A microwave, solvothermal synthesis of hydroxyapatite (HAp) nanopowder with a programmed material resorption rate was developed. The aqueous reaction solution was heated by a microwave radiation field with high energy density. The measurements included powder X-ray diffraction (PXRD) and the density, specific surface area (SSA), and chemical composition as specified by the inductively coupled plasma optical emission spectrometry technique (ICP-OES). The morphology and structure were investigated using scanning electron microscopy (SEM) and transmission electron microscopy (TEM). A degradation test in accordance with norm ISO 10993-4 was conducted. The developed method enables control of the average grain size and chemical composition of the obtained HAp nanoparticles by regulating the microwave radiation time. As a consequence, it allows programming of the material degradation rate and makes possible an adjustment of the material activity in a human body to meet individual resorption rate needs. The authors synthesized a pure, fully crystalline hexagonal hydroxyapatite nanopowder with a specific surface area from 60 to almost 240 m2/g, a Ca/P molar ratio in the range of 1.57–1.67, and an average grain size from 6 nm to over 30 nm. A 28-day degradation test indicated that the material solubility ranged from 4 to 20 mg/dm3.
The number of cases of bone defects requiring replacement has increased rapidly in recent years [
One potential solution marketed as filler for small bone gaps and defects is a bioactive ceramic made of micro- and sub-microhydroxyapatite. Due to their strong similarity to the mineral phase of native bone, called apatite, bioactive ceramics display appropriate levels of osteoconductivity and biocompatibility [
A second important feature which influences the HAp bioactivity is its stoichiometry. Bone mineral has a Ca/P molar ratio near 1.50, which is close to the tricalcium phosphate (TCP) stoichiometry, but structurally and chemically it is similar to the stoichiometric HAp with a Ca/P molar ratio equal to 1.67 [
The literature presents various methods for hydroxyapatite nanoparticle synthesis; these include processes conducted by mills [
The purpose of this work was to develop a microwave solvothermal synthesis method (MSS) based on high energy density microwave radiation [
The hydroxyapatite nanocrystals were obtained by the following method, described in detail in [
The hydroxyapatite nanopowder NanoXIM201 (FLUIDINOVA, ENGENHARIA DE FLUIDOS, SA TECMAIA-Parque de Ciência e Tecnologia da Maia Rua Eng° Frederico Ulrich, 2650 4470-605 Moreira da Maia, Portugal) was used as a reference material in the degradation test and in the PXRD analysis. To the authors’ knowledge, this commercially available hydroxyapatite powder had the lowest available particle size on the market. Its characteristics were a density of 2.93 g/dm3, an SSA of 120 m2/g, an average particle diameter calculated from SSA of 17 nm, and a Ca/P molar ratio declared by the producer and confirmed by the authors of 1.66. Figure
SEM micrograph of NanoXIM201 nanopowder produced by the Fluidinova Company.
Additionally, an XRD analysis using a plate from a pig’s shank bone was used as a reference for natural apatite.
The density measurements were performed using a helium pycnometer (Micromeritics AccuPyc, model 1330) using an in-house procedure [
The phase composition of the reaction products was analyzed by powder X-ray diffraction (Panalytical X’Pert PRO diffractometer, Cu Ka1 radiation). The patterns were collected at room temperature in the 2 theta range 10–150° and with a step of 0.03°. The pattern analysis was performed by whole pattern fitting (the Rietveld method) using the DDM software suite [
The morphology of the nanopowder samples was examined with SEM (ZEISS LEO 1530) and TEM (JEOL JEM2000EX). The TEM investigations, high-resolution TEM (HRTEM), and selected area electron diffraction (SAED) were conducted at 200 kV. The specimens for the TEM observations were prepared by dropping the methanol particle dispersion, created by an ultrasonic technique, on a carbon film supported on a 300 mesh copper grid. Additionally, TEM studies were used to determine the nanoparticle size distribution. The grain size histograms were obtained by considering a region of a sample having about 250 nanocrystals and approximating the shape of each nanocrystal by a sphere. The obtained histograms were fitted to either normal or log-normal distributions (Chi-square test and Person’s coefficient).
The chemical composition of the powders was determined by inductively coupled plasma optical emission spectrometry (ICP-OES), with induction in argon plasma (Jobin-Yvon, model 138 Ultrace).
The determination of material solubility was performed according to norm ISO 10993-14: biological evaluation of medical devices—identification and quantification of degradation products from ceramics. The material was tested in the form of a 6 mm disk (each disk was around 35 mg and was created from the nanopowder using a laboratory hydraulic press under 5 MPa pressure) and was placed in 200 mL of buffer solution (TRIS-HCl buffer with pH
With the shortest reaction of 1.5 min of microwave radiation, the obtained GoHAP had a density equal to 2.91 g/cm3, which is 4% lesser than the value given in the literature for hydroxyapatite, 3.05 g/cm3 [
Effect of the microwave radiation time on the material specific surface area, the density, the grain size calculated on the basis of SSA measurements, and the Ca/P ratio established by the ICP-OES measurements.
Radiation time | SSA | Density | Grain size | Ca/P |
---|---|---|---|---|
(min) | (m2/g) | (g/cm3) | (nm) | |
1.5 | 236 | 2.91 | 9 | 1.57 |
2.5 | 174 | 2.94 | 12 | 1.65 |
5.0 | 99 | 2.96 | 21 | 1.66 |
7.5 | 81 | 3.00 | 25 | 1.67 |
10.0 | 63 | 3.03 | 32 | 1.67 |
Effect of the microwave radiation time on the specific surface area.
The ICP-OES analysis indicated that for 1.5 min reaction, the Ca/P molar ratio was equal to 1.57, which is close to the molar ratios of the natural bone calcium and phosphor elements [
Effect of the microwave radiation time on the Ca/P molar ratio.
The XRD analysis was conducted on GoHAP powders obtained from 1.5-, 2.5-, 5.0-, and 10.0-minute reactions and using a plate from a pig’s shank bone as a reference for natural apatite (Figure
Effect of the microwave radiation time on the lattice parameters determined by the XRD analysis.
Radiation time | Lattice parameter |
Lattice parameter |
---|---|---|
(min) | (±0.001 Å) | (±0.0007 Å) |
1.5 | 9.433 | 6.8745 |
2.5 | 9.439 | 6.8775 |
5.0 | 9.429 | 6.8772 |
7.5 | 9.425 | 6.8767 |
10.0 | 9.423 | 6.8777 |
XRD patterns of HAp particles in pig bone and GoHAP powders synthesized with microwave radiation times of 1.5, 2.5, 5.0, and 10.0 minutes.
The morphology investigation was conducted with SEM and TEM techniques. Figure
SEM micrographs of GoHAP synthesized in (a) 1.5-minute reaction, (b) 2.5-minute reaction, (c) 5.0-minute reaction, and (d) 10.0-minute reaction.
A TEM examination showed that for all microwave radiation times, the obtained hydroxyapatite had the space group P.P63/m of a hexagonal crystal structure with the parameters
SAED with the identified planes.
The dark field TEM image of GoHAP synthesized with a 1.5-minute reaction, and the histogram of the particle size distribution.
The dark field TEM image of GoHAP synthesized with a 7.5-minute reaction, and the histogram of the particle size distribution.
A degradation test was conducted for GoHAP obtained during reactions with microwave radiation times of 1.5, 2.5, 5.0, and 10.0 minutes. Commercially available HAp nanopowder, NanoXIM, was used as a reference material. The initial pH of the test solution was 7.47, the conductivity was 6.82 mS/cm, and the calcium ion concentration was 0.479
Calcium ion concentration changes for GoHAP synthesized with microwave radiation times of 1.5, 2.5, 5.0, and 10.0 minutes, and for the commercial nanopowder, NanoXIM used as a reference material.
The degradation test was additionally followed by determining the weight loss after 28 days of tests. During the test, NanoXIM lost 4% of its original weight, resulting in 7.0 mg/dm3 of solubility, and GoHAP obtained with 1.5-minute reaction time lost over 22% of its initial weight, equal to 38.5 mg/dm3. GoHAP from the 2.5-minute reaction had a solubility of 25.6 mg/dm3, from the 5-minute reaction had solubility of 14.1 g/dm3, and from the 10 min reaction had solubility of 8.7 mg/dm3, as determined from weight loss measurements. The solubility measured by the gravimetric method was in all cases approximately twice as large as the solubility measured by changes in the calcium ion concentration, probably due to nanopowder dispersion. The samples of GoHAP synthesized in 1.5 min reaction were deeply cracked on whole sample surface, but the test disk still was keeping its original shape, when the NanoXIM sample was cracked only in few places and was very weak. In case of the rest GoHAP samples, the density and depth of cracks were decreasing with powder reaction time increase—the GoHAP obtained in 10-minute reaction presented similar cracking to the NanoXIM disk, but it was much more stable during the mechanical operations, like disk transport, and so forth.
Such a high solubility rate should ensure a short degradation time of future scaffold. In degradation time longer than 28 days of conducted ISO test, GoHAP samples should keep or even accelerate their degradation rate, but in case of the real bone implants, the scaffolds degradation rate for time perspective longer than 28 days will depend on many other factors like new tissue formation process, and so forth, so that for long-time periods it should be checked by in vivo tests.
The degradation test showed that the developed method has the potential to adjust the material solubility according to the individual case needs in range from 4 to 20 mg/dm3, according to the ISO 10993-14 norm. The synthesis of this material with a programmable degradation rate was possible due to the implementation of a unique microwave heating technology (MSS) with very high energy density, which enables precise control of the material grain size growth. Compared to conventional heating, microwaves transmit energy directly to the entire volume almost without causing temperature gradients in the reaction vessel. The microwave radiation time may be regulated with 1 s precision, which enables precise grain size growth control.
The rapid microwave heating process leads to overheating of the reaction solution. From the overheated solution, a fine dispersion of nano-HAp precipitates starts to crystallise. Most likely, for short crystalisation times, kinetic processes dominate its growth, and nonstoichiometric crystallites grow with an nonequilibrium structure. Our experimental data show that this is the case for extremely short reaction times of 1.5 minutes. With increase of microwave radiation time the precipitates structure approaches the equilibrium one, and a fully stoichiometric structure is achieved for radiation times longer than 5 minutes (Figure
Precise grain size growth control which determines material specific surface area, together with material stoichiometry control both available via microwave radiation time regulation made the programming of hydroxyapatite solubility possible. The microwave solvothermal synthesis (MSS) with the high energy density allowed obtaining GoHAP powder with unique features which is a promising material for resorbable ceramic bone implants fabrication [
A fully crystalline hydroxyapatite nanopowder with programmed solubility rate was successfully synthesized by a novel MSS method using high-density microwave radiation as a heating mechanism. The material degradation rate was regulated by the amount of applied microwave radiation, which determined the particle size and stoichiometry of the obtained hydroxyapatite nanopowder and consequently enabled the material solubility to be programmed. The obtained nanopowder has unique properties. It is a pure crystalline, hexagonal hydroxyapatite nanopowder with a specific surface area ranging from 60 to almost 240 m2/g and a Ca/P molar ratio in the range of 1.57–1.67; these values are fully regulated by the applied microwave radiation time. The average particle size estimated by the TEM investigation was regulated between 6 nm to over 30 nm. As the degradation study demonstrated, the developed method was able to control the material solubility in conditions simulating the human body in the range from 4 mg/dm3—which is close to NanoXIM, a commercial HAp nanopowder—to 20 mg/dm3. The presented material has the potential to significantly improve the properties of ceramic bone scaffolds by allowing the implant degradation rate to be adjusted to individual situations.
This work was financed by the European Regional Development Fund within the Innovative Economy Operational Programme in the frame of the BIO-IMPLANT project “Bioimplants for the treatment of bone tissue lesions in oncological patients” (