Development and characterisation of novel Ce‐doped hydroxyapatite–Fe₃ O₄ nanocomposites and their in vitro biological evaluations for biomedical applications

Abstract

Hydroxyapatite (HAP: Ca₁₀ (PO₄)₆ (OH)₂) is extensively used in biomedical field because of its biocompatibility, osteoconductivity and non‐toxicity properties. However, HAP exhibits poor mechanical strength and bacterial restriction behavior. To overcome these drawbacks, various metal ions such as Ag⁺, Zn²⁺, Cu²⁺, Ti⁴⁺ and Ce^4+/3+ are incorporated in HAP matrix to increase the mechanical and biological properties. Among these, Cerium (Ce) is selected as antibacterial agent due to its high thermal stability and its applications in dental fillings, bone healing and catheters. Fe₃ O₄ nanoparticles were used in hyperthermia treatment, magnetic fluid recordings and catalysis. In this present study, we have synthesized nanocomposites consisting of 1.25% Ce doped HAP with various concentrations of Fe₃ O₄ NPs as 90:10 (C‐1), 70:30 (C‐2) and 50:50 wt% (C‐3) using ball milling technique. The obtained Ce@HAP‐Fe₃ O₄ nanocomposites were characterized by ATR‐FTIR, XRD, VSM, SEM‐EDAX and TEM analysis. Further, the fabricated Ce@HAP‐Fe₃ O₄ nanocomposites were tested for its antibacterial activity towards Staphylococcus aureus (S. aureus) and Escherichia coli (E.coli), where C‐3 composites exhibit the excellent pathogen inhibition towards E.coli. In addition, the cytotoxicity evaluation on C‐3 nanocomposites by in vitro biocompatibility study using MG‐63 cells shows the prominent viable cell enhancement up to 400µg/mL concentrations.

1 Introduction

Hydroxyapatite [HAP:Ca₁₀ (PO₄)₆ (OH)₂] is a type of Ca₃ (PO₄)₂ ‐based bioactive ceramic which is commonly used in bone tissue engineering due to its structural and chemical correlation with natural bone and teeth [1, 2]. HAP exhibits excellent biocompatibility, osteoconductivity, bioactivity and chemical stability [3]. These characteristic features of HAP made us to use it as coatings on the implant surface to assist osteointegration without rejection to the surrounding tissues and also powder forms in bone and tooth fillings [4]. However, the tremendous usage of HAP in clinical applications is limited because of its poor mechanical property, brittleness, low biodegradation and weak in microbial restriction behaviour [5]. To overcome these drawbacks, substitution of Ca²⁺ ions in the HAP crystal lattice with trace metal elements like Ag⁺, Cu²⁺, Zn²⁺, Na⁺, Ce^3+/4+, Si⁴⁺, Sr²⁺ etc., can enhance its properties such as crystallinity, solubility, thermal stability and antibacterial activity [6, 7].

Among these metal ions, cerium (Ce) is the most abundant rare earth element broadly used in biomedical applications like dental fillings, bone healing and catheters owing to its biological properties [8]. Ce acts as an antibacterial agent with high thermal stability and is present in the human bone, which stimulates the function of bone metabolism, it plays a crucial role in the prevention of cavities and reduction in demineralisation of enamel [9, 10]. Ce is present in two forms like cerous (Ce³⁺) and ceric (Ce⁴⁺) which exhibit superior antioxidant and pathogen inhibition efficiency for medical applications [11, 12]. The ionic radius of Ce³⁺ (0.107 nm) and Ce⁴⁺ (0.087 nm) is almost similar to the Ca in HAP, therefore integration of HAP with Ce will lead to the development of promising biomaterials with increased solubility, biodegradability and bacteriostatic properties for applications towards drug delivery, imaging, development of artificial bone and dental materials [13]. Kaygili et al. have employed the sol–gel technique to prepare HAP with Ce³⁺ ions and examined the phase pure formation and crystallinity [14]. Yingguang et al. investigated the antibacterial potential of Ce³⁺ ‐substituted HAP towards Lactobacillus, E. coli and S. aureus pathogens which exhibited excellent microbial restriction [15]. Feng et al. used the hydrothermal method for the fabrication of Ce³⁺ ‐substituted HAP and evaluated the effect of Ce³⁺ integration in the HAP crystal structure [16]. Ciobanu et al. synthesised the HAP with the inclusion of Ce⁴⁺ ions and evaluated its bacterial restriction behaviour against S. aureus, E. coli pathogens and found that E. coli exhibits more inhibition efficiency when compared with S. aureus [17]. Qiuha Yuan et al. reported that Ce‐HAP with polylactic acid (PLA) composite coatings on metal surfaces exhibited high purity and good thermal stability with a uniform compact surface topography [18]. Morais et al. studied Ce‐glass reinforced hydroxyapatite and found that the developed composites exhibit antibacterial activity and also augment the osteoblast cell response [19].

Magnetic nanoparticles (NPs) are promising materials in the biomedical field due to their unique properties like electrical, superparamagnetism and low toxicity [20]. Magnetic NPs with these properties have many applications in various fields like cellular therapy, drug delivery, tissue repair, magnetic resonance imaging and hyperthermia for treatment of cancer, catalysts and gene manipulations [21]. Recently, magnetite (Fe₃ O₄) and maghemite (γ‐Fe₂ O₃) with highly saturated magnetisation have been considered as favourable materials for clinical applications [22]. The magnetite NPs (Fe₃ O₄ NPs) are well‐known materials with excellent biocompatibility and in the presence of an external magnetic field, they tend to align with the growth of osteoblast cells [23]. Hence, Fe₃ O_4­ –HAP composites reduce the toxic effects and improve the radiopacity, osteoblast proliferation compared with pure HAP [24]. The Fe₃ O₄ –HAP composites exhibit improved solubility under physiological conditions [25]. The Fe₃ O₄ NPs with coatings of HAP were highly recommended to use in osteoporosis and bone defected areas [26]. Therefore, these composites can act as alternate materials for cancer treatment in biomedical applications.

Currently, our research group has mainly focused on the preparation of Ce⁴⁺ ‐doped HAP and examined its antibacterial, haemocompatibility and biocompatibility properties to be used as an alternative biomaterial for orthopaedic applications [27]. Recently, much interest has been attracted towards the development of novel composites with enhanced mechanical, chemical and biological properties for better bone remodelling applications. However, to the best of our knowledge no research reports have been published on the combination of Ce⁴⁺ ‐doped HAP with Fe₃ O₄ NPs. Hence, in this study, we have developed sol–gel‐based Ce‐doped HAP powder and Fe₃ O₄ NPs, respectively, by using the co‐precipitation method. These individual powders were made into nanocomposites using ball milling techniques with different ratios of Ce@HAP–Fe₃ O₄ such as 90 : 10, 70 : 30 and 50 : 50 wt%, respectively. The fabricated nanocomposites were characterised by Fourier transform infrared with attenuated total reflectance spectroscopy (ATR–FTIR), powder‐X‐ray diffraction (XRD), vibrating sample magnetometry (VSM), scanning electron microscopy–energy dispersive spectroscopy (SEM–EDAX) and transmission electron microscopy (TEM) analysis. Furthermore, the antibacterial activity was examined for Ce@HAP–Fe₃ O₄ composites against S. aureus and E. coli bacteria using the colony forming unit (CFU) methods. In addition, we have performed an in vitro biocompatibility study using MG‐63 osteoblast cells at different concentrations of composites such as 200–1000 µg/ml for 24 and 48 h.

2 Experimental procedures

2.1 Materials

Calcium nitrate tetrahydrate [Ca(NO₃)₂. 4H₂ O – SDFCL 99%], ammonium cerium nitrate [(NH₄)₂ Ce(NO₃)₆ – SDFCL 98.5%], triethyl phosphite, aqueous ammonia (aq. NH₃), double distilled water [DD H₂ O], iron sulphate (FeSO₄), iron chloride anhydrous (FeCl₃), aqueous ammonia (aq. NH₃), phosphate buffer solution (PBS) and dimethyl sulphoxide were used.

2.2 Sol–gel synthesised 1.25% of Ce@HAP

1.25% of Ce was doped into HAP by the refluxing‐based sol–gel method and represented in a chemical formula as Ca_1−x Ce _x (PO₄)₆ (OH)₂ [x  = 1.25%]. The stoichiometric molar ratio of Ca + Ce/P was maintained at 1.67 for phase pure formations. Firstly, 0.9875 M calcium (Ca²⁺) and 0.0125 M ceric (Ce⁴⁺) were dissolved in DD H₂ O and stirred for 10 min using a magnetic stirrer. Then 0.6 M of the prepared P source was added to the above Ca + Ce solution dropwise and aged for 24 h at room temperature. After the ageing process, the solution mixture was refluxed at 85°C for 16 h to hydrolyse the reactants. The obtained sol was transferred into a beaker and vapourised, during the evaporation process the viscous gel was obtained due to the polycondensation of ions by the formation of Ca–O–P bonds. The gel was kept in an oven for drying and sintered at 700°C for 2 h to prepare 1.25% Ce@HAP.

2.3 Co‐precipitation derived Fe₃ O₄ NPs

In this preparation method, FeSO₄ and FeCl₃ were mixed in a molar ratio of 2 : 1. FeSO₄ and FeCl₃ reagents were dissolved in DD H₂ O and stirred for 30 min using a magnetic stirrer. To this mixture of solution we have added the aq. NH₃ to maintain the pH as 10 and the obtained solution was turned to black precipitate at 80°C in 1 h. The formed Fe₃ O₄ NPs were separated by applying an external magnetic field and the mixture was washed several times with DD H₂ O to neutralise the pH ∼ 7. The formed Fe₃ O₄ NPs were kept in an oven at 100°C for 1 h.

2.4 Development of Ce@HAP–Fe₃ O₄ composites

The powders of Ce@HAP, Fe₃ O₄ NPs were taken in various ratios such as 90 : 10 (C‐1), 70 : 30 (C‐2) and 50 : 50 (C‐3), respectively. These different ratios of powders were homogenously mixed in an agate mortar and pestle for 1 h. After this step, the developed C‐1, C‐2 and C‐3 powders were subjected to the planetary ball milling technique for 2 h using silicon carbide balls (10 mm) at a revolution speed of 450 rpm with a ball to powder ratio of 8 : 1. The obtained C‐1, C‐2 and C‐3 nanocomposite powders were investigated for their biological studies such as antibacterial studies and in vitro biocompatibility.

2.5 Antibacterial activity

The bacterial restriction behaviour of Ce@HAP‐Fe₃ O₄ nanocomposites was quantified towards Gram positive Staphylococcus aureus and Gram negative Escherichia coli bacteria. For this experimental analysis, inoculum medium was cultured by transferring a loop of bacterial strains into a 10 ml of sterilised broth and incubated overnight at 37°C. From this overnight culture, 2% of inoculum was transferred into a side arm flask containing 100 ml of sterilised nutrient broth and incubated at 37°C in an orbital shaker. The growth of bacteria in a liquid medium was measured by an optical density (0.5) using a ultraviolet–visible spectrophotometer at 600 nm.

Then 2 ml of 0.5 optical density (OD) culture was centrifuged at 6000 rpm for 5 min to obtain the cell pellets. These cell pellets were washed with PBS solution and the culture with PBS solution was transferred to separate falcon tubes which contain 5 mg/ml concentration of 1.25% Ce@HAP, Fe₃ O₄ NPs, C‐1, C‐2, and C‐3. The bacteria treated samples were incubated at 37°C for 4 h and the culture without the composite material serves as the control. After incubation, the cultures were serially diluted to 10⁻³ dilution for individual and composites. From this culture 0.1 ml of the sample was swabbed on to the culture plate containing an agar solid medium. The swabbed plates were incubated at 37°C for 24 h and the number of colonies formed on the agar plates was measured by using a digital colony counter. The CFU was calculated by using the below mentioned formula

$$\mathrm{CFU}=\frac{\mathrm{No}.\mathrm{of}\mathrm{colonies}\times \mathrm{dilution}\mathrm{factor}}{\mathrm{volume}}$$ (1)

2.6 In vitro cell culture experiment

Based on the antibacterial activity, we have selected C‐3 nanocomposites for evaluation of biocompatibility using MG‐63 osteoblast cells derived from human osteosarcoma cells which were cultured in α‐minimal essential medium and contains 10% foetal bovine serum under a 5% CO₂ atmosphere at 37°C. MG‐63 cells were seeded at a cell density of 2 × 10⁻³ cells/cm in a 96‐well plate for 24 and 48 h on C‐3 nanocomposites using various concentrations such as 200, 400, 600, 800 and 1000 µg/ml. The MG‐63 cultured plate without composite powder was served as the control.

2.6.1 In vitro cell viability assay

The cell viability/proliferation was evaluated by MTT [3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide] tetrazolium reduction assay. After incubation of the nanocomposite with culture (24 and 48 h), 0.45 mg/ml of 10 µL MTT solution was added to the well and incubated for 4 h at 37°C. Followed by the addition of 100 µL solubilisation buffer [40% (v/v) dimethylformamide in 2% (v/v) glacial acetic acid and 16% (w/v) sodium dodecyl sulphate] to each well to dissolve formazan crystals and mixed thoroughly to ensure solubilisation. Viable cells with active metabolism convert MTT into a purple coloured formazan product with an absorbance at 570 nm using a microplate reader and viable MG‐63 cells were observed by using a phase contrast microscope. The experiment was performed in triplicates. The percentage of cell viability was calculated by using the following formula:

$$\mathrm{Percentage}\mathrm{of}\mathrm{viability}=\mathrm{OD}\mathrm{of}\mathrm{sample}/\mathrm{OD}\mathrm{of}\mathrm{control}\times 100$$ (2)

2.7 Characterisation

1.25% Ce@HAP, Fe₃ O₄ NPs, C‐1, C‐2 and C‐3 powders were characterised by ATR‐FTIR (Shimadzu) to identify the functional groups in the range of wavenumbers from 4000 to 400 cm⁻¹. The phase purity, degree of crystallinity and phase composition were determined by Bruker D8 XRD with Cu‐Kα radiation (1.5406 Å). Scanning was performed in the range of 20–80° at the 2θ angle. The surface morphology was examined by SEM and elemental composition was confirmed by EDAX analysis. A transmission electron microscope was used to assess the particle size and morphology of the particles which were carried out by high resolution‐TEM (HR‐TEM: Tecnai, G2 20 Twin). For TEM sample preparation, the powders were dispersed in ethanol and sonicated for 10 min prior to loading on the carbon–copper grid. The magnetic property of the sample was determined using a vibrating magnetometer at room temperature.

3 Results and discussion

3.1 ATR‐FTIR analysis

The characteristic functional groups present in 1.25% Ce@HAP, Fe₃ O₄, C‐1, C‐2, and C‐3 were confirmed by ATR‐FTIR spectroscopy and shown in Fig. 1. Fig. 1 a shows the FTIR spectrum of 1.25% Ce@HAP and exhibits the significant vibrational bands for the formation of pure HAP at 1094, 1016, 951, 603 and 560 cm⁻¹ which correspond to the P–O bonds in the PO₄ ³⁻ group and also bands at 3573 and 633 cm⁻¹ attribute to the O–H bands in the OH⁻ group, respectively [28]. The asymmetrical and symmetrical stretching modes of P–O bonds were found at 1094–1016 and 951 cm⁻¹ whereas the bending mode of the O–P–O bond was found at 603–560 cm⁻¹, which are major evidence for the presence of a phosphate moiety in the HAP crystal structure [29]. Also, it was found that there is no other characteristic bands were observed at 871 and 1442 cm⁻¹ which denotes the absence of carbonate groups in the HAP structure. Hence, this spectrum confirms that 1.25% of Ce⁴⁺ was doped into the HAP lattice without impurities. The FTIR spectrum of Fe₃ O₄ NPs (Fig. 1 b) shows the characteristic broad band at 537 cm⁻¹ which reveals the stretching mode of the Fe–O group and confirms the formation of Fe₃ O₄ NPs [30]. The ATR–FTIR spectra of C‐1 to C‐3 nanocomposites are shown in Figs. 1 c –e, in these spectra composites existed in all the significant bands of PO₄ ³⁻ and OH⁻ groups for the presence of 1.25% Ce@HAP. In these spectra, the vibrational bands at 560–633 cm⁻¹ for C‐1 to C‐3 were found to become broad due to the inclusion of Fe₃ O₄ NPs and also the band intensities were observed to reduce from C‐1 to C‐3 nanocomposites with the increased Fe₃ O₄ NP content in the nanocomposites from 10 to 50 wt%, respectively. Therefore, ATR‐FTIR spectra confirm the existence of functional groups for 1.25% Ce@HAP and Fe₃ O₄ NPs in nanocomposites which were further proved by XRD analysis.

Fig. 1.

ATR‐FTIR spectroscopy of

(a) 1.25% Ce@HAP, (b) Fe₃ O₄ NPs, (c) C‐1, (d) C‐2, (e) C‐3

3.2 Powder‐XRD analysis

The XRD pattern of 1.25% Ce@HAP is shown in Fig. 2 a, which exhibits the characteristic diffraction peaks for the formation of phase pure 1.25% Ce@HAP without any other secondary phases such as tricalcium phosphate, cerium oxide and exactly matched with the parent pattern of HAP (JCPDS: 09‐432) [31]. The strong diffracted peaks were observed at 2θ  = 26.06, 28.27, 29.21, 32.05, 32.26, 33.15, 34.25, 40.08, 46.85 and 49.68°_­, which were a major support for the presence of the crystalline nature of 1.25% of Ce‐doped HAP. Fig. 2 b presents the XRD paradigm of Fe₃ O₄ NPs which shows the broad diffraction peaks at 30.12, 35.79, 43.52, 57.43 and 62.93° which denotes the formation of phase pure nanosized spinal structured Fe₃ O₄ NPs (JCPDS: 85‐1436) [32] with the size of particles in the range of 52–68 nm. The XRD patterns of C‐1 to C‐3 nanocomposites are shown in Figs. 2 c –e, in these combinations of patterns of 1.25% Ce@HAP and Fe₃ O₄ NPs diffraction peaks were observed at 2θ  = 25.9–34.02 and 30.12–62.93°, respectively. These combined diffraction peaks proved the formation of nanocomposites with a mixture of 1.25% Ce@HAP and Fe₃ O₄ NPs. From these patterns, it was found that the crystallinity of 1.25% Ce@HAP gradually decreased from C‐1 to C‐3 due to the decrease in the addition of the 1.25% Ce@HAP content from 90 to 50 wt% whereas the peak intensity of Fe₃ O₄ was steadily raised from C‐1 to C‐3 because of its increment in the composites from 10 to 50 wt%, respectively, and which is mostly substantiated with theoretical ratios of nanocomposites. Also, it was observed that the XRD patterns from C‐1 to C‐3 were slightly amorphous in nature with less crystallinity owing to the inclusion of Fe₃ O₄ NPs, the crystallite size and crystallinity were calculated by the following formulas. Therefore, XRD patterns confirm the fabrication of nanocomposites with a combination of 1.25% Ce@HAP and Fe₃ O₄ NPs (Table 1)

$$D=\frac{0.9\lambda }{\beta (1/2)\cos \theta }$$ (3)

$${\chi }_c={\left(0.24{\mathrm{A}}^{\circ }/{\beta }_{1/2}\right)}^3$$ (4)

Fig. 2.

XRD patterns of

(a) 1.25% Ce@HAP, (b) Fe₃ O₄ NPs, (c) C‐1, (d) C‐2, (e) C‐3

Table 1.

Calculation of crystallite size and crystallinity of 1.25% Ce@HAP, Fe₃ O₄ NPs, C‐1, C‐2 and C‐3

S.No.	Crystallite size D (nm)		Crystallinity (X c)
	1.25% Ce@HAP	Fe3 O4 NPs	1.25% Ce@HAP	Fe3 O4 NPs
pure 1.25%Ce@HAP	85 ± 2.2	—	2.6305	—
pure Fe3 O4 NPs	—	59 ± 2.8	—	1.8905
C‐1	63 ± 1.9	52 ± 2.3	2.4803	1.2308
C‐2	58 ± 1.5	46 ± 1.5	1.8235	1.5605
C‐3	49 ± 1.2	32 ± 1.8	1.4580	1.6320

3.3 SEM‐EDAX analysis

The surface morphology of 1.25% Ce@HAP, Fe₃ O₄ NPs, C‐1, C‐2 and C‐3 is shown in Fig. 3. The SEM micrograph of 1.25% Ce@HAP (Fig. 3 a) powder exhibits a rod‐shaped morphology with high agglomeration and particles were of non‐uniform size. Fig. 3 b displays the SEM image of Fe₃ O₄ NPs and shapes of the particles were highly dense in nature with agglomeration and size was in the nano to submicron range. Figs. 3 c and d show the C‐1 to C‐3 nanocomposites which demonstrated that particles were agglomerated and similar to spherical‐shaped morphology. These SEM images conclude that rod‐shaped 1.25% Ce@HAP particles were altered into spherical‐shaped clusters due to the ball milling process for 2 h at 450 rpm. Also, it was found that from C‐1 to C‐3, the particles became more agglomerated due to the increase of the Fe₃ O₄ NP content in the composites from 10 to 50 wt%, respectively. However, the sizes of the C‐1, C‐2 and C‐3 nanocomposites were in the nano regime with a larger surface area.

Fig. 3.

SEM micrographs of

(a) 1.25% Ce@HAP, (b) Fe₃ O₄ NPs, (c) C‐1, (d) C‐2, (e) C‐3

The EDAX spectra of individual and composites are presented in Fig. 4. The EDAX spectra (Figs. 4 a and b) of 1.25% Ce@HAP and Fe₃ O₄ NPs confirm the presence of Ca, P, O, Ce and Fe, O elements, respectively. Figs. 4 c –e show the presence of Ca, P, Ce, O and Fe atoms for the C‐1, C‐2 and C‐3 nanocomposites, these EDAX spectra confirm the atomic percentage of Fe was found to increase for C‐1, C‐2 and C‐3 at 9.45, 28.62 and 42.92%, respectively, and corroborate with the experimental addition of Fe₃ O₄ NPs into the development of composites.

Fig. 4.

EDAX patterns of

(a) 1.25% Ce@HAP, (b) Fe₃ O₄ NPs, (c) C‐1, (d) C‐2, (e) C‐3

3.4 HR‐TEM analysis

HR‐TEM (Fig. 5 a) reveals that 1.25% Ce@HAP powder was in the shape of rods with agglomeration. The size of 700 C for 2 h sintered 1.25% Ce@HAP was in the range of 60–85 nm. Fig. 5 b shows that particles of Fe₃ O₄ exhibited spherical geometry with high agglomeration and the size of the particles is around 62 nm. Figs. 5 c –e present the surface topography of C‐1, C‐2 and C‐3 nanocomposites, respectively. These micrographs disclose that 1.25% Ce@HAP rod‐shaped particles were changed to elongated spherical‐shaped particles, which correlates with SEM analysis. In the TEM images of these composites, small‐sized lesser dense Fe₃ O₄ NPs were uniformly distributed over the 1.25% Ce@HAP particles. It was found that C‐1 to C‐3 composites exhibited more agglomeration due to the increase of Fe₃ O₄ NP (10–50 wt%) content in the nanocomposites and the crystallite size of C‐1, C‐2 and C‐3 nanocomposites were determined as 89, 106, and 118 nm, respectively.

Fig. 5.

HR‐TEM images of

(a) 1.25% Ce@HAP, (b) Fe₃ O₄ NPs, (c) C‐1, (d) C‐2, (e) C‐3

3.5 VSM analysis

Fe₃ O₄ NPs, C‐1, C‐2 and C‐3 nanocomposites were evaluated for their magnetic properties at room temperature using a vibrating sample magnetometer with an applied external magnetic field ranging from +1 to −1 kOe. The VSM data of nanocomposite powders are shown in Fig. 6, which confirms that the developed composites exhibited superparamagnetic behaviour at 32°C. From these hysteresis loops, saturation magnetisation (M _s) was calculated as 69.70, 26.09, 20.08 and 16.74 emu/g for Fe₃ O₄ NPs, C‐3, C‐2 and C‐1, respectively, the coercivity (H _c) was zero for all nanocomposites. However, this study proves that M _s was found to increase from C‐1 to C‐3 due to the increase in the addition of Fe₃ O₄ into composites and also the size of particles was gradually increased which is substantiated by TEM analysis [33].

Fig. 6.

VSM analysis of

(a) 1.25% Ce@HAP, (b) Fe₃ O₄ NPs, (c) C‐1, (d) C‐2, (e) C‐3

3.6 Antibacterial activity

Among the various bacterial identification methods, the CFU has been used as a conventional method to quantify the bacterial culture in the solid agar medium. In this study, the bacterial restriction behaviour of 1.25% Ce@HAP, Fe₃ O₄, C1, C‐2 and C3 nanocomposites was examined against the most common human pathogenic bacteria such as E. coli and S. aureus which have much association with general bone or wound infections. The bacterial cultures (10³ cells/ml) at 0.5 OD of both E. coli and S. aureus were subjected to the individual and nanocomposite powders at a concentration of 5 mg/ml (Fig. 7). The antibacterial activity of 1.25% Ce@HAP exhibits excellent pathogen restriction towards E. coli than S. aureus due to the lesser dense cell wall, whereas Fe₃ O₄ NPs showed significant effects on the both pathogens when compared with the controls. The Fe₃ O₄ NPs display more reduction in the formation of colonies for E. coli than S. aureus. The 1.25% Ce@HAP‐Fe₃ O₄ nanocomposites (C‐1 to C‐3) demonstrated the superior pathogen inhibition efficiency against E. coli and S. aureus pathogens than the individual materials. The microbial restriction behaviour was found to enhance from C‐1 to C‐3 nanocomposites due to the presence of a 50 : 50 ratio of 1.25% Ce@HAP and Fe₃ O₄ NPs, which have a higher influence on the inhibition of both viable cell growth. However, these results confirm that C‐1 to C‐3 nanocomposites have more antibacterial activity towards the lesser dense cell wall of E. coli than the rigid cell wall of S. aureus.

Fig. 7.

Antibacterial activity of

(a) 1.25% Ce@HAP, (b) Fe₃ O₄ NPs, (c) C‐1, (d) C‐2, (e) C‐3 against S. aureus and E. coli pathogens

The possible mechanism illustrated as the interaction of liberated ions such as Ce and Fe from the composite powders with the outer membrane layer of microorganisms. The released ions can be diffused into the cell wall of pathogens and lead to the destruction of the cell wall by plasmolysis. After the cell wall breakage, the ions will directly interrupt the DNA replication and cause the damage to the DNA strands and prevent the respiration process of pathogens [34, 35]. Hence, this study proves that the developed nanocomposites can be used for the restriction of bone infection causing microbes.

3.7 In vitro biocompatibility study

Human osteosarcoma derived MG‐63 osteoblast cell lines were used to assess the in vitro biocompatibility study of C‐3 nanocomposites using different concentrations such as 200, 400, 600, 800 and 1000 µg/ml for 24–48 h. Based on the superior pathogen restriction behaviour, the C‐3 nanocomposite was selected for the evaluation of its cell viability and cytotoxicity behaviour for bone regeneration applications. The MTT assay result is presented in Fig. 8, which shows the percentage of cell viability for control was raised from 24 to 48 h from 102 to 106%, respectively. The C‐3 nanocomposites were exposed to the MG‐63 cells using various concentrations (200–1000 µg/ml) and exhibit the enhancement of viable cells from 24 to 48 h. The C‐3 composites at 200 and 400 µg/mL concentrations revealed increased percentage of cell viability when compared to the control. It was noticed that for 24 h treatment, the concentrations of 200–400 µg/mL displays a cell viability as 108–112% and 48 h shown as 111–116% respectively. The concentrations from 600 to 1000 µg/mL demonstrated the slight toxic effect on the MG‐63 cells. The MG‐63 viable cell percentage was found to reduce to 82, 71, 62% for 24 h, whereas to 88, 76 and 64% for 48 h. Therefore, the MTT assay confirms that C‐3 composites at higher concentrations are toxic towards the MG‐63 osteoblast cells due to the release of Ce and Fe ions which can cause the effect on the viability of cells. This was further proved by inverted phase contrast microscopy (Fig. 9), which shows the prominent cell response in terms of cell attachment, spreading over the 200–400 µg/ml concentrations with the cell morphology of elongated spindle shape (green notation) whereas at 600–1000 µg/ml exhibit the more non‐adherent non‐viable cells (red notation).

Fig. 8.

Percentage of MG‐63 cell viability at 200–1000 µg/ml concentrations of C‐3 nanocomposites for 24 and 48 h

Fig. 9.

Phase contrast microscopy images of MG‐63 cells at 200–1000 µg/ml concentrations of C‐3 nanocomposites for 24 and 48 h

Hence, this in vitro MTT assessment proves that at lower concentrations C‐3 nanocomposites can be recommended for use in biomedical applications.

4 Conclusions

In the present investigation, we have successfully developed nanocomposites with a combination of 1.25% Ce@HAP and Fe₃ O₄ NPs in the ratio of 90 : 10, 70 : 30, 50 : 50, respectively, using planetary ball milling technique.

• The ATR‐FTIR analysis confirms that with an increase in the Fe₃ O₄ content from C‐1 to C‐3, the peak broadness was found to be slightly increased in the range of wavenumbers (560–630 cm⁻¹). The XRD patterns evidenced that the existence of phase composition of composites with a combination of 1.25% Ce@HAP and Fe₃ O₄, it was observed from these patterns that the crystallinity of 1.25% Ce@HAP and Fe₃ O₄ were gradually decreased and increased with respect to the theoretical ratios of composites 90 : 10, 70 : 30 and 50 : 50, respectively.

• The SEM micrographs reveal that the rod‐shaped morphology of 1.25% Ce@HAP powder was altered into spherical‐shaped clusters with high agglomeration due to the ball milling process for 2 h at 450 rpm. The EDAX spectra confirm that the atomic percentage of Fe was 9.45, 28.62 and 42.92% for C‐1, C‐2 and C‐3 nanocomposites. TEM analysis confirms that Fe₃ O₄ NPs were uniformly distributed on the surface of 1.25% Ce@HAP and more agglomeration arise from C‐1 to C‐3 due to the increased addition of Fe₃ O₄ NPs into the nanocomposites and the size was in the range of 89–118 nm, respectively. From the VSM study, saturation magnetisation (M _s) was calculated as 69.70, 26.09, 20.08 and 16.74 emu/g for Fe₃ O₄ NPs, C‐3, C‐2 and C‐1, respectively, the coercivity (H _c) was zero for all nanocomposites.

• The antibacterial activity was found to enhance from C‐1 to C‐3 nanocomposites due to the increase of Fe₃ O₄ NPs, which have a higher influence on the inhibition of both viable cell growths because of released Ce and Fe ions. Hence, it confirms that C‐1 to C‐3 nanocomposites have more bacterial inhibition efficiency towards E. coli than S. aureus owing to lesser cell wall density.

• Based on the antibacterial activity, an in vitro biocompatibility study was performed on the C‐3 nanocomposites using MG‐63 osteoblast cells and it revealed high cytocompatible up to 400 µg/ml concentration and exhibited the enhancement of percentage of cell viability for 24 and 48 h with prominent cell attachment, adhesion and proliferation. For 600–1000 µg/ml concentrations, it demonstrated slight toxicity to the MG‐63 cells due to the leaching out of ions from the composites such as Fe and Ce.

Therefore, these in vitro biological evaluations prove that the developed nanocomposites can act as a promising biomaterial for future bone regeneration and remodelling applications.