Nano-Hydroxyapatite vs. Xenografts: Synthesis, Characterization, and In Vitro Behavior

Abstract

This research focused on the synthesis of apatite, starting from a natural biogenic calcium source (egg-shells) and its chemical and morpho-structural characterization in comparison with two commercial xenografts used as a bone substitute in dentistry. The synthesis route for the hydroxyapatite powder was the microwave-assisted hydrothermal technique, starting from annealed egg-shells as the precursor for lime and di-base ammonium phosphate as the phosphate precursor. The powders were characterized by Fourier-transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), scanning electron microscopy (SEM), energy-dispersive X-ray analysis (EDAX), transmission electron microscopy (TEM), X-ray fluorescence spectroscopy (XRF), and cytotoxicity assay in contact with amniotic fluid stem cell (AFSC) cultures. Compositional and structural similarities or differences between the powder synthesized from egg-shells (HA1) and the two commercial xenograft powders-Bio-Oss^®, totally deproteinized cortical bovine bone, and Gen-Os^®, partially deproteinized porcine bone-were revealed. The HA1 specimen presented a single mineral phase as polycrystalline apatite with a high crystallinity (X_c 0.92), a crystallite size of 43.73 nm, preferential growth under the c axes (002) direction, where it mineralizes in bone, a nano-rod particle morphology, and average lengths up to 77.29 nm and diameters up to 21.74 nm. The surface of the HA1 nanoparticles and internal mesopores (mean size of 3.3 ± 1.6 nm), acquired from high-pressure hydrothermal maturation, along with the precursor's nature, could be responsible for the improved biocompatibility, biomolecule adhesion, and osteoconductive abilities in bone substitute applications. The cytotoxicity assay showed a better AFSC cell viability for HA1 powder than the commercial xenografts did, similar oxidative stress to the control sample, and improved results compared with Gen-Os. The presented preliminary biocompatibility results are promising for bone tissue regeneration applications of HA1, and the study will continue with further tests on osteoblast differentiation and mineralization.

Keywords: apatite; biomaterial; bone substitute; microwave-assisted hydrothermal synthesis.

Figure 1

XRD plot for: (left) hen egg-shell powder before calcination; (right) HA1, Bio-Oss, and Gen-Os powders.

Figure 2

FTIR absorption spectra for xenograft Bio-Oss, Gen-Os, and HA1 powder: (*) absorption bands for protein functional groups.

Figure 3

SEM images for: (a–c) Gen-Os (200×, 10,000×, and 40,000×); (d–f) Bio-Oss (200×, 10,000×, and 40,000×); (g–i) HA1 synthesized from egg-shells (1000×, 20,000×, and 100,000×).

Figure 4

EDS spectra for: (red) Bio-Oss; (green) Gen-Os; (blue) HA1 powders.

Figure 5

TEM images and SAED patterns for: (A–C) Bio-Oss, (C.1) SAED pattern Bio-Oss; (D,E) Gen-Os (rod-like particles- red arrows), (E.1) SAED pattern Gen-Os; (F–H) HA1 (intra-particle pores-yellow arrows), (H.1) SAED pattern HA1.

Figure 6

HA1 (blue), Bio-Oss (red), Gen-Os (green) particles length distribution (A) and width distribution (B); internal pores size distribution for HA1 sample (C).

Figure 7

HRTEM images for specimens: (a) Bio-Oss (inset, inverse fast Fourier transform); (b) Gen-Os (inset, inverse fast Fourier transform); (c) HA1 (inset, inverse fast Fourier transform).

Figure 8

(A) MTT assay results (after 72 h incubation) and (B) GSH assay showing the oxidative stress of AFSCs cultured in the presence of HA1, Gen-Os, Bio-Oss powders, and CTRL sample (only cells); the results are presented as the mean ± S.D. of 3 replicates; different letters indicate significant differences between each sample; p < 0.05/n (n = 6)-based ANOVA statistical analysis, followed by a two-tailed t-test with Bonferroni post hoc correction (A).

Figure 9

Fluorescent microscopy images of Bio-Oss, Gen-Os, HA1, and CONTROL colored with CMTPX fluorophore.