 µm (Fig. 8(d)). The suitable porosity is created according to the scaffold requirement for osteoblast cells' growth, adhesion, and proliferation [48].The optimal coating is integral and dense on the surface of the scaffold with a thickness of about 1.67 µm (Fig. 8(e)).

Surface roughness was inspected using AFM. This method possesses a three-dimensional ability allowing the quantification of the surface roughness. This is typically accomplished using the AFM software. Figure 9 (a and c) show three-dimensional AFM scans of the CS and optimal coating surfaces, respectively. The scans were taken on a small coating surface area directly behind the pre-existent roughness. The two-dimensional profiles were quantified using the AFM image processing software. The higher values of the maximum height of the CS and optimal coatings peaks were 0.95 and 1.73 µm, respectively (Fig. 9 (b and d)).

Compared to the CS coating, the surface of the optimal coating was rougher. This is consistent with the findings in the literature indicating that GO nanosheets can disperse in the CS matrix. Here, it is essential to highlight that the nanomaterial coatings with notable enhancement in surface roughness are always favorable for positive utilization in biological investigations [49].

3.8. In Vitro Biocompatibility Of Coatings

The MTT method was further used to quantify the proliferation of MG63 cells cultured onto the Mg-2Zn scaffold, CS, and optimal coating after three days (Fig. 10 (a)). The cell viability percentage on the Mg-2Zn scaffold, CS, and the optimal coating were 83, 95, and 98%, respectively. It is clear that the cells on optimal coating proliferated better than Mg-2Zn and CS coating, mainly due to the simultaneous effect of CS and GO nanosheets.

Suo et al. investigated the enhancement of osseointegration using HA, CS, and GO/CS/HA composite coatings on titanium fabricated by electrophoretic deposition. The results show that HA and CS coating considerably enhanced the bone marrow stromal cell (BMSCs) interactions in vitro. In addition, this GO/CS/HA coating might increment osseointegration in vivo. Consequently, GO/CS/HA may have potential applications in dental implants [50].

The cell morphology of the MG63 cultured cells on Mg-2Zn, CS, and optimal coating after 5 hours was indicated in (Fig. 10 (b, c, and d)). The spindle-shaped cells could be seen on the samples (Zones with red circles). However, the MG63 cells on optimal coating exhibited better stretch with more Filopodia extensions (Zones with blue circles) than the Mg-2Zn and CS coating. The presence of multiple Filopodia expanded coating provides solid proof that the cells are well attached to the optimal coating. Therefore, the optimal coating can be used as a suitable coating for Mg-2Zn in bone tissue engineering.

In this study, the CS/GO nanocomposite coatings with the PED method were fabricated to improve the corrosion properties and biocompatibility of Mg-2Zn porous scaffolds. The Taguchi method was used to explore the effect of GO contents and PED process factors, including (CD, DC, and pH) to optimize the nanocomposite coatings. The nanocomposite coating was obtained under optimal conditions of 2wt% GO, CD = 20 mA/cm², DC = 0.5, and pH = 5. The electrochemical tests indicated that the corrosion rate of the Mg-2Zn, CS, and the optimal coating was calculated as 2.1065, 0.4058, and 0.1442 mm/year, respectively. Also, thermal analysis curves showed that the nanocomposite coatings suggested by Taguchi's design had higher thermal stability than the CS and GO coatings, possibly attributed to the strong H-bonding between CS and GO. In-vitro biocompatibility evaluations using a human osteosarcoma cell line (MG-63 osteoblast) indicated that the optimal nanocomposite coating could supply sufficient support for cell growth and proliferation of MG-63 cells. Moreover, this study recommends that the PED method is an alternative process for fabricating the nHA-CS nanocomposite coating on the Mg-2Zn. This coating can be applied to regenerate bone tissue.

6.1. Competing Interests

The authors declare no conflict of interest.

6.2. Author Contributions Statement

Arash montazeri: wrote the main manuscript text.

Mohammad Ranjbar: Participation in experiments and preparation of materials.

Mahla Sadat Nezhadfard: Participation in experiments and preparation of materials.

Ali Yeganeh Kari: Participation in experiments and preparation of materials.

6.3. Funding

No funds were used in this paper.

6.4. Data Availability Statements

The data that support the findings of this study are not openly available due to privacy and ethical concerns and are available from the corresponding author upon reasonable request.

Song G., and Song S., A Possible Biodegradable Magnesium Implant Material, Adv. Eng. Mater., 2007, vol. 9, pp. 298-302. https://doi.org/10.1002/adem.200600252
Wolf F.I., and Cittadini A., Chemistry and biochemistry of magnesium, Mol. Aspects Med., 2003, vol. 24, pp. 3-9. https://doi.org/10.1016/s0098-2997(02)00087-0
Staiger M.P., Pietak A.M., Huadmai J., and Dias G., Magnesium and its alloys as orthopedic biomaterials: A review, Biomaterials, 2006, vol. 27, pp. 1728-1734. https://doi.org/10.1016/j.biomaterials.2005.10.003
Witte F., Kaese V., Haferkamp H., Switzer E., Meyer-Lindenberg A., Wirth C.J., and Windhagen H., In vivo corrosion of four magnesium alloys and the associated bone response, Biomaterials, 2005, vol. 26, pp. 3557-3563. https://doi.org/10.1016/j.biomaterials.2004.09.049
Li L., Zhang M., Li Y., Zhao J., Qin L., and Lai Y., Corrosion and biocompatibility improvement of magnesium-based alloys as bone implant materials: a review, Regenerative Biomaterials, 2017, vol. 4, pp. 129-137. https://doi.org/10.1093/rb/rbx004
Wu G., Ibrahim J.M., and Chu P.K., Surface design of biodegradable magnesium alloys — A review, Surf. Coat. Technol., 2013, vol. 233, pp. 2-12. https://doi.org/10.1016/j.surfcoat.2012.10.009
Hahn B.-D., Park D.-S., Choi J.-J., Ryu J., Yoon W.-H., Choi J.-H., Kim H.-E., and Kim S.-G., Aerosol deposition of hydroxyapatite–chitosan composite coatings on biodegradable magnesium alloy, Surf. Coat. Technol., 2011, vol. 205, pp. 3112-3118. https://doi.org/10.1016/j.surfcoat.2010.11.029
Solìs Moré Y., Panella G., Fioravanti G., Perrozzi F., Passacantando M., Giansanti F., Ardini M., Ottaviano L., Cimini A., Peniche C., and Ippoliti R., Biocompatibility of composites based on chitosan, apatite, and graphene oxide for tissue applications, Journal of Biomedical Materials Research Part A, 2018, vol. 106, pp. 1585-1594. https://doi.org/10.1002/jbm.a.36361
Hu Q., and Luo Y., Chitosan-based nanocarriers for encapsulation and delivery of curcumin: A review, Int. J. Biol. Macromol., 2021, vol. 179, pp. 125-135. https://doi.org/10.1016/j.ijbiomac.2021.02.216
Xue J., Feng C., Xia L., Zhai D., Ma B., Wang X., Fang B., Chang J., and Wu C., Assembly Preparation of Multilayered Biomaterials with High Mechanical Strength and Bone-Forming Bioactivity, Chem. Mater., 2018, vol. 30, pp. 4646-4657. https://doi.org/10.1021/acs.chemmater.8b01272
Vlasceanu G.M., Șelaru A., Dinescu S., Balta C., Herman H., Gharbia S., Hermenean A., Ionita M., and Costache M., Comprehensive Appraisal of Graphene–Oxide Ratio in Porous Biopolymer Hybrids Targeting Bone-Tissue Regeneration, Nanomaterials, 2020, vol. 10, pp. 1444. https://doi.org/10.3390/nano10081444
Montazeri A., Saeedi F., Bahari Y., and Ahmadi Daryakenari A., Preclinical assessment of chitosan–polyvinyl alcohol–graphene oxide nanocomposite scaffolds as a wound dressing, Polym. Polym. Compos., 2021, vol. 29, pp. S926-S936. https://doi.org/10.1177/09673911211029242
Jiang L., Chen D., Wang Z., Zhang Z., Xia Y., Xue H., and Liu Y., Preparation of an Electrically Conductive Graphene Oxide/Chitosan Scaffold for Cardiac Tissue Engineering, Applied Biochemistry and Biotechnology, 2019, vol. 188, pp. 952-964. https://doi.org/10.1007/s12010-019-02967-6
Arnaldi P., Carosio F., Di Lisa D., Muzzi L., Monticelli O., and Pastorino L., Assembly of chitosan-graphite oxide nanoplatelets core shell microparticles for advanced 3D scaffolds supporting neuronal networks growth, Colloids Surf. B. Biointerfaces, 2020, vol. 196, pp. 111295. https://doi.org/10.1016/j.colsurfb.2020.111295
Liu X., Wu Y., Zhao X., and Wang Z., Fabrication and applications of bioactive chitosan-based organic-inorganic hybrid materials: A review, Carbohydr. Polym., 2021, vol. 267, pp. 118179. https://doi.org/10.1016/j.carbpol.2021.118179
Francolini I., Perugini E., Silvestro I., Lopreiato M., Scotto d’Abusco A., Valentini F., Placidi E., Arciprete F., Martinelli A., and Piozzi A., Graphene Oxide Oxygen Content Affects Physical and Biological Properties of Scaffolds Based on Chitosan/Graphene Oxide Conjugates, Materials, 2019, vol. 12, pp. 1142. https://doi.org/10.3390/ma12071142
Nayak T.R., Andersen H., Makam V.S., Khaw C., Bae S., Xu X., Ee P.-L.R., Ahn J.-H., Hong B.H., Pastorin G., and Özyilmaz B., Graphene for Controlled and Accelerated Osteogenic Differentiation of Human Mesenchymal Stem Cells, ACS Nano, 2011, vol. 5, pp. 4670-4678. https://doi.org/10.1021/nn200500h
Bressan E., Ferroni L., Gardin C., Sbricoli L., Gobbato L., Ludovichetti F.S., Tocco I., Carraro A., Piattelli A., and Zavan B., Graphene based scaffolds effects on stem cells commitment, J. Transl. Med., 2014, vol. 12, pp. 296. https://doi.org/10.1186/s12967-014-0296-9
Feng W., and Wang Z., Biomedical applications of chitosan-graphene oxide nanocomposites, iScience, 2022, vol. 25, pp. 103629. https://doi.org/10.1016/j.isci.2021.103629
Hermenean A., Codreanu A., Herman H., Balta C., Rosu M., Mihali C.V., Ivan A., Dinescu S., Ionita M., and Costache M., Chitosan-Graphene Oxide 3D scaffolds as Promising Tools for Bone Regeneration in Critical-Size Mouse Calvarial Defects, Sci. Rep., 2017, vol. 7, pp. 16641. https://doi.org/10.1038/s41598-017-16599-5
Qiu X., Wan P., Tan L., Fan X., and Yang K., Preliminary research on a novel bioactive silicon doped calcium phosphate coating on AZ31 magnesium alloy via electrodeposition, Materials Science and Engineering: C, 2014, vol. 36, pp. 65-76. https://doi.org/10.1016/j.msec.2013.11.041
Li L.-Y., Cui L.-Y., Zeng R.-C., Li S.-Q., Chen X.-B., Zheng Y., and Kannan M.B., Advances in functionalized polymer coatings on biodegradable magnesium alloys – A review, Acta Biomater., 2018, vol. 79, pp. 23-36. https://doi.org/10.1016/j.actbio.2018.08.030
Hornberger H., Virtanen S., and Boccaccini A.R., Biomedical coatings on magnesium alloys – A review, Acta Biomater., 2012, vol. 8, pp. 2442-2455. https://doi.org/10.1016/j.actbio.2012.04.012
Zhang J., Dai C., Wei J., Wen Z., Zhang S., and Chen C., Degradable behavior and bioactivity of micro-arc oxidized AZ91D Mg alloy with calcium phosphate/chitosan composite coating in m-SBF, Colloids Surf. B. Biointerfaces, 2013, vol. 111, pp. 179-187. https://doi.org/10.1016/j.colsurfb.2013.05.040
Park K.H., Kim S.-J., Hwang M.-J., Song H.-J., and Park Y.-J., Pulse electrodeposition of hydroxyapatite/chitosan coatings on titanium substrate for dental implant, Colloid. Polym. Sci., 2017, vol. 295, pp. 1843-1849. https://doi.org/10.1007/s00396-017-4166-x
Wang H.X., Guan S.K., Wang X., Ren C.X., and Wang L.G., In vitro degradation and mechanical integrity of Mg–Zn–Ca alloy coated with Ca-deficient hydroxyapatite by the pulse electrodeposition process, Acta Biomater., 2010, vol. 6, pp. 1743-1748. https://doi.org/10.1016/j.actbio.2009.12.009
Gheysour M., and Faghihi S., Biologically Modified Titanium Substrates for Improved Surface Bioactivity, Journal of Applied Biotechnology Reports, 2017, vol. 4, pp. 625-631
Karimi N., Kharaziha M., and Raeissi K., Electrophoretic deposition of chitosan reinforced graphene oxide-hydroxyapatite on the anodized titanium to improve biological and electrochemical characteristics, Materials Science and Engineering: C, 2019, vol. 98, pp. 140-152. https://doi.org/10.1016/j.msec.2018.12.136
Ordikhani F., Ramezani Farani M., Dehghani M., Tamjid E., and Simchi A., Physicochemical and biological properties of electrodeposited graphene oxide/chitosan films with drug-eluting capacity, Carbon, 2015, vol. 84, pp. 91-102. https://doi.org/10.1016/j.carbon.2014.11.052
Seyedraoufi Z.S., and Mirdamadi S., Effects of pulse electrodeposition parameters and alkali treatment on the properties of nano hydroxyapatite coating on porous Mg–Zn scaffold for bone tissue engineering application, Materials Chemistry and Physics, 2014, vol. 148, pp. 519-527. https://doi.org/10.1016/j.matchemphys.2014.06.067
Seyedraoufi Z.S., and Mirdamadi S., In vitro biodegradability and biocompatibility of porous Mg-Zn scaffolds coated with nano hydroxyapatite via pulse electrodeposition, Transactions of Nonferrous Metals Society of China, 2015, vol. 25, pp. 4018-4027. https://doi.org/10.1016/S1003-6326(15)64051-1
Hamghavandi M.R., Montazeri A., Ahmadi Daryakenari A., and Pishvaei M., Preparation and characterization of chitosan/graphene oxide nanocomposite coatings on Mg–2 wt% Zn scaffold by pulse electrodeposition process, Biomedical Materials, 2021, vol. 16, pp. 065005. https://doi.org/10.1088/1748-605x/ac1f9f
Rosa J.L., Robin A., Silva M.B., Baldan C.A., and Peres M.P., Electrodeposition of copper on titanium wires: Taguchi experimental design approach, J. Mater. Process. Technol., 2009, vol. 209, pp. 1181-1188. https://doi.org/10.1016/j.jmatprotec.2008.03.021
Rao R.S., Prakasham R.S., Prasad K.K., Rajesham S., Sarma P.N., and Rao L.V., Xylitol production by Candida sp.: parameter optimization using Taguchi approach, Process Biochem., 2004, vol. 39, pp. 951-956. https://doi.org/10.1016/S0032-9592(03)00207-3
Rao R.S., Kumar C.G., Prakasham R.S., and Hobbs P.J., The Taguchi methodology as a statistical tool for biotechnological applications: A critical appraisal, Biotechnol. J., 2008, vol. 3, pp. 510-523. https://doi.org/10.1002/biot.200700201
Lawal D., Bin Ali A., and Mohammed A.S., Tribological investigations of carbon nanotube-reinforced polymer (UHMWPE) nanocomposites using Taguchi methodology, J. Appl. Polym. Sci., 2016, vol. 133, pp. https://doi.org/10.1002/app.44018
Biological evaluation of medical devices. International Continence Society, 2009.
Yan T., Zhang H., Huang D., Feng S., Fujita M., and Gao X.-D., Chitosan-Functionalized Graphene Oxide as a Potential Immunoadjuvant, Nanomaterials, 2017, vol. 7, pp. 59. https://doi.org/10.3390/nano7030059
Pati M.K., Pattojoshi P., and Roy G.S., Synthesis of Graphene-Based Nanocomposite and Investigations of Its Thermal and Electrical Properties, Journal of Nanotechnology, 2016, vol. 2016, pp. 5135420. https://doi.org/10.1155/2016/5135420
Zuo P.-P., Feng H.-F., Xu Z.-Z., Zhang L.-F., Zhang Y.-L., Xia W., and Zhang W.-Q., Fabrication of biocompatible and mechanically reinforced graphene oxide-chitosan nanocomposite films, Chem. Cent. J., 2013, vol. 7, pp. 39. https://doi.org/10.1186/1752-153X-7-39
Pan Y., Bao H., and Li L., Noncovalently Functionalized Multiwalled Carbon Nanotubes by Chitosan-Grafted Reduced Graphene Oxide and Their Synergistic Reinforcing Effects in Chitosan Films, ACS Applied Materials & Interfaces, 2011, vol. 3, pp. 4819-4830. https://doi.org/10.1021/am2013135
Rajesh R., Sujanthi E., Senthil Kumar S., and Venkatesan R., Designing versatile heterogeneous catalysts based on Ag and Au nanoparticles decorated on chitosan functionalized graphene oxide, Physical Chemistry Chemical Physics, 2015, vol. 17, pp. 11329-11340. https://doi.org/10.1039/C5CP00682A
Rana V.K., Choi M.-C., Kong J.-Y., Kim G.Y., Kim M.J., Kim S.-H., Mishra S., Singh R.P., and Ha C.-S., Synthesis and Drug-Delivery Behavior of Chitosan-Functionalized Graphene Oxide Hybrid Nanosheets, Macromolecular Materials and Engineering, 2011, vol. 296, pp. 131-140. https://doi.org/10.1002/mame.201000307
Liu L., Li C., Bao C., Jia Q., Xiao P., Liu X., and Zhang Q., Preparation and characterization of chitosan/graphene oxide composites for the adsorption of Au(III) and Pd(II), Talanta, 2012, vol. 93, pp. 350-357. https://doi.org/10.1016/j.talanta.2012.02.051
Dinescu S., Ionita M., Pandele A.M., Galateanu B., Iovu H., Ardelean A., Costache M., and Hermenean A., In vitro cytocompatibility evaluation of chitosan/graphene oxide 3D scaffold composites designed for bone tissue engineering, Bio-Medical Materials and Engineering, 2014, vol. 24, pp. 2249-2256. https://doi.org/10.3233/BME-141037
Bonilla J., Fortunati E., Atarés L., Chiralt A., and Kenny J.M., Physical, structural and antimicrobial properties of poly vinyl alcohol–chitosan biodegradable films, Food Hydrocolloids, 2014, vol. 35, pp. 463-470. https://doi.org/10.1016/j.foodhyd.2013.07.002
Travlou N.A., Kyzas G.Z., Lazaridis N.K., and Deliyanni E.A., Graphite oxide/chitosan composite for reactive dye removal, Chem. Eng. J., 2013, vol. 217, pp. 256-265. https://doi.org/10.1016/j.cej.2012.12.008
Lock J., Nguyen T.Y., and Liu H., Nanophase hydroxyapatite and poly(lactide-co-glycolide) composites promote human mesenchymal stem cell adhesion and osteogenic differentiation in vitro, J. Mater. Sci. Mater. Med., 2012, vol. 23, pp. 2543-2552. https://doi.org/10.1007/s10856-012-4709-0
Nagarajan S., Mohana M., Sudhagar P., Raman V., Nishimura T., Kim S., Kang Y.S., and Rajendran N., Nanocomposite Coatings on Biomedical Grade Stainless Steel for Improved Corrosion Resistance and Biocompatibility, ACS Applied Materials & Interfaces, 2012, vol. 4, pp. 5134-5141. https://doi.org/10.1021/am301559r
Dhivya S., Saravanan S., Sastry T.P., and Selvamurugan N., Nanohydroxyapatite-reinforced chitosan composite hydrogel for bone tissue repair in vitro and in vivo, Journal of Nanobiotechnology, 2015, vol. 13, pp. 40. https://doi.org/10.1186/s12951-015-0099-z

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Chitosan/Graphene Oxide Nanocomposite Coatings on Mg alloy: Corrosion and Biocompatibility Properties

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Abstract

Figures

1. Introduction

2. Material And Methods

2.1. Materials

2.2. Fabrication Of Scaffolds

2.3. Coating Process

2.4. Characterization

2.5. Taguchi Method

3. Results And Discussion

3.1. Particle size distribution and zeta potential

3.2. Characteristics Of The Nanocomposite Coatings

3.3. Determination Of The Optimal Coating By Taguchi Method

3.4. Determination Of The Highest Corrosion Resistance Of Cs/go Coatings By The Taguchi Method

3.5. Evaluation Of Corrosion Resistance Of The Optimal Coating

3.6. Thermal Stability Analysis

3.7. Surface Morphology Of The Optimal Coating

3.8. In Vitro Biocompatibility Of Coatings

4. Conclusions

Declarations

References

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