Cytocompatibility and Mechanical Properties of Hydroxyapatite Composite Reinforced with Multi-Walled...

Fatemeh Gholami, Sharif Hussein Sharif Zein, Suzylawati Binti Ismail, Soon Huat Tan

  Open Access OPEN ACCESS  Peer Reviewed PEER-REVIEWED

Cytocompatibility and Mechanical Properties of Hydroxyapatite Composite Reinforced with Multi-Walled Carbon Nanotubes and Bovine Serum Albumin

Fatemeh Gholami1,, Sharif Hussein Sharif Zein2, Suzylawati Binti Ismail1, Soon Huat Tan1

1School of Chemical Engineering, Engineering Campus, Universiti Sains Malaysia, Nibong Tebal, Pulau Pinang, Malaysia

2School of Engineering, University of Hull, Cottingham Road, Hull, HU6 7RX, UK

Abstract

In this study, HA/MWCNTs/BSA composites with highly improved mechanical properties for use as bone replacement materials were developed. The HA/MWCNTs/BSA composites powders with different types of MWCNTs (MWCNTs-OH, MWCNTs) were successfully prepared. Compressive strength tests and cell toxicity test on human fibroblasts cell lines (CCD-18Co) were performed to evaluate the mechanical and biological properties of the developed composites. The results show that the HA composites which are prepared using MWCNTs-OH and BSA with compressive strength of 18.92 MPa has the highest results in comparison with HA composites which are prepared using non-functionalized MWCNTs. The obtained compressive strength is higher than values that reported for the strength of trabecular bone (2-12 MPa). Cell culture experiments showed that at low concentrations (6.25 and 12.5 mg/ml), HA/MWCNT/BSA composites led to cell proliferative rather than cytotoxic effects on fibroblasts, evidenced by high cell viability of approximately 250%.

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Cite this article:

  • Gholami, Fatemeh, et al. "Cytocompatibility and Mechanical Properties of Hydroxyapatite Composite Reinforced with Multi-Walled Carbon Nanotubes and Bovine Serum Albumin." Chemical Engineering and Science 2.1 (2014): 1-4.
  • Gholami, F. , Zein, S. H. S. , Ismail, S. B. , & Tan, S. H. (2014). Cytocompatibility and Mechanical Properties of Hydroxyapatite Composite Reinforced with Multi-Walled Carbon Nanotubes and Bovine Serum Albumin. Chemical Engineering and Science, 2(1), 1-4.
  • Gholami, Fatemeh, Sharif Hussein Sharif Zein, Suzylawati Binti Ismail, and Soon Huat Tan. "Cytocompatibility and Mechanical Properties of Hydroxyapatite Composite Reinforced with Multi-Walled Carbon Nanotubes and Bovine Serum Albumin." Chemical Engineering and Science 2, no. 1 (2014): 1-4.

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1. Introduction

The similarity of the chemical composition of HA to the mineral phase of bone and their excellent biocompatibility meets the requirement of materials designed for bone repair and augmentation purposes. However, the application of HA in load bearing devices is limited by its poor mechanical properties [1]. CNTs, with their outstanding stiffness and strength, have good potential applications in tissue engineering [2, 3]. Several studies on reinforcement of metal, ceramic, and polymer composites using CNTs have successfully demonstrated its capability of improving the structural properties, such as strength, elastic modulus, fracture toughness, and wear resistance [4, 5]. Their strength and stiffness, combined with their small size and large interfacial area, suggest that they may have great potential as a reinforcing agent for HA [6, 7]. A few studies showed the biocompatibility of CNTs in orthopedic application and established that CNTs may accelerate bone growth and increase proliferation and differentiation of osteoblasts [8, 9, 10]. HA-CNT composites, combining HA and CNTs, can improve mechanical properties of HA for use in a wider range of biomedical applications. Xu et al. [11] reported on the mechanical properties and response of osteoblast-like cells to HA–MWCNTs composites densified by spark plasma sintering. HA powder and MWCNTs were dry mixed and then consolidated using spark-plasma sintering at temperatures ranging from 900°C to 1200°C. The mechanical properties reportedly increased in hardness and Young’s modulus compared with HA. They also reported an increase in cellular response to the composites compared with HA alone [11]. The aim of this study is to investigate the mechanical properties and the cytotoxicity of the HA composites, incorporated with different types of MWCNTs.

2. Materials and Methods

The scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR) and cell culture experiments with human CCD-18Co fibroblasts cell lines were performed to evaluate the mechanical, structural and biological properties of the HA/MWCNTs /BSA composites.

2.1. Sample Preparation

HA powder was mixed with de-ionized water, 15 wt.% bovine serum albumin (BSA), and 0.5 wt.% of different types of MWCNTs (MWCNTs-OH, purity: 99.9% MWCNTs*, purity: 99.9%, MWCNTs and purity: 95%) to produce HA/MWCNTs/BSA composites. The deionized water was added to a mixture of HA, BSA and MWCNTs and blended with a mechanical overhead stirrer until a homogeneous paste was obtained. The paste was packed into a cylindrical stainless steel mold (diameter: 6 mm, length: 12 mm), and then it was stored in an incubator (Gyro-Rocker Incubator Model: S170) at 37 °C and 97% humidity for 24 hr until further experiments with the samples were performed. Immediately before testing, the samples were taken out and dried at room temperature (25°C to 28°C).

2.2. Cell Seeding and Treatment of Cells

Prior to inoculation with HA/MWCNTs/BSA composites, the fibroblasts were seeded in 96-well plates with a density of 1.5×105 cells per well and allowed to attach for 12 h before further treatment. The ground HA/MWCNTs/BSA powder was diluted with cell culture medium to obtain the desired concentrations of 6.25, 12.5, 25, 50, 100 and 200 µg/ml. Into each well containing the cells, 100 mL of fresh medium supplemented with CPC/MWCNT/BSA particle concentrations between 6.25 mg/ml and 200 µg/ml was added. To the control wells, only 100 ml of cell culture medium was added.

2.3. MTT Assay

The mitochondrial respiratory activity of the fibroblasts treated with HA/MWCNTs/BSA composite particles was determined colourimetrically using MTT ((3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assays [12]. MTT salt solution (5 µg/ml) was added 4 h prior to the end of the 72 h sample incubation to allow cellular tetrazolium metabolism. After 4 h, cell culture medium and MTT solution was aspirated, and the MTT lysis solution (DMSO) was added into the wells to solubilize formazan crystals, and the plates were incubated for 5 min before absorption was read at a wavelength of 570 nm with a reference wavelength of 620 nm (ELISA microplate reader, Ascent Multiskan). For each analysis, the samples were examined in triplicates, and the average and standard deviation were calculated. Results were reported as percentage of cell viability and expressed as the mean ± standard deviation. The percentage of viable cells was calculated from the optical density (O.D.) values using Eqs. (1) and (2) respectively:

(1)
(2)

Data were recorded and analyzed to determine the inhibitory effects of the test substance on cell proliferation or viability in three to six independent measurements.

2.4. Mechanical Testing

The compressive strength of the cylindrical specimens (diameter = 6 mm, height = 12 mm) was tested using an Instron 3367 universal testing machine at a crosshead speed of 1.0 mm/min. The compressive strength was determined on the basis of maximum compressive force and the nominal cross-sectional area of the scaffolds. Three samples of each scaffold composition were investigated.

3. Result and Discussion

The FTIR spectra of the HA/MWCNTs/BSA composites are summarized in Figure 1. The absorption bands at 3297 cm-1 to 3307 cm-1 and 3400 cm-1 to 3500 cm-1 corresponded to the strong characteristic peak of the stretching mode of the –OH group . Subsequently, the sputtering target primarily comprised crystalline HA. As shown in Figures 4.6 (a) to 4.6 (d), the peaks pertaining to the HA phase were the –OH bands at 3298, 3409, and 3411 cm-1, and this sputtering target comprised primarily crystalline HA. The peaks observed at 604 and 551 cm-1 belong to P–O bands (ν4 mode) [13, 14, 15].

Figure 1. FTIR patterns of (a) HA/MWCNTs*/BSA, (b) HA/MWCNTs/BSA, (c) HA/MWCNTs-OH/BSA

Figure 2 shows the compressive strength of HA/MWCNTs-OH/BSA, HA/MWCNTs/BSA, HA/MWCNTs*/BSA composites. The CPC/MWCNTs-OH/BSA composite has the highest compressive strength (18.92 MPa) compare to other composites which are prepared using non-functionalized MWCNTs. As shown in SEM micrographs of composites in Figure 3, there are some holes in the structures of HA/MWCNTs*/BSA and HA/MWCNTs/BSA composites and smaller pores in the HA/MWCNTs-OH/BSA composites that might cause to reduce the compressive strength of them.

The lower compressive strength results of HA/MWCNTs/BSA and HA/MWCNTs*/BSA (13.64 and 6.99 MPa respectively) are might be due to the weak bonding between particles and the presence of more holes in their structures. The MWCNTs are insoluble in water and organic solvent, and have a tendency to agglomerate and form clusters and bundles [16]. Furthermore, MWCNTs are naturally having a highly hydrophobic surface, so their low chemical wettability lead to have nonuniform dispersion in HA matrix and consequently low mechanical strength. Functionalization of CNTs surface cause to increase nucleation sites and improve dispersion of CNTs in HA matrix. Using modified CNTs shown a level of improvements in the properties of the final composites [17, 18]. The compressive strength results confirmed that there is stronger bonding between MWCNTs-OH with HA particles in composites and lead to better results for these composites. Introducing of BSA in HA matrix can improve the mechanical properties. The compressive strength of cortical bone is in the range between 100 to 230 MPa and for trabecular bone in the range 2-12 MPa [19]. The developed composites are applicable for use as bone replacement material to the level of trabecular bone. Further improvement is necessary to make it appropriate to use as a bone filler or bone replacement at high load-bearing anatomical sites.

Figure 2. Compresseiv strength of (a) HA/MWCNTs*/BSA, (b) HA/MWCNTs/BSA, (c) HA/MWCNTs-OH/BSA

The cell culture experiments with human CCD-18Co fibroblasts were performed to evaluate the biological properties of a novel multi-walled carbon nanotube reinforced hydroxyapatite composite.

Figure 3. SEM micrographs of composites (a) HA/MWCNTs-OH/BSA, (b) HA/MWCNTs/BSA and (c) HA/MWCNTs*/BSA

Figure 4 shows the effect of HA composites on CCD-18Co fibroblast cells, as measured by MTT assay. Among the samples tested (HA/MWCNTs-OH/BSA, HA/MWCNTs/BSA, and HA/MWCNTs*/BSA), any of them did not elicit cytotoxic effects on cell proliferation. HA/MWCNTs-OH/BSA, and HA/MWCNTs/BSA showed proliferative effects on the cells. At low concentration, all samples were found to be non-cytotoxicity when treated to the human fibroblast cells, whereas when the concentration of samples was increased, we observed a reduction in cell viability. There was a significant reduction in the viability of CCD-18Co human fibroblast cells by increasing the concentration of 6.25 µg/ml to 200 µg/ml (p < 0.001). For composites with different type of MWCNTs, MTT assays revealed significantly decreasing human fibroblast cell viability with increasing HA composite powder concentration. At a lower concentration (6.25 µg/ml), the percentage of cell viability was 204%, 249% and 106%, for the composites with MWCNTs-OH, MWCNTs and MWCNTs* (95% purity) respectively, and the HA/MWCNTs (99.9 % purity) composite had the highest cell viability (249%) at this concentration. Thus, at low concentrations (6.25 and 12 µg/ml), there was a tendency towards higher cell viabilities, indicating a cell proliferative effect of the HA/MWCNTs/BSA composite as evidenced by a higher metabolic activity of the fibroblasts.

Figure 4. Influence of HA/MWCNTs /BSA composites on CCD-18Co fibroblast metabolic activity as measured using MTT assay (data are presented as the means ±2 standard deviations).

4. Conclusion

In this study, HA/MWCNTs/BSA composites prepared with various types of MWCNTs (MWCNTs-OH, 99.9% purity, MWCNTs with 99.9% purity and MWCNTs* with 95% purity). The novel HA/MWCNTs/BSA and HA/MWCNTs-OH/BSA composites showed favourable cytocompatibility (with 204% and 249% cell viability respectively) with compressive strength of 13.64 and 18.92 MPa respectively, and it is therefore, considered an attractive bone replacement and bone filling material. The biological effect of the compound in cell proliferation hints potential wound healing effect, which adds further benefits to this composite. Further studies are required to find the mechanism of affecting cells by these composites, and more research is needed for increasing the compressive strength of composite to make it applicable in clinical purpose, under load-bearing.

Acknowledgements

A Research University grant from Universiti Sains Malaysia (USM-RU grant) to support this research work is gratefully acknowledged.

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