Contrasting In Vitro Apatite Growth from Bioactive Glass Surfaces with that of Spontaneous Precipitation

Abstract

Body-fluid-exposed bioactive glasses (BGs) integrate with living tissues due to the formation of a biomimetic surface layer of calcium hydroxy-carbonate apatite (HCA) with a close composition to bone mineral. Vast efforts have been spent to understand the mechanisms underlying in vitro apatite mineralization, as either formed by direct precipitation from supersaturated solutions, or from BG substrates in a simulated body fluid (SBF). Formally, these two scenarios are distinct and have hitherto been discussed as such. Herein, we contrast them and identify several shared features. We monitored the formation of amorphous calcium phosphate (ACP) and its crystallization into HCA from a Na 2 O⁻CaO⁻SiO 2 ⁻P 2 O 5 glass exposed to SBF for variable periods out to 28 days. The HCA growth was assessed semi-quantitatively by Fourier transform infrared spectroscopy and powder X-ray diffraction, with the evolution of the relative apatite content for increasing SBF-exposure periods evaluated against trends in Ca and P concentrations in the accompanying solutions. This revealed a sigmoidal apatite growth behavior, well-known to apply to spontaneously precipitated apatite. The results are discussed in relation to the prevailing mechanism proposed for in vitro HCA formation from silicate-based BGs, where we highlight largely simultaneous growth processes of ACP and HCA.

Keywords: X-ray diffraction; apatite growth mechanism; bioactive glass; biomimetic mineralization; infrared spectroscopy; quantification of apatite content; simulated body fluid.

Figure 1

Powder XRD patterns recorded from the pristine BG45${}_{4.0}$ glass (top trace), as well as the SBF-exposed BG45${}_{4.0}$–${\tau }_{\mathrm{SBF}}$ specimens, with the as-indicated SBF-soaking periods (${\tau }_{\mathrm{SBF}}$) increasing from top to bottom. Also shown (red sticks) is a generic diffractogram representative of well-crystalline hydroxyapatite (International Centre for Diffraction Data: data-set 00-09-0432). The dotted vertical lines indicate a selection of Bragg peaks and their associated Miller indices.

Figure 2

(a) FTIR spectra recorded from the BG45${}_{4.0}$ glass powder before and after SBF exposure for 24 h, shown together with the results from a highly crystalline hydroxyapatite reference powder (HAref); the relative intensities are plotted in arbitrary units (a.u.). The spectra are zoomed over the wavenumber range relevant for discriminating IR responses from PO${}_4^{3-}$ groups in disordered (BG45${}_{4.0}$) and ordered (BG45${}_{4.0}$–24 h and HAref) structural environments. (b,c) FTIR spectra from BG45${}_{4.0}$–${\tau }_{\mathrm{SBF}}$ glass powders exposed to SBF for the as-indicated ${\tau }_{\mathrm{SBF}}$ periods. Note that the data are normalized relative to the integrated intensity of BG45${}_{4.0}$–72 h in (b), but to equal spectral intensities at 590 cm${}^{-1}$ in (c) to better convey the more pronounced band splitting that develops for increasing ${\tau }_{\mathrm{SBF}}$.

Figure 3

(a) IR-derived relative HCA contents in BG45${}_{4.0}$–${\tau }_{\mathrm{SBF}}$ specimens plotted against ${\tau }_{\mathrm{SBF}}$. (b–d) Results of (b) pH, (c) [Ca] and (d) [P] measured in the accompanying solutions. Note the usage of a logarithmic (${log}_{10}$) horizontal scale. The dotted vertical lines and the shaded domains highlight the corresponding induction, proliferation, and maturation stages normally discussed in the context of spontaneous apatite formation, while red dotted horizontal lines in (b–d) indicate the respective values of the pH, [Ca] and [P] in the pristine SBF solution (i.e., for ${\tau }_{\mathrm{SBF}}=0$). Note that error bars are only displayed when outside of the symbols. The largest relative amount of HCA (observed at ${\tau }_{\mathrm{SBF}}$ = 72 h) corresponds to 29 ± 3 wt% HCA out of all solid phases.

Figure 4

Schematic illustration of the Hench mechanism [2] for HCA formation from a melt-prepared Na–(Ca)–Si–O–(P) glass exposed to SBF; the five HM stages are identified with the induction, proliferation, and maturation stages associated with sigmoidal growth (arrows; left panel). The first three HM steps involve (1) exchange of Na${}^{+}$/Ca${}^{2+}$ cations with protons from the solution, and (2) hydrolysis of Si–O–Si bonds, together leading to a high abundance of silanol (SiOH) surface groups, a portion of which (3) form Si–O–Si linkages by water removal. As depicted in the right panel, the HM stages (1)–(3) together produce a silica-gel layer, which comprises SiOH groups and water, but is nearly devoid of Na${}^{+}$/Ca${}^{2+}$ species. Next follows (4) a heterogenous nucleation of ACP, which then (5) crystallizes into HCA. The two last HM steps proceeds in parallel, with co-existing ACP/HCA components of the CaP layer (bottom, right), where HCA crystallizes from the interior of the ACP particles [14,15].