2.1

Preparation of dentin powder

One hundred noncarious freshly unerupted human third molars were collected after obtaining informed consent under a protocol approved by the Human Assurance Committee of the Medical College of Georgia. The teeth were ground with coarse diamond burs in a high-speed handpiece with air–water spray to remove the enamel. The roots were removed at the cementoenamel junction using an Isomet saw (Buehler Ltd., Lake Bluff, IL, USA). The pulpal soft tissues were removed with a spoon excavator and the predentin was removed with a diamond bur. The resulting crown segments were cut into small fragments (4 mm × 4 mm× 3 mm) that were placed in 25 mL stainless steel screw-top jars, submerged in liquid nitrogen for 10 min and triturated at 30 Hz for 9 min in a ball-mill (MM301, Retsch, Newtown, PA, USA). This treatment reduced the dentin fragments to a fine powder (mean particle size <50 μm). The powder was stored at −70 °C until required.

2.2

Preparation of demineralized dentin powder

Half of the dentin powder was kept mineralized (MD), while the other half was divided in 10 g batches that were transferred to centrifuge tubes containing 30 mL of 0.1 M formic acid/sodium formate buffer, pH 2.5. The centrifuge tubes were tumbled at room temperature for 5 days, replacing the demineralizing solution with fresh solution every day. Complete demineralization was confirmed radiographically and by calculating the density of the powder that fell from 2.1 to 1.05 g/mL at the end of demineralization . After centrifuging the demineralized dentin (DD) powder at 3000 rpm for 30 min, the supernatant demineralizing solution was removed and the powder was resuspended with 30 mL of phosphate buffered saline (pH 7.4) three times to rinse away all traces of formic acid/formate.

2.3

Rinsing of dentin powder to remove UV absorbing material

Both mineralized and especially demineralized dentin powder releases products that may give UV absorption at 225 nm, the wavelength that was used to quantitate CHX uptake. This “background” absorbance was removed to <0.05 absorbance at 225 nm by multiple rinses with water. That is, 3 g of powder was suspended in 30 mL of water and tumbled for 1 h, centrifuged and the absorbance of the supernatant measured against water in UV transparent 96-well plate reader (Costar 3635, UV Plate, Corning, NY, USA) in a Synergy HC plate reader (Biotek, Winooski, VT, USA). This was repeated 5–6 times/day for up to 10 days, until the absorbance of the supernatant was less than 0.05 A at 225 nm. Then the powder was immediately used for the CHX binding experiments.

2.4

Chlorhexidine binding experiments

Following the method of Blackburn et al. , 0.05 g of dentin powder were transferred to microcentrifuge tubes and mixed with 1 mL of standard solutions containing 0, 0.04, 0.10, 0.2, 0.39, 3.9, 19.7 or 29.6 mM of chlorhexidine digluconate (CHX) in 0.05 M phosphate buffer, pH 7.4. Preliminary work demonstrated that CHX binding was similar irrespective of whether the dentin powder was treated for 1 or 30 min; so it was decided to use 30 min for convenience. The microcentrifuge tubes were then capped and tumbled for 30 min at 37 °C. Then, they were centrifuged at 3000 rpm and the supernatant was removed. Three hundred microlitre of supernatant were placed in a UV transparent 96-well plate for measurement of the absorbance at 225 nm against water, in duplicate in the Synergy HC plate reader. From a standard CHX curve, the absorbance of the supernatant was converted to CHX concentration. If there was no dentin powder in the tube, there was no change in the CHX concentration of the standard. In the presence of dentin powder, the CHX concentration of the supernatant was always less than that of the standard solution, indicating that the dentin powder bound CHX. This was expressed in μmol CHX/g dry powder. This value was designated as the bound CHX at equilibrium. No further binding occurred at longer incubation times. The binding of CHX by dentin powder was calculated as:

where CHX _Bound = μmol CHX/g dry weight of powder, CHX _STD = CHX concentration in standard solutions before exposure to powder (μmol/mL) and CHX _equil = equilibrium CHX concentration in solution (μmol/mL) after exposure to powder, divided by the weight of the dry dentin powder.

2.5

Chlorhexidine debinding experiments

The excess remaining CHX solution was removed from the microcentrifuge tubes with dry paper points and the powder pellet was then resuspended in 1 mL of water, 100% hydroxyethyl methacrylate (HEMA), 100% ethanol or 0.5 M NaCl. The use of HEMA was meant to serve as a representative hydrophilic monomer that is commonly used in many adhesive blends. High sodium chloride concentrations are generally used to displace electrostatistically-bound materials from their substrates . The microcentrifuge tubes were hand shaken at 3 Hz for 3 min and then centrifuged to repellent the powder. Two 300 μL aliquots of the supernatant were transferred to 96-well plates for quantitation of the amount of CHX that could be displaced or extracted from the dentin powder by these solvents.

2.6

Hydroxyapatite (HA) binding and debinding experiments

Pure hydroxyapatite powder was used as a model binding substrate to simulate the expected binding characteristics of mineralized dentin powder. If binding of CHX to HA was similar to that of mineralized dentin powder then that binding would be predominately due to the mineral phase of dentin instead of the organic phase. Pure hydroxyapatite (reagent grade, Sigma–Aldrich, St. Louis, MO, USA, Catalog no. 289396) was used as a reference binding material. Fifty milligram of HA was placed in separate microcentrifuge tubes and suspended in 1 mL of CHX standards for 30 min to obtain maximum CHX binding. As pilot studies showed that similar CHX binding occurred at 1 min versus 30 min, we used 30 min for these studies. The decrease in the CHX concentration of the standards (measured by decreases in absorbance at 225 nm) was used to calculate the degree of CHX binding to HA. Debinding experiments using water, HEMA, 100% ethanol or 0.5 M NaCl were done as described above in chlorhexidine debinding experiments.

2.7

Hoy’s solubility parameters for HEMA, ethanol, water and demineralized dentin

We have used Hoy’s solubility parameters in our previous work to rank the ability of solvents to hydrogen bond (H-bond) by their Hoy’s solubility parameters for hydrogen bonding ( δ _h ) . For the substrates (DD and MD), the Hoy’s solubility parameters for hydrogen bonding ( δ _h ) for HEMA, ethanol and water were plotted against the CHX binding concentration yielded at different CHX-applied concentrations.

2.8

FTIR absorption spectra of dentin powder

To determine whether there was any interaction between CHX and dentin substrates, the FTIR absorption spectra of these moist substrates were obtained before and after exposure to CHX. This was accomplished using a Nicolet 6700 FTIR spectrophometer (Thermo Scientific Inc., Waltham, MA, USA) with a single-reflection diamond attenuated total reflection (ATR) set-up (Smart OMNI-Sampler). The anvil of the back pressure tower of the Smart OMNI-Sampler was pressed against the dentin powder and the slip clutch of the back pressure tower was turned clockwise until an audible click was heard. This ensured that correct pressure was applied to each powder aliquot, independent of the size, shape or mineralization status of the powder particles. Infrared spectra were obtained over the range of 4000–700 cm ⁻¹ at 4 cm ⁻¹ resolution using 32 scans.

2.9

Statistical analyses

Polynomial regression was used to model the relationship between medium CHX concentration and CHX uptake separately for the three substrates: demineralized dentin (DD), mineralized dentin (MD) and hydroxyapatite (HA) powder. The maximum CHX uptake ( B _max ) was estimated based on the fitted curve (or line) and the interpolation method was used to estimate the concentration K _1/2 that would yield an uptake of B _max /2. K _1/2 is the concentration of CHX required to half-saturate each binding substrate. Since B _max is the maximum binding capacity on the y axis, B _max /2 represents half-saturation. Extrapolation of B _max /2 to the binding curve permits determination of the CHX medium concentration by drawing a vertical line to intersect the x axis (CHX medium concentration). This permits comparisons of the concentration of CHX required to half-saturate the three different binding substrates. Approximate standard errors were then calculated and used to determine an approximate 95% confidence interval for K _1/2 . The Wald test was used to compare K _1/2 values across the three media. Two-tailed tests with a significance level of 0.05 were used for all statistical comparisons.