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Article

Preparation of Hydroxyapatite-Titanium Dioxide Composite from Eggshell by Hydrothermal Method: Characterization and Antibacterial Activity

by
Atiek Rostika Noviyanti
1,*,
Efa Nur Asyiah
1,
Muhamad Diki Permana
1,2,
Dina Dwiyanti
1,
Suryana
3 and
Diana Rakhmawaty Eddy
1
1
Department of Chemistry, Faculty of Mathematics and Sciences, Universitas Padjadjaran, Jl. Raya Bandung-Sumedang km. 21, Jatinangor, Sumedang 45363, Indonesia
2
Center for Crystal Science and Technology, University of Yamanashi, Yamanashi 400-8511, Japan
3
Department of Biology, Faculty of Mathematics and Sciences, Universitas Padjadjaran, Jl. Raya Bandung-Sumedang km. 21 Jatinangor, Sumedang 45363, Indonesia
*
Author to whom correspondence should be addressed.
Crystals 2022, 12(11), 1599; https://doi.org/10.3390/cryst12111599
Submission received: 29 September 2022 / Revised: 7 November 2022 / Accepted: 7 November 2022 / Published: 10 November 2022
(This article belongs to the Special Issue Hydroxyapatite Base Nanocomposites (Volume II))
Browse Figures
Versions Notes

Abstract

:
Hydroxyapatite (HA) has been widely used in biomedical applications. HA is prepared from natural sources of eggshell. The obtained HA is composited with TiO2 using the hydrothermal method at a temperature of 230 °C. The structure and morphology of HA-TiO2 composites are characterized by X-ray diffraction (XRD), Fourier-transform infrared spectroscopy (FTIR), and a scanning electron microscope (SEM). Meanwhile, its antibacterial activity was tested on Staphylococcus aureus and Escherichia coli bacteria. The formation of the HA-TiO2 composite is evidenced by typical peaks on the XRD pattern for HA and TiO2. The FTIR spectrum shows that no bond formed between TiO2 and HA which indicates the formation of composites. The smallest crystallite size and the highest specific surface area were obtained from the composite with the composition of HA-TiO2 30:70. In addition, the composition of the composite also shows the smallest particle size distribution. Therefore, the presence of TiO2 plays a significant role in determining the HA properties formed. Furthermore, the HA-TiO2 composite showed good antibacterial activity using disk diffusion and optical density (OD) methods. These results indicate that the synergistic combination of HA from eggshell with TiO2 has favorable properties for antibacterial activity.

1. Introduction

Hydroxyapatite (HA) is one of the inorganic compounds with the chemical formula Ca10(PO4)6(OH)2 [1,2]. HA has been widely used in biomedical applications, especially in bone and dental implants [3,4]. This is because HA is a component of bone minerals and a constituent of teeth in humans that has good bioactivity and biocompatibility [5,6]. HA is usually synthesized from chemicals in the form of H2SO4 and Ca(OH)2, but in general, the process is quite complicated and the product lacks biocompatible properties [7]. The biocompatibility properties of the material for the purposes of biomedical application are conditions that must be met. Some researchers have modified the sources of synthesis materials used, as sourcing materials from nature is more often cheaper, easier to obtain, and most importantly demonstrates biocompatible properties [8]. HA from natural sources also contains ions such as cationic Na+, Zn2+, Mg2+, K+, Si2+, Ba2+, and anionic F−, Cl−, SiO44−, CO32− [9,10]. Some sources of HA from natural materials are cow bones, blood clam shells, and fish bones [11]. Another study revealed the synthesis of HA from chicken eggshells [12]. Eggshells are the easiest natural source to obtain, are relatively inexpensive, and are easy to prepare [13,14]. Currently, many researchers are conducting research on eggshells as an alternative treatment to replace bone damage in humans, since the eggshell itself contains a fairly high element of CaCO3 which is useful as a biomaterial [15]. In addition, the eggshell produced in a year weighs 138,956 tons, and the CaCO3 content of eggshells can reach 94% [16]. Thus, the chicken eggshell has a high potential for the synthesis of high-quality hydroxyapatite.
HA has the advantage that it can absorb bacteria by adsorbing molecules on its surface [17,18]. However, HA cannot decompose a molecule [19,20]. In addition, HA has properties that are resistant to ultraviolet radiation and X-rays. Thus, it is resistant to interference, especially to radiation [21,22]. To overcome the shortcomings, HA needs to be modified by adding other materials. Many studies have observed an increase in the effectiveness of HA due to the formation of composites with polymers [23], metals [24], or metal oxides [25]. The metal oxide that is commonly used is titanium dioxide (TiO2), which is non-toxic, environmentally friendly, and has high mechanical stability, high photocatalytic activity, and antibacterial activity [26,27]. TiO2 has the ability to deactivate Gram-positive and Gram-negative bacteria [28].
Based on the results of previous studies, the increase in the performance of HA composites is strongly influenced by the composition and modification of the structure as well as the synthesis method. Therefore, many researchers have prepared HA-TiO2 composites by various methods such as sol-gel, solid state, and hydrothermal methods [29,30,31]. Ortiz et al. [32] synthesized TiO2/HA composite by the sol-gel method followed by carbon dioxide supercritical drying. In this study, the precursors used were Ti(OBu)4 and 2–5% mol HA. In addition, other studies showed that the addition of 15 and 30% TiO2 to HA showed changes in the behavior of the hydroxyapatite matrix such as the crystallinity, morphology, and bioactivity [33,34]. In another study, HA-TiO2 composite with the addition of TiO2 by 25% can significantly increase the mechanical properties of HA [35,36].
In addition, several researchers have succeeded in synthesizing HA-TiO2 composites to inactivate bacteria through photocatalytic degradation mechanisms [37]. By modifying HA with TiO2, HA can adsorb molecules which will then be broken down by TiO2. HA-TiO2 composites with a ratio of 50–50 showed a bacterial decomposition of about 50% after 2 h of UV exposure [38,39]. In other research, the HA-TiO2 composite with 60% TiO2 showed good antibacterial activity [40]. Thus, the synthesis of HA and the formation of its composite with TiO2 with the right composition can be used as an antibacterial material and is the focus of this study.
However, although several studies have succeeded in synthesizing various HA/TiO2 composites [41], the degradation efficiency still needs to be improved because the research does not fully discuss the effect of compositional variations of the two materials. In addition, no reports have shown the synthesis of HA/TiO2 composites from natural sources, especially from chicken egg shells. Therefore, in this study, we conducted the HA synthesis via a one-step hydrothermal method using the variant source of CaO from chicken eggshells [14]. The synthesized HA was then composited with various molar variations of TiO2 by the hydrothermal method and observed for its antibacterial activity against Gram-positive and Gram-negative bacteria. The use of the hydrothermal method has the advantage that the synthesis results have high purity and crystallinity. In addition, the yield is more than 90% with a homogeneous particle size distribution [42]. The effect of TiO2 addition on structure, bond type, and morphology was observed using X-ray diffraction (XRD), Fourier-transform infrared spectroscopy (FTIR), and scanning electron microscopy (SEM). The use of different sources and synthesis methods is very likely to affect the structure, bonding, morphology, particle size, and effectiveness of the composite as an antibacterial agent [23,43].

2. Materials and Methods

2.1. Synthesis of HA-TiO2 Composites by Hydrothermal Method

HA was synthesized by methods from our previous study [14,44]. HA was synthesized from CaO obtained from eggshells. Chicken eggshells were washed with water to remove contaminants and crushed using a planetary ball mill. After that, it was calcined at 1000 °C for 5 h with an increase in temperature of 15 °C/min to produce CaO. The CaO obtained is then used as a precursor for the synthesis of HA by mixing it with diammonium hydrogen phosphate ((NH4)2HPO4, 99.0%, Merck, Kenilworth, NJ, USA) with a Ca:P mole ratio of 1.67. The pH of the solution with acetic acid (CH3COOH, 99.5%, Merck, Kenilworth, NJ, USA) was adjusted to pH 9. Then, the mixture was put into an autoclave at 120 °C for 48 h. After that, the formed HA was dried to obtain HA crystals.
Composites were prepared by mixing HA and TiO2 (Anatase, 99.0%, Merck, Kenilworth, NJ, USA) with various weight variations, namely HA:TiO2 with ratios of 30:70 (H3T7), 40:60 (H4T6), 50:50 (H5T5), 60:40 (H6T4), and 70:30 (H7T3). The mixture was dispersed and stirred with 60 mL of deionized water using a magnetic stirrer, then put into a 100 mL autoclave, and then heated at 230 °C for 48 h to obtain a HA-TiO2 composite [14,43]. The prepared synthesis process for HA-TiO2 crystals is shown in Figure 1.

2.2. Composite Characterization

Composite structures (H3T7, H4T6, H5T5, H6T4 and H7T3) were determined with X-ray diffraction (XRD, PANalytical PW3040/X0 X’Pert PRO, Malvern, UK). Measurements were made in the range of 10–90° (2θ) with a step size of 0.01° and rate of 10°/min using Cu Kα radiation (λ = 0.15406 nm) at room temperature. The XRD pattern was analyzed using HighScore Plus software (PANalytical 3.0.5) [45]. The crystallite size of the composite crystal is calculated by the Debye-Scherrer equation as in Equation (1) [46].
D = (Kλ)/(Bcosθ)
where D is the crystal size (nm), K is the crystal form factor (0.9), λ is the wavelength of X-rays (0.15406 nm), B is the value of Full Width at Half Maximum (FWHM) (rad), and θ is angle of diffraction (rad). The crystallite size of HA was calculated from peaks at 2θ = 31.8°(211), while TiO2 was calculated from peaks at 2θ = 25.3°(101). The crystallinity of synthesizing HA/TiO2 was calculated from the XRD data using Equation (2) below [47]:
Crystallinity (%) = [(I300 − V112/300)/I300] × 100%
where I300 is the intensity of the diffraction peak at the (300), V112/300 is the intensity of the (112) and (300). In this study, the calculated SSA is crystalline SSA with the definition as surface area (SA) of crystals per mass of crystals [48]. SSA (m2/g) was calculated using Sauter’s formula (Equation (3)), where D is the crystallite size (m) and ρ is the density of crystals (g/m3).
SSA = 6/(D × ρ)
Ascertainment of the functional groups contained in HA-TiO2 is by Fourier-transform infrared spectroscopy (FTIR, PerkinElmer Spectrum 100, Waltham, MA, USA) using the KBr disc technique, at a frequency interval of 400–4000 cm−1 with a step size of 1 cm−1. Each of the collected spectrums is an average of 16 FTIR scans. A few micrograms of the HA/TiO2 composites were mixed with KBr, with a ratio KBr and composites 1:100, then pressed for structural analysis. To determine its morphology, composite samples were characterized using a scanning electron microscope (SEM, JEOL JSM-6360LA, Tokyo, Japan) with a voltage of 20 kV at an ×1000 magnification. Several milligrams of the composite were fixed in a sample holder and coated using gold. Then, the morphology and size were examined and analyzed using ImageJ 1.52a software [49]. A total of approximately 300 particles were analyzed for the area of each sample. The pixel area in the image is converted to the particle area. After that, the length/diameter of each particle was calculated. In addition, the mean and standard deviation values are also calculated.

2.3. Determination of Antibacterial Activity

2.3.1. Disc Diffusion Method

HA-TiO2 composites are suspended to test their antibacterial properties on E. coli (Gram-negative bacteria) and S. aureus (Gram-positive bacteria). The method used is the disc diffusion method. All tools used for antibacterial testing are sterilized first by washing followed by heating using an autoclave at 121 °C for 120 min. After sterilization, the provision of test bacteria is then carried out, the test bacteria that have been inoculated are taken 1 isolate using sterile nichrome wire, then suspended into a test tube containing 5 mL of liquid media. Subsequently, the suspension of the test bacteria was incubated for 24 h at a temperature of 37 °C. Turbidity of the bacterial suspension was standardized to the equivalent of 1 McFarland. The media for nutrient agar (NA) is to be put in a petri dish and allowed to harden. The suspension of the test bacteria is taken and applied to the agar NA medium. Disc paper is placed regularly on the substrate so that it is then incubated for 24 h at a temperature of 37 °C. After that, observations are made and measured inhibition zones are formed using calipers. The inhibitory power is determined by reducing the diameter of the formed inhibition zone by the diameter of the disc paper (ø = 6 mm).

2.3.2. Optical Density Method

Antibacterial activity test using the optical density (OD) method measured the decrease in absorbance value at a wavelength of 600 nm. The composite sample, HA from eggshell, pure TiO2, and positive control (4.5% amoxicillin), were prepared in distilled water. Then 3 mL of S. aureus and E. coli cultures (∼106 cfu/mL) were added to each. The mixed suspension was kept in an incubator with continuous shaking for different time periods. The results of incubation were measured for the absorption value (OD) at a wavelength of 600 nm using a UV-visible spectrophotometer [50]. The graph was plotted of OD at 600 nm versus time to predict the growth of bacteria after interacting with materials.

3. Results and Discussions

3.1. XRD Characterization

The XRD pattern of the HA-TiO2 composite shows peaks corresponding to the Inorganic Crystal Structure Database (ICSD) 98-005-6311 for HA (hexagonal, P63/m) [51] and ICSD 98-015-4602 for TiO2 anatase (tetragonal, I41/amd) [52], which is shown in Figure 2. The conformity of the sample peaks by reference confirms the presence of merging or composting [53]. The diffraction peaks at 2θ = 25.9°(002), 31.8°(211), 46.7°(222), and 49.5°(213) are typical peaks from pure HA, while the peak of anatase TiO2 was evident at 2θ = 25.3°(101), 37.8°(004), and 48.0°(200). The highest peak of HA composite HA-TiO2 resulted from the synthesis of H3T7, H4T6, H5T5, H6T4, dan H7T3 is the same as the highest peak of pure HA (31.8°). The peak intensity of HA increased with increasing HA concentration, as well as the peak of TiO2 which increased with increasing TiO2 content [17]. Based on the XRD pattern, all composites did not obtain other phase peaks. Thus, it can be concluded that there is no impurity in the HA-TiO2 composite. This indicates that no chemical reaction occurred that caused other products to form between the HA and TiO2 phases.
The percentage of crystal structure was calculated by the Rietveld refinement method using HighScore Plus software (PANalytical 3.0.5). The goodness of fit (GoF) is calculated to confirm the accuracy of the Rietveld refinement. Figure S1 shows the Rietveld refinement of XRD pattern. The results show that the percentage of the phase formed in the XRD pattern corresponds to the amount of precursor added (Table 1). In addition, in this calculation, an accurate GoF value is obtained with a value close to 1 [54]. Then, the crystallinity of the sample was calculated based on the peaks in the XRD pattern. This crystallinity value is important because it determines its properties in further applications. It is known that HA-TiO2 50:50 composites have the highest crystallinity compared to other composites (Table 1). However, H3T7, H4T6, H5T5, and H6T4 composites had higher crystallinity than pure HA (56%) [44]. High crystallinity will cause the material to be more stable and have increased mechanical strength when applied [55,56].
The crystallite size was calculated using the Debye-Scherrer equation. The crystallite size of HA was calculated from peaks at 2θ = 31.8°(211), while TiO2 was calculated from peaks at 2θ = 25.3°(101). In Table 2, it can be seen that the crystallite size of the TiO2 decreases with the increasing amount of TiO2 in the composite. As for the crystallite size of HA, the highest crystallite size is in the H4T6 composite and tends to decrease in other composites. However, the difference is not significant. The calculation results show a relatively large measurement error with the difference between samples that are not too far apart. The smallest crystallite size for HA is in the H3T7 composite [57]. The crystallinity size will affect the specific surface area (SSA) properties. SSA is a very important material property for adsorption, heterogeneous catalysis, and reactions at the surface [58,59,60]. The H3T7 composite has the highest SSA value compared to other composites. This SSA value is directly proportional to the size of the crystallite obtained. The SSA values for HA and TiO2 crystals are shown in Table 2.
The HA crystal from the synthesized composite had a smaller lattice volume as the amount of TiO2 was added (Table 3). This indicates that TiO2 causes shrinkage of the lattice volume of HA. This phenomenon complies with another study we found demonstrating that the presence of TiO2 can decrease the length of the lattice constant in the c direction [32]. The smallest HA lattice volume was obtained on the H3T7 composite with a volume of 528.211 Å3. In addition, in the anatase TiO2, the smallest lattice volume value is also shown in the H3T7 composite, which means that the small amount of TiO2 gives the effect of decreasing the lattice volume. The volume of the TiO2 anatase lattice in the H3T7 composite is 135.834 Å3. The crystal structure of HA and anatase TiO2 was shown in Figure 3.

3.2. FTIR Analysis

FTIR analysis was carried out to analyze the functional groups in the HA-TiO2 composite. The FTIR spectrum of HA, TiO2, and variations in the HA-TiO2 are shown in Figure 4. The peak at wavenumber 3399–3571 cm−1 for all HA-TiO2 composites appears with a pointed shape indicating the presence of a –OH group in HA [44]. Meanwhile, the peak at wavenumber 567–1091 cm−1 is a response to the absorption for PO43− [61]. In the wavenumber range of 500–700 cm−1, peak widening is seen, which is observed as the concentration of HA decreases [17]. Meanwhile, the peak at the wavenumber 1420–1458 cm−1 is characteristic of the presence of a carbonate group (CO32−). Carbonates identified on the FTIR spectrum can be atmospherically derived and adsorbed with HA [62,63]. The addition of TiO2 to HA did not significantly show a shift in the FTIR spectrum. There is no bond between TiO2 and HA, but that only indicates the presence of HA and TiO2 that do not bind to each other, which is a characteristic of the formation of composites of both. HA-TiO2 composite FTIR spectrum data for ratio variations can be seen in Table 4.

3.3. SEM Analysis

Figure 5 shows the morphology of pure HA from eggshells and HA-TiO2 composite. The surface structure of HA samples from chicken eggshells has a surface shape in the form of agglomeration of fine sub-particles. After adding TiO2, relatively less agglomeration occurs. All composite morphologies have spherical and porous particle shapes. The entire composite sample looks inhomogeneous to each other. In addition, in each composite sample, there is a very large particle size that is agglomerated and fused with each other. The addition of TiO2 led to an increase in the density of the composite layer [53,57].
From the particle size analysis using ImageJ 1.52a software (Figures S2 and S3) [65], all composites have different particle sizes between 90–1000 nm (Figure 6). Data regarding the value of the mean, mode, median, standard deviation, and polydispersity index (PI) are shown in Table 5. The amount of addition of TiO2 to the composite causes the average particle size to be smaller. Mode values of each composite variation H3T7, H4T6, H5T5, H6T4, and H7T3 were 90.8, 193.7, 194.1, 196.5, and 193.7 nm, respectively.
Furthermore, it is known that if the value of the polydispersity index (PI) is more than 1, it indicates that the sample has a wide nanoparticle distribution and non-uniform nanoparticle diameter size [66]. It is very difficult to make particles of uniform size (monodispersion). The PI values for the H3T7, H4T6, H5T5, H6T4, and H7T3 composites were 1.42, 1.42, 1.32, 1.33, and 1.31, respectively. The H3T7 composite has the smallest average particle size. It is understood that the amount of TiO2 affects the sample size, whereas the addition of TiO2 can cause a decrease in particle size. Size reduction can increase the redox rate for electrons and holes during surface photocatalytic processes. It also decreases photoelectron and hole recombination thereby increasing reactivity [66].

3.4. Antibacterial Activity

The results obtained from this antibacterial test are inhibitory diameter which can be seen in Figure 7. The inhibition zone showed bactericidal activity on the Gram-positive bacteria (S. aureus) and Gram-negative bacteria (E. coli). The highest inhibition zone is found in the H3T7 composite, where Gram-positive bacteria are more extensive than Gram-negative bacteria. These results correspond to previous studies that reported that the antibacterial activity of the HA-TiO2 composite is more pronounced against Gram-positive bacteria due to the plasmolysis of the cell wall or the separation of the cytoplasm from the cell wall [40]. Some studies have reported that Gram-negative bacteria are usually more resistant. This resistance is associated with the cell walls of Gram-negative bacteria that are more complex than those of Gram-Positive bacteria. However, under certain conditions, Gram-negative bacteria can be more resistant to chemical agents than to Gram-positive bacterial cells [67,68]. Another reason is that the external membrane of Gram-negative bacteria consists mainly of a strong layer of lipopolysaccharides, which are considered a barrier. Thus, Gram-positive bacteria have a high sensitivity due to differences in the composition of their cell walls compared to Gram-negative bacteria [69,70]. The antibacterial test image can be seen in Figure S4.
Antibacterial testing on TiO2 does not produce an inhibitory zone. In contrast to TiO2, HA in the test produces an inhibitory zone, this is because HA can adsorb bacteria, in other words, HA deactivates bacteria by an adsorption mechanism. Adhered bacteria are believed to be killed due to the disruption of their cell membrane by the composites, which reach across the microbial membrane [71]. However, the combination of compounds, namely the HA-TiO2 composite, makes the inhibition zone vary. Since this antibacterial activity is a surface reaction [72,73], this activity is determined by surface area and particle size. Samples with higher surface area and small particle sizes showed greater antibacterial activity.
After that, the composite antibacterial activity test was carried out using the optical density method. The positive control used was amoxicillin and the negative control was S. aureus and E. coli without the addition of composites. Amoxicillin was chosen as a positive control in the antibacterial activity test because amoxicillin is an antibiotic that is widely used in the treatment of various infectious diseases caused by pathogenic bacteria [74]. In this study, the difference between the OD method and the disc diffusion method is that in the OD method, light is used to initiate the photocatalytic process. Figure 8 shows the photocatalytic antibacterial activity based on the percentage of inhibition of E. coli and S. aureus bacteria. All composites applied to each bacterium were recorded in time intervals. Based on the test results, the antibacterial activity of the composite was higher than pure HA, pure TiO2, and the positive control of amoxicillin. The greatest inhibition was obtained from the H3T7 composite for E. coli bacteria. In addition, the H3T7 composite also has the smallest particle size and the largest specific surface area, thereby increasing the active site and surface reactivity. H3T7 composites have high antibacterial activity compared to other composites, this is supported by XRD data showing a smaller crystalline size and high surface area, and by SEM data which shows smaller particle sizes [75,76]. A large specific surface area will lead to an increase in the active site and surface reactivity [72]. However, for S. aureus bacteria, the best antibacterial activity was shown by the H7T3 composite followed by H3T7. This is in accordance with the antibacterial data using the disk diffusion method that the H3T7 and H7T3 composites provide the largest zone of inhibition. The growth of bacteria decreased drastically over the period. This study demonstrates that both materials’ synergistic effect in a short time is well proven.

4. Conclusions

HA-TiO2 composites from eggshells have been successfully synthesized using the hydrothermal method. It can be concluded that the composition of the HA-TiO2 composite from eggshell affects the crystallinity, crystal size, specific surface area, and particle distribution. The H3T7 composite had the smallest crystallite size and the highest specific area, namely 32.5 ± 2.9 nm and 58.4 m2/g for HA, and 13.5 ± 2.2 nm and 113.7 m2/g for TiO2, respectively. The FTIR spectrum shows that there is no chemical bond formed between TiO2 and HA which indicates the formation of a composite. The morphology of the HA-TiO2 composite has an irregular particle shape, and agglomerates, and is not homogeneous for all compositions. The size distribution shows that the H3T7 composite has the smallest particle size. This indicates that the addition of TiO2 to HA significantly reduces the particle size.
Antibacterial activity using the disk diffusion method showed that the H3T7 composite had the highest antibacterial activity against Staphylococcus aureus and Escherichia coli bacteria compared to other composites. In addition, the optical density (OD) method also showed that all composites had better antibacterial activity than pure HA and pure TiO2. These results indicate that the synergistic combination of HA from eggshell with TiO2 has favorable properties for antibacterial activity.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/cryst12111599/s1, Figure S1: Rietveld refinement of XRD pattern of (a) H3T7; (b) H4T6; (c) H5T5; (d) H6T4; and (e) H7T3; Figure S2: SEM image with ×10,000 magnification of (a) H3T7; (b) H4T6; (c) H5T5; (d) H6T4; and (e) H7T3; Figure S3: SEM Image after processing in ImageJ 1.52a software for particle distribution analysis of (a) H3T7; (b) H4T6; (c) H5T5; (d) H6T4; and (e) H7T3; Figure S4: Antibacterial test results against Escherichia coli (1) and Staphylococcus aureus (2) bacteria using disc diffusion method of (a) HA; (b) TiO2; (c) H3T7; (d) H4T6; (e) H5T5; (f) H6T4; and (g) H7T3.

Author Contributions

Conceptualization, A.R.N. and D.R.E.; methodology, E.N.A.; software, E.N.A. and M.D.P.; validation, A.R.N. and D.R.E.; formal analysis, E.N.A.; D.D. and M.D.P.; investigation, E.N.A.; D.D. and M.D.P.; resources, E.N.A.; data curation, E.N.A.; writing—original draft preparation, E.N.A.; writing—review and editing, E.N.A. and M.D.P.; visualization, E.N.A. and M.D.P.; supervision, A.R.N. and D.R.E.; project administration, A.R.N. and S.; funding acquisition, A.R.N. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by Riset Data Pustaka dan Daring (RDPD) 2021, Universitas Padjadjaran, grant number 1959/UN6.3.1/PT.00/2021.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Not applicable.

Acknowledgments

The authors thank to Kiki Maesaroh for preparing the materials. We also thank to Annisa Luthfiah and Lintang Kumoro Sakti for discussing the manuscript.

Conflicts of Interest

The authors declare no conflict of interest.

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Crystals 12 01599 g001 550
Figure 1. Synthesis preparation for HA-TiO2 composites by hydrothermal method.
Figure 1. Synthesis preparation for HA-TiO2 composites by hydrothermal method.
Crystals 12 01599 g001
Crystals 12 01599 g002 550
Figure 2. XRD pattern of (a) TiO2 ICSD 01-071-1166; (b) H3T7; (c) H4T6; (d) H5T5; (e) H6T4; (f) H7T3 and; (g) HA ICSD 00-009-0432.
Figure 2. XRD pattern of (a) TiO2 ICSD 01-071-1166; (b) H3T7; (c) H4T6; (d) H5T5; (e) H6T4; (f) H7T3 and; (g) HA ICSD 00-009-0432.
Crystals 12 01599 g002
Crystals 12 01599 g003 550
Figure 3. Crystal structure of HA and anatase TiO2.
Figure 3. Crystal structure of HA and anatase TiO2.
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Crystals 12 01599 g004 550
Figure 4. Infrared spectra of (a) TiO2; (b) H3T7; (c) H4T6; (d) H5I; (e) H6T4; (f) H7T3; and (g) HA.
Figure 4. Infrared spectra of (a) TiO2; (b) H3T7; (c) H4T6; (d) H5I; (e) H6T4; (f) H7T3; and (g) HA.
Crystals 12 01599 g004
Crystals 12 01599 g005 550
Figure 5. SEM image of (a) HA from chicken eggshell; (b) H3T7; (c) H4T6; (d) H5T5; (e) H6T4; and (f) H7T3.
Figure 5. SEM image of (a) HA from chicken eggshell; (b) H3T7; (c) H4T6; (d) H5T5; (e) H6T4; and (f) H7T3.
Crystals 12 01599 g005
Crystals 12 01599 g006 550
Figure 6. Particle size distribution from SEM image using ImageJ 1.52a software of (a) H3T7; (b) H4T6; (c) H5T5; (d) H6T4; and (e) H7T3.
Figure 6. Particle size distribution from SEM image using ImageJ 1.52a software of (a) H3T7; (b) H4T6; (c) H5T5; (d) H6T4; and (e) H7T3.
Crystals 12 01599 g006
Crystals 12 01599 g007 550
Figure 7. Inhibition zone test of HA-TiO2 composite against Escherichia coli and Staphylococcus aureus bacteria.
Figure 7. Inhibition zone test of HA-TiO2 composite against Escherichia coli and Staphylococcus aureus bacteria.
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Crystals 12 01599 g008 550
Figure 8. Optical density measurements of bacteria at a wavelength of 600 nm (OD 600) for (a) Escherichia coli; and (b) Staphylococcus aureus.
Figure 8. Optical density measurements of bacteria at a wavelength of 600 nm (OD 600) for (a) Escherichia coli; and (b) Staphylococcus aureus.
Crystals 12 01599 g008
Table
Table 1. Percentage of phase composition, Rietveld refinement parameters, and crystallinity of the samples.
Table 1. Percentage of phase composition, Rietveld refinement parameters, and crystallinity of the samples.
SampleCrystal Phase (%) *Rietveld Refinement ParametersCrystallinity (%)
HATiO2RexpRwpGoF
H3T729.3 ± 0.070.7 ± 2.222.6016.291.9260
H4T640.1 ± 0.059.9 ± 2.622.5916.971.7761
H5T552.6 ± 0.047.4 ± 2.221.7016.631.7063
H6T461.2 ± 0.038.8 ± 2.823.3317.641.7558
H7T373.0 ± 0.027.0 ± 1.418.5215.601.4136
* Mean ± standard deviation.
Table
Table 2. Crystallite size and specific surface area (SSA) calculated using Scherrer method from all peaks of HA and TiO2.
Table 2. Crystallite size and specific surface area (SSA) calculated using Scherrer method from all peaks of HA and TiO2.
SampleCrystallite Size (nm) *SSA (m2/g)
HATiO2HATiO2
H3T732.5 ± 2.913.5 ± 2.258.4113.7
H4T642.8 ± 3.816.5 ± 3.444.493.0
H5T535.7 ± 3.813.6 ± 2.353.1112.8
H6T439.2 ± 3.616.6 ± 3.348.692.4
H7T335.3 ± 7.117.5 ± 3.554.088.4
* Mean ± standard deviation.
Table
Table 3. Crystal lattice parameters of the samples.
Table 3. Crystal lattice parameters of the samples.
SampleHA *Anatase TiO2 *
a = b (Å)c (Å)V (Å3)a = b (Å)c (Å)V (Å3)
H3T79.420(3)6.873(3)528.2113.786(1)9.475(5)135.834
H4T69.422(3)6.875(2)528.5773.785(2)9.482(6)135.871
H5T59.422(2)6.875(2)528.5783.786(2)9.477(7)135.835
H6T49.425(2)6.876(2)528.9813.786(2)9.477(10)135.878
H7T39.428(2)6.879(2)529.5253.788(2)9.487(8)136.112
* Values in parentheses represent estimated standard deviations in the last quoted place.
Table
Table 4. Types of vibrations in HA, TiO2, and HA/TiO2 composites based on the peaks that appear in each wavenumber.
Table 4. Types of vibrations in HA, TiO2, and HA/TiO2 composites based on the peaks that appear in each wavenumber.
Vibration TypeWavenumber (cm−1)Ref.
HATiO2H3T7H4T6H5T5H6T4H7T3
Ti–O–Ti-800–450800–450800–450800–450800–450800–450[64]
PO43−1095–472-1091–7441089 5671089–6001089–5701089–567[61]
O–H (HA)3573-3571–33993570–34193571–34003571–34353571–3413[44]
OH free-163516351635163416361639[64]
CO32−1456–1403-1456–14031456–14041456–14041456–14071456–1410[62,63]
Table
Table 5. Statistical data calculation from particle size distribution using ImageJ from SEM image.
Table 5. Statistical data calculation from particle size distribution using ImageJ from SEM image.
SampleMeasurement Parameters
Mean (nm)Mode (nm)Median (nm)Standard deviation (nm)PI
H3T7220.490.8194.1143.91.42
H4T6344.2193.7293.7223.11.42
H5T5313.5194.1294.2176.71.32
H6T4292.2196.5290.1167.71.33
H7T3353.6193.7296.9196.71.31
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Noviyanti, A.R.; Asyiah, E.N.; Permana, M.D.; Dwiyanti, D.; Suryana; Eddy, D.R. Preparation of Hydroxyapatite-Titanium Dioxide Composite from Eggshell by Hydrothermal Method: Characterization and Antibacterial Activity. Crystals 2022, 12, 1599. https://doi.org/10.3390/cryst12111599

AMA Style

Noviyanti AR, Asyiah EN, Permana MD, Dwiyanti D, Suryana, Eddy DR. Preparation of Hydroxyapatite-Titanium Dioxide Composite from Eggshell by Hydrothermal Method: Characterization and Antibacterial Activity. Crystals. 2022; 12(11):1599. https://doi.org/10.3390/cryst12111599

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Noviyanti, Atiek Rostika, Efa Nur Asyiah, Muhamad Diki Permana, Dina Dwiyanti, Suryana, and Diana Rakhmawaty Eddy. 2022. "Preparation of Hydroxyapatite-Titanium Dioxide Composite from Eggshell by Hydrothermal Method: Characterization and Antibacterial Activity" Crystals 12, no. 11: 1599. https://doi.org/10.3390/cryst12111599

APA Style

Noviyanti, A. R., Asyiah, E. N., Permana, M. D., Dwiyanti, D., Suryana, & Eddy, D. R. (2022). Preparation of Hydroxyapatite-Titanium Dioxide Composite from Eggshell by Hydrothermal Method: Characterization and Antibacterial Activity. Crystals, 12(11), 1599. https://doi.org/10.3390/cryst12111599

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