In vitro and in vivo characterization of mineralized hydroxyapatite/polycaprolactone-graphene oxide based bioactive multifunctional coating on Ti alloy for bone implant applications

2.1 Materials

Materials include Titanium (Ti) alloy (99.9% purity), polycaprolactone (PCL), calcium chloride dihydrate (CaCl₂·2H₂O), graphite, potassium permanganate (KMnO₄), magnesium chloride hexahydrate (MgCl₂·6H₂O), zinc chloride hexahydrate (ZnCl₂·6H₂O), ethanol (C₂H₆O), sulfuric acid (H₂SO₄), phosphoric acid (H₃PO₄), hydrogen peroxide (H₂O₂), dipotassium hydrogen phosphate (K₂HPO₄), hydrochloric acid (HCl), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide tetrazodium (MTT), streptomycin, and penicillin were purchased from Sigma-Aldrich, India. All chemicals were of analytical grade and used without further purification. Aqueous solutions were prepared using deionized water from a Milli-Q purification system.

2.2 Synthesis of graphene oxide (GO)

The GO was synthesized from graphite by using a simplified Hummers method (Lim et al., 2011). Briefly, 60 mL of con.H₂SO₄ and 7 mL H₃PO₄ were added to a beaker containing 0.5 g of expanded graphite solution. Afterward, 3 g of KMnO₄ was slowly added into the mixture solution under continuous stirring at room temperature. Complete oxidation of the graphite was ensured after 3 days with a constant stirring. The suspension was cooled and diluted with 200 mL of ice (cold) water. Then, 30% H₂O₂ was added until the gas evolution reaction ceased to remove any KMnO₄. Observations of this reaction include a plethora of bubbles and suspension changing color from a dark brownish to a yellow color. The suspension was then purified and diluted with ultrapure deionized water and centrifugation at 10,000 rpm for 15 min. The precipitate was collected and dried for 5 h in hot air-oven to get the GO powder (Baradaran et al., 2014).

2.3 Specimen preparation

Titanium alloy (Ti-6Al-4V) (purity = 99.9%) were cut into small pieces of 10 × 10×5 mm³ size and inserted in epoxy resin by leaving an area of 1 cm² for exposure to the solution. The specimens were polished and roughened through electrodeposition process by using various grades of silicon carbide paper (SiC) ranging from 180 to 2000 grit. After polishing, all the specimens were cleaned by ethanol and acetone. Finally, the specimens were rinsed with the ultrapure deionized water and dried in flowing air for future use.

2.4 Preparation of M-HAP electrolyte

To obtain M-HAP, 0.1 M MgCl₂·6H₂O, 0.1 M ZnCl₂·6H₂O and 0.3 M CaCl₂·2H₂O were dissolved in ultrapure water to get a mixture solution in the ratio of 8:1:1, respectively. Next, 0.3 M K₂HPO₄ solution was added drop wise into the reaction mixture under constant magnetic stirring for 5 h at room temperature (25 °C) using thermostat to produce the target (Ca + Mg+Zn) and P ratio of 1:67. Finally, the pH of the product was adjusted to 4.7 by using either HCl or NH₄ OH.

2.5 Preparation of M-HAP/GO electrolyte

To prepare M-HAP/GO electrolyte, GO (1 wt%) powder was suspended in 2 mL of ultrapure distilled water. Then, the solution was sonicated for 15 min to prepare a stable aqueous dispersion. After that, the GO dispersion was added into the aqueous solution of M-HAP with vigorous stirring at 25 °C. The pH of the electrolyte was maintained at 4.7.

2.6 Preparation of M-HAP/PCL/GO electrolyte

For the preparation of M-HAP/PCL/GO electrolyte, the different concentrations of PCL (5, 10 and 15 wt%) were dissolved in chloroform under vigorous stirring for 5 h to get a clear solution. Then, it was added drop wise into the M-HAP/GO electrolyte under magnetic stirring for 5 h at 25 °C. The pH of the electrolyte was also maintained at 4.7 and used for the electrodeposition process.

2.7 Electrodeposition of M-HAP/GO and M-HAP/PCL/GO composite on Ti alloy

The electrodeposition of M-HAP/GO and M-HAP/PCL/GO composite coating on Ti alloys at various concentrations were performed by using three-electrode system with an electrochemical workstation (Model: CHI 760C). The platinum (Pt) disk, Ti alloys (the specimen) and saturated calomel electrodes (SCE) were used as the counter, working and reference electrode respectively. The electrodeposition was performed at −1.5 V for duration of 30 min at room temperature as shown in Scheme 1. After the deposition, the coated samples were rinsed with ultrapure water and dried under air for future use.

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Scheme 1 Schematic representation of electrolyte preparation and electrodeposition of M-HAP/PCL/GO composite coating on Ti alloy.

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2.8 Mechanical characterization

The adhesion strength of the M-HAP/GO and M-HAP/PCL/GO (with different weight percentage of PCL) composite film coatings adhered to Ti alloy was determined by the pull-out test. Using the international standard machine (Instron 5565) and the ASTM F 1044-05 standard, a minimum of five indentations were made on each sample to measure and the average value was recorded. All the coated specimens were kept in oven at 100 °C for about 60 min prior to testing and the fixtures were subjected to the pull-out test at a crosshead speed of 1 mm/min.

The microhardness (Hv) of M-HAP, M-HAP/PCL and M-HAP/PCL/GO composite materials was performed by Vickers micro-hardness tester system (Akashi AAV-500 series). The applied load was 0.49 N with an indentation time of 10 s, five replicates in each group was performed per sample and the average microhardness strength was recorded.

2.9 Characterization methods

Fourier-transform Infrared spectrophotometer (FT-IR; NICOLET380) was used to analyze the functional groups present on the composite materials using the KBr pellet technique. The crystalline structures and phase compositions of the M-HAP/PCL/GO composite was carried out by X-ray diffraction spectroscopy (XRD-6000, Shimadzu, Japan). The structural morphologies of the composites and coated films were assessed by using field-emission scanning electron microscope (FE-SEM 1430 VPLEO, UK) at an acceleration voltage of 15 kV.

2.10 Assessment of antimicrobial activity

The two experimental bacterial strains were obtained and then maintained at the Department of Microbiology, University of Madras, Chennai, India. The antimicrobial activities of M-HAP, M-HAP/GO and M-HAP/PCL/GO composites were tested with two bacterial strains: Escherichia coli and Staphylococcus aureus by agar disc diffusion method. For the disc diffusion technique, nutrient agar was poured into petriplates and allowed to solidify. After solidification, the overnight incubated bacterial culture was brought out and introduced to the agar by using an inoculation loop. After inoculation, the bacterial containing petriplates were covered and kept in an incubator at 37 °C for 24 h. After incubation, the M-HAP, M-HAP/GO and M-HAP/PCL/GO composites with different concentration (25 µL, 50 µL, 75 µL, 100 µL and 125 µL) discs of filter paper were dipped in the solutions and then placed onto the bacteria culture. Again, the plates were kept overnight in incubator at 37 °C for 24 h. The zone of inhibition was quantified using a ruler and expressed in millimeters (mm) around the disc. Digital images were also captured to assist in the evaluation of the observable antibacterial activity of the composites. This procedure was followed in triplicate and results recorded.

2.11 Cell viability and cell proliferation assay

The study determines the cell viability and proliferation effects of M-HAP, M-HAP/PCL and M-HAP/PCL/GO composites on osteoblast cells. Human osteoblast cells (MG63) were obtained from National Centre for Cell Sciences (NCCS), Pune, India. The MG63 cells were maintained in Dulbecco's Modified Eagle Medium (DMEM) media supplemented with 1% L-glutamine, 10% fetal bovine serum (FBS), 1% streptomycin and 1% penicillin at 37 °C in 5% CO₂ humidified incubator. Culture Media was changed every 2 days and the cells were subcultured by trypsinization. The cell viability study was performed with a MTT assay (Murugan et al., 2017). In brief, MG63 osteoblast cells were added to a 96-well plate (1 × 10⁵ cells/each well) in 90% DMEM medium for a fixed time at 37 °C (5% CO₂). After being incubated for 24 h, M-HAP, M-HAP/PCL and M-HAP/PCL/GO (10 mg) were added and incubated for 1 day, 3 days and 5 days, respectively. Then, 3 mg/mL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, 100 μL) solution was poured and permitted to incubate for 3 h. Finally, the produced formazan was diluted in 200 μL of dimethyl sulfoxide (DMSO) and viewed using an Enzyme-Linked Immunosorbent Assay (ELISA) analyzer (SPR-960) at 540 nm. Percentage (%) of cell proliferation and cell viability were measured with respect to control at 1 day, 3 days and 5 days. All experiment was performed in triplicates for each set of samples. The cell viability was calculated by the following formula; $$(\%)\text{Cell viability}=\text{Test}/\text{Control}\times 100$$

2.12 In vivo study and surgical procedure

To evaluate the tissue and cell behavior effects of M-HAP/PCL and M-HAP/PCL/GO composites coated Ti alloys, the composites were implanted into the femur bone in Wistar rats and then observed by histological study. All the animal experimental procedures were approved by the Institutional Animal Ethical Committee (IAEC) meeting held at Periyar University, Salem, India (PU/IAEC/085/PO/OC/07/CPCSEA/ZOOC/02/2016–2019). Six male Wistar rats (weight 250–300 g) were used for the experimental study. The rats were housed under normal conditions at 20 °C with a dark cycle (12 h) and light cycle (12 h). The Wistar rats were randomly divided into two groups: M-HAP/PCL coated (group I, n = 3), and M-HAP/PCL/GO (group II, n = 3). All of the Wistar rats were individually anesthetized by given intravenous injections of xylazine (1.5 mg/kg) and ketamine (15 mg/kg) and were provided with a combination of 20% v/v isoflurane and propylene glycol for inhalation. Afterward, the target location in the femur region was shaved and wiped with iodine. Then, the muscular regions were disconnected at implant region over the femur to expose the periosteum. Next, a 2 mm hole at the femur bone was made using a drilling machine, using sufficient irrigation with saline solution to minimize temperature increase in the bone. Following surgery, the penicillin antibiotic was given to all the experimental Wistar rats for two post-operative days. The Wistar rats were placed aside on nurturing during surgical period (14 and 28 days). After 14 and 28 days of implantation, the rats were sacrificed and the implant materials were carefully gathered for the histological observations.

For histological examinations, the implants with rat bones were cut into a thickness of 2 mm. The sliced sections were fixed in 20% formalin and decalcified inside 10% acetic acid solution for 4 days. Afterward, the decalcified sample sections were embedded in paraffin and a 70 nm sections were taken by microtome. Then, the thick sections were stained with Mallory and hematoxylin with eosin staining solution and observed under the light microscopy.

2.13 Statistical analysis

All the experimental values were shown the mean ± standard deviation (SD). One-way analysis of variance (ANOVA) with p^∗ < 0.05 as the level of significance was utilized for statistical analysis. All of the experimental groups were performed in triplicate.

3.1 FT-IR analysis

FT-IR spectra of M-HAP, M-HAP/PCL and M-HAP/PCL/GO composite coated Ti alloy were measured. As shown in Fig. 1a, the characteristic peaks at 485 cm⁻¹, 560 cm⁻¹, 610 cm⁻¹ and 959 cm⁻¹ indicated the appearance of PO₄³⁻ groups in M-HAP coating; additionally, the absorption bands at 3485 cm⁻¹ point out the stretching vibration of hydroxyl (OH⁻) groups. All the M-HAP peaks showed a slight shift when compared with the peaks obtained for HAP (Murugan et al., 2015). As shown in Fig. 1b, the spectra of M-HAP/PCL, the strong absorption band located at 3480 cm⁻¹ is due to the presence of OH group, whereas, the signal peaks at 484 cm⁻¹, 563 cm⁻¹, 607 cm⁻¹, 957 cm⁻¹ and 1094 cm⁻¹ clearly confirmed the presence of PO₄³⁻ groups in M-HAP/PCL. On the other hand, the major peak observed at 1245 cm⁻¹ represented the asymmetric C-O stretching of carbonyl groups, besides corresponding to amorphous and crystalline nature of PCL (Rezaei and Mohammadi 2013). The M-HAP/PCL/GO composite coating in the Fig. 1c confirmed the presence of the signal peak at 3488 cm⁻¹ indicating the OH^¯stretching vibrations. The appearance of additional absorption peaks at 490 cm⁻¹, 565 cm⁻¹, 604 cm⁻¹, 960 cm⁻¹ and 1092 cm⁻¹ correspond to the (PO₄³⁻) groups in M-HAP/PCL/GO. The new absorption peak of carboxyl groups (C⚌O) in GO at 1620 cm⁻¹ was also distinct. These findings indicated the successful formation of M-HAP/PCL/GO composite coating on Ti alloy.

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Fig. 1 FT-IR spectra of (a) M-HAP (b) M-HAP/PCL and (c) M-HAP/PCL/GO composite coated Ti alloys.

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3.1.1

3.1.1 XRD pattern

Fig. 2a–c shows the XRD spectra of M-HAP, M-HAP/GO and M-HAP/PCL/GO coated films. As shown in Fig. 2a, the XRD pattern of M-HAP exhibited the diffraction peaks (2θ) at 28.8°, 29.8°, 30.4°, 31.1°, 34.5°, 41.0° and 53.4° respectively which could be assigned to the M-HAP (ICDD card no. 09-0432) (Uskoković et al., 2017). The XRD peaks confirmed the formation of M-HAP and comparable with the JCPDS card, 09-0432. Subsequently, the XRD spectrum obtained for M-HAP/GO composite coated Ti alloy (Fig. 2b) showed peaks at 2θ values of 14.9°, 28.1°, 29.5°, 30.3°, 31.3°, 34.7°, 40.9° and 53.2°. The peaks range at 28.1°, 29.5°, 30.3°, 31.3°, 34.7°, 40.9° and 53.2 were corresponding to crystalline nature of M-HAP. A peak observed at 14.9° can be assigned to the diffraction of (0 0 2) planes of graphite (ICDD card no.: 13-0148), which confirms the incorporation of GO into M-HAP. The XRD pattern of M-HAP/PCL/GO composites was further investigated. In Fig. 2c, the peaks were identified at 2θ values of 14.8°, 20.1°, 23.3° 28.3°, 29.6, 30.1, 31.2°, 34.6°, 41.1° and 53.6°. The defined peak signals at 2θ values of 28.3°, 29.6, 30.1, 31.2°, 34.6°, 41.1° and 53.6°, correspond to M-HAP. The peaks at 14.8°, 20.1° and 23.3° represented GO/PCL (PCL (10 wt%) in GO (1 wt%). Since, PCL is a semi-crystalline polymer: the XRD peaks for PCL can be recognized at 2θ = 20.1° and 23.3° which can be represented to (1 1 0) and (2 0 0), respectively (ICDD card no. 74-566). It is noteworthy that the peak range becomes slightly broader than the peak signals observed for HAP, which indicates that the M-HAP/PCL/GO composite had a low crystalline nature. The XRD data confirmed the incorporation of PCL with M-HAP/GO coating. In addition, the peaks point out the formation of the M-HAP/PCL/GO composite on the Ti alloy.

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Fig. 2 XRD pattern of (a) M-HAP (b) M-HAP/GO and (c) M-HAP/PCL/GO composite coated Ti alloys.

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3.1.2

3.1.2 Surface morphology analysis

FE-SEM images show the surface morphologies of M-HAP/GO and M-HAP/PCL/GO composite coating obtained with various concentration of PCL (5 wt%, 10 wt% and 15 wt%). The morphology of M-HAP/GO (GO 1 wt%) appeared flake like structure with uneven pores in between them (Fig. 3a). As shown in Fig. 3b, the morphological feature of M-HAP/PCL/GO (PCL 5 wt%) composite coated Ti alloy exhibits the well-defined flake like layered film around micro-structure arrangement with few irregular pores in between them. Similarly, the morphology of the M-HAP/PCL/GO composite coated Ti alloy surface (Fig. 3c) showed obvious evidence that PCL (10 wt%) uniformly covered on GO and mineralized HAP with porous-like structure. The GO allows the surface to have the adequate hydrophilicity and numerous functional groups as nucleation sites. With the higher concentration of PCL up to 15 wt%, this porous-like structure externally forming on the surface paves the way for the detachment of the composite coating from the Ti alloy surface (Fig. 3d). The surface morphological evaluation of the M-HAP/PCL/GO (10 wt% of PCL) composite coating displayed an entire surface coverage and uniform arrangement. It was evident that the porous M-HAP/PCL/GO structure can offer a considerable effectiveness in the mechanical strength of the coated substrate. The minerals substituted HAP and GO could be deposited evenly with the PCL molecules along the surface. Moreover, the interconnected network like porous structure seemed to be sufficient for orthopedic applications.

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Fig. 3 FE-SEM images of (a) M-HAP/GO composite coatings on Ti alloy and M-HAP/PCL/GO composite at different concentrations of PCL (b) 5 wt%, (c) 10 wt% and (d) 15 wt% composite coatings on Ti alloys.

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3.2 Mechanical characterization of the coatings

3.2.1

3.2.1 Adhesion strength

The mechanical strength of the composite coating on the implants is one of the most significant properties for orthopedic applications. Here, the adhesion strength of the M-HAP/PCL and different concentrations of PCL in GO reinforced M-HAP/PCL composite on Ti alloy was evaluated. Fig. 4 shows the adhesion strength for M-HAP/PCL (PCL 10 wt%), M-HAP/PCL/GO (PCL 5 wt%, GO 1 wt%), M-HAP/PCL/GO (PCL 10 wt%, GO 1 wt%) and M-HAP/PCL/GO (PCL 15 wt%, GO 1 wt%) coatings were (21.3 ± 0.4 MPa), (27.5 ± 0.5 MPa), (29.6 ± 0.4 MPa) and (26.8 ± 0.6 MPa), respectively. These results indicated that GO could effectively increase the binding strength of the coatings. The high adhesion strength of coated M-HAP/PCL/GO (PCL 10 wt%, GO 1 wt%) (29.6 ± 0.4 MPa) composite can be considered a suitable biomaterial for high load bearing applications. These results conclusively proved that even low concentrations of GO may improve the mechanical property of M-HAP/PCL.

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Fig. 4 Adhesion strength of M-HAP/PCL and M-HAP/PCL/GO (with different wt.% of PCL) composite coatings on Ti alloys.

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3.2.2

3.2.2 Vickers micro-hardness

The mechanical properties of the composite coated samples were examined by micro-hardness tester. The micro-hardness (Hv) values for the M-HAP, M-HAP/PCL (PCL 10 wt%) and M-HAP/PCL/GO (PCL 10 wt%) composite coatings are shown in Fig. 5. The micro-hardness value of M-HAP, M-HAP/PCL and M-HAP/PCL/GO material was found to be (315 ± 4 Hv), (370 ± 3 Hv) and (455 ± 5 Hv), respectively. The M-HAP/PCL/GO composite had higher micro-hardness (Hv) value compared to M-HAP and M-HAP/PCL composite. This may be due to the presence GO which comprises epoxide and hydroxyl groups on the basal plane as well as carboxyl and carbonyl moieties on its edges. These functional groups favored the interfacial bonding between HAP and polymeric matrices. It proved that GO could improve the mechanical property of M-HAP/PCL composite coating (Peng et al., 2017). The M-HAP/PCL/GO composite may be employed for biomedical applications. The overall synthetic process of M-HAP/PCL/GO composite is shown in Fig. 6.

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Fig. 5 Micro-hardness of M-HAP, M-HAP/PCL and M-HAP/PCL/GO composite coatings on Ti alloys.

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Fig. 6 The schematic illustration for the formation mechanism of M-HAP/PCL/GO composite.

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3.3 Biological studies

3.3.1

3.3.1 Antimicrobial test

To investigate the antibacterial activity of composite, varying doses (25 µL, 50 µL, 75 µL, 100 µL and 125 µL) of control, M-HAP, M-HAP/GO (1 wt% GO) and M-HAP/PCL/GO (10 wt% PCL) composite were tested against both Escherichia coli (Gram-negative) and Staphylococcus aureus (Gram-positive) bacteria the dominant bacterial causative agents for postsurgical infections associated with the implants. In Fig. 7(a and b), the graphs show the inhibition zone of different composite treatment. Among these, M-HAP/PCL/GO composite treated sample induced higher antibacterial activity. The antibacterial activity with the agar disc diffusion method (at 37 °C for 24 h) can be seen in Fig. 8, which shows the inhibition zones for the M-HAP/PCL/GO composite coated sample with both E. coli (lower panel) and S. aureus (upper panel) at different doses (25 μL, 50 μL, 75 μL, and 100 μL). It was noted from the images that for M-HAP/PCL/GO composite coatings, the inhibitory zone on E. coli strain was 10.5, 12, 14.5, 16.5 and 20.5 mm and S. aureus strain was 10, 11, 14 and 16 mm respectively. At the final concentration (100 μL), the M-HAP/PCL/GO composite coated sample revealed the superior rate of antimicrobial activity when compared with other doses. In addition, the antibacterial activity of the M-HAP/PCL/GO composite coating against E. coli was slightly higher than S. aureus bacteria. This is likely due to variation in the structure of the bacteria’s’ cell wall. The gram positive S. aureus cell wall had thickness ranges from 20 to 80 nm owing to the presence of double layer of peptidoglycan and the cell wall of gram negative E. coli strain had a reduced thickness ranges from 1.5 to 10 nm because of single layer peptidoglycan (Kaviya et al., 2011). The earlier studies suggests that Mg²⁺ and Zn²⁺ may have contributed in targeted killing of S. aureus including strains that are antibiotic-resistant (Xie and Yang 2016). The results of this test confirmed that these ions improve the antibacterial effect and furthermore, that the M-HAP/PCL/GO composite has a greater level of antibacterial activity against both E. coli and S. aureus at 37 °C for 24 h.

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Fig. 7 Antibacterial activity of control, M-HAP, M-HAP/GO and 10 wt% PCL/M-HAP/GO composite coatings on Ti alloy samples with different concentrations of 25 µL, 50 µL, 75 µL, 100 µL and 125 µL against S. aureus (a) and E. coli (b).

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Fig. 8 Photographic images for the zone of inhibition of 10 wt% PCL/M-HAP/GO composite coating against S. aureus and E. coli.

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3.3.2

3.3.2 Cell proliferation

In order to assess the cell proliferation or cell viability of human MG63 osteoblast cells in conjunction with M-HAP, M-HAP/PCL (10 wt% PCL) and M-HAP/PCL/GO (10 wt% PCL) composites, samples were quantitatively evaluated by MTT assay. As shown in Fig. 9, the nontoxic features of all M-HAP, M-HAP/PCL and M-HAP/PCL/GO composite coatings were revealed. The cell proliferative effects of M-HAP, M-HAP/PCL and M-HAP/PCL/GO composites were tested for 24 hr. at the intervals of 1 day, 3 days and 5 days. The MG63 osteoblast cells cultured on the M-HAP/PCL/GO composite (10 mg/mL) coated sample displayed outstanding viability after 3 and 5 days of incubation at pH 7.4, which indicates the dramatic increase in cell viability. These comparative results confirmed that M-HAP/PCL/GO composite coated Ti alloy would be a good candidate material for additional research in orthopedic applications such as bone implant scaffolding. With the success of the MG63 cell proliferation at the 5 days of incubation. The M-HAP/PCL/GO was deemed a suitable material for the in vitro biocompatibility study.

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Fig. 9 Bar diagrams showing the% cell viability of HOS MG63 cells with M-HAP, M-HAP/PCL and 10 wt% PCL/M-HAP/GO composite coatings on Ti alloy for 1, 3 and 5 days of incubation.

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3.3.3

3.3.3 In vivo biocompatibility study of the composite coating

To evaluate the in vivo biocompatibility and new bone formation nature of the M-HAP/PCL (PCL 10 wt%) and M-HAP/PCL/GO (PCL 10 wt%) composite coated Ti alloy implant was made and used on male Wistar rats using the hematoxylin and eosin (H&E) staining histological study. The M-HAP/PCL and M-HAP/PCL/GO composite coated Ti alloy was implanted into the surgical site for 14 and 28 days. After the insertion, the surgical places were healed in both M-HAP/PCL and M-HAP/PCL/GO composites. After the period of 14 & 28 days, the implants were successfully removed from the bone of experimental Wistar rats using forceps. To study the interaction between the implant and surrounding tissue after 14 and 28 days, both the M-HAP/PCL composite and the M-HAP/PCL/GO composite coated Ti alloy were assessed. From Fig. 10(a and b), the formation of connective fibrous tissue encompassing fibroblastic cells along with the rim around the implants was found significantly induce newly formed spongy bone (trabecular bone) at 14 days and 28 days in M-HAP/PCL. In addition, the Wistar rats implant with M-HAP/PCL/GO composite coated (Group II) showed no obvious clinical infection at 14 days and 28 days (Fig. 10c and d). Hence, these results implied that the Histological observation showed no occurrence of infection in the M-HAP/PCL/GO composite coated Ti alloy implant samples. The group II implants exhibited the most constructive healing response by the presence of newly formed spongy bone and no presence of inflammation was observed. It was found that there was new bone formation after 28 days post-operation, which suggests that the area of newly formed bone gradually increased between 14 and 28 days. Besides, the ingrowths of abundant osteoblast like cells detected around the implants which suggest that M-HAP/PCL/GO composite coated materials offer the scaffolding or the development of new bone and tissue response. The findings also implied that GO not only possess in vitro osteogenesis enhancing ability, but also excellent in vivo bone-forming ability (Peng et al., 2017). The results displayed no unfavorable local effects such as marked hematoma or edema with the composites up to 28 days postoperatively. These results further support that the M-HAP/PCL/GO composite coated Ti alloy has positive outcomes in the orthopedic application as a better bone regenerator in bone replacement material.

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Fig. 10 Histological analysis of in vivo bone formation ability for M-HAP/PCL (10 wt% PCL) composite coated Ti alloy group (I) (a, b) and M-HAP/PCL/GO (PCL 10 wt%) composite coated Ti alloy group (II) (c, d) at 14 and 28 days of implantation.

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