Fabrication of Hydroxyapatite Nanofibers with High Aspect Ratio via Low-Temperature Wet Precipitation Methods Under Acidic Conditions
HAp nanofibers (or whiskers) have been attracted considerable attention for their application as adsorbents and reinforcing fillers owing to their unique morphologies. However, fabrication of HAp nanofibers has been limited to high-temperature and/or long-term methods. Herein, we report that HAp nanofibers with more than 5 μm in length (aspect ratio >100) can be easily obtained by a simple wet precipitation method without additives at relatively low temperature (80 °C) under acidic conditions (initial pH of 6.5 and final pH of 3.9), without pH control during the precipitation.
KeywordsHydroxyapatite Nanofiber Wet chemical precipitation Acidic condition Crystal growth
Hydroxyapatite (HAp) is recognized as a major inorganic component of human hard tissues (bones and teeth). Synthetic HAp, a type of bioceramics, exhibits biocompatibility (i.e., nontoxicity) (Lawton et al. 1989; Abdel-Gawad and Awwad 2010) and excellent cell adhesion properties (Dorozhkin 2010). Therefore, HAp and its composites with polymers or metals have been widely used in orthopedic and dental tissue engineering fields (Choi et al. 2010; Honda et al. 2010; Okada and Matsumoto 2015). Other important applications of HAp include their use as drug delivery carriers (Matsumoto et al. 2004; Bouladjine et al. 2009; Tomoda et al. 2010) and in liquid chromatographic packing materials (Kawasaki 1991) by utilizing the favorable adsorption capacity of the HAp surface for biomolecules, such as cell adhesion proteins (Kilpadi et al. 2001; Lee et al. 2010). HAp belongs to a hexagonal crystal system and possesses different properties on its a and c planes, and hence, its morphology strongly affects protein adsorption properties (Kilpadi et al. 2001; Lee et al. 2010). Therefore, the control of HAp morphology is of fundamental importance to improve its adsorption properties.
HAp nanofibers (or whiskers) have been attracted considerable attention, owing to their unique morphologies, for application as adsorbents and reinforcing components in biomedical composites (Qi et al. 2017). Nevertheless, previous reports have demonstrated the synthesis of HAp nanofibers only at high temperature, during long periods, or by adding additives, such as hydrothermal methods (e.g., 180–200 °C) (Sadat-Shojai et al. 2012; Chen and Zhu 2016), homogeneous precipitation methods (e.g., 72 h) (Aizawa et al. 2005; Zhan et al. 2005), and wet precipitation methods with surfactants (Liu et al. 2002; Chen and Zhu 2016). Therefore, synthesis of HAp nanofibers at mild conditions could reduce costs and enable more diverse applications of HAp. However, the synthesis of HAp nanofibers at low temperature without additives is still challenging.
Herein, we report that HAp nanofibers could be easily obtained by a simple wet precipitation method without additives at relatively low temperature (80 °C) under acidic conditions, without pH control during the precipitation. The formation process of the HAp nanofibers by the simple wet precipitation method was also evaluated.
22.2 Materials and Methods
Unless otherwise mentioned, all materials were of reagent grade and were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). All materials were used as received. Milli-Q water (Millipore Corp., Bedford, MA, USA) with a specific resistance of 18.2 × 106 Ω·cm was used.
An aqueous solution of Ca(NO3)2·H2O (10 g/L = 42.3 mM; 160 mL) was poured into a 500-mL conical flask equipped with an inlet for nitrogen and a magnetic stirrer. After the reactor was heated at a predetermined temperature (30, 50, or 80 °C), initial pH of the solution was adjusted to 6.5 (i.e., acidic condition) or 10.0 (i.e., alkaline condition) by adding a 28% ammonia solution. After the temperature and the initial pH were equilibrated for 5 min, an aqueous solution of (NH4)2HPO4 (101.6 mM; 40 mL; pH 8.0) was added at a feed rate of 8.0 mL/h into the conical flask, and the resultant mixture was stirred for another 12 h at a constant temperature, with variations within 0.1 °C. During the reaction, although the pH was not controlled, the changes in pH were recorded with a pH meter (HM-31P; pH resolution, 0.01; DKK-TOA Corp., Tokyo, Japan) at a time interval of 2 min. The resulting product was then centrifugally washed three times with distilled water and then dried at room temperature under reduced pressure for 1 day.
The dispersed sample was dried on an aluminum stub and coated using an osmium coater Neoc-Pro (Meiwafosis Co. Ltd., Tokyo, Japan) before particle morphology observation by scanning electron microscopy (SEM) using a JSM-6701F microscope (JEOL Ltd., Tokyo, Japan) operated at 5 kV or the sample was dried on a collodion-coated grid before particle morphology observation and electron diffraction measurement by transmission electron microscopy (TEM) using a JEM-2100F microscope (JEOL Ltd.) operated at 200 kV. The number-averaged size (N = 50) was determined from SEM photographs with image analysis software (Image J; National Institutes of Health, Bethesda, MD, USA). Fourier-transform infrared (FTIR) spectra were obtained using an IRAffinity-1S system (Shimadzu Corp., Kyoto, Japan) with a KBr pellet method at a resolution of 4 cm−1 with 32 scans. Product identification was also conducted by X-ray diffraction (XRD) measurements (RINT2500HF; Rigaku Corp., Tokyo, Japan) equipped with a Cu-Kα radiation source.
22.3 Results and Discussion
Taken together, these results indicate that needle-like HAp was firstly precipitated at the initial pH of 6.5 at 80 °C, which is consistent with the phase diagram (Fig. 22.1b). The firstly formed HAp acted as a seed crystal during the following feeding and ripening periods even at acidic conditions. The unexpected stability of enamel apatite (i.e., ribbon-like apatite crystal elongated extremely in its c-axis direction) at acidic conditions has been also reported; that is, enamel apatite did not transformed to DCPD or other calcium phosphates even at pH 4 for 2 months due to a decrease in the ion product of enamel apatite with the decrease in pH (Larsen and Jensen 1989). The acidic condition would be preferable for preventing both secondary nuclei formation (due to an increase in the saturated solubility of HAp (Matsumoto et al. 2007) and CO32− contamination (due to a decrease in the saturated solubility of CO2), which are known to inhibit HAp crystal growth (i.e., elongation).
The HAp nanofibers prepared by the simple wet precipitation method without additives at relatively low temperature (80 °C) have potential applications as adsorbents and reinforcing components in biomedical composites because of their unique morphologies (Qi et al. 2017). The preparation method described here accompanies with the crystal growth of HAp (without secondary nuclei formation, under appropriate conditions). Further optimization of this low-temperature wet precipitation method could enable (1) the preparation of more uniform size distribution of HAp nanocrystals for the pre-prepared seed crystals with controlled initial size and number of seeds (or by developing a modified wet precipitation method with a stepwise pH control from alkaline to acidic condition during the reaction) and (2) the development of brush-like HAp coating for the substrates pre-coated with HAp seed crystals. Note that brush-like HAp coating of titanium substrate showed superior osteoconductivity compared with other morphologies (i.e., needle-like, plate-like, net-like, and spherical) of HAp (Kuroda and Okido 2012). Nevertheless, brush-like HAp coating has been limited to some metallic substrates because the previously developed methods require electroconductive and thermally stable substrates due to their coating conditions [e.g., electrochemical and/or high temperature conditions such as above 140 °C (Kuroda and Okido 2012) for wet methods and above 300 °C (Teshima et al. 2012) for dry methods]. The fabrication of brush-like HAp coated substrate at low temperature and its application will be reported in the near future.
This work was supported partly by the Japan Society for the Promotion of Science KAKENHI (grant numbers: JP16H05533, JP15K1572307, and JP25220912), the Matching Planner Program (MP27115663113) from Japan Science and Technology Agency, and the Cooperative Research Project of Research Center for Biomedical Engineering, Ministry of Education, Culture, Sports, Science and Technology of Japan.
- Abdel-Gawad EI, Awwad SA (2010) Biocompatibility of intravenous nano hydroxyapatite in male rats. Nat Sci 8:60–68Google Scholar
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