Abstract
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.
1. Introduction
When a human bone fracture occurs, various types of internal fixation 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 effects 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 stiffness for bone plate fixations, 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. The biological internal fixation employing the internal fixator principle exhibits an indirect healing pattern and a low infection rate, which was firmly established with a very high follow-up rate of 97% [4]. Bonfield et al. introduced hydroxyapatite (HA)-reinforced high-density polyethylene (HDPE), and there has been a continuous effort to develop bone-analogue composites for biomedical applications. However, these composites’ lower strength and stiffness to the cortical bone have restricted their use as load-bearing bone replacements [5]. In a recent study, scientists created new plate fixation using thermoplastic composite polymer. Due to their biocompatibility and degradation rates, which are readily modified 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 (A12O3, TiO2, quartz, wollastonite, kaolin, CaCO3, etc.), carbons (carbon black, carbon fiber, carbon nanotubes, graphite), and polymers (polyurethane, phenyl p-hydroxyzoate, etc.) can improve the mechanical properties of UHMWPE composites [8–10]. Hydroxyapatite (HA, Ca10(PO4)6(OH)2), the main inorganic component of hard tissues, has a variety of applications in bone fillers and replacements due to its excellent bioactivity and osteoconductivity [11]. Celebi Efe et al. investigated whether UHMWPE–TiO2 composite films meet basic requirements for biological applications of artificial hip joint acetabular liner materials [12]. In this research analysis, wettability through contact angle measurement enhanced the antibacterial activity of n-HA and n-TiO2 biocomposite and CF and KF hybrid biocomposite plates fixation and studied the cytotoxicity of UHMWPE biocomposites with human fibroblast cells.
2. Materials and Methods
UHMWPE polymer powder with molecular weight 600–700 (104 g/mol.) and density (0.093–0.94) (g/cm3) 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. The 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-TiO2. Secondly, the UHMWPE is added to the nanoparticles simultaneously, followed by mechanical mixing for 30 min to n-HA and 15 min to n-TiO2 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. Thereafter, 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. Then, 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 fibers (Kevlar and carbon) as one layer. Then, the hot press was achieved by the same procedures which were previously mentioned for the preparation of particulate biocomposite material.
2.1. Antibacterial Activities
The 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 diffusion 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. The 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].
2.2. Contact Angle (Wettability Test)
Many biological, chemical, and physical processes depend on the wettability of a surface. The 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]. The contact angle was measured according to the ASTM stander (D5946-04) using optical contact angle and interface tension meter [18]. The 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. Then, 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.
2.3. 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. This 24-hour incubation period was involved. The cells were then treated for 4 hours with a fresh, serum-free culture medium that contained serial dilutions of the sample. Then, 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 final MTT concentration of 0.5 mg/ml. The 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. The 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].
3. Result and Discussions
3.1. Characterization
The purpose of this test was to evaluate the thermal behavior and physical changes that occurred when pure UHMWPE and UHMWPE biocomposite specimens were heated. Differential scanning calorimetry (DSC) measurements were carried out according to ASTM D3418-03 under a nitrogen gas atmosphere. The 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]. Thermal properties of biocomposite materials (UHMWPE + n-HA wt. %) and (UHMWPE + n-TiO2 wt. %) with variation weight fraction of NPs using the DSC inspection are illustrated in Figure 2. The melting and crystallization temperatures of polymer composites were measured using differential scanning calorimetry (DSC), as shown by the curves. Percentages are summarized in Table 1.
(a)
(b)
(c)
(d)
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-TiO2, respectively, for weight fraction of 4.5%. It can be noticed that the melting temperature increase with the increase (HA and TiO2) in nanoparticles but the higher values was obtained in (UHMWPE/n-HA) biocomposite compared to (UHMWPE/n-TiO2) biocomposite due to better compatibility [10, 21]. This indicates that the 4.5% wt. of NPs has the strongest nucleation effect that promotes the formation of microcrystalline zones within the biocomposite [22, 23]. However, the effect of fiber 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 fiber within the matrix and also to their thermal properties of the carbon fibers [24]. Moreover, crystallization temperature was increasing when the addition of NPs. The 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.
3.2. Contact Angle
Figure 3 illustrates the contact angle of (n-HA) and (n-TiO2) particulate biocomposites. It can be seen from the figure that the incorporation of HA and TiO2 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-TiO2, respectively. This may be explained by the fact that the presence of nanoparticles in excess of a threshold quantity may cause HA and TiO2 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 fiber represents a high reduction in contact angle and has a hydrophilic character compared to 4.5% n-HA and 4.5% n-TiO2 particulate biocomposites. Carbon fiber when added to particulate biocomposites shows excellent wettability as compared to Kevlar fiber which has (32.57) for (4.5% n-HA + CF) whenever (45.96) for (4.5% n-HA + KF). The hydrophilicity is a direct relation to biocompatibility; the smallest the contact angle the better the biocompatibility [27].
3.3. Antibacterial Activity
The 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 biocomposites samples against E. coli and S. aureus bacteria. The positions (1–7) at the top correspond to UHMWPE, HA, HA + KF, and HA + CF and at the bottom TiO2, TiO2 + KF, and TiO2 + CF biocomposites, respectively. The highest inhibition zone values are obtained by n-TiO2 biocomposite. It was explained that n-TiO2 shows its antibacterial activity. The anatase crystalline structure of TiO2 presents its highest antibacterial activity among other crystalline structures which is an important condition that affects its physicochemical properties, which in turn affects its antibacterial activity [28, 29]. Depending on the result, it can be noticed that the antibacterial efficiency of UHMWPE biocomposites to the S. aureus bacteria is more than that of antibacterial efficiency to E. coli bacteria [30]. Furthermore, when particulate biocomposite was combined with fibers, it was shown that (UHMWPE+n-TiO2+CF) hybrid biocomposite exhibited antibacterial activities, which assisted in bacterial cell death.
3.4. MMT Assay (Cell Availability)
Biocompatibility was evaluated by assessing the cell viability that can be defined 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 viabilities at 24, 48, and 72 h, respectively. When HA and TiO2 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 fiber than with Kevlar fiber 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 significant toxicity toward human fibroblast cells. The result can be explained by hybrid biocomposites maintaining superior aviability after culturing, making them very good candidates for bone plate fixation in vivo.
4. Conclusion
This study aimed at the development of UHMEPE nanobiocompsite materials which firstly used as internal bone plate fixation. According to the obtained data, it was found that(i)The 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 fixation containing multiple reinforcement titanium dioxide (n-TiO2) and carbon fiber.(ii)Contact angle decreased with an increase in the nanoparticles content, which led to an increase in hydrophobicity. Whereas hybrid biocomposites for bone plate fixation enhance wettability, a hybrid biocomposite (UHMWPE/CF) with n-TiO2 would be more hydrophilic and therefore have high surface energy.(iii)The in vitro investigation with MTT assay reveals a high cytocompatibility of the prepared biocomposite specimens whereas the incorporation of n-HA and n-TiO2 in UHMWPE matrix became more active after 72 hr. of exposure in human fibroblast cells, and there was a remarkable increase in cell viability when hybrid biocomposites bone plate fixation containing multiple reinforcement hydroxyapatite (n-HA) and carbon fiber. It seems that a material composition enhances cells’ growth and activity without any toxic effects on the cells.(iv)DSC analysis results revealed an improvement in the thermal stability of bionanocomposites, and the melting temperature Tm and crystallization temperature Tc have been enhanced by the addition of n-HA and carbon fiber.
Data Availability
The data that support the findings of this study can be obtained from the corresponding author upon request.
Conflicts of Interest
The authors declare that they have no conflicts of interest.