Excella e24

The concentration of Sr²⁺ ions released from the composite cement was evaluated under physiologic conditions (37 °C, pH 7.4) for up to 28 days by soaking 13.5 × 10 mm (D × H) solid samples in Tris-HCl solution at a concentration of 1 g of materials/20 mL of buffer. Briefly, samples were soaked at 37 °C with an agitation speed of 120 rpm (Excella E24, Eppendorf, Hamburg, Germany). At specific timepoints (3 h, 1, 3, 7, 14, 21 and 28 days), the supernatant was collected, stored at 4 °C until analysis and a complete refresh of the solution was performed. The Sr²⁺ concentration was measured through the inductively coupled plasma atomic emission spectrometry (ICP-AES) technique (iCPA RQ ICP-MS, Thermoscientific, Waltham, MA, USA) after appropriate aqueous dilutions of the collected supernatants (1:100) to not saturate the instrument detector. All the experiments were conducted in triplicate and results were reported as mean value ± standard deviation. Prior to release experiments, the concentration of strontium ions initially present in the CSH/Sr-MBG/ZrO₂ cements was determined. For this purpose, 15 mg of cement were dissolved in a mixture of nitric and hydrofluoric acids, heated for 12 h at 70 °C and the resulting solutions were analyzed via ICP-AES analysis. The experiments were completed in triplicate and the results were reported as mean value ± standard deviation.

The release kinetics of adsorbed HRP was determined by soaking 10 mg of LPMS_HRP in 2 mL of PBS, pH 7.4 [21 (link),79 (link)]. The resulting suspension was placed in an orbital shaker (Excella E24, Eppendorf, Hamburg, Germany) at 37 °C with an agitation rate of 150 rpm. At specified time points (1 h, 3 h, 5 h, 7 h, 24 h), the entire volume of supernatant was withdrawn after a centrifugation step (6000 rpm, 3 min), stored at −20 °C and fully replaced with fresh buffer.
The amount of released HRP was assessed by a MicroBCA Assay Kit as described above. All measurements were carried out in triplicate.

In vitro degradation tests were conducted in accordance with standard ISO 10993-14 by soaking cylindrical samples (13.5 × 10 mm, D × H) in a Tris-HCl solution (0.1 M, pH 7.4) at a concentration of 1 g of materials/20 mL of buffer. In detail, after a setting period of 24 h in an incubator at 37 °C (100% humidity), samples were weighed and measured in terms of height and diameter. Once immersed in the respective volume of Tris-HCl solution, the samples were placed in an orbital shaker (Excella E24, Eppendorf, Hamburg, Germany) at 37 °C with an agitation rate of 120 rpm until their complete degradation. Every 7 days, the Tris-HCl solution was fully refreshed and the samples corresponding to the specific time point were washed twice with ddH₂O, dried in an oven at 70 °C for 2 h and their weight and dimension were recorded. The residual weight percentage (residual weight) of each sample was calculated as follows: $$\mathrm{Residual}\mathrm{weight}(\%)=\left(1-\frac{w_{\mathrm{o}}-w_{\mathrm{t}}}{w_{\mathrm{o}}}\right)\times 100$$
where w_o is the initial weight of the sample (g) and w_t is the weight recorded in the specific time point t (g). The experiments were conducted in duplicate, and results were reported as mean ± standard deviation.