Improving the Biological Properties of UHMWPE Biocomposite for Orthopedic Applications

Bone plates are essential for bone fracture healing because they modify the biomechanical microenvironment at the fracture site to provide the necessary mechanical fixation for fracture fragments. The objective of this study was to determine cell availability, antibacterial activity, and wettability through a contact angle test. However, biocomposites that involve UHMWPE reinforced with n-HA and n-TiO2 particles at different fractions (0, 1.5, 2.5, 3.5, and 4.5%) and 5% from carbon and Kevlar fibers were fabricated by hot pressing technique. In vitro studies revealed good cell viability on the surface of the hybrid biocomposite even after 72 hr. The UHMEPE nanocomposite reinforced with carbon showed better cell attachment for fibroblasts than other UHMWPE nanocomposite materials reinforced with Kevlar fiber. The results of the contact angle measurements indicated that the incorporation of nanoparticles and the fiber reinforcement increased the wettability due to the hydrophilic character of nanobiocomposite, and also (UHMWPE-4.5% wt. TiO2–CF) biocomposite was the best wettability (∼48% as compared to neat UHMWPE). Antibacterial experiments involving Gram-positive bacteria, Staphylococcus aureus, confirm excellent bactericidal property for (UHMWPE-4.5% wt. TiO2–CF) biocomposite. Thermal analysis of the produced nanocomposites revealed that they had higher melting and crystallinity temperatures than pure UHMWPE.


Introduction
When a human bone fracture occurs, various types of internal fxation devices, such as bone plates, are placed at the fracture site to help stabilize the bone structure [1]. Metals such as stainless steel, titanium, and their alloys are not the best material for a bone plate because of the negative efects on callus formation and fracture healing caused by the high modulus of elasticity and biomechanical mismatch to the bone [2,3]. Polymer-based composite, which has reduced stifness for bone plate fxations, can be used as an alternative to metal materials to solve these problems. Due to its high chemical resistance, biocompatibility, and mechanical and tribological properties, ultrahigh molecular weight polyethylene (UHMWPE) is a polymer that is frequently employed in medical applications. Te biological internal fxation employing the internal fxator principle exhibits an indirect healing pattern and a low infection rate, which was frmly established with a very high follow-up rate of 97% [4]. Bonfeld et al. introduced hydroxyapatite (HA)-reinforced high-density polyethylene (HDPE), and there has been a continuous efort to develop bone-analogue composites for biomedical applications. However, these composites' lower strength and stifness to the cortical bone have restricted their use as load-bearing bone replacements [5]. In a recent study, scientists created new plate fxation using thermoplastic composite polymer. Due to their biocompatibility and degradation rates, which are readily modifed by changing the composition and production method, thermoplastic polymers are more advantageous than thermoset polymers [6,7]. According to previous researchers, second phases of ceramics (A1 2 O 3 , TiO 2 , quartz, wollastonite, kaolin, CaCO 3 , etc.), carbons (carbon black, carbon fber, carbon nanotubes, graphite), and polymers (polyurethane, phenyl p-hydroxyzoate, etc.) can improve the mechanical properties of UHMWPE composites [8][9][10]. Hydroxyapatite (HA, Ca 10 (PO 4 ) 6 (OH) 2 ), the main inorganic component of hard tissues, has a variety of applications in bone fllers and replacements due to its excellent bioactivity and osteoconductivity [11]. Celebi Efe et al. investigated whether UHMWPE-TiO 2 composite flms meet basic requirements for biological applications of artifcial hip joint acetabular liner materials [12]. In this research analysis, wettability through contact angle measurement enhanced the antibacterial activity of n-HA and n-TiO 2 biocomposite and CF and KF hybrid biocomposite plates fxation and studied the cytotoxicity of UHMWPE biocomposites with human fbroblast cells.

Materials and Methods
UHMWPE polymer powder with molecular weight 600-700 (104 g/mol.) and density (0.093-0.94) (g/cm 3 ) was supplied by the Luoyang Max Pipe Industry as a matrix. Hydroxyapatite nanopowder and titanium oxide (anatase phase) nanopowder were obtained from Xian Real and Hangzhou Union in Biotechnology Company, China, as reinforcement materials. Te materials were weighed by weight fraction (0, 1.5, 2.5, 3.5, and 4.5%). Firstly, the powder particles are dispersed in ethanol with an ultrasonic device for 45 min for n-HA and 30 min for n-TiO 2 . Secondly, the UHMWPE is added to the nanoparticles simultaneously, followed by mechanical mixing for 30 min to n-HA and 15 min to n-TiO 2 at 1500 rpm. To violate the ethanol, we then simply place the mixture in an oven at 60°C for 2 hours and allowed it to stand for 48 hours, tightly dry. Tereafter, the mixture was placed in a mold and pressed in a hydraulic press at a temperature of 180°C and a pressure of 12 MPa for one hour. Ten, the mold left to cool in air up to room temperature to get the sheet of composite material as shown in Figure 1. After achieved all tests on the prepared particulate biocomposite materials, it is found that the best composite materials are (UHMWPE+4.5% n-HA) and (UHMWPE+4.5% n-TiO2) and it is chosen to fabricate the hybrid biocomposites by the addition of two types of fbers (Kevlar and carbon) as one layer. Ten, the hot press was achieved by the same procedures which were previously mentioned for the preparation of particulate biocomposite material.

Antibacterial Activities.
Te antibacterial potential of the prepared samples (1, 2, 3, 4, 5, 6, 7) was investigated against Gram's negative and Gram's positive bacterial strains using an agar well difusion assay [13,14]. About 20 mL of Muller-Hinton (MH) agar was aseptically poured into sterile Petri dishes. Muller-Hinton (MH) agar was aseptically placed in the amount of 20 mL onto sterile Petri plates. From their stock cultures, the bacterial species were separated using a sterile wire loop [14]. After the organisms had been cultivated, 6-mm-diameter wells on the agar plates were bored using sterile points. Te samples (1, 2, 3, 4, 5, 6, 7) were injected into the bored wells at a variety of concentrations. Prior to calculating and recording the average zones of inhibition diameter, the cultivated plates containing the samples (1, 2, 3, 4, 5, 6, 7) and the test organisms were incubated overnight at 37°C [15,16].

Contact Angle (Wettability Test).
Many biological, chemical, and physical processes depend on the wettability of a surface. Te contact angle, which is the angle created by the tangent to the liquid-vapor interface and the solid surface at the three-phase contact line, is frequently used to describe wetting [17]. Te contact angle was measured according to the ASTM stander (D5946-04) using optical contact angle and interface tension meter [18]. Te specimen was placed on a glass slide; the tissues were then inserted into the instrument specimen holder after being tightened for better observation during the contact angle measurements. Ten, a distilled water droplet with a volume of 8 ml was dropped onto the biocomposite surface. After dipping, the contact angle measurement was taken, and a video camera captured the droplet shape.

MMT Assay (Cell Availability).
In order to quantify the cell growth, 100 L of DMEM/F12 supplemented with 10% heat-inactivated fetal bovine serum (FBS) was added to the cells before they were seeded in a 96-well tissue culture plate at a density of 104 cells per well. Tis 24-hour incubation period was involved. Te cells were then treated for 4 hours with a fresh, serum-free culture medium that contained serial dilutions of the sample. Ten, for a second 24 hours, the media were changed with 100 L of brand-new full media. Cells were then incubated for a further 4 hours at 37°C after the medium was replaced with 100 L of fresh medium containing MTT, giving a fnal MTT concentration of 0.5 mg/ml. Te medium was aspirated after 4 hours, and each well's absorbance (570 nm) was measured using a microplate reader after the MTT formazan produced by living cells was dissolved in 100 L of DMSO. Te absorbance values of the sample-treated wells and control wells (the control cells grown in a medium without the CDs), respectively, were used to calculate the relative cell viability (%). Data are provided as average SD (n � 3) [19].

Result and Discussions
3.1. Characterization. Te purpose of this test was to evaluate the thermal behavior and physical changes that occurred when pure UHMWPE and UHMWPE biocomposite specimens were heated. Diferential scanning calorimetry (DSC) measurements were carried out according to ASTM D3418-03 under a nitrogen gas atmosphere. Te prepared samples with weight of (8-10) ± 0.5 mg were mounted in aluminum pans and heats up from −40 to 250°C with a heating rate of 10°C/min [20]. Termal properties of biocomposite materials (UHMWPE + n-HA wt. %) and (UHMWPE + n-TiO 2 wt. %) with variation weight fraction of NPs using the DSC inspection are illustrated in Figure 2. Te melting and crystallization temperatures of polymer composites were measured using diferential scanning calorimetry (DSC), as shown by the curves. Percentages are summarized in Table 1.
From the DSC analysis represented in Figure 2, it is clear that the melting point of UHMWPE increased from 136.29°C for neat UHMWPE polymer to 145 and 144.82 for n-HA and, n-TiO 2 , respectively, for weight fraction of 4.5%. It can be noticed that the melting temperature increase with the increase (HA and TiO 2 ) in nanoparticles but the higher values was obtained in (UHMWPE/n-HA) biocomposite compared to (UHMWPE/n-TiO 2 ) biocomposite due to better compatibility [10,21]. Tis indicates that the 4.5% wt. of NPs has the strongest nucleation efect that promotes the formation of microcrystalline zones within the biocomposite [22,23]. However, the efect of fber reinforcement on the melting points of samples was more remarkable and reaches 148.27°C at (UHMWPE + 4.5% HA + CF) due to the uniform distribution of the fber within the matrix and also to their thermal properties of the carbon fbers [24]. Moreover, crystallization temperature was increasing when the addition of NPs. Te higher thermal stability of the biocomposites as compared with the neat UHMWPE is attributed to the formation of a cross-linked network upon chain scission and improved compactness of the polyethylene. Figure 3 illustrates the contact angle of (n-HA) and (n-TiO 2 ) particulate biocomposites. It can be seen from the fgure that the incorporation of HA and TiO 2 NPs caused the contact angle to decrease with an increase in wt.% of nanoparticles, from (62.66) for pure UHMWPE to (53.54), (52.04) for 4.5% n-HA and 4.5% n-TiO 2 , respectively. Tis may be explained by the fact that the presence of nanoparticles in excess of a threshold quantity may cause HA and TiO 2 deposited on the surface of nanobiocomposites to reduce surface roughness, making the surfaces more compact and reducing their hydrophobicity [25,26]. Figure 4 illustrates the contact angle of hybrid biocomposites, and the wetting behavior for Kevlar and carbon fber represents a high reduction in contact angle and has a hydrophilic character compared to 4.5% n-HA and 4.5% n-TiO 2 particulate biocomposites. Carbon fber when added to particulate biocomposites shows excellent wettability as compared to Kevlar fber which has (32.57) for (4.5% n-HA + CF) whenever (45.96) for (4.5% n-HA + KF). Te hydrophilicity is a direct relation to biocompatibility; the smallest the contact angle the better the biocompatibility [27].

Antibacterial Activity.
Te antibacterial activity of the samples is evaluated against two bacterial types S. aureus and E. coli. Figures 5 and 6 presented the inhibition zone for   Te anatase crystalline structure of TiO 2 presents its highest antibacterial activity among other crystalline structures which is an important condition that afects its physicochemical properties, which in turn afects its antibacterial activity [28,29]. Depending on the result, it can be noticed that the antibacterial efciency of UHMWPE biocomposites to the S. aureus bacteria is more than that of antibacterial efciency to E. coli bacteria [30]. Furthermore, when particulate biocomposite was combined with fbers, it was shown that (UHMWPE+n-TiO 2 +CF) hybrid biocomposite exhibited antibacterial activities, which assisted in bacterial cell death.

MMT Assay (Cell Availability).
Biocompatibility was evaluated by assessing the cell viability that can be defned as a time-dependent phenomenon. Figures 7 and 8 show the cell viability of all biocomposites, and results revealed that pure UHMWPE exhibited 91.37%, 91.65%, and 91.92    International Journal of Biomaterials viabilities at 24, 48, and 72 h, respectively. When HA and TiO 2 nanoparticles were added, the results showed a considerable increase in cell availability, which increased as the number of days increased [22]. In addition, the viability results showed higher cell viability when particulate biocomposite reinforced with carbon fber than with Kevlar fber for both types of nanoparticle samples at 24, 48, and 72 hr. [31]. Furthermore, 4.5% n-HA + CF hybrid biocomposites showed excellent cell availability (99.29%) at 72 hr. Also, none of the specimens show any signifcant   Inhibition zone (mm)

Conclusion
Tis study aimed at the development of UHMEPE nanobiocompsite materials which frstly used as internal bone plate fxation. According to the obtained data, it was found that (i) Te study of the antibacterial activity against E. coli and S. aureus bacteria recorded an improvement in bone plates biocomposite compared with pure UHMWPE, and the best result obtained with hybrid biocomposites bone plate fxation containing multiple reinforcement titanium dioxide (n-TiO 2 ) and carbon fber. (ii) Contact angle decreased with an increase in the nanoparticles content, which led to an increase in hydrophobicity. Whereas hybrid biocomposites for bone plate fxation enhance wettability, a hybrid biocomposite (UHMWPE/CF) with n-TiO 2 would be more hydrophilic and therefore have high surface energy. (iii) Te in vitro investigation with MTT assay reveals a high cytocompatibility of the prepared biocomposite specimens whereas the incorporation of n-HA and n-TiO 2 in UHMWPE matrix became more active after 72 hr. of exposure in human fbroblast cells, and there was a remarkable increase in cell viability when hybrid biocomposites bone plate fxation containing multiple reinforcement hydroxyapatite (n-HA) and carbon fber. It seems that a material composition enhances cells' growth and activity without any toxic efects on the cells. (iv) DSC analysis results revealed an improvement in the thermal stability of bionanocomposites, and the melting temperature T m and crystallization temperature T c have been enhanced by the addition of n-HA and carbon fber.

Data Availability
Te data that support the fndings of this study can be obtained from the corresponding author upon request.

Conflicts of Interest
Te authors declare that they have no conficts of interest.