Modifying of the Biocompatible HA/Mwcnts/BSA Composites with TiO2 for Using as a Bone Replacement Materials

Bone is a natural nano-structured composite composed of organic compounds, which is mainly collagen, reinforced with inorganic compounds like hydroxyapatite (HA) [1]. HA is very similar in composition in the mineral phase of bone; it has good biocompatibility in vitro and in vivo and it is an excellent material for use in bone replacement purpose [2]. However, due to its low tensile strength and brittleness, HA alone could not make up as great bone replacement material due to its poor mechanical properties. In order to increase the mechanical strength of HA, variety of reinforcing elements ranging from particular bioceramic inclusions to polymer fibers and carbon nanotubes (CNTs) have been considered [3]. The reinforcement could significantly affect the mechanical strength and toughness of the bone replacement material [4,5].

Bone is a natural nano-structured composite composed of organic compounds, which is mainly collagen, reinforced with inorganic compounds like hydroxyapatite (HA) [1]. HA is very similar in composition in the mineral phase of bone; it has good biocompatibility in vitro and in vivo and it is an excellent material for use in bone replacement purpose [2]. However, due to its low tensile strength and brittleness, HA alone could not make up as great bone replacement material due to its poor mechanical properties. In order to increase the mechanical strength of HA, variety of reinforcing elements ranging from particular bioceramic inclusions to polymer fibers and carbon nanotubes (CNTs) have been considered [3]. The reinforcement could significantly affect the mechanical strength and toughness of the bone replacement material [4,5].
Since CNTs entered the world materials stage, their mechanical properties have been praised as some of the best present. Their strength and stiffness, combined with their small size and large interfacial area, suggest they may have great potential as reinforcing agent for HA. CNTs are a promising new material owing to its unique internal structure, low mass density, remarkable chemical stability and electronic conductivity [4,5]. The addition of metal nanoparticles to organic materials is known to increase the surface hydrophobicity and to reduce adherence to biomolecules. By using titanium dioxide (TiO 2 ) as an additional component in the composite, it is expected to improve mechanical strength and antimicrobial properties of the composites.
Coating of stronger material on the CNTs has been introduced to enhance the mechanical strength of CNTs. Basically, CNTs coated with metal oxides are expected to exhibit different or better physical properties than those of neat nanotubes. Metal is chosen to be coated with CNTs due to its superior mechanical properties, which allow for load-bearing situations [6]. CNTs and TiO 2 composite materials are attractive materials for researchers in relation to the treatment of contaminated water and air by heterogeneous photo-catalysis, hydrogen evolution, CO 2 photo-reduction, and dye sensitized solar cells. There are many methods could be employed to coat the TiO 2 such as plasma spraying, electrophoretic deposition (EPD) and solgel. Plasma spraying technique is widely used because of its process feasibility [7]. It is ease to operation, but it state that the thick coating produced often exhibit porosity. The porosity will weaken the interfacial strength and leads to adhesion failure [7]. EPD method is a cost-effective method (low equipment costs, simplicity in setup) and able to fabricate free standing objects and coatings from particulate materials. It offers rigid control of film thickness, uniformity and deposition rate [8]. Sol-gel technique is a famous processing route in fabricating optical quality film, forming planar optical wave guides and most surface coating on glass. The inhomogeneity TiO 2 coating on the CNTs surface, the damage of the CNTs surface structure after coating and the thermal stability of the TiO 2 layer deposited on CNTs have been reported. This might be because of the deficient purification or lack of sufficient functionalization of multi-walled carbon nanotubes (MWNTs) before coating [9]. Generally, sol-gel method leads to a heterogeneous, nonuniform coating of CNTs by TiO 2 , showing bare CNT surfaces and random aggregation of TiO 2 onto the CNT surface [10]. The most common precursors in TiO 2 coating is titanium tetraisopropoxide (TTIP). It is chosen due to it can be readily dissolves in alcohol and is not overly sensitive to humidity [11]. As for the solvent, methanol had been chosen as it could condense to generate water to hydrolyze TTIP under supercritical state [12]. Basically, the formation of TiO 2 coating is by the hydrolysis-polycondensation of titanium alkoxides, which in this case is TTIP [13].
This study aims to investigate the effect of coating different types of MWCNTs (MWCNTs, Hydroxylated multi-walled carbon nanotubes (MWCNTs-OH) and Carboxylated multi-walled carbon nanotubes (MWCNTs-COOH)) on mechanical properties of the composite. Furthermore, antimicrobial properties of the composite were studied. Briefly, conventional sol-gel preparation method was performed for the coating process. Hydrolysis and condensation reactions were performed by adding in methanol and titanium (IV) isopropoxide solution. The MWCNTs were added to the solution, and then the resulting gel was filtered and dried for 3 hours at 80°C before being air-calcined in a preheated furnace (400°C, 5 hours). HA was physically mixed with BSA and TiO 2 -MWCNTs. The prepared paste was packed into a cylindrical stainless steel mold (diameter=6 mm, height=12 mm) and incubated for 24 hours (37°C and 97% humidity). The disc diffusion test was carried out on nutrient agar medium by following the guidelines standardized by the National Committee  Figure 2 shows that among the three types of MWCNTs, TiO 2 with pure MWCNTs gave the best mechanical strength. As for MWCNTs-OH and MWCNTs-COOH coated with the similar weight percentage of TiO 2 , both showed quite a similar mechanical strength in this test. The mechanical strength results of TiO 2 coated functionalized MWCNTs were relatively weaker than TiO 2 coated non-functionalized MWCNTs [14].The compressive strength of the HA could not be measured because it was too weak to form the required shape for compressive test purposes. The compressive strength of HA/BSA composite is higher than pure HA, it indicates that BSA can improve the mechanical properties of the composite [9].
Basically the antimicrobial tests were done on microorganisms that can cause implant-related infection to investigate the antimicrobial activities of the samples [15]. The type of microorganism being chosen for the test was Staphylococcus aureus. It is a multipurpose and dangerous pathogen exists in human bodies [16]. This gram-positive bacterium is one of the most common microorganisms in human bodies' infection [17]. By using the published Clinical and Laboratory Standards Institute (CLSI) guidelines, we can determine the susceptibility or resistance of the microorganism to each sample tested. The antimicrobial tests showed that the samples have a little antimicrobial effect on the microorganism. All three of the samples showed similar results based on the observation made. It was suggested that the antimicrobial effect of TiO 2 was suppressed due to coating on MWCNTs, however, the thin TiO 2 biofilm managed to form a protective layer and fell under 'Resistant' category according to CLSI definition.
Between the novel HA/TiO 2 -MWCNTs/BSA composites with different types of MWCNTs (functionalized and non-functionalized MWCNTs) presented in this study, the composite which was prepared by using TiO 2 -MWCNTs appeared to be most effective in term of increasing the mechanical strength of the composite. TEM observations showed that the coating of TiO 2 on MWCNTs was better as compared to functionalized MWCNTs. Diffusion disc test results showed that the antimicrobial effect of composite fell under 'Resistant' category according to CLSI definition.