Solutions

Deionized water (Milli-Q, Merck, Rahway, NJ) was used as a control in all the experiments. The endodontic NaOCl solution under investigation was CanalPro 3% (Coltène, Altstätten, Switzerland, LOT 20222889 and 20222937). The etidronate powder that is advocated as a decalcifying add-on to NaOCl solutions was Dual Rinse HEDP (Medcem, Weinfelden, Switzerland, LOT DR230628) The combined NaOCl & HEDP solutions were mixed freshly before the experiments, by adding the adequate amount of powder (wt/wt) to the liquid necessary for the individual assessment. Powder to liquid mixtures were prepared using a precision balance (PM300, Mettler-Toledo, Greifensee, Switzerland). All the experiments described below were performed at an ambient temperature of 22–23 °C. The NaOCl solution that had be stored in the refrigerator was warmed in a water bath to 20 °C before use.

To assess whether there was a specific influence of the etidronate powder (Dual Rinse HEDP, Medcem) dissolved in the NaOCl solution, a similar mixture (9.1 g of NaOCl solution & 0.9 g of powder) was prepared using table salt (NaCl). Moreover, to then assess the impact of dissolved etidronate (HEDP) beyond clinical dosages on physico-chemical solution properties and cavitation bubble features, a series of solution was prepared containing (wt/wt) 0%, 5%, 10% and 20% Dual Rinse HEDP (Medcem) in the NaOCl solution (Canal Pro 3%, Coltène).

Chemical and physical assessments

The content of available chlorine in the solutions was measured by iodometric titration. The solution was spiked with potassium iodide (PanReac AppliChem, Darmstadt, Germany), and rendered acidic by adding HCl (Emsure 1.00316.1011, Merck). The liberated iodine was then titrated immediately using a 0.1 M sodium thiosulfate solution (PanReac AppliChem 186987.1211) in a titration apparatus (665 Dosimat, Metrohm, Herisau, Switzerland). Hydrogen ion activity was assessed using a calibrated electrode (6.0228.010, Metrohm) attached to a pH measuring device (727 pH lab, Metrohm).

Surface tension was measured in a pendant drop shape analyzer (DSA100, KRÜSS GmbH, Hamburg, Germany) at an ambient temperature of 22 °C. Surface tension was calculated based on the drop shape in relation to needle diameter (0.98 mm) and density of the liquid by the proprietary software. Liquid density was assessed using a high-precision balance (AT261, Mettler Toledo) by measuring the weight of 1.0 mL of the solution.

Viscosity of the solutions was determined in a rotational viscometer for low viscosity assessments (ViscoQC 300, Anton Paar, Buchs, Switzerland) equipped with a proprietary measuring bob (B-DG26) in a corresponding measuring cup (C-DG26). Viscosity measurements were performed at a rotational speed of 100 rpm in a controlled temperature environment of 20 °C (Checktemp 1, Hanna Instruments, Smithfield, RI) in a water bath (M3, Lauda, Königshofen, Germany).

Analysis of primary cavitation at the laser tip

A 2940 nm erbium-doped yttrium aluminium garnet (Er: YAG) laser (Skypulse, Fotona, Lljubliana, Slovenia) was equipped with a H14 handpiece holding either a flat or a conical fiber tip (SWEEPS 400/14, Fotona). The tip was placed in an optical glass cuvette (28 × 28 × 35 mm, Hellma, Müllheim, Germany) containing the test or control solutions. It was placed 9.5 mm below the liquid surface. For calibration purposes, a silicone stopper was attached to the fiber tip. Its diameter was measured with a digital caliper. The laser was used in SSP mode (pulse length of 50 µs) at a frequency of 15 Hz, and pulse energy of 40 mJ. A high-speed camera system (Photron SA-X, Tokyo, Japan) was mounted in front of the cuvette and high-speed recordings of 5 s were made. A lens system allowed zooming in on the fiber tip and surrounding liquid. The high-speed camera settings were as follows: frame rate: 81,000 frames per s, exposure time: 1/93,237 s, image resolution: 512 × 256 pixels. The proprietary FASTCAM viewer software PFV4 (Photron) was used to record and analyze the primary cavitation. The following primary cavitation bubble features were determined:

time to maximal dimension: time (in ms) from start formation to its maximum dimension;

lifetime: time (in ms) from formation to implosion of the primary bubble;

distance between the laser tip and the distal end of the bubble: the portion of the bubble (in mm) in front of the laser tip.

the maximum length (in mm).

the maximum width (in mm).

the total area (in mm²).

One parameter, bubble expansion speed, denoting the speed of expansion (in mm/ms) of the primary bubble, was calculated as follows: distance between the laser tip and the distal end of the bubble divided by time to maximal dimension.

Distance measurements were all performed at maximum bubble dimension, using the measuring tool of the PFV software. For surface measurements, the frame with the maximum bubble dimension was imported in Fiji and analyzed using the polygon selection tool and area measurement function. In both instances, the silicone stopper was used for image calibration. Duration was measured based on the time (in ms) as indicated per frame in the FastCam Viewer. All measurements were carried out on 3 consecutive primary cavitation bubbles per condition.

Data presentation and analysis

All measurements were done in triplicate (n = 3). The data presented here all represent means and standard deviations, indicating the measurement error. The strength of the linear correlation between HEDP concentration in the endodontic NaOCl irrigant and physical parameters of the resulting solutions as well as primary cavitation bubble features was explored by linear regression analysis using a software program (JMP Pro 17, SAS Institute, Cary, NC), and the Pearson correlation coefficients (PCCs) are reported.