Technical buffer solutions

Of each freshly prepared sealer (n = 12, 3 per group with the experiment performed in triplicates), 200 μL was placed at the base of individual wells of a sterile 12-well flat bottom plate (Star Lab, Milton Keynes, UK) using a 1-mL sterile syringe. 10 mL of HBSS (Gibco®, Grand Island, NY, USA) was immediately added to each well and stored at 37 °C. The HBSS was replaced at each endpoint (15, 30, 60, 120 min and 1, 3, 7, 14, 28 days). The collected HBSS solution was transferred to a glass vial (Fisherbrand, Loughborough, UK) and vortexed for one minute. Determination of the HBSS solution pH was performed at room temperature using a pH meter (FiveEasy Cond Meter F30, Mettler Toledo, Greifensee, Switzerland) and a temperature compensated pH electrode (pH electrode InLab Easy, Mettler Toledo, Greifensee, Switzerland) previously calibrated at five points (pH 2.00, pH 4.01, pH 7.00, pH 9.21, pH 12.00) using buffer solutions (Technical Buffer Solutions, Mettler Toledo, Greifensee, Switzerland). The mean and standard deviation of each were calculated.

Immunohistological analyses were performed as described previously^{14 (link)}. A pH-sensing chemical probe (pHocas-3) dissolved in PBS was injected subcutaneously at 6 mg per kg body weight daily for 3 days before sacrifice. Immediately after sacrifice, the samples were frozen in chilled hexane (Wako) using dry ice, and 12-μm-thick sections of non-decalcified knee joints were prepared using a Multi-Purpose Cryosection Preparation Kit^{34 (link)} (Section-Lab and Leica Microsystems). Then, the samples were stained with propidium iodide and observed using a TCS-SP5 confocal microscope (Leica Microsystems). Collagen-enriched bone matrices were visualized using the second harmonic generation (SHG) with infrared lasers of a TCS-SP5 multi-photon laser microscope driven by a Chameleon XR Sapphire laser (Coherent). Technical buffer solutions (Mettler-Toledo) were used to control the pH of the sections.