Hydrothermally processed 1D hydroxyapatite: Mechanism of formation and biocompatibility studies

Abstract

Recent developments in bone tissue engineering have led to an increased interest in one-dimensional (1D) hydroxyapatite (HA) nano- and micro-structures such as wires, ribbons and tubes. They have been proposed for use as cell substrates, reinforcing phases in composites and carriers for biologically active substances. Here we demonstrate the synthesis of 1D HA structures using an optimized, urea-assisted, high-yield hydrothermal batch process. The one-pot process, yielding HA structures composed of bundles of ribbons and wires, was typified by the simultaneous occurrence of a multitude of intermediate reactions, failing to meet the uniformity criteria over particle morphology and size. To overcome these issues, the preparation procedure was divided to two stages: dicalcium phosphate platelets synthesized in the first step were used as a precursor for the synthesis of 1D HA in the second stage. Despite the elongated particle morphologies, both the precursor and the final product exhibited excellent biocompatibility and caused no reduction of viability when tested against osteoblastic MC3T3-E1 cells in 2D culture up to the concentration of 2.6mg/cm(2). X-ray powder diffraction combined with a range of electron microscopies and laser diffraction analyses was used to elucidate the formation mechanism and the microstructure of the final particles. The two-step synthesis involved a more direct transformation of DCP to 1D HA with the average diameter of 37nm and the aspect ratio exceeding 100:1. The comparison of crystalline domain sizes along different crystallographic directions showed no signs of significant anisotropy, while indicating that individual nanowires are ordered in bundles in the b crystallographic direction of the P63/m space group of HA. Intermediate processes, e.g., dehydration of dicalcium phosphate, are critical for the formation of 1D HA alongside other key aspects of this phase transformation, it must be investigated in more detail in the continuous design of smart HA micro- and nano-structures with advanced therapeutic potentials.

Keywords: Biomedical; Hydrothermal; Hydroxyapatite; Nanowires; Particle size distribution.

Figure 1

The sketch of “one-pot” route for the synthesis of 1D HAstructures (a), and the two-stage 1D HA synthesis route via DCP platelets (b).

Figure 2

XRD patterns of precursor for samples 1 and 2 and for samples from 1 to 5 within the whole 2Θ measurement range (a) and with a zoom on a selected interval (b). The diffractograms are compared to the reflections of the reference AMCSD code 0008880 and code 0009357 corresponding to DCPD (brushite) and pure HA, respectively.

Figure 3

SEM images of 1D HA along with the particle size distributions for Samples 2 (a) and 4 (b), including the mixing proportion and other parameters for the convoluted components of the distributions. The μ-s and σ-s are given in microns. Scale bars are a) 50 and 10 μm and b) 50 and 5 μm respectively.

Figure 4

XRD patterns of the precursors for the hydrothermal synthesis of HA following the second, two-step route (a) for the whole 2Θ measurement range and (b) with a zoom on a selected interval. The star (*) denotes an unidentified impurity in sample 6 (b). The patterns are compared with the reference cards for brushite – DCPD code no. 0008880 [45] and monetite – DCPA code no. 0009584 [46].

Figure 5

SEM and TEM images of platelets as precursors for the hydrothermal synthesis of Samples 6 (a) (scale bars 1 μm and 500 nm) and 7 (b) (scale bars 1 μm, 500 nm and 200 nm), revealing laminated microstructure.

Figure 6

XRD patterns of Samples 6, 7 and 8 compared to the reference AMCSD code 0009357 corresponding to pure HA.

Figure 7

FE-SEM, TEM images and electron diffractions of Samples 6 (a) (scale bars 200 nm) and 7 (b) (scale bars 200 and 50 nm). SAED patterns are identified as HA reflections.

Figure 8

Distribution of the diameters and lengths of HA nanowires comprising Sample 7, resulting from the comparative analysis of several TEM and optical microscopy images. The mean diameter and length of HA nanowires are 37 nm and 3.7 microns respecively.

Figure 9

FTIR spectra of precursors for the hydrothermal synthesis of HA (a) and of the products (Samples 2, 6, 7, 8) (b).

Figure 10

MTT assay absorbance indicative of the viability ofosteoblastic MC3T3-E1 cells incubated with various calcium phosphate powders at the concentrations of 0.5 and 2.6 mg/cm² (1 and 5 mg per well and per ml of media in the standard 24-well plate, respectively). All the data are represented as averages of three independent cell/particle analyses. Error bars represent the standard deviation (for Sample 7 HA at 5 mg/ml it is invisible to the eye). Data points significantly different from the “cell only” control (p < 0.05) are marked with an asterisk.

Figure 11

Confocal optical micrographs of MC3T3-E1 cells incubated either with identical amounts (5 mg/well) of Sample 7 HA particles (a) or Sample 7 precursor DCPA particles (b) and culturedfor 7 days. Green – calcium phosphate particles; blue – MC3T3-E1 cell nuclei; red – f-actin cytoskeletal microfilaments.