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Accepted ManuscriptOrthopaedicbioactiveglass/chitosancompositescoated316Lstainless steel by green electrophoretic co-depositionZainabM.Al-Rashidy,M.M.Farag,N.A.AbdelGhany,A.M.Ibrahim, Wafa I. Abdel-FattahPII:S0257-8972(17)31199-4DOI:doi:10.1016/j.surfcoat.2017.11.052Reference:SCT 22900To appear in:Surface & Coatings TechnologyReceived date:1 August 2017Revised date:23 October 2017Accepted date:18 November 2017Pleasecitethisarticleas:ZainabM.Al-Rashidy,M.M.Farag,N.A.AbdelGhany,A.M.Ibrahim,WafaI.Abdel-Fattah,Orthopaedicbioactiveglass/chitosancompositescoated316Lstainlesssteelbygreenelectrophoreticco-deposition.Theaddressforthecorrespondingauthorwascapturedasaffiliationforallauthors.Pleasecheckifappropriate. Sct(2017), doi:10.1016/j.surfcoat.2017.11.052ThisisaPDFfileofanuneditedmanuscriptthathasbeenacceptedforpublication.Asaservicetoourcustomersweareprovidingthisearlyversionofthemanuscript.Themanuscriptwillundergocopyediting,typesetting,andreviewoftheresultingproofbeforeitispublishedinitsfinalform.Pleasenotethatduringtheproductionprocesserrorsmaybediscoveredwhichcouldaffectthecontent,andalllegaldisclaimersthatapplytothejournal pertain.
ACCEPTED MANUSCRIPT1 Orthopaedic bioactive glass/chitosan composites coated 316L stainless steel by green electrophoretic co-deposition Zainab M. Al-Rashidy1, M. M. Farag2, N. A. Abdel Ghany3*, A. M. Ibrahim4, and Wafa I. Abdel-Fattah1* 1-Refractories and Ceramics Dept., National Research Centre, Dokki, Cairo, Egypt 2- Glass Research Dept., National Research Centre, Dokki, Cairo, Egypt 3-Physical Chemistry Dept., National Research Centre, Dokki, Cairo, Egypt 4-University College for Girls, Ain Shams University, Cairo, Egypt *Corresponding authors: N. A. Abdel Ghany Physical Chemistry Dept., National Research Centre, Dokki, 12622, Cairo, Egypt na_manakhly@yahoo.co.uk, na.abdelghany@nrc.sci.eg Wafa I. Abdel-Fattah, Postal address: National Research Centre, Refractories and Ceramics Dept, Tahrir St.,33 El Bohouth St.(former El-Tahrir St.) Dokki, POB 12622, Cairo, Egypt. E-mail: nrcfifi@yahoo.com Co-authors: Z. M. Al-Rashidy Refractories and Ceramics Dept., National Research Centre, Dokki, 12622, Cairo, Egypt zmalrashidy@gmail.com M. M. Farag Glass Research Dept., National Research Centre, Dokki, 12622, Cairo, Egypt mmfarag_nrc@yahoo.com A.M. Ibrahim University College for Girls, Ain Shams University, Cairo, Egypt alaa.ellaban@gmail.com ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT2 Graphical abstract ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT3 Abstract Single-step electrophoretic co-deposition (EPD) technique was used to enhance the bioactivity of the 316L stainless steel (316L SS) surface by bioactive glass/chitosan composite coatings. Two modified glass compositions of Bioglass? by the addition of boron were utilized to prepare such composite layers, and the Bioglass? was synthesized for comparison. Different EPD coating factors were studied. Namely, voltage, time, glass powder, and chitosan concentrations, to obtain crack-free and good adhesive coatings. The achieved surfaces were characterized by SEM/EDX, TGA, XRD and FTIR and their wettability and roughness were investigated. The in vitro bioactivity of the coated metals was compared in simulated body fluid (SBF) and Dulbecco’s modified eagle medium (DMEM) for up to 15 days. The electrochemical corrosion behavior of the coated metals was evaluated using potentiodynamic polarisation and impedance techniques in the two biological solutions SBF and DMEM at 37 ?C. The results showed that the achieved coatings were uniform, homogenous and crack-free with desirable thickness. Moreover, the coating layers were exhibited by very high wettability, and their roughness values ranged from 170 ? 18 to 234 ? 17 ?m. The in vitro bioactivities proved that the coating layers represented a better ability to form hydroxyapatite crystals on their surfaces in SBF than in DMEM. The corrosion resistance of 316L SS was improved by the bioactive composite coatings. Keywords: glass/chitosan, 316L SS, orthopedic, coating, electrophoretic, corrosion ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT4 1. Introduction Interest significantly increased in the field of coating of metallic implants by bioactive materials. Widely used implants for orthopedic purposes are made mainly of metal alloys, such as stainless steel and titanium alloys. Importantly, 316L stainless steel has been used extensively in the orthopedic field.It is due to its high mechanical strength and low price. Despite the advantages of such metal alloy, it has a shortage in implantation field. Some toxic ions may be released into the body from the metal surface, and the metal is likely encapsulated by fibrous tissue [1, 2], which could lead to the movement and loosening of the metal implant [1, 3]. Nevertheless, a coating of the metal implant surface with bioactive materials has been applied as a practical solution for these problems. Such bioactive layers are characterized by their ability to induce osteointegration of the metal surface, as well as, decreasing or even suppressing the ions released from the metal. There have been numerous bioactive materials used as coatings for metal implants, such as hydroxyapatite (HA) [4-6], bioactive glass and glass-ceramic [7-10] and composite materials based on biopolymers and ceramic materials [11-14]. Recently, composite inorganic-organic materials have attracted the interest in the orthopedic field. Such materials mimic the bone structure which is composed of collagen as organic phase and nano-HA crystals as the inorganic phase [15]. Therefore, the composite coatings based on biopolymer (as the organic phase, e.g., alginate and chitosan) and ceramic particles (as inorganic phase, e.g., HA and bioactive glasses) combine the bioactivity of the ceramic component and the mechanical properties of the polymer [16]. Furthermore, active biomolecules, such as antibiotics, proteins, and enzymes can be included throughout the polymer matrix during the coating process [16, 17]. Moreover, a room temperature coating process can be achieved by the use of a polymer phase. Bioactive glasses are an essential class of material for biomedical applications. They can form a chemical bond with surrounding tissue via the formation of HA layer. The bioactive glass was initially discovered by Hench and coworkers in the late 1960s and early 1970s, which was encoded later as 45S5 or Bioglass? [18]. A new research field for using glasses as implants and bone tissue engineering applications was established. After Hench`s glass, other types of bioactive glasses were developed and used in different biomedical applications. Such glasses were widely utilized as bioactive coatings for metal implants. Depending on preparation method, two types of bioactive glass used as a bioactive coating of different metal surfaces; melt-derived ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT5 bioactive glasses [8, 19-21] and sol-gel-derived bioactive glasses [22-27]. Nevertheless, the incorporation of boron in bioactive glass compositions proved to enhance the bioactivity and biocompatibility of the glass. Boron showed a potential effect on stimulation angiogenesis in vivo [28], as well as, it proved that it enhanced the growth of bone tissue [29, 30]. Moreover, it was confirmed that boron compounds demonstrated powerful anti-inflammatory effect with minimum side effects [31]. These anti-inflammatory influences come from the defeat of serine proteases that are released by inflammation-activated white blood cells. As well as, boron-containing compounds can reduce the reactive oxygen species which produced in the course of neutrophils respiratory burst along with T-cell activity [29, 31]. Consequently, the incorporation of boron in bioactive glasses is of particular interest for biomedical applications. Biopolymers showed a substantial impact on the enhancement of composite coatings properties. Different types of biopolymers were used with bioactive glass particles for coating purpose of metal implants. E.g., polyetheretherketone polymer utilized with 45S5 glass as a composite coating on shape memory alloy (NiTi, Nitinol?) [32]. Furthermore, bioactive glass-alginate composite coatings have been investigated for metal coating application [33]. Moreover, different composite layers based on bioactive glass particles and chitosan have been studied [25, 34-38]. Chitosan has been widely used as an organic component of organic-inorganic composite coatings. It is a natural cationic polysaccharide polymer derived from chitin, which is the second most abundant natural polymer after cellulose. It has numerous advantages to be used in different biomedical applications, such as its antibacterial properties [39]. Additionally, it represented cytocompatibility with varying types of cells [40]. Moreover, desirable mechanical strength, biocompatibility, and biodegradability of chitosan are beneficially controllable [41]. The process of organic-inorganic coatings is a room temperature technique. Following this concept electrophoretic deposition (EPD) in the aqueous electrolyte is considered a superior and green route in this regard. It is characterized by its simplicity being cost-effectiveness with short processing time and achievement of uniform coatings with the possibility for a coat of complex shapes. The EPD process is carried out by passing an electric current between two conductive electrodes immersed in a colloidal suspension. Under an electric field, the particles are charged and moved to the oppositely charged electrode to deposit and co-deposit achieving an adhesive layer on the surface of such electrode [42-45]. EPD technique has been successfully ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT6 used to produce composite coatings based on bioactive glass and biopolymers for orthopedic and dental application [32, 34-36, 46]. The present work aimed to prepare composite coatings for 316L SS based on chitosan and new glass compositions using the simple and cost-effective eco-friendly EPD method. The new glasses contained boron, was prepared. Nevertheless, it was targeted to tailor different properties of the suggested composite coatings by changing glass compositions and glass to polymer ratio. Moreover, the surfaces acquired the advantage of chitosan, such as antimicrobial activity, enhancement of the coating adhesion on the metal. Finally, the electrochemical tests of different coated metallic substrates were performed to determine the corrosion behavior of such samples. 2. Experimental procedures 2.1. Materials The following chemicals with high purity were used in the present work. Chitosan with high molecular weight, 310-375 kDa (Sigma-Aldrich), acetic acid (98%) (Acros Organics, Belgium), SiO2 (99.8%, quartz sand, Egypt), Na2CO3 (99.5%, ADWIC, Egypt), CaCO3 (98.5%, BDH, UK), H3BO3 (99.5%, ADWIA, Egypt) and NH4H2PO4 (ARABLAB Company, UAE) to fabricate glasses and composites coatings. 2.2. Synthesis of bioactive glass Table (1) represents glass compositions used in this work in weight percents. Bioglass? (45S5) was synthesized in parallel for comparison. High purity chemicals, SiO2, Na2CO3, CaCO3, H3BO3, and NH4H2PO4, were used as glass precursors. Glass batch components were mixed well to ensure powder homogeneity. To get rid of the gases resulting from heating the glass components at a temperature above 600 ?C the mixed powders were transformed to a platinum crucible and heated to 1000 ?C in a muffle furnace for one h. After that, the glass batch was melted at temperature range 1350-1450 ?C. The molten glass was poured into water to achieve glass frits. These frits were ground in a ball mill and sieved to get particle sizes less than 63 ?m. Moreover, the particle size distribution and zeta potential were evaluated for the achieved glass grains in an aqueous medium using dynamic light scattering analysis (Nicomp N3000 ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT7 particle size analyzer, Santa Barbara, Calif., USA). The particles were dispersed in the water by sonication probe before the measurement. Table 1. Three different glass compositions (wt%) used in this study SiO2 Na2O CaO B2O3 P2O5 40S 40.0 25.0 20.0 5.0 10.0 HB5 40.0 24.5 24.5 5.0 6.0 H 45.0 24.5 24.5 – 6.0 2.3. EPD coating process A high molecular weight chitosan polymer (0.5, 1.0 or 1.5 g) was dissolved in 1000 ml of 1% acetic acid (98%) solution for one day at room temperature. After complete dissolution of the polymer, glass particles were suspended in the solution by stirring for two hours and sonication (WiseClean, WUC-D03H) for 15 min. Additionally, pH of suspensions was measured. Stainless steel 316L electrode plates (15 x 10 x 0.2 mm) were polished by 600 and 1200 silicon carbide paper and washed with distilled water and acetone in an ultrasonic bath for 15 min and dried immediately before the electrophoretic coating process. The two electrode coating cell was composed of stainless steel anode and cathode with 1 cm distance with subjected coating area of 10 x 10 mm. Then the two electrodes were immersed in the glass suspension, and an electric voltage was applied by DC power supply (GW Model No: GPC-3060). The samples were carefully and slowly pulled out from the suspension after EPD. Finally, the samples were dried at least for 24 h at ambient conditions. Different parameters were applied to achieve the optimum conditions for the electrophoretic deposition process. Such parameters for coating were applied voltages (20, 30, 40 and 50 V), time (3, 5, 7, 10 and 15 min.), glass concentrations (2, 4, 6 and 8 g/l), and chitosan concentrations (0.5, 1 and 1.5 g/l). The wt% of the polymer in the composite coating can be calculated from the following equation (1): Polymer wt% = Wp * 100/(Wp+Wg) (1) Where, Wp and Wg are the weight of polymer and glass, respectively. 2.3.1. Coating Characterization ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT8 Fourier transform infrared spectroscopy (model FT/IR-6100 type A) was used at room temperature (~ 20 ?C) within the wave number range of 4000??400 cm-1 at a resolution of 2 cm-1. The functional groups and chemical structure of the prepared borate glass particles and coatings were determined. The thermal behavior of the borate glass was characterized by thermo-gravimetric analysis (TGA) under the nitrogen atmosphere using DSC-TGA instrument model STD Q600 at a heating rate of 10 ?C/min. The surface morphologies and compositions were determined using scanning electron microscopy coupled with Energy Dispersive X-ray analysis (SEM/EDX) (model JEOL JXA-840A, Electron probe microanalyzer). X-ray diffractometer (model, BRUKER as, D8 ADVANCE) was used to study the crystal structure of as-prepared glass and glass coatings using Ni-filtered Cu radiation with a tube voltage of 40 kV. A current of 25 mA was utilized. The wettability of the achieved borate glass coatings was evaluated by measuring the contact angle using horizontal plate camera perpendicular to liquid droplet plane using Compact Video Microscope (CVM), manufactured by SDL-UK. About 250 ?l of distilled water was used as a liquid for measuring the wettability according to ASTM D724-99 and ASTM D5946-96 method. Furthermore, the determination of the surface roughness was carried out by Elcometer 224 surface profile meter, UK. 2.4. In vitro degradation test 2.4.1. Biological fluids In vitro degradation tests of the glass-coated substrates were followed in SBF solution which prepared according to Kokubo and Takadama protocol [47] and DMEM solution by measuring the ionic concentrations of the released species from glass coatings and stainless steel substrates. The boron, calcium, phosphorus, silicon, and iron ions along with the total protein levels released into the immersion liquids were measured. The predetermined times of 3, 6, 24, 72, 168 and 336 h using either ICP (model, Agilent5100 Synchronous Vertical Dual View (SVDV)) or colorimetric kits (BIODIGNOSTIC, Egypt)were followed. Ionic boron and silicon concentrations were measured by ICP, and the other ones were measured by colorimetric kits. The pH of incubated solutions was also measured. 2.4.2. Inorganic ionic concentrations ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT9 Calcium ions react with methyl thymol, in an alkaline medium to form colored compound (blue color) that absorbed at λ = 585 nm. Inorganic phosphorus reacts with molybdic acid to form phosphomolybdate complex (blue color) that is absorbed at λ = 640 nm. The iron is dissociated by hydrochloric acid then reduced to ferrous by thioglycolic acid. The colored compound which the metal forms with bathophenanthroline was measured colorimetrically at 535 nm. The concentrations of calcium, phosphorus, and iron were calculated by applying the following equation: Concn. = (Asample ̸ Astandard) x concn. of standard Where, concentrations of Ca and P ions were in mmol/l and concentration of Fe ion was in ?mol/l. The protein molecule when treated with an alkaline cupric sulphate, form a violet colour that absorbs at 550 nm. The concentration of protein is calculated by the following equation: Concn. = (Asample ̸ Astandard) x 5 g/dl 2.4.3. Corrosion behaviour evaluation The electrochemical behavior of both the uncoated and coated substrates in SBF and DMEM solutions were investigated by potentiodynamic polarisation test, and electrochemical impedance spectroscopy (EIS). Before each test, the specimens surface area of the working electrode that contact with the electrolyte was cleaned and the working area was measured. The electrochemical three-electrode cell was used for in vitro potentiodynamic corrosion tests. The platinum sheet was used as a counter electrode and saturated calomel electrode (SCE) as a reference electrode. SBF and DMEM solutions were used as an electrolyte to evaluate and compare the corrosion behavior of 316L SS with and without composite coating. The potentiodynamic polarization experiments were run; where each of 316L SS substrate and composite coated 316L SS samples were immersed for 15 min in physiological solutions at the temperature of 37 ??C before measurement to attain the steady state potential. Potentiodynamic polarisation curves were performed using Autolab 302N electrochemical workstation (Metrohm). Dynamic polarisation curves were recorded at a potential scanning rate of 1 mV/S, at open air. The reversed current density for cyclic polarisation was one mA/cm2. The anodic and cathodic polarisation curves were obtained for each specimen starting from steady-state potential. The corrosion current density, corrosion rate and corrosion potential ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT10 of various samples (316L SS substrate and composite coated 316L SS samples) were determined by Tafel extrapolation. For reducibility, five sets of the uncoated and coated samples obtained under the optimum coating parameters were measured. The electrochemical impedance spectroscopy (EIS) data were recorded from 20 kHz to 0.01 Hz with a ten mV sinusoidal perturbing signal at open-circuit potential. To reduce the time and potential noise interference the lowest frequency was set to 0.01 Hz. The EIS measurement may be affected by phase shifts from the potentiostat in the high-frequency region, and so the upper-frequency limit was set at 20 kHz. In this method, the samples were immersed in SBF and DMEM solutions at 37 ? one ?C. In the analysis of the impedance data, the appropriate equivalent circuit EC which did not only match the physical structure of measured electrode system but was also able to produce similarly proposed spectrum diagrams for the examined samples. 2.5. Statistical analysis All experimental data stated in this work were expressed as the average ? standard deviation (SD) for n = 3 and were analyzed using standard analysis of Students t-test. The level of significance (P value) is set at < 0.05. 3. Results and discussion 3.1. Glass characterization Figure 1a represents XRD patterns of the as-casted three glass samples (the 40 S, Ws, HB5, and H). The figure shows no diffraction peaks for all the samples, which proved their amorphous structure. Moreover, the particle sized distribution analysis was carried out for glass particles used in EPD coating process which showed in Figure 1b. The figure showed a monomodal, and narrow particle size distribution for all glasses ranged from 0.3 ?m to 10 ?m. Three cumulative % points (D10, D50, and D90) were used herein to represent the extremes and the mean particle sizes (Table 2). The size diameters, D10 of 0.47, 0.49 and 0.44 ?m, and D90 of 2.63, 1.77 and 2.10 ?m were measured for the 40s, HB5 and H samples, respectively. Moreover, D50 was represented the mean particle size distribution were measured for the 40s, HB5 and H samples as 1.37, 1.10 and 1.20 ?m, respectively. On the other hand, the zeta potential of different glass particles was measured in an aqueous medium (Table 2). The results showed that H glass ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT11 (Bioglass?) particles demonstrated higher negatively charged particles (-41.13 mV) than the 40s (-18.63 mV) and HB5 (-24.78 mV) glass particles in an aqueous medium. These likely attributed to higher SiO2 wt% in H glass and consequently higher number of silanol groups which created in aqueous solution on the glass surface as a result of non-bridging oxygens hydration of Si-O groups in glass network. Table 2. Different cumulative % points of particle size (?m) and zeta potential (mV) of different glasses Cumulative % point of diameter (?m) Zeta potential (mV) D10 D50 D90 40S 0.47 1.37 2.63 -18.63 HB5 0.49 1.10 1.77 -24.78 H 0.44 1.20 2.10 -41.13 Figure 1. XRD patterns (a) and particle size distribution (b) of as-prepared glass particle (40S, HB5 and H). 3.2. Effect of different coating parameters The chitosan solutions containing glass particles were deposited on the cathode electrode. As chitosan polymer is dissolved in an acidic medium, it becomes a cationic polyelectrolyte at pH < 5 because it can be protonated and so it gains a positive charge as shown in Figure 2 [48]. As mentioned before from zeta potential measurements, all glass particles showed negatively charged particles in the aqueous medium. Therefore, attraction forces can be created between ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT12 these negatively charged glass particles and positively charged chitosan, and consequently, each glass particle was likely surrounded by chitosan layer and formed the stable suspension. Figure 2. Protonation of chitosan polymer in acidic medium A variety of different factors of the coating process did not change the deposited electrode. Therefore, the deposition of composite coating depended mainly on the chitosan polymer that was because the chitosan shifted the zeta potential of glass particles to the positive value. These results agreed with the previous study [49]. Nevertheless, several coating conditions of voltage, coating time, glass, and polymer concentrations were examined to achieve good adhesive, homogeneous and crack free coatings with desirable thickness. The results showed that the composite layers were produced successfully at optimum conditions of; 20 V, deposition time of 5 min, 0.5 g/l polymer concentration and 6 g/l glass concentration. Any lower values of these parameters produced inhomogeneous and thin layers, while, higher ones resulted in thick and bad-adhesive, extraordinarily porous and easily cracked coatings. On the other hand, pH values measured for chitosan solution and glass/chitosan suspension were 3.43 for chitosan solution, 4.77, 4.75 and 4.67 for suspensions contained the 40s, HB5 and H glass particles, respectively. Therefore, different glass suspensions possessed pH 40S > HB5, which likely attributed to the water adsorption affinity of each glass type. Moreover, the recorded weight loss of the composite coatings can be ascribed to the evaporation of the adsorbed water molecules on the coatings, as well as, the thermal decomposition of chitosan backbone. It can be observed from the figure that the weight loss occurred in two steps for 40S (at temperature ranges; 25-230 ?C and 230-550 ?C) composite sample, while it occurred in four steps for the HB5 (at temperature ranges; 25-146 ?C, 146-228 ?C, 228-650 ?C and 650-700 ?C) and H (at temperature ranges; 25-146 ?C, 146-230 ?C, 230-580 ?C and 680-640 ?C) composites. The total weight loss for the 40S, HB5 and H, was 28.5%, 25% and 31.5%, respectively. Consequently, the glass content was 71.5%, 75% and 68.5%, respectively. Interestingly, the weight loss order of composite coatings was the same order for the corresponding glasses. However, the experimental weight losses were ranged from 25% to 31.5% which were higher than the theoretical starting chitosan polymer (~7.7 %) calculated according to Equation (1). This difference might be originated from the evaporation of water molecules adsorbed on the glass particle surfaces and that incorporated into the chitosan polymer. From the other side, during the EPD process, precipitation of glass particles was occurred to some degree, which affected the final glass-to-polymer weight ratio. ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT14 Figure 3.Thermogravimetric analysis (TGA) of as-prepared glass and the derived composite coatings (40S, HB5 and H samples). 3.4. SEM-EDX analysis of coated substrates Figure 4 represents SEM micrographs of the metal coated surface (upper), its cross section (middle) and EDX analysis (lower) of the 40s, HB5 and H composite coating samples. The figure for all composite coatings showed that the glass particles were dispersed homogeneously within the chitosan matrix. Moreover, suitable adhesive and crack-free layers are demonstrated. Nevertheless, the composite coating thicknesses measured from SEM micrographs varied according to the type of glass filler. H sample showed larger coating thickness (246 ? 47 ?m) than HB5 (105 ? 16 ?m) and 40S (143 ? 33 ?m) samples. Again, these differences in the coating thicknesses were likely assigned to the difference of zeta potential of the three glass powders in an aqueous medium. A higher negative charge of H glass can be prospective formed a more stable suspension which achieved optimal conditions for depositing a thicker layer. Moreover, high roughness morphologies of the coating surfaces were recorded from the cross-section of the samples and high standard deviation values of thickness measurements. On the ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT15 other hand, EDX analyses of different coating surfaces showed the same elemental components of the glass particles. Figure 4. SEM micrographs of metal coated surface (upper) and its cross section (middle) and corresponding EDX analysis (lower) for the surfaces of40S, HB5 and H composite coatings. 3.5. Wettability and roughness of coated substrates The wettability of the bioactive coatings for metal implants is essential issues from the view of cell viability principle. The hydrophilic surface of bioactive materials is one of the crucial marks for cell attachment and proliferation on such surface [50]. The wettability is measured from the contact angle between the tangent of a water drop and substrate plane. Figure 5 demonstrates optical photos of water drop before (upper) and after (lower) contacting with the different coated metal substrate surfaces (the 40s, HB5 and H samples). The figure showed that it was not possible to measure the contact angle because the water drop wholly diffused and absorbed by the coating surface, it was almost zero. This result indicated the high wettability of composite coating layers, which mainly resulted from the hydrophilic nature of chitosan polymer and bioactive glass particles. These results agreed with a previous study [51]. Interestingly, the ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT16 new glasses (the 40s and HB5 glasses) prepared in this study had hydrophilic properties like 45S5 glass. Therefore, different composite coatings transformed the substrate surface to be wholly wettable and the hydrophilic surface giving a positive mark for the cell attachment and interaction [50]. Figure 5. Optical photos of water drop before (upper) and after (lower) contacting with different coated metal substrate surfaces, 40S, HB5 and H samples. Figure 6 represents the average roughness values of the composite coatings compared to 316L SS substrate. The roughness values of 316L SS, the 40s, HB5 and H composite coatings were five ? 0.7, 170 ? 18, 234 ? 17 and 201 ? 26 ?m respectively. All the coats (P << 0.01) increased the roughness of 316L SS significantly. HB5 sample showed substantially higher roughness than the 40s and H samples acquiring values of P ~ 0.0001 and 0.03, respectively.The 40S sample possessed lower value than each HB5 and H samples (P ~ 0.0001 and 0.04, respectively). The coatings showed micro-topographies which enhances cell adhesion as demonstrated in a previous study [52]. In other words, the samples demonstrated desirable roughness which can be advantageous for bioactivity, where it promotes an adsorption of organic components and enhance the cell attachment inside the body. ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT17 Figure 6. Roughness values of different composite coating compared to 316L SS substrate. P-value obtained by Students t-test (*P<0.05, **P< 0.01 and ***P<1×10-7) 3.6. In vitro degradation and bioactivity tests 3.6.1. pH measurements The pH of the incubated media (SBF and DMEM) of the studied composite coated substrates as a function of time are represented in Figure 7a, b. The pH values of samples after immersion in SBF reached its maximum values ranging from 7.4 to 7.5 after one-day incubation, and then they decreased continuously throughout the rest of incubation period to reach to values around 7.0. Such decrease is attributed to the depletion of Ca and P ions present in SBF for the hydroxyapatite crystals formation as confirmed by the degradation curve of Ca and P ions (Figures 6c and d). On the other hand, pH values of samples incubated in DMEM showed different behavior, where, fluctuation of pH values was recorded. pH values reached their maxima to become around 8.0 post six h of incubation, and then they decreased to their minimum values to reach 7.1 after three days of immersion. Further, they increased again and continuously during the rest of the immersion time to reach values between 7.4 and 7.9. Such variation in pH values was due to the non-buffered nature of such fluid (unlike SBF) [53]. ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT18 3.6.2. Ionic release concentrations The levels of Ca, P, Si, B, Fe and total proteins released from different composite coatings into SBF and DMEM are shown in Figures 6 c-l. Mostly, the release of all ions in SBF and DMEM showed a two-stage release. The first stage occurred in the first day of in incubation and it showed a relatively high release rate. The second stage was steady state release which occurred during the rest of soaking time (1-14 d), and it characterized by a lower release rate. Figures 7c and d represent the cumulative release curves of Ca ion in both solutions. Generally, Ca ions concentration in SBF was slightly higher than that in DMEM solution. This was because of a higher initial Ca ionic concentration in SBF (2.5 mmol/dm3) compared to that in DMEM (1.8 mmol/dm3) [53]. On the other hand, the concentration of Ca ions in SBF was increased during the first day of incubation with rates 20.0 x 10-2, 21.4 x 10-2 and 22.2 x 10-2 mmol/l.h-1 for 40S, HB5 and H samples, respectively. Thereafter, the release rates were decreased throughout the rest of incubation period w

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