Highly biocompatible, nanocrystalline hydroxyapatite synthesized in a solvothermal process driven by high energy density microwave radiation

Abstract

Dariusz Smolen1, Tadeusz Chudoba1, Iwona Malka1, Aleksandra Kedzierska1, Witold Lojkowski1, Wojciech Swieszkowski2, Krzysztof Jan Kurzydlowski2, Malgorzata Kolodziejczyk-Mierzynska3, Malgorzata Lewandowska-Szumiel31Polish Academy of Science, Institute of High Pressure Physics, Warsaw, Poland; 2Faculty of Materials Engineering, Warsaw University of Technology, Warsaw, Poland; 3Department of Histology and Embryology, Center of Biostructure Research, Medical University of Warsaw, Warsaw, PolandAbstract: A microwave, solvothermal synthesis of highly biocompatible hydroxyapatite (HAp) nanopowder was developed. The process was conducted in a microwave radiation field having a high energy density of 5 W/mL and over a time less than 2 minutes. The sample measurements included: powder X-ray diffraction, density, specific surface area, and chemical composition. The morphology and structure were investigated by scanning electron microscopy as well as transmission electron microscopy (TEM). The thermal behavior analysis was conducted using a simultaneous thermal analysis technique coupled with quadruple mass spectrometry. Additionally, Fourier transform infrared spectroscopy tests of heated samples were performed. A degradation test and a biocompatibility study in vitro using human osteoblast cells were also conducted. The developed method enables the synthesis of pure, fully crystalline hexagonal HAp nanopowder with a specific surface area close to 240 m2/g and a Ca/P molar ratio equal to 1.57. TEM measurements showed that this method results in particles with an average grain size below 6 nm. A 28-day degradation test conducted according to the ISO standard indicated a 22% loss of initial weight and a calcium ion concentration at 200 µmol/dm3 in the tris(hydroxymethyl)aminomethane hydrochloride test solution. The cytocompatibility of the obtained material was confirmed in a culture of human bone derived cells, both in an indirect test using the material extract, and in direct contact. A quantitative analysis was based on the 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide. Viability assay as well as on DNA content measurements in the PicoGreen test. Indirect observations were performed at one point in time according to the ISO standard for in vitro cytotoxicity (ie, after 24 hours of cell exposure to the extracts). The direct contact tests were completed at three time points: after 24 hours, on day 7, and on day 14 of a culture in an osteogenic medium. All of the tests revealed good tolerance of cells toward the material; this was also shown by means of live/dead fluorescent staining. Both quantitative results and morphological observations revealed much better cell tolerance toward the obtained HAp compared to commercially available HAp NanoXIM, which was used as a reference material.Keywords: bone regeneration, bone substitute, microwave, HAp