Changes in physicochemical and biological properties of porcine bone derived hydroxyapatite induced by the incorporation of fluoride

Abstract

As the main inorganic component of xenogenic bone graft material, bone-derived biological apatite (BAp) has been widely used in implant dentistry, oral and maxillofacial surgery and orthopedics. However, BAp produced via calcination of animal bones still suffers from some drawbacks, such as insufficient mechanical strength and inadequate degradation rate, which impede its application. Fluoride is known to play important roles in both physiological and pathological processes of human hard tissues for its double effects on bones and teeth. In order to understand the effects of fluoride on the properties of BAp, as well as the mechanism behind them, porcine bone derived hydroxyapatite (PHAp) was prepared via thermal treatment, which was then fluoride incorporated at a series concentrations of sodium fluoride, and noted as 0.25-FPHAp, 0.50-FPHAp, and 0.75-FPHAp respectively. The physicochemical characteristics of the materials, including crystal morphology, crystallinity, functional groups, elemental composition, compressive strength, porosity and solubility, were then determined. The biological properties, such as protein adsorption and cell attachment, were also evaluated. It was found that the spheroid-like crystals of PHAp were changed into rod-like after fluoride substitution, resulting in a fluoride concentration-dependent increase in compressive strength, as well as a decreased porosity and solubility of the apatite. However, even though the addition of fluoride was demonstrated to enhance protein adsorption and cell attachment of the materials, the most favorable results were intriguingly achieved in FPHAp with the least fluoride content. Collectively, low level of fluoride incorporation is proposed promising for the modification of clinically used BAp based bone substitute materials, because of its being able to maintain a good balance between physicochemical and biological properties of the apatite.

Keywords: 102 Porous / Nanoporous / Nanostructured materials; 107 Glass and ceramic materials; 211 Scaffold / Tissue engineering / Drug delivery; 30 Bio-inspired and biomedical materials; 302 Crystallization / Heat treatment / Crystal growth; Fluoride; biological apatite; biological properties; physicochemical properties; porcine bone.

Graphical abstract

Figure 1.

TEM images (scale bar = 100 nm) and magnified section (scale bar = 5 nm) with corresponding SAED patterns (scale bar = 5 1/nm) of (a, e) PHAp; (b, f) 0.25-FPHAp; (c, g) 0.50-FPHAp; (d, h) 0.75-FPHAp. SEM images (scale bar = 1 μm) of (i) PHAp; (j) 0.25-FPHAp; (k) 0.50-FPHAp; (l) 0.75-FPHAp. EDS spectra, as well as the distribution of calcium and fluoride by EDS mapping of (m) PHAp; (n) 0.25-FPHAp; (o) 0.50-FPHAp; (p) 0.75-FPHAp.

Figure 2.

(a) FTIR spectra, (b) expanded water stretching region and (c) OH libration region of PHAp, 0.25-FPHAp, 0.50-FPHAp, and 0.75-FPHAp. (d) XRD patterns and (e) expanded X-ray reflections of PHAp, 0.25-FPHAp, 0.50-FPHAp, and 0.75-FPHAp (all spectra are vertically offset for clarity).

Figure 3.

Box plot showing the compressive strength of PHAp, 0.25-FPHAp, 0.50-FPHAp, and 0.75-FPHAp.

Figure 4.

(a) Mercury intrusion cumulative curves of PHAp, 0.25-FPHAp, 0.50-FPHAp, and 0.75-FPHAp. (b) Incremental intrusion in relate to pore diameter showing the pore diameter distribution of PHAp, 0.25-FPHAp, 0.50-FPHAp, and 0.75-FPHAp.

Figure 5.

Solubility isotherms of PHAp, 0.25-FPHAp, 0.50-FPHAp, and 0.75-FPHAp 100 mM KCl solution at 37.0 ± 0.1°C by solid titration. Solubility data of stoichiometric HAp were shown for comparison.

Figure 6.

SEM images (scale bar = 20 μm) and inserted magnified view (scale bar = 10 μm) showing the adhesion of human osteoblastic-like MG63 cell on the surfaces of (a) PHAp, (b) 0.25-FPHAp, (c) 0.50-FPHAp, and (d) 0.75-FPHAp. (e) Quantitative evaluation of cell attachment showed only 0.25-FPHAp favored increased attachment of MG63, while 0.75-FPHAp was inferior than PHAp for cell attachment. (f) Protein absorption assay demonstrated that the incorporation of fluoride resulted in significant higher protein absorption of the apatite.