Preparation of simulated body fluid solutions

Simulated body fluid (SBF) solutions were prepared using the method proposed by Kokubo et al.⁶² to mimic major ionic compounds in human body plasma⁶³. American Chemical Society grades of NaCl (7.996 g, BDH Chemicals), NaHCO₃ (0.350 g, BDH Chemicals), KCl (0.224 g, BDH Chemicals), MgCl₂·6H₂O (0.305 g, EMD Millipore), 1 M HCl (40 ml, BDH Chemicals), Na₂SO₄ (0.071 g, Alfa Aesar), and tris(hydroxymethyl)aminomethane (Tris, 6.057 g, Alfa Aesar) were added to 900 ml of deionized water (18.2 MΩ-cm). Either 0 or 10 mg of pAsp (sodium salt, Mw: 5,000 Da, LANXES) was added to the solution, depending on the experimental condition. Then the solution was equally separated into two 500 ml polyethylene bottles, and either 0.604–0.684 g of K₂HPO₄·3H₂O (Alfa Aesar) or 0.974–1.103 g of CaCl₂·2H₂O (Alfa Aesar) was added to each bottle. The pH of the solutions was adjusted to 7.25 with 1 M HCl, followed by filling up the volume with water to 500 ml. To prevent any precipitation of calcium phosphate minerals prior to the start of the experiment, the two stable solutions containing either Ca²⁺ or HPO₄²⁻ precursors (SBF-Ca or SBF-P) were prepared separately and mixed just before the reaction (Supplementary Fig. ^1b). The concentrations of Ca and P in the reactor, after the two solutions were mixed, were 2.65 to 3.0 times higher than in the SBF solution by Kokubo’s method (2.65–3.0 × SBF), however, the Ca/P molar ratio was constantly fixed to 2.5. The continuous flow-through reaction system (Supplementary Fig. ^1b) allowed maintaining constant concentrations of ionic components and pH in the reactor for each set of experiment (details listed in Supplementary Table ¹). The addition of 10 mg l⁻¹ pAsp, a nucleation inhibitor for EM, effectively promoted IM for up to 15 h during the mineralization of collagen matrices in the 3.0 × SBF²³. The use of a buffer in the SBF solution was unavoidable in order to maintain the desired pH. This buffer allowed us to use a constant supersaturation value for each SBF solution for the application of CNT. In this study, tris-buffer was used because it has been widely and effectively used for biomimetic CaP synthesis and intrafibrillar collagen mineralization^23,58. More details about the potential influence of buffers in physiological solutions were summarized in a recent review paper⁵⁸.

Supersaturation (σ) values of the SBF solutions were calculated for both HA (σ_HA) and ACP (σ_ACP). The ion activity product of hydroxyapatite (IAP_HA) was defined as (αCa2+)5(αPO43-)3(αOH-)⁶⁴. The activity of an ionic compound i, α_i, is the product of its activity coefficient, γ_i, and concentration, C_i. We calculated γ_i values at 37°C from the modified Debye–Hückel equation, logγi=-0.5211zi2[I121+I12-0.3I],^64,65, where I is the total ionic strength (I = ½∑ C_iz_i) and z_i is the charge number. To calculate C_i of all the ionic components in SBF solutions, MINEQL + was used, with consideration of pAsp based on its dissociation and calcium binding constants as reported by Wu and Grant⁵². Using values from the literature^66,67, we set K_sp (HA) = 2.35 × 10⁻⁵⁹ and set the molecular volume, v_m (HA) = 2.63 × 10^–22 cm³. σ_ACP was calculated based on Ca₂(HPO₄)₃²⁻ (IAP_ACP = (αCa2+)2(αHPO42-)3) with an estimated K_sp (ACP) value of (6.04 × 10⁻⁴)⁵ and v_m (ACP)  =  5.0 × 10⁻²³ cm³, as recently suggested by Habraken et al.²⁵. The concentrations of ionic compounds and calculated σ values are summarized in Supplementary Table ¹.