In vitro corrosion resistance and cytocompatibility of minerals substituted apatite/biopolymers duplex coatings on anodized Ti for orthopedic implant applications

2.1 Preparation of TNT coatings on RBA

The commercially pure Ti sheets were procured from Sigma- Aldrich Chemie, GmbH, Schnell dorf, Germany. The Ti substrates were cut into dimensions of 10 × 10 × 0.5 mm³. Prior to the anodization process, the working electrode (Ti) substrates of 1 cm² surface were mechanically grounded with silicon carbide sheets of different grade size ranging from 400 to 1200 grits. These metallic Ti substrates were thoroughly washed in 1:1 ratio of ethanol/acetone mixture in an ultrasonic bath, followed by polishing solution containing HF, HNO₃, and H₂O with a volume ratio 1:4:5 for 5 min at room temperature to remove unwanted impurities and then rinsed with distilled water, dried in air for improved the coating adhesion onto the substrate. After chemical polishing, the anodization was performed in a two electrode cell connected to a DC power supply using Ti substrate as the anode and graphite as cathode at 40 V for 60 min in an electrolyte containing 0.5 wt% NH₄F and 1 M NH₄H₂PO₄ under thermostatically controlled manner. After anodization, TiO₂ formed on Ti substrates were washed with tap water and dried by annealing at 450 °C for 2 h.

2.2 Synthesis of polymers coatings on TiO₂ substrate.

1 g of chitosan was dissolved in 1% acetic acid containing 100 ml of water, and the prepared CS solution was stirred overnight to form clear solution. To prepare a casein solution at various concentrations, 1%, 2%, and 3% of casein powder prepared separately by adding 1 M NaOH solution was stirred and clear solution was obtained by maintaining at pH 10.

Briefly, 50 ml of 1% (w/v) chitosan solution and 50 ml of casein solutions at various concentrations were mixed together and stirred for 3 h. The electrophoretic deposition of polymer blends on TiO₂ substrate was carried out by using a DC power supply kept at a constant potential 2 V with a deposition time of 45 min.

2.3 Electrodeposition of M-HAP on chitosan-casein coated TiO₂ substrate.

The electrodeposition of M-HAP on CS-CA/TNT coated Ti specimen was performed potentiostatically by using an electrochemical workstation (CHI 760C, CH Instruments, USA) at constant current density of 9 mA cM-² for 30 min). Three categories of M-HA electrolytes were utilized for the development of M-HAP.

Category 1 M-HAP (I): 0.4 M CaCl₂·2H₂O, 0.05 M La (NO₃)₃·6H₂O, 0.05 M Tb(NO₃)₃·6H₂O and 0.3 M(NH₄)₂HPO₄.

Category 2 M-HAP (II): 0.375 M CaCl₂·2H₂O, 0.05 M La(NO₃)₃·6H₂O, 0.075 M Tb(NO₃)₃·6H₂O and 0.3 M(NH₄)₂HPO₄.

Category 3 M-HAP (III): 0.375 M CaCl₂·2H₂O, 0.05 M La(NO₃)₃·6H₂O, 0.1 M Tb(NO₃)₃·6H₂O and 0.3 M(NH₄)₂HPO₄.

The above solutions were prepared to produce the target molar ratio of (Ca + La + Tb)/P as 1.67. After that time, slowly anionic solution was added dropwise to cationic solution and pH of the electrolyte was adjusted to 10.0 by using NH₄OH solution. During the deposition process, minerals substituted HA electrolyte was deaerated with N₂ atmosphere for 30 min, in order to reduce the formation of calcium carbonate (CaCO₃) deposits and thus to diminish the carbon dioxide (CO₂).

The deposition process of TNT/CS-CA@M-HAP (I) to TNT/CS-CA@M-HAP (III) coated on Ti substrate were carried out from all the above three categories of the solutions under magnetic stirring for 5 h, then the coating on Ti specimen were rinsed with deionized water and then dried for overnight at room temperature.

2.4 Characterization of TNT/CS-CA @M-HAP duplex coatings on Ti substrates

The Fourier Transform- infrared spectroscopy (FT-IR, Perkin Elmer RX1 Spectrometer) was used to confirm the functional groups of the as-formed TNT, TNT/CS-CA, TNT/CS-CA@M-HAP-I, TNT/CS-CA@M-HAP-II and TNT/CS-CA@M-HAP-III duplex coatings on Ti substrates in the reflectance absorption mode in the range 4000–400 cm⁻¹. The coatings samples scraped from the surface of Ti were mixed with KBr were pressed into discs and made them into pellet form and then analyzed.

The phase identification and crystallinity of the TNT/CS-CA@M-HAP nanocomposite samples were analyzed by XRD experiments using PANalytical X’ Pert PRO diffractometer with monochromatic CuKα radiation (1.5406 Å) generated at 40 kV and 30 mA in the range between 20° ≤ 2θ ≤ 80° with a scan rate of 0.02° at a scanning angle of 10 min⁻¹, respectively by comparing the XRD patterns with standard data compiled by the JCPDS software. The average crystallite size of the synthesized samples were determined using the Debye-Scherrer approximation (Sasireka et al., 2019). $$D=K\lambda /\left(\beta cos\theta \right)$$ where D is the average crystallite size calculated for the (hkl) reflectance, λ is the wavelength of radiation (λ = 1.5406°) in A^o, β is the full width at half maximum approximation (FWHM) in radian, θ is the angle of diffraction as represented in degrees and K is the broadening constant.

To investigate the morphological structure of the samples (TNT, TNT/CS-CA, TNT/CS-CA@M-HAP-I, TNT/CS-CA@M-HAP-II and TNT/CS-CA@M-HAP-III duplex layer coated on Ti were scrutinized by using high resolution scanning electron microscope (HRSEM, JEOL, JSM-840A) and cross- sectional analysis of all coated samples was also carried out. Before handling HRSEM analysis, the specimens were coated by gold sputtering and all coated samples were recorded using the secondary electron detectors. The elemental composition and elemental mapping of the deposited duplex layer coatings on Ti was investigated by using an energy dispersive X-ray (EDAX) analysis. The coated specimens were immersed in fresh Ringer’s solution for 1 h to leach out mineral ions characteristics of the TNT, TNT/CS-CA, TNT/CS-CA@M-HAP-I, TNT/CS-CA@M-HAP-II and TNT/CS-CA@M-HAP-III duplex coatings on Ti and they were evaluated using the inductively coupled plasma atomic emission spectroscopy (ICP-AES, Instruments, Wrexham, UK).

2.5 Mechanical properties of the coatings

2.6 Antibacterial activity

The antibacterial activity of the TNT/CS-CA@M-HAP (II) duplex layer coated on Ti substrate have been tested against two prokaryotic strains S. aureus (ATCC 25923) and E. coli (ATCC 25922) using agar well diffusion technique, in performed at Muller-Hinton agar plates by pouring agar for thick dense layers and adding inoculated of the test microorganisms of two bacterial strains via. Gram positive bacteria and Gram negative bacteria and then incubated at 37 °C for overnight in Trypticase Soy Broth (TSB) cultures to obtain better growth. Petri discs of 6 mm diameter were left for 10 min in the laminar flow and were immersed into various concentrations (25, 50, 75, 100 μL) of different samples (Pishbin et al., 2014). The antibacterial activity was evaluated by measuring the zone of inhibition around the disc produced by the coating samples against the two bacterial strains.

2.7 Invitro biological studies

2.8 Electrochemical evaluation of composite coatings on Ti

The corrosion resistivity of uncoated Ti and TNT/CS-CA, TNT/CS-CA@M-HAP (I), TNT/CS-CA@M-HAP (II), TNT/CS-CA@M-HAP (III) (at optimum concentration of La³⁺, Tb³⁺) duplex coated on Ti specimens was determined by Tafel polarization and Electrochemical impedance spectroscopy (EIS) in Ringeŕs solution (with composition 8.6 g/l NaCl; 0.66 g/l CaCl₂·2H₂O and 0.6 g/l KCl) at pH 7.4 and commonly accepted human body temperature, 37 ± 1 °C. Generally, the electrochemical corrosion experiments were done in the three-electrode system using the electrochemical workstation model CHI 760 (CH INSTRUMENTS, USA) with platinum and saturated calomel electrode (SCE) as counter and reference electrodes, respectively. The coated Ti specimens were used as the working electrodes of dimension 1 cm² area and its corrosion potential was measured related to SCE. Before the electrochemical studies, working electrodes were allowed to become stable until it reached a steady potential denoted as the open circuit potential (OCP).

The potentiodynamic polarization experiments were performed as the potential range from −0.4 V to + 1.2 V vs SCE for uncoated Ti and TNT/CS-CA, TNT/CS-CA@M-HAP (I), TNT/CS-CA@M-HAP (II), TNT/CS-CA@M-HAP (III) duplex coated samples on Ti at a sweep rate of 1.0 mV s⁻¹. During the cyclic polarization studies, the potential was raised in noble direction, until the breakdown potential (E_b) was reached. The breakdown potential (E_b) was obtained at the potential where there was a monotonic raise in the current density. The repassivation potential (E_pp), is one at which the scan meets reverse the passive region. Likewise, the electrochemical impedance spectra (EIS) were recorded over the frequency ranging from 10⁻¹ Hz to 10⁵ Hz and with applied AC perturbation amplitude of 5 mV. To confirm the reproducibility, all electrochemical experiments were repeated at least three times and the obtained data was recorded using the internally available software.

3.1 Functional group analysis

Representative Fourier Transform infrared (FTIR) spectra have been used for determining functional groups or chemical bonds that exist in a material. Fig. 1 (a-e) illustrates the FTIR spectra of anodized Ti, CS and CA blending on TNT and M-HAP (M-La³⁺, Tb³⁺) deposited on CS-CA/Titania nanotubes and under at various concentrations of trivalent ions. From Fig. 1a, it can be observed that the peaks at 3429 cm⁻¹ and 2990 cm⁻¹ are attributed to the O-H stretching vibration modes of water molecule adsorbed on the surface and bending vibrations of OH groups respectively. This indicates that a significant fraction of H₂O and OH groups exist in as anodized sample and intensity of peak is very low due to annealing. The bands at 696 cm⁻¹ and 540 cm⁻¹ are assigned to the vibration of Ti-O and Ti-O-Ti bond in TNT (Mohan Raj et al., 2018).

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Fig. 1

FT-IR spectra of (a) TNT (b) TNT/CS-CA (c) TNT/CS-CA@ M-HAP (I) (d) TNT/CS-CA@ M-HAP (II) (e) TNT/CS-CA@ M-HAP (III) coatings on Ti substrate.

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Fig. 1b shows the sequential binary coatings with chitosan (CS) and casein (CA) coated on TNT. It shows strong IR absorption bands at 3440 cm⁻¹ and 1742 cm⁻¹ corresponds to –OH stretching vibration and amino group of chitosan that are shifted to 1609, 1539 and 1220 cm⁻¹ that arise from N-H stretching and amide bending vibration bands and peak at 1641 cm⁻¹ is due to C⚌O stretching vibration of casein (Singh et al., 2015).

Fig. 1(c–e) shows that the FT-IR spectra of M-HAP characteristic vibrations of phosphate groups occurs at 1017 cm⁻¹ and 605 cm⁻¹ respectively. The broad band of stretching vibration modes of OH groups can be located at 3429 cm⁻¹, due to presence of hydrogen bonded water molecule absorbed during apatite formation (Farzadi et al., 2014, Chang et al., 2013). The sharp peak indicates two stretching vibration modes of La-OH and Tb-OH at 653 cm⁻¹ and 720 cm⁻¹ present in M- HAP composite coating respectively (Hosseini et al., 2014).

3.2 Phase identification and structure

Fig. 2(a–e) shows the XRD patterns of TNT, TNT/CS-CA, TNT/CS-CA@M-HAP (I), TNT/CS-CA@M-HAP (II) and TNT/CS-CA@M-HAP (III) duplex layer coatings fabricated on Ti alloy. Fig. 2(a) illustrates the XRD pattern of TNT which shows diffraction peaks with planes at 2θ positions of 25.2° (1 0 1), 37.9° (1 0 3), 48.6° (2 0 0), 53.8°(1 0 5), 63.7° (2 1 3), 75.3° (1 0 7) that corresponds to anatase form of TiO₂ (JCPDS card no: 21–1272). The very sharp peak of anatase phases of titania formed during anodization indicate the highly crystalline nature of the coating (Raj and Mohan Raj, 2016).

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Fig. 2

XRD spectra of (a) TNT (b) TNT/CS-CA (c) TNT/CS-CA@ M-HAP (I) (d) TNT/CS-CA@ M-HAP (II) (e) TNT/CS-CA@ M-HAP (III) coatings on Ti substrate.

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The strong interaction was observed between with addition of chitosan and casein molecules as those of mechanical blending. XRD pattern of TNT/CS-CA composite coating depicted in Fig. 2b shows the new diffraction peaks at 2θ values of 12.6°, 18.5° are assigned to CS, CA composite coating, respectively and the results are in accordance with the earlier reports (Surmeneva et al., 2013). The high crystallinity is appeared due to the strong interaction of chitosan with casein through hydrogen bonding between hydroxyl groups and amine groups.

The XRD patterns for M-HAP coatings on TiO₂/CS-CA substrates show similar diffraction peaks representing the HAP formation which indicates that all coated samples exhibit a similar crystallographic nature as depicted in Fig. 2(c–e). The peaks of M-HAP correspond to the 2θ values of 25.8°, 27.2°, 30.4°, 31.9°, 32.5° and 35.9° without any secondary phases are found.

XRD peaks identified for M-HAP well match with the standard XRD data (JCPDS No: 09-0432, JCPDS No: 01-083-2038). However, the 2θ values for M-HAP peak positions are shifted towards the lower angles which might have aroused due to influence of the lanthanum and terbium mineral ions substitution that leads to the distortion in the HAP lattice (Sohn, 2014, Stancheva and Bojinov, 2012). This could be recognized due to the presence of M-HAP in the duplex coatings.

3.3 Morphological observation

Fig. 3a depicts SEM images of highly ordered close packed titania nanotube arrays after annealing at 450 °C for 3 h prepared by electrochemical anodization of Ti using the 1 M (NH₄)₂HPO₄ + 0.5% NH₄F electrolyte solution for 90 min at 40 V. The SEM top- view image (Fig. 3a) reveals a wide distribution of average diameter of 20 nm pores. In these experiments, the fluoride concentration was varied from 0.5 to 2 wt%. Indeed, that source of fluoride is typically water soluble and more ionic nature. Therefore, an increase in fluoride concentration yields an increase in overall ionic concentration of the solution which results in an increase in electrolyte conductivity and higher current response. The fluoride ions are more rich species in these electrolytes that can be assumed to be prone to chemical dissolution in aqueous environment (Lee et al., 2009).

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Fig. 3

HRSEM micrographs of (a) TNT (b) TNT/CS-CA (c) TNT/CS-CA@ M-HAP (I) (d) TNT/CS-CA@M-HAP (II) (e) TNT/CS-CA@ M-HAP (III) coatings on Ti substrate (f) EDAX spectrum of the TNT/CS-CA@ M-HAP (II) coating.

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Fig. 3b demonstrates the morphology of coatings (CS-CA) that exhibit less porous structure. The phase separation of polymeric hydrogel synthesized using CS-CA has lesser porosity with various irregular segregated structures as shown in Fig. 3b. In this study, the polymer adhere onto TNT were used to enhance the number of pores and pore size was decreased which might be due to significant formation of tightly packed network structure. The embedding of this polymer resulted in apparently reduction in pore size (Govindaraj et al., 2015).

The structural morphologies of the electrodeposited of M-HAP-I, M-HAP-II and M-HAP-III coatings on CS-CA/TNT composite coating samples are depicted in Fig. 3c-e. The M-HAP-I (Fig. 3(c)) coating is heterogeneous with agglomerated granules over the surface. Similarly, the M-HA-II coating (Fig. 3(d)) and M-HAP-III coating (Fig. 3(e)) exhibit the interconnected granular structure, but Fig. 3d exhibits uniform coverage of interconnected granular structure and the small pores that exist in the coating is taken as optimum condition for good anticorrosion and improved cell attachment. In Fig. 3(f) shows the EDS spectrum of TNT/CS-CA@M-HAP coated surfaces which shows Ca, P, C, O, Ti, La and Tb elements and the spectrum supports for the presence of trivalent ions of the as-developed coating.

Fig. S1 shows the elemental mapping of TNT/CS-CA@M-HAP(II) composite coating indicating the ubiquitous presence of Ca, P, C, O, Ti, La and Tb elements. Thus, the homogeneously distributed mineral ion is well evidenced from the EDS mapping results.

3.4 Leach out analysis

The release of mineral ions from the TNT/CS-CA@M-HAP (II) composite coated Ti was evaluated from the ICP-AES analysis after soaking in Ringeŕs solution for 1, 4 and 7 days and the results are shown in Fig. 4. The average Ca and P ion concentration ions released in the solution increased at day 1. Likewise, the release of other minerals such as La and Tb ions in the solution also slightly increased as increase immersion time from 0 to 1 day. Moreover, as the immersion time increases from 4 to 7 days, the release of mineral ions slightly decreased, which may be due to formation of apatite growth on the surface of the coating. From the resultant coatings, the released mineral ions such as Ca, P, La and Tb will be effectively take part in the antibacterial and bioactive properties. (Sasireka et al., 2019; Sohn, 2014; Devi et al., 2016).

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Fig. 4

ICP-AES analysis of TNT/CS-CA@ M-HAP (II) bilayer coated on Ti samples at different immersion days.

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3.5 Thickness, hardness and adhesion strength

Figs. 5 and 6 shows the thickness, hardness and adhesion strength values of Ti alloy, TNT, TNT/CS-CA, TNT/CS-CA@M-HAP (I), TNT/CS-CA@M-HAP (II) and TNT/CS-CA@M-HAP (III) composite coatings. The mechanical properties of TNT arrays such as thickness, hardness and adhesion strength were almost same when compared to the Ti substrate and these values increased when CS-CA was coated on TNT and it is further increased for TNT/CS-CA@M-HAP (II) composite coating. Thus TNT/CS-CA@M-HAP (II) coating shows maximum thickness, hardness and adhesion strength than any other coatings.

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Fig. 5

Thickness, Hardness of various coated samples on Ti specimen.

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Fig. 6

Adhesion strength of various coated samples on Ti specimen.

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In this particular case, presence of CS-CA allowed good mixing of TNT, CS-CA, La, Tb and HAP layers by modifying their respective surface charges, thereby permitting the formation of uniform multi layers on Ti substrate enabling good mechanical strength to the substrate. Due to the presence of these multi layers, the overall thickness of the coating is higher which obviously increase the mechanical properties of the coatings. In the same manner, the mechanical properties of the coating and thickness are high for TNT/CS-CA@M-HAP (II) coating compared to other coatings but adhesion strength remains the same. This could be due to decrease in the porosity of the coatings as the addition of minerals increases.

3.6 Antibacterial activity

The antibacterial activity of as-synthesized TNT/CS-CA, TNT/CS-CA@M-HAP (I), TNT/CS-CA@M-HAP (II) and TNT/CS-CA@M-HAP (III) composite coatings was studied and the zone of inhibition at four different concentration such as 25 μL, 50 μL, 75 μL and 100 μL against the pathogenic bacteria, namely S. aureus (G⁺) and E. coli (G⁻) by the disc diffusion method and depicted in Figs. 7 and 8. These data revealed, the substitution of La³⁺ and Tb³⁺ minerals in the HAP concentration in the coating. The zone of inhibition for the TNT/CS-CA@M-HAP (II) sample against S. aureus and E. coli were found to be 11 & 20 mm for 25 μL, 14 & 22 mm for 50 μL, 18 & 25 mm for 75 μL and 22 & 27 mm for 100 μL concentrations, respectively. At 100 μL, the TNT/CS-CA@M-HAP (II) duplex layer sample recorded superior antibacterial activity against E. coli by exhibiting the highest diameter of inhibition zone (Pogrebnjak et al., 2019). The antibacterial activity of TNT/CS-CA@M-HAP (II) duplex layer coatings against E. coli strain was slightly higher than that of S. aureus strain due to the differences observed in the structure of cell wall. The reason may be ascribed to the substitution of minerals such as La³⁺ and Tb³⁺ in HAP and also polymer composite incorporation in TNT plays a very important role in enhancing the antibacterial activity.

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Fig. 7

Qualitative antibacterial activity of sample TNT/CS-CA@ M-HAP (II) coated on Ti specimen against gram positive and gram negative bacteria.

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Fig. 8

Antibacterial activity of sample TNT/CS-CA@ M-HAP (II) coated on Ti specimen against gram positive and gram negative bacteria.

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3.7 Apatite formation on the composite in SBF solution

Researchers have widely used SBF test to evaluate the capability of test samples to form an apatite layer on their surfaces. By performing SBF immersion, the apatite layer forming ability, or bioactivity of our TNT/CS-CA@M-HAP (II) nanocomposite can be assessed. The morphology of TNT/CS-CA@M-HAP (II) bioactive composite coatings were confirmed by the SBF immersion study for various days such as 1 day, 4 days, 7 days, 14 days and 21 days of immersion for the forming apatite layer are seen in SEM micrograph in the Fig. 9.

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Fig. 9

HRSEM images of apatite formed on TNT/CS-CA@ M-HAP (II) bilayer coated on Ti samples dipped in SBF for several days (a) day 1, (b) day 4 (c) day 7 (d) day 14 and (e) day 21.

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The apatite layer forming ability is major constituent mechanism of the chemical bioactivity. The growth of apatite on the nanocomposite coatings differ at each day. At day 1, there is no formation of apatite layer and after day 4, the started to grow on lesser number of apatite formation. At day 7, apatite growth is seen on the surface of flower petal as displayed in Fig. 9c. At day 14, the growth of apatite was formed like spongy layer over the surfaces of nanocomposite coatings as shown in Fig. 9d. At day 21, some spongy flower like structure was observed as shown in Fig. 9e. In the sample, the formation of apatite layer usually takes place through an arrangement of chemical reactions. When coated samples were submerged in SBF solution, then the surface gains the positively charged ions that can attract negatively charged ions such as phosphate and hydroxyl group and forming Ca²⁺ poor amorphous in nature. This process indicates the formation of bone like apatite growth on the nanocomposite surface (Alqarni et al., 2018). Thus, the HRSEM micrographs displayed the apatite mineralization ability at 7, 14 and 21 days of immersion and revealed that the resultant nanocomposite coatings improve the bioactivity in SBF solution.

3.8 Electrochemical analysis

Electrochemical performances are one of the most universal tests to estimate the in vitro corrosion behavior of the formed coatings, and it is generally composed of potentiodynamic polarization (Tafel exploitation plots) and Nyquist impedance plots. Fig. 10 displays the representative potentiodynamic polarization curves of the nanocomposite coatings of TNT/CS-CA and TNT/CS-CA@M-HAP, as well as TNT and uncoated Ti substrate in the Ringers physiological solution at 37 °C, and the related electrochemical parameters were determined from the polarization curves are summarized in Table 1. Potentiodynamic polarization measurements were done in the range from −0.4 V to 1.2 V vs. SCE, at a scan rate of 1.0 mV s⁻¹ after reaching a steady state potential. The corrosion potential (E_corr, V), corrosion current density (i_corr, μA cm⁻²), anodic as well as cathodic Tafel slopes (β_a, β_c) values were calculated at the points after E_corr values ± 100 mV using a computer least-squares method due to the degree of nonlinearity and eventually investigated uniform metal corrosion rate through Faraday's law (Fekry, 2016). The values of E_corr for tested six samples revealed a shift towards more positive values as obtained from the open circuit potential in the following order: TNT/CS-CA@M-HAP-II (0.501 V) > TNT/CS-CA@M-HAP-III (0.242 V) > TNT/CS-CA@M-HAP-I (0.059 V) > TNT/CS-CA (0.011 V) > TNT (-0.046 V) > uncoated Ti (-0.142 V). Also, the I_corr values increased in the same order, indicating that the TNT/CS-CA@M-HAP-II nanocomposite coating has the lowest I_corr value (0.61 A cm⁻²) and compared to the uncoated Ti electrode which has the highest I_corr value (10 A cm⁻²) with highest corrosion rate. However, M-HAP has decreased corrosion rate as the macromolecule is adsorbed well on the surface through electron pairs (Liu et al., 2017). When chitosan-casein polymer blended into TNT decreases the corrosion rate by increasing the film strength and then adding M-HAP into Chi-CA/TNT gives the lowest corrosion rate. This is due to minerals substituted HAP increasing the adsorption of both Chi-CA and Titania nanotubes to the Ti surface. The TNT increases the adhesion strength between the polymers and M-HAP composite matrix and the Ti surface. This leads to an increase in film stability, strength and adsorption on the Ti surface. Also, phosphate buffer solution forming ions helps in decreasing the lower corrosion rate of the Ti surface.

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Fig. 10

Potentiodynamic polarization curves of uncoated Ti, anodized Ti, TNT/CS-CA and TNT/CS-CA@M-HAP (I) to TNT/CS-CA@M-HAP (III) coatings on Ti samples obtained in Ringer’s solution.

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Finally, the M-HAP@Chi-CA/TNT nanostructured dense coating acts as a barrier to the transport of electrons and ions between the Ti alloy and the PBS solution. So, this novel synthesized nanocomposite coating significantly improved the corrosion resistance of the Ti alloy. Finally, the results of polarization curves are in the same trend with more positive corrosion potential compared to uncoated Ti surface (Li et al., 2014).

EIS results can give valuable information about the electrochemical behavior of both uncoated and coated Ti substrates in PBS solution. The impedance parameters derived from these plots using the equivalent electrical circuit are displayed in Fig. 11 and Table-1. The best circuit model was used to fit the EIS data for the corrosion behavior of uncoated and coated Ti substrates that consists of solution resistance (R_s), polarization resistance (R_p), charge transfer resistance of the coated substrate (R_ct) and double layer capacitance (C_dl).

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Fig. 11

Nyquist plots of uncoated Ti, anodized Ti, TNT/CS-CA and TNT/CS-CA@M-HAP (I) to TNT/CS-CA@M-HAP (III) coatings on Ti samples obtained in Ringer’s solution.

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Besides Bode and phase angle plots were also used to analyze the corrosion process. The high impedance modulus in lower frequency region and one time constant revealed that the film has effective barrier property. In phase angle plots, TNT/CS-CA@M-HAP-II coating has higher phase angle and low frequency impedance modulus indicate the better corrosion resistance and passivity of stable structure are shown in Fig. S2(i, ii). At the same time, it is believed that the coated samples have higher phase angle and better corrosion resistance when compared to uncoated Ti surface. Because it has inert and homogenous film deposition on the surface.

3.9 Cell viability

The in vitro biocompatibility of cells over the TNT/CS-CA@M-HAP-II as-synthesized composite coatings is one of the most significant factors to assess the biological performance of the coating. The cell viability of MG 63 human osteoblast cells on TNT/CS-CA@M-HAP-II coatings was determined using an MTT assay for 1, 4 and 7 days of culture as shown in Fig.14. After the 24 h of incubation period, MTT solution (20 µL of 5 mg/ml serum free DMEM medium) was added into each well and the cells were incubated for another 2–4 h at 37 °C in a humidified 5% CO₂ atmosphere. The percentage of cell viability of various coatings is displayed as bar diagram in Fig. S3.

The absorbance of cell viability at a wavelength of 570 nm is directly proportional to the number of live cells in the present of MG63 human osteoblast cells culture medium are shown in Fig. S4. The optical images of the number of viable cells is increased as increasing after 7 days of incubation time as displayed in Fig. 12 (a-c). The MG63 cells were started to spread on the control over the surface on day 1. When compared to control at day 4, the cells were spread well through cytoskeletal processes over the composite coating. The TNT/CS-CA@M-HAP-II composite coatings exhibited more number of viable cells at day 7 compared with control as illustrated in the Fig. 14d, that does not display any toxicity of cells and also has good cytocompatibility. The cell viability results revealed that the as-developed coating can be served as a bio implant for orthopedic applications.

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Fig. 12

Optical images showing the cell viability of MG63 human osteoblast cells on TNT/CS-CA@M-HAP (II) coatings for (a) 1, (b) 4 days and (c) 7 days of incubation.

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3.10 Live/dead staining

Fig. 13(a–d) shows the results of live/dead cells on the TNT/CS-CA@M-HAP-II nanocomposite coating at various days of incubation as determined by fluorescent microscopy.

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Fig. 13

Fluorescence microscopic images showing Live/dead staining MG63 human osteoblast cells on TNT/CS-CA@M-HAP (II) coatings for (a) control, (b) 1, (c) 4 days and (d) 7 days.

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The fluorescent microscopic images (Fig. 13a–d) denote the both live and dead stains indicating the green and red color. The evaluation of live/dead cells was carried out by culturing in medium for 1, 4 and 7 days. Fig. 13b indicated that the lesser number of live cells and more number of dead cells were spread well on the surface of TNT/CS-CA@M-HAP-II, respectively for 1 day of incubation when compared with control. The cells are spreading and growth with highly elongated morphology on TNT/CS-CA@M-HAP-II coated samples for 4 days of incubation as shown in fluorescent images in the Fig. 13c (Favi et al., 2013), (sathishkumar et al., 2016) (Li et al., 2014). The growth of MG 63 human osteoblast cells clearly exhibits that the cell proliferation on the TNT/CS-CA@M-HAP-II nanocomposite coated samples are well distributed throughout the surface of the coated material at higher absorbance of 7 days of incubation in cell culture (Fig. 13d), which is mainly due to the presence of mineral ions and polymer in the nanocomposite.