Effect of a novel quaternary ammonium silane on dentin protease activities

Abstract

Objectives

Demineralized dentin collagen release C-terminal cross-linked telopeptide (ICTP) and C-terminal peptide (CTX) during degradation. The present study evaluated the effects of dentin pre-treatment with K21, a quaternary ammonium silane (QAS), on matrix metalloproteinase (MMP) and cathepsin K-mediated collagen degradation.

Methods

Dentin beams were demineralized with 10% H 3 PO 4 for 24 h. After baseline dry mass measurements, the beams were divided into 5 groups (N = 10) according to protease inhibitors. The beams were pre-treated for 2 min with 2% chlorhexidine (CHX), 2%, 5% or 10% QAS; no pre-treatment was performed for the control group. The beams were subsequently incubated in calcium- and zinc-containing medium for 3, 7 or 14 days, after which changes in dry mass were measured and incubation media were examined for ICTP and CTX release. The MMP-2 and cathepsin K activities in QAS-treated dentin powder were also quantified using ELISA.

Results

The two factors (disinfectants and time) had a significant effect on dry mass loss, ICTP and CTX release (p < 0.001). The percentage of dry mass loss increased with time and was significantly lower in all experimental groups when compared to the control at 14 days (p < 0.001). Conversely, the rate of ICTP and CTX release was significantly lower in the experimental groups, compared to the uninhibited control at 7 and 14 days (p < 0.001). Dentinal MMP-2 and cathepsin K activities were significantly reduced after demineralized dentin was pre-treated with QAS.

Conclusion

The experimental QAS is a good inhibitor of MMP and cathepsin K activities in demineralized dentin.

Clinical significance

The newly developed antibacterial quaternary ammonium silane increases the resistance of dentin collagen to degradation by inhibiting endogenous matrix metalloproteinases and cysteine cathepsins. The quaternary ammonium silane cavity disinfectant is promising for use as a protease inhibitor to improve durability of resin-dentin bonds.

Introduction

Dentin contains type I collagen and non-collagenous proteins embedded in a matrix of carbonated apatite . Matrix metalloproteinases (MMPs) and cysteine cathepsins are found extensively in sound , caries-infected and caries-affected dentin . These endogenous dentin enzymes are entrapped within the mineralized collagen matrix during dentinogenesis. Matrix metalloproteinases are responsible for degradation of extracellular matrix components, especially the highly cross-linked triple helical type I collagen . Whereas cathepsin B and cathepsin L cleave only the non-helical telopeptide extensions of collagen, cathepsin K is the only cysteine cathepsin that cleaves collagen in the triple helical region .

The hybrid layer is produced by acid etching and infiltration of solvated methacrylate resin comonomers into the demineralized dentin collagen matrix. Treatment of dentin with 37% phosphoric acid does not denature the endogenous MMP and cathepsin activities of dentin matrices . Subsequent application of acidic resin reactivates endogenous MMPs . Similar to MMPs, cysteine cathepsins may be activated in mildly acidic environments; acid activation of dentin-bound cathepsins may further result in activation of the matrix-bound MMPs. A highly significant correlation between cysteine cathepsin and MMP activities in intact and carious dentin suggests that both proteases may be responsible for the breakdown of resin-sparse, unprotected collagen fibrils within the hybrid layer, eventually resulting in debonding of the overlying restorations.

Incomplete removal of caries-infected dentin during cavity preparation results in entrapment of bacteria within the cavity . Open restoration margins enable the oral microflora to invade the resin-dentin interface. These microorganisms continue to multiply and cause secondary caries formation, which is one of the major causes for failure of resin-based restorations . Disinfection of the cavity using an antimicrobial prior to restorative procedures has been recommended to prevent secondary caries .

Chlorhexidine (CHX) is commonly used as a cavity disinfectant to eliminate residual bacteria in caries-affected dentin and dentinal tubules after mechanical caries removal . This bisbiguanide is also a potent, non-specific inhibitor of MMP −2, −8 and −9 and cysteine cathepsins . Chlorhexidine is water-soluble and binds only electrostatically to the demineralized dentin matrix. Thus, it may be displaced by competing cations from the dentinal fluid and saliva, compromising its long-term protease inhibitory effect. In the presence of water at the bonded interface, CHX may debind from collagen and slowly leach out of the hybrid layer over time . Recent studies have shown that CHX pre-treatment of demineralized dentin has limited effect in preventing bond degradation after 9–12 months . In addition, CHX has recently been reported to exert dose-dependent, mild transdentinal toxic effects on odontoblast-like cells , as well as inhibiting the mineralization potential of multipotent stem cells derived from human exfoliated deciduous teeth . Therefore, there is a need to look for other antibacterial cavity cleansers of lower toxicity to inhibit oral biofilms and caries.

Quaternary ammonium compounds are commonly used as disinfectants of intact skin, non-critical surfaces and mucous membranes . However, concerns of resistance development, potential toxicities and loss of antimicrobial activity over time as a result of leaching from the bound surfaces have limited their clinical use. The organosilicon quaternary ammonium chloride, 3-(trimethoxysilyl)-propyldimethy-octadecyl ammonium chloride (Si-QAC; C 26 H 58 ClNO 3 Si; CAS number 27668-52-6), possesses potent antibacterial activities. Being an antimicrobial quaternary ammonium silane (QAS), Si-QAC has been used for antimicrobial coatings of fabrics and medical devices . The antimicrobial property of Si-QAC is attributed to its long, lipophilic C18 alkyl chain that penetrates bacterial cell membranes and causes cell death of bacteria by direct contact and leaching of intracellular components . Being a trialkoxysilane, SiQAC possesses hydrolyzable and condensable methoxy groups which enable it to covalently attach to other alkoxysilanes or silanol-containing substrate surfaces via the formation of siloxane bridges .

Because methanol is produced during the hydrolysis and condensation reactions of SiQAC, the molecule is potentially toxic for intraoral use . Hence, SiQAC has been substituted with 3-(triethoxysilyl)-propyldimethyloctadecyl ammonium chloride (i.e. the ethoxy version of SiQAC, abbreviated as Et-SiQAC) for coupling with tetraethoxysilane (TEOS) via sol-gel synthesis. This resulted in the generation of an ethanol- or acetone-soluble, fully-hydrolysed, partially-condensed QAS (1-octadecanaminium, N,N’-[[3,3-bis[[[3-(dimethyloctadecylammonio) propyl]dihydroxysilyl]oxy]-1,1,5,5,-tetrahydroxyl-1,5-trisiloxanediyl]di-3,1-propanediyl] bis[N, N-dimethyl] chloride (1:4); CAS number 1566577-36-3; codenamed K21). Because of the elimination of methanol, K21 may be used, without further purification, as an intraoral disinfectant. This ethoxylated QAS molecule has been shown to be effective against Porphyromonas gingivalis and Enterococcus faecalis when used as coatings for surgical sutures and dental flosses .

Because quaternary ammonium compounds possess MMP and cysteine cathepsin inhibitory effects, it is speculated that the ethoxylated QAS may also inhibit endogenous dentin proteases. Nevertheless, the inhibitory effect of ethoxylated QAS on MMPs and cysteine cathepsins in dentin has not been investigated. The combined antimicrobial and anti-collagenolytic effects of QAS may be beneficial in preventing degradation of resin-dentin bonds and development of secondary caries. Thus, the objective of the present study was to evaluate the effects of different concentrations of an experimental ethoxylated QAS, K21, in inactivating dentin proteases. The null hypotheses tested were that (i) pre-treatment of demineralized dentin matrices with QAS has no effect on their loss of dry mass by collagen degradation; and (ii) pre-treatment of demineralized dentin with QAS has no effect on inhibition of dentinal MMP or cathepsin K activities.

Materials and methods

Synthesis and characterization of K21

The experimental QAS (codenamed K21) was synthesized by sol-gel reaction between 1 mol of TEOS (Mw 208) and 4 mol of Et-SiQAC (Mw 538). The chemical structure of K21 and the processes of hydrolysis and condensation are shown in Fig. 1 A. In a typical synthesis, 2.08 g of TEOS (Millipore Sigma, St. Louis, MO, USA) was blended with 29.89 g of Et-SiQAC (72 wt% of Et-SiQAC dissolved in ethanol; Gelest Inc., Morrisville, PA, USA) and 5 mL of ethanol (to render the blend more homogeneous). Hydrolysis was initiated by the addition of 10.08 g of 0.02 M HCl-acidified water (pH 1.66, representing 3.5 times the stoichiometric molar concentration of water required, to ensure complete hydrolysis). Hydrolysis and condensation of the two ethoxysilanes were monitored by attenuated total reflection-Fourier transform infrared spectroscopy (ATR-FTIR; Nicolet 6700, Thermo Fisher Scientific, Waltham, MA, USA) at a resolution of 4 cm −1 and an average of 32 scans per spectrum.

Fig. 1
A. Proposed chemical formula of the QAS (K21) molecule. B. A yellow solution was obtained after compete hydrolysis of the reaction mixture. C. The partially-condensed solid after heating of the completely hydrolyzed solution. (For interpretation of the references to colour in text, the reader is referred to the web version of this article.)

A yellow solution mixture was obtained after completion of the hydrolysis reaction ( Fig. 1 B). The yellow solution mixture was then maintained at 80 °C for 6 h to remove as much as possible the reaction by-products (ethanol and water), until a pale yellow rubbery solid material was produced ( Fig. 1 C). This yellow, partially-condensed solid was characterized using 1 H → 29 Si cross polarization-magic angle spinning (CP-MAS) solid-state nuclear magnetic resonance spectroscopy (NMR). 29 Si solid-state NMR was performed at ambient temperature using a 270 MHz spectrometer (JEOL, Tokyo, Japan) equipped with a 7 mm MAS probe. Spectra were acquired in the 1 H → 29 Si CP mode, using a MAS frequency of 4 kHz, with a 45 ° pulse angle of 5 s. The 1 H Larmor frequency for 29 Si was 53.76 MHz. Chemical shifts were referenced to external tetramethylsilane at 0 ppm. The yellow, partially-condensed K21 solid was immediately dissolved in absolute ethanol to produce solutions containing 2 wt%, 5 wt% or 10% of the QAS to be used as the experimental protease inhibitors in the present study.

Demineralized dentin beams

Fifty extracted non-carious human third molars were obtained from patients (18–21 year-old) with their informed consent under a protocol reviewed and approved by Institutional Review Board of the University of Hong Kong/Hospital Authority Hong Kong West Cluster (UW14-406). All the experiments were performed in accordance with the approved guidelines and regulations. The teeth were stored in 0.9% NaCl with 0.02% sodium azide at 4 °C to prevent bacterial growth and were used in within 1 month after extraction. Demineralized dentin beams were prepared by removing the occlusal enamel and superficial dentin of each tooth with an Isomet saw (Buehler Ltd., Lake Bluff, IL, USA) under water cooling. A 1-mm thick mid-coronal dentin disk was prepared from each tooth. One dentin beam with dimensions 6 mm × 2 mm × 1 mm was then sectioned from the center of each disk and a total of 50 beams were prepared. The dentin beams were completely demineralized in 10 wt% H 3 PO 4 for 24 h at 25 °C. The demineralized beams were thoroughly rinsed in deionized water with constant stirring at 4 °C for 1 h. Absence of residual minerals in each beam was verified using digital radiography.

The demineralized beams were randomly allocated to each of the five groups (N = 10) so that there was no statistically significant difference in the mean dry mass among the groups. Group 1 was the negative control, which was not treated with any disinfectant. Groups 2, 3, 4 and 5 were treated respectively with 2% CHX (Millipore Sigma), 2% QAS, 5% QAS and 10% QAS, by dipping the beams in the respective medium for 2 min each. After treatment with the disinfectants, each beam was blot-dried using Kimwipes (Kimberly Clark Corp, Roswell, GA, USA) and placed separately in a labeled polypropylene tube containing 1 mL of complete storage medium (CM). The CM is a calcium- and zinc-containing storage medium containing 5 mM HEPES, 2.5 mM CaCl 2 ·H 2 O, 0.05 mM ZnCl 2 and 0.3 mM NaN 3 (pH 7.4).

Loss of dry mass over time

Loss of dry mass was used as an indirect measurement of dissolution of the demineralized dentin matrix by endogenous matrix-bound proteases after each incubation period (3, 7 or 14 days) . Prior to treatment with the respective disinfectant, the beams were transferred to individually labeled polypropylene tubes and placed in a vacuum desiccator chamber containing anhydrous calcium sulfate (Drierite, W.A. Hammond Drietite Co. Ltd, Xenia, OH, USA). With the vial cap off, each beam was desiccated to a constant mass for 72 h. The initial dry mass was measured to the nearest 0.001 mg using an analytical balance (XP6 Microbalance, Mettler Toledo, Hightstown, NJ, USA). After dry mass measurement, the beams were rehydrated in deionized water at 4 °C for 1 h before treatment with the designated disinfectants. The sealed tubes were placed in a shaking-water bath (Thermo Fisher Scientific) with a shaking speed of 60 cycles/min at 37 °C for 3, 7 or 14 days. The dry mass was re-measured using the same conditions after each incubation period. At the end of each incubation period, the storage medium was replaced with fresh incubation medium. The medium obtained after each incubation period was stored in a freezer (−80 °C) and subsequently analyzed for C-terminal cross-linked telopeptide (ICTP) and C-terminal peptide (CTX) fragments.

Solubilized collagen peptides

Degradation of the demineralized collagen matrix by MMPs was determined by measuring the amount of solubilized type I collagen ICTP fragments using an ICTP enzyme-linked immunosorbent assay (ELISA) kit (UniQ EIA, Orion Diagnostic, Finland). The rationale for this analysis was based on the finding that ICTP telopeptide fragments are solely derived from the telopeptidase activity of MMPs in a degrading dentin collagen matrix . The collagen matrix degradation activity of cathepsin K was determined by measuring the amount of CTX fragments within the incubation medium, using a Serum Crosslaps ELISA kit (Immunodiagnostic System, Farmington, UK). The rationale for this analysis was based on the finding that CTX fragments are solely derived from the enzymatic activity of endogenous cathepsin K within a degrading collagen matrix . At the end of each incubation period, the sealed tubes were retrieved from the shaker-water bath and the entire volume (i.e. 1 mL) of the medium was removed. Ten to twenty μL aliquots of the incubation medium were used to measure solubilized ICTP and CTX collagen fragments.

Protease detection from demineralized collagen

Specimen preparation

Thirty human third molars (obtained from 19 to 35 year-old subjects) were ultrasonically cleaned and stored in 1% thymol solution at 4 °C. The roots were removed and occlusal enamel was sectioned perpendicular to the longitudinal axis of each tooth with the Isomet saw using water cooling, exposing a flat mid-coronal dentin surface (1 mm below the dentinoenamel junction). The tooth segments were meticulously rinsed with deionized water after the pulpal tissues were removed from each of the cut tooth section and rinsed with deionized water.

The cleaned tooth segments were pulverized in liquid nitrogen into a fine powder using a steel mortar/pestle (Reimiller, Reggio Emilia, Italy). Five one-gram aliquot of dentin powder were demineralized with 0.5 M EDTA (pH 7.0). The demineralized dentin powder was rinsed with deionized water for 5 times, dried and divided into five groups. Group 1 was left untreated and served as control. The remaining four groups of dentin powder were treated respectively with 2% CHX (Group 2), 2% QAS (Group 3), 5% QAS (Group 4) and 10% QAS (Group 5) for 2 min. The treated dentin powder was re-suspended in extraction buffer [50 mM Tris-HCl at pH 7.5 containing 0.2% Triton X-100, 5 mM CaCl 2 , and 100 mM NaCl] for 24 h to extract the proteases. The vials were centrifuged at 20,000 rpm for 30 min at 4 °C. The supernatants were collected, dialyzed in bags with 30-kDa molecular cut-off overnight, lyophilized and frozen at −20 °C until they were analyzed.

MMP-2 and cysteine cathepsin ELISAs

The concentrations of endogenous MMP-2 and cathepsin K in the supernatants derived from different groups of dentin powder were quantified using ELISA (Human MMP2 ELISA Kit – Lot #5619 for MMP-2; Human CTSK/Cathepsin K ELISA Kit – Lot #5614 for cathepsin K, both from Lifespan Biosciences, Seattle, WA, USA). The kits were used according to the manufacturer’s instructions. A calibration curve correlating protease concentration with absorbance intensity was prepared for each protease. The dialyzed supernatants were incubated in the respective assay buffers for MMP-2 and cathepsin K for 1.5 h at 37 °C. Detection reagents were added after several rinses and the absorbance was recorded with a spectrophotometer at 405 nm (Bio-Rad Laboratories, Inc. Hercules, CA, USA). All tests were conducted in a triplicate and the protease concentrations were expressed as ng/mL.

Statistical analyses

The percent loss of dry mass over incubation time and the rate of release of ICTP and CTX (ng telopeptide/mg dry dentin/unit time) from all groups were compared for normality (Shapiro-Wilk test) and equality of variance (modified Levine test). When the normality and equality variance assumptions of the original data were violated, the data were nonlinearly transformed to satisfy those assumptions prior to the use of parametric statistical methods. Two-factor analysis of variances (ANOVA) was employed to examine the effects of incubation time and disinfectants, and the interaction of those two factors on the respective parameters investigated. Post-hoc multiple comparisons were performed with the Tukey statistic. Dentinal MMP-2 and cathepsin K concentrations in the five groups were separately analyzed using one-way ANOVA and Tukey multiple comparison test. For all tests, statistical significance was preset at α = 0.05.

Materials and methods

Synthesis and characterization of K21

The experimental QAS (codenamed K21) was synthesized by sol-gel reaction between 1 mol of TEOS (Mw 208) and 4 mol of Et-SiQAC (Mw 538). The chemical structure of K21 and the processes of hydrolysis and condensation are shown in Fig. 1 A. In a typical synthesis, 2.08 g of TEOS (Millipore Sigma, St. Louis, MO, USA) was blended with 29.89 g of Et-SiQAC (72 wt% of Et-SiQAC dissolved in ethanol; Gelest Inc., Morrisville, PA, USA) and 5 mL of ethanol (to render the blend more homogeneous). Hydrolysis was initiated by the addition of 10.08 g of 0.02 M HCl-acidified water (pH 1.66, representing 3.5 times the stoichiometric molar concentration of water required, to ensure complete hydrolysis). Hydrolysis and condensation of the two ethoxysilanes were monitored by attenuated total reflection-Fourier transform infrared spectroscopy (ATR-FTIR; Nicolet 6700, Thermo Fisher Scientific, Waltham, MA, USA) at a resolution of 4 cm −1 and an average of 32 scans per spectrum.

Fig. 1
A. Proposed chemical formula of the QAS (K21) molecule. B. A yellow solution was obtained after compete hydrolysis of the reaction mixture. C. The partially-condensed solid after heating of the completely hydrolyzed solution. (For interpretation of the references to colour in text, the reader is referred to the web version of this article.)

A yellow solution mixture was obtained after completion of the hydrolysis reaction ( Fig. 1 B). The yellow solution mixture was then maintained at 80 °C for 6 h to remove as much as possible the reaction by-products (ethanol and water), until a pale yellow rubbery solid material was produced ( Fig. 1 C). This yellow, partially-condensed solid was characterized using 1 H → 29 Si cross polarization-magic angle spinning (CP-MAS) solid-state nuclear magnetic resonance spectroscopy (NMR). 29 Si solid-state NMR was performed at ambient temperature using a 270 MHz spectrometer (JEOL, Tokyo, Japan) equipped with a 7 mm MAS probe. Spectra were acquired in the 1 H → 29 Si CP mode, using a MAS frequency of 4 kHz, with a 45 ° pulse angle of 5 s. The 1 H Larmor frequency for 29 Si was 53.76 MHz. Chemical shifts were referenced to external tetramethylsilane at 0 ppm. The yellow, partially-condensed K21 solid was immediately dissolved in absolute ethanol to produce solutions containing 2 wt%, 5 wt% or 10% of the QAS to be used as the experimental protease inhibitors in the present study.

Demineralized dentin beams

Fifty extracted non-carious human third molars were obtained from patients (18–21 year-old) with their informed consent under a protocol reviewed and approved by Institutional Review Board of the University of Hong Kong/Hospital Authority Hong Kong West Cluster (UW14-406). All the experiments were performed in accordance with the approved guidelines and regulations. The teeth were stored in 0.9% NaCl with 0.02% sodium azide at 4 °C to prevent bacterial growth and were used in within 1 month after extraction. Demineralized dentin beams were prepared by removing the occlusal enamel and superficial dentin of each tooth with an Isomet saw (Buehler Ltd., Lake Bluff, IL, USA) under water cooling. A 1-mm thick mid-coronal dentin disk was prepared from each tooth. One dentin beam with dimensions 6 mm × 2 mm × 1 mm was then sectioned from the center of each disk and a total of 50 beams were prepared. The dentin beams were completely demineralized in 10 wt% H 3 PO 4 for 24 h at 25 °C. The demineralized beams were thoroughly rinsed in deionized water with constant stirring at 4 °C for 1 h. Absence of residual minerals in each beam was verified using digital radiography.

The demineralized beams were randomly allocated to each of the five groups (N = 10) so that there was no statistically significant difference in the mean dry mass among the groups. Group 1 was the negative control, which was not treated with any disinfectant. Groups 2, 3, 4 and 5 were treated respectively with 2% CHX (Millipore Sigma), 2% QAS, 5% QAS and 10% QAS, by dipping the beams in the respective medium for 2 min each. After treatment with the disinfectants, each beam was blot-dried using Kimwipes (Kimberly Clark Corp, Roswell, GA, USA) and placed separately in a labeled polypropylene tube containing 1 mL of complete storage medium (CM). The CM is a calcium- and zinc-containing storage medium containing 5 mM HEPES, 2.5 mM CaCl 2 ·H 2 O, 0.05 mM ZnCl 2 and 0.3 mM NaN 3 (pH 7.4).

Loss of dry mass over time

Loss of dry mass was used as an indirect measurement of dissolution of the demineralized dentin matrix by endogenous matrix-bound proteases after each incubation period (3, 7 or 14 days) . Prior to treatment with the respective disinfectant, the beams were transferred to individually labeled polypropylene tubes and placed in a vacuum desiccator chamber containing anhydrous calcium sulfate (Drierite, W.A. Hammond Drietite Co. Ltd, Xenia, OH, USA). With the vial cap off, each beam was desiccated to a constant mass for 72 h. The initial dry mass was measured to the nearest 0.001 mg using an analytical balance (XP6 Microbalance, Mettler Toledo, Hightstown, NJ, USA). After dry mass measurement, the beams were rehydrated in deionized water at 4 °C for 1 h before treatment with the designated disinfectants. The sealed tubes were placed in a shaking-water bath (Thermo Fisher Scientific) with a shaking speed of 60 cycles/min at 37 °C for 3, 7 or 14 days. The dry mass was re-measured using the same conditions after each incubation period. At the end of each incubation period, the storage medium was replaced with fresh incubation medium. The medium obtained after each incubation period was stored in a freezer (−80 °C) and subsequently analyzed for C-terminal cross-linked telopeptide (ICTP) and C-terminal peptide (CTX) fragments.

Solubilized collagen peptides

Degradation of the demineralized collagen matrix by MMPs was determined by measuring the amount of solubilized type I collagen ICTP fragments using an ICTP enzyme-linked immunosorbent assay (ELISA) kit (UniQ EIA, Orion Diagnostic, Finland). The rationale for this analysis was based on the finding that ICTP telopeptide fragments are solely derived from the telopeptidase activity of MMPs in a degrading dentin collagen matrix . The collagen matrix degradation activity of cathepsin K was determined by measuring the amount of CTX fragments within the incubation medium, using a Serum Crosslaps ELISA kit (Immunodiagnostic System, Farmington, UK). The rationale for this analysis was based on the finding that CTX fragments are solely derived from the enzymatic activity of endogenous cathepsin K within a degrading collagen matrix . At the end of each incubation period, the sealed tubes were retrieved from the shaker-water bath and the entire volume (i.e. 1 mL) of the medium was removed. Ten to twenty μL aliquots of the incubation medium were used to measure solubilized ICTP and CTX collagen fragments.

Protease detection from demineralized collagen

Specimen preparation

Thirty human third molars (obtained from 19 to 35 year-old subjects) were ultrasonically cleaned and stored in 1% thymol solution at 4 °C. The roots were removed and occlusal enamel was sectioned perpendicular to the longitudinal axis of each tooth with the Isomet saw using water cooling, exposing a flat mid-coronal dentin surface (1 mm below the dentinoenamel junction). The tooth segments were meticulously rinsed with deionized water after the pulpal tissues were removed from each of the cut tooth section and rinsed with deionized water.

The cleaned tooth segments were pulverized in liquid nitrogen into a fine powder using a steel mortar/pestle (Reimiller, Reggio Emilia, Italy). Five one-gram aliquot of dentin powder were demineralized with 0.5 M EDTA (pH 7.0). The demineralized dentin powder was rinsed with deionized water for 5 times, dried and divided into five groups. Group 1 was left untreated and served as control. The remaining four groups of dentin powder were treated respectively with 2% CHX (Group 2), 2% QAS (Group 3), 5% QAS (Group 4) and 10% QAS (Group 5) for 2 min. The treated dentin powder was re-suspended in extraction buffer [50 mM Tris-HCl at pH 7.5 containing 0.2% Triton X-100, 5 mM CaCl 2 , and 100 mM NaCl] for 24 h to extract the proteases. The vials were centrifuged at 20,000 rpm for 30 min at 4 °C. The supernatants were collected, dialyzed in bags with 30-kDa molecular cut-off overnight, lyophilized and frozen at −20 °C until they were analyzed.

MMP-2 and cysteine cathepsin ELISAs

The concentrations of endogenous MMP-2 and cathepsin K in the supernatants derived from different groups of dentin powder were quantified using ELISA (Human MMP2 ELISA Kit – Lot #5619 for MMP-2; Human CTSK/Cathepsin K ELISA Kit – Lot #5614 for cathepsin K, both from Lifespan Biosciences, Seattle, WA, USA). The kits were used according to the manufacturer’s instructions. A calibration curve correlating protease concentration with absorbance intensity was prepared for each protease. The dialyzed supernatants were incubated in the respective assay buffers for MMP-2 and cathepsin K for 1.5 h at 37 °C. Detection reagents were added after several rinses and the absorbance was recorded with a spectrophotometer at 405 nm (Bio-Rad Laboratories, Inc. Hercules, CA, USA). All tests were conducted in a triplicate and the protease concentrations were expressed as ng/mL.

Statistical analyses

The percent loss of dry mass over incubation time and the rate of release of ICTP and CTX (ng telopeptide/mg dry dentin/unit time) from all groups were compared for normality (Shapiro-Wilk test) and equality of variance (modified Levine test). When the normality and equality variance assumptions of the original data were violated, the data were nonlinearly transformed to satisfy those assumptions prior to the use of parametric statistical methods. Two-factor analysis of variances (ANOVA) was employed to examine the effects of incubation time and disinfectants, and the interaction of those two factors on the respective parameters investigated. Post-hoc multiple comparisons were performed with the Tukey statistic. Dentinal MMP-2 and cathepsin K concentrations in the five groups were separately analyzed using one-way ANOVA and Tukey multiple comparison test. For all tests, statistical significance was preset at α = 0.05.

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Jun 19, 2018 | Posted by in General Dentistry | Comments Off on Effect of a novel quaternary ammonium silane on dentin protease activities
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