Inhibition of matrix metalloproteinase activity in human dentin via novel antibacterial monomer

Highlights

  • A new monomer DMADDM was tested as an inhibitor against MMPs for its potential to combat degradation of dentin collagen and the dentin–resin bond.

  • DMADDM exhibited a strong anti-MMP effect, reaching more than 90% inhibition on rhMMP-8 and rhMMP-9 with 5% or more of DMADDM.

  • The use of DMADDM preserved the mechanical stiffness and hindered the dissolution of demineralized human dentin.

  • DMADDM is promising for use in bonding agents to prevent dentin collagen degradation in the hybrid layer and prolong the dentin–restoration bond. .

Abstract

Objective

Dentin–composite bond failure is caused by factors including hybrid layer degradation, which in turn can be caused by hydrolysis and enzymatic degradation of the exposed collagen in the dentin. The objectives of this study were to investigate a new antibacterial monomer (dimethylaminododecyl methacrylate, DMADDM) as an inhibitor for matrix metalloproteinases (MMPs), and to determine the effects of DMADDM on both soluble recombinant human MMPs (rhMMPs) and dentin matrix-bound endogenous MMPs.

Methods

Inhibitory effects of DMADDM at six mass% (0.1% to 10%) on soluble rhMMP-8 and rhMMP-9 were measured using a colorimetic assay. Matrix-bound endogenous MMP activity was evaluated in demineralized human dentin. Dentin beams were divided into four groups ( n = 10) and incubated in calcium- and zinc-containing media (control medium); or control medium + 0.2% chlorhexidine (CHX); 5% 12-methacryloyloxydodecylpyridinium bromide (MDPB); or 5% DMADDM. Dissolution of dentin collagen peptides was evaluated by mechanical testing in three-point flexure, loss of dentin mass, and a hydroxyproline assay.

Results

Use of 0.1% to 10% DMADDM exhibited a strong concentration-dependent anti-MMP effect, reaching 90% of inhibition on rhMMP-8 and rhMMP-9 at 5% DMADDM concentration. Dentin beams in medium with 5% DMADDM showed 34% decrease in elastic modulus (vs. 73% decrease for control), 3% loss of dry dentin mass (vs. 28% loss for control), and significantly less solubilized hydroxyproline when compared with control ( p < 0.05).

Significance

The new antibacterial monomer DMADDM was effective in inhibiting both soluble rhMMPs and matrix-bound human dentin MMPs. These results, together with previous studies showing that adhesives containing DMADDM inhibited biofilms without compromising dentin bond strength, suggest that DMADDM is promising for use in adhesives to prevent collagen degradation in hybrid layer and protect the resin–dentin bond.

Introduction

Dental resin composites are popular filling materials because of their excellent esthetics, direct-filling capability, and improved load-bearing properties . After being bonded to the tooth structure via adhesive , it is desirable to have a long-lasting restoration-tooth bonded interface. Although most adhesives exhibit excellent short-term bonding strength, the durability of the bonded interface still remains a challenge . Nearly half of all dental restorations fail within 10 years, and replacing them accounts for 50–70% of all restorations performed . This is costly, considering that the annual cost for tooth cavity restorations was about $46 billion in 2005 in the United States alone . The cost is increasing rapidly with an aging population, longer life expectancy, and increased tooth retention in seniors . Therefore, there is an urgent need to improve the dentin–resin interfacial bond to reduce failure and increase the longevity of dental restorations .

The degradation of the hybrid layer at the dentin–adhesive interface was believed to be the primary reason for failure, which was caused by several mechanical and chemical factors, including the hydrolysis and enzymatic degradation of the exposed collagen and the adhesive resin . Water sorption in vivo was inevitable due to the polar ether-linkages and/or hydroxyl groups in adhesive , and it may result in hydrolysis of the hydrophilic resin components . Recently, it was reported that host-derived matrix metalloproteinases (MMPs) were involved in hybrid layer degradation . MMPs are a group of zinc- and calcium-dependent host-derived proteases and exist in mineralized dentin . They can be released and activated by the acidic etchants of dentin bonding , and by lactic acid from oral pathogenic bacteria . The activated collagen-bound MMPs and/or non-collagen-bound MMPs may progressively degrade the uncovered collagen fibrils in the bonded dentin. The breakdown of collagen may increase the water content, cause further collagen degradation, and deteriorate the dentin–restoration bond .

Chlorhexidine (CHX) had MMP inhibitory and anti-enzyme properties . Collagen degradation of demineralized dentin was almost completely inhibited via CHX . However, CHX is water-soluble and electrostatically binds to demineralized dentin matrix. When incorporated into adhesive, CHX may diffuse out of the dentin collagen matrix via a competitive desorption mechanism in the presence of other cations, leading to a decrease in its long-term anti-MMP effectiveness . In contrast, the bond strength of an antibacterial adhesive containing 12-methacryloyloxydodecyl-pyridinium bromide (MDPB) did not decrease with time in a one-year period . Another study showed that MDPB was effective in inhibiting both soluble MMPs and matrix-bound dentin MMPs . Recently, a new antibacterial monomer dimethylaminododecyl methacrylate (DMADDM) was synthesized and incorporated into a bonding agent, which showed no reduction in bonding strength from 1 day (d) to 6 months of water-aging . While the dentin shear bond strength of a commercial adhesive control decreased from about 30 MPa at 1 d to 20 MPa at 6 months, that of the DMADDM adhesive stayed at 30 MPa . However, the anti-MMP properties of DMADDM have not been reported.

The objectives of this study were to investigate for the first time the effects of DMADDM on soluble rhMMP-8 and rhMMP-9 and human dentin matrix-bond endogenous MMPs, and on dentin elastic modulus and dentin dissolution and mass loss. It was hypothesized that: (1) DMADDM will have potent inhibitory effects against soluble rhMMP-8 and rhMMP-9 and matrix-bond endogenous MMPs; (2) The use of DMADDM will greatly reduce the elastic modulus loss in demineralized dentin, dentin mass loss, and the dissolution of collagen peptides from dentin, compared to control without DMADDM.

Materials and methods

Synthesis of antibacterial monomer

The synthesis of DMADDM was detailed elsewhere . A modified Menschutkin reaction method was used where a tertiary amine group was reacted with an organo-halide. A benefit of this method is that the reaction products are generated at virtually quantitative amounts and require minimal purification . Briefly, 10 mmol of 1-(dimethylamino)docecane (Sigma, St. Louis, MO) and 10 mM of 2-bromoethyl methacrylate (BEMA, Monomer-Polymer and Dajec Labs, Trevose, PA) were combined with 3 g of ethanol in a 20 mL scintillation vial. The vial was stirred at 70 °C for 24 h for the reaction to be completed. Then the solvent was removed via evaporation, yielding DMADDM as a clear, colorless and viscous liquid . The reaction and products were verified via Fourier transform infrared spectroscopy (FTIR) in a previous study .

Inhibition of soluble recombinant human matrix metalloproteinases (rhMMPs)

Purified rhMMP-8 (catalog No. 72008), rhMMP-9 (catalog No. 55576-1) and the Sensolyte Generic MMP colorimetric assay kit (catalog No. 72095) were obtained from AnaSpec Inc. (San Jose, CA). MMP-8 is the major collagenase in human dentin . MMP-9 is the most studied gelatinase in dentin . The Sensolyte Generic MMP kit contains a thiopeptide that can be cleaved by MMPs to release a sulfhydryl group. The sulfhydryl group reacts with 5,5′-dithiobis(2-nitrobenzoic acid) to produce a colored reaction product 2-nitro-5-thiobenzoic acid, which can be detected spectrophotometrically at 412 nm .

DMADDM was dissolved in deionized water at DMADDM/(DMADDM + water) = 0.1%, 1%, 2.5%, 5%, 7.5% and 10% by mass. The thiopeptolide substrate solution was diluted to 0.2 mM with the supplied assay buffer in a 1:50 volume ratio. The assay was performed in a 96-well plate using four replicate wells for each DMADDM solution. For example, for rhMMP-9, each well contained 2 μL of rhMMP-9 (19.6 ng/well), 10 μL of a DMADDM solution at various concentrations (0.1, 1, 2.5, 5, 7.5 and 10 mg of DMADDM per well, respectively), and 50 μL of thiopeptolide substrate solution. Additional buffer was added to achieve a total of 100 μL for each well. As this resulted in a 10-fold dilution of the anti-MMP agent DMADDM, the DMADDM solutions at the aforementioned concentrations were prepared to 10 times their designated final concentrations. The DMADDM solutions were pre-incubated with each MMP enzyme for 20 min to prevent a burst of uninhibited enzyme activity, based on a previous study .

The control groups included: (1) a positive control containing rhMMP only without anti-MMP agent; (2) an inhibitor control containing rhMMP and 10 μL of the 20 μM GM6001, a known MMP inhibitor supplied in the assay kit by the manufacturer; (3) a test compound control containing assay buffer and the GM6001 or the DMADDM solution at each concentration to be tested; (4) a substrate control containing assay buffer. Additional assay buffer was added to bring the total volume of all control wells to 50 μL prior to adding 50 μL of the thiopeptolide substrate solution to obtain the 100 μL/well.

The reagents were mixed by shaking the plate gently for 30 s. Then they were read kinetically at every 10 min for 60 min by measuring the absorbance at 412 nm using a plate reader (SpectraMax M5, Molecular Devices, Sunnyvale, CA) . Background absorbance was determined using the mean absorbance readings from the “substrate control” wells and subtracted from the readings of other wells containing the thiopeptolide substrate .

The potency of MMP inhibition by the proprietary MMP inhibitor GM6001 (inhibitor control) and the DMADDM at six concentrations was calculated as the percentage of the adjusted absorbance of the positive control . Percentage of MMP inhibition = 1 − (absorbance of test compound group − absorbance of test compound control)/(absorbance of the positive control − absorbance of substrate control). The test compound control was used for subtraction of background absorbance from the compound group. The substrate control was used for subtraction of background absorbance from the positive control group.

For statistical analysis, as the normality and homoscedasticity assumptions of the data appeared to be valid, the percentages of inhibition in the six groups (for the aforementioned six DMADDM concentrations) were analyzed using one-way ANOVA on ranks and Tukey’s multiple comparison tests at α = 0.05.

Preparation and incubation of demineralized human dentin beams

Extracted human third molars were collected with approval by the University of Maryland. The teeth were stored at 4 °C in 0.9% NaCl supplemented with 0.02% sodium azide to prevent bacterial growth and used within 1 month after extraction. For each tooth, the enamel and superficial dentin were removed by horizontal sectioning at 1 mm below the central fissures using a water-cooled cutting saw (Isomet, Buehler, Lake Bluff, IL). A 1-mm thick dentin disk consisting of midcoronal dentin was prepared from each tooth. Two dentin beams of 6 × 2 × 1 mm were sectioned from the middle of each disk under water cooling . Prior to demineralization, for each dentin beam, a small dimple was made at the end of one of the two 6 × 2 mm surfaces to allow subsequent measurements to be performed on the same surface. The beams were submerged in 10% phosphoric acid for 18 h to completely demineralize the dentin . Absence of residual mineral was confirmed using digital radiography. Then, the elastic modulus of each demineralized dentin beam was determined using three-point flexure (Section 2.4 ), and this is termed the elastic modulus at 0 d.

Demineralized dentin beams were randomized into four groups for aging in four types of solutions ( n = 10). A calcium- and zinc-containing complete storage medium was used which contained 5 mM HEPES, 2.5 mM CaCl 2 ·H 2 O, 0.05 mM ZnCl 2 , and 0.3 mM NaN 3 (adjusted to pH 7.4 with 1 M HCl), and this is referred to as control medium. The four solutions were: control medium; control medium plus 5% DMADDM; control medium plus 0.2% CHX (Sigma); and control medium plus 5% MDPB. MDPB powder was kindly provided by Kuraray Medical Inc. (Tokyo, Japan), included here since it was a well-known and potent quaternary ammonium methacrylate. For calculation of the mass%, for example, DMADDM/(DMADDM + control medium) = 5%. The 5% DMADDM was based on Section 2.2 which showed that the inhibition of rhMMP-8 and rhMMP-9 in the 5% DMADDM group was excellent and exceeded 90%. Furthermore, the result of our previous study showed that an adhesive containing 5% DMADDM had strong antibacterial activity after being cured . Each demineralized dentin beam was placed in a polypropylene tube containing 1 mL of one of the four solutions. The sealed tubes were incubated in a shaker at 60 cycles/min and 37 °C for 30 d . Elastic modulus of dentin beams were determined using three-point flexure (Section 2.4 ), and this is termed the elastic modulus at 30 d.

Elastic modulus of demineralized dentin beams

Elastic modulus of demineralized dentin beams were measured using three-point flexure with a span of 4 mm, suitable for testing small-sized human tooth samples. A universal testing machine (model V1000, John Chatillon & Sons, Greensboro, NC) was used with a 1 N load-cell (Transducer Techniques, Temecula, CA). Each beam was loaded to a maximum displacement of 200 μm at a rate of 60 μm/min, which was determined in preliminary study to be suitable for measuring elastic modulus of demineralized dentin in the elastic region. After reaching maximum displacement, the load was reduced to 0 N within 15 s . The dentin beam was taken out of the immersion; it remained saturated with solution and wet during testing. Elastic modulus ( E ) was calculated as: E = mL 3 /4 bd 3 , where m is slope of load-displacement curve, L is span, b is dentin beam width, and d is dentin beam thickness . As this test used relatively small dentin beams, the elastic modulus was approximate, to determine the ranking of elastic modulus of dentin for the different treatment groups .

The data were analyzed using a two factor repeated measures analysis of variance to examine the effects of the four solutions and the repeated factor storage time (0 d vs. 30 d), and the interaction of the two variables on elastic modulus. Pair-wise multiple comparisons were performed using the Tukey’s test. Statistical significance was set at α = 0.05.

Loss of mass versus immersion time for demineralized dentin

Demineralized dentin beams at 0 d were placed in uncapped polypropylene tubes and placed in a container with anhydrous calcium sulfate (Drierite, Hammond Drierite, Xenio, OH), to be desiccated until a constant weight was reached. The dry mass of each beam was measured to an accuracy of 0.1 mg via an analytical balance (AB204, Mettler Toledo, Switzerland). After the measurement, each dried and shrunken dentin beam was placed in its original polypropylene tube with incubation medium. This completely rehydrated the dried beam, and reversibly recovered its original dimensions . After 30 d, the beam was dried again as described above, and the dry mass was again measured. Percentage of mass loss in dentin = (dry mass at 0 d − dry mass at 30 d)/dry mass at 0 d. The data were analyzed using Kruskal–Wallis one-way ANOVA and Dunn’s multiple comparison tests at α = 0.05.

Dissolution of collagen peptides from demineralized dentin beams

When the demineralized dentin beams were incubated in the calcium and zinc-containing medium, the activated matrix-bound MMPs slowly digested the demineralized collagen and released the solubilized peptide fragments . A hydroxyproline assay (Sigma) was used to evaluate the collagen degradation. At 30 d, 100 μL of the medium was collected from each vial and transferred to a labeled polypropylene tube. An aliquot of 100 μL concentrated hydrochloric acid (HCl, 12 M) was added into the tube and the mixture was hydrolyzed at 120 °C for 3 h. Then, 5 mg of activated charcoal was added, mixed and centrifuged at 13 kg for 2 min. An aliquot of 10 μL supernatant was transferred to a 96 well plate. At the same time, hydroxyproline standards for colorimetric detection were prepared by diluting 10 μL of the 1 mg/mL hydroxyproline standard solution with 90 μL of water to prepare a 0.1 mg/mL standard solution. An amount of 0.1 mg/mL hydroxyproline standard solution, at 0, 2, 4, 6, 8, or 10 μL, was added into a 96 well plate, generating corresponding standards of 0 (blank), 0.2, 0.4, 0.6, 0.8, and 1.0 μg/well. The plate was placed in a 60 °C oven to evaporate all the wells. Then, 100 μL Chloramine T/Oxidation Buffer Mixture was added to each sample well and standard well. After incubation at room temperature for 5 min, 100 μL 4-(dimethylamino) benzaldehyde (DMAB) reagent was added to each sample well and standard well, and incubated for 90 min at 60 °C. Then the absorbance was measured at 560 nm using the plate reader. The background for the assay was the value obtained for the 0 (blank) hydroxyproline standard. The blank value was subtracted from all the readings. The amount of hydroxyproline (μg/mL) was determined from a pre-established calibration curve derived from a linear regression equation of the absorbance of hydroxyproline against the known concentrations of hydroxyproline in the standards, following the manufacturer’s instructions for the hydroxyproline assay.

The resulting amount of hydroxyproline was used to estimate the percentage of solubilized collagen fragments in the medium. This estimate was based on the assumption that 90% of the dry mass of demineralized dentin consisted of type I collagen according to a previous study , and that the dentin collagen contained 9.6 mass% of hydroxyproline . Hence, the total hydroxyproline mass in the dentin beam at 0 d = dentin dry mass at 0 d × 0.9 × 0.096. Therefore, the percentage of solubilized collagen at 30 d = the measured mass of solubilized hydroxyproline at 30 d/the total hydroxyproline mass in the dentin beam at 0 d = the measured mass of solubilized hydroxyproline at 30 d/(dentin dry mass at 0 d × 0.9 × 0.096). However, in order to compare with previous study , the dissolved collagen from the demineralized dentin beam was expressed as μg of solubilized hydroxyproline per mg dry mass of demineralized dentin at 0 d. The amounts of dissolved collagen from the demineralized dentin beams in the four groups were evaluated using Kruskal–Wallis one-way ANOVA and Dunn’s multiple comparison tests at α = 0.05.

Materials and methods

Synthesis of antibacterial monomer

The synthesis of DMADDM was detailed elsewhere . A modified Menschutkin reaction method was used where a tertiary amine group was reacted with an organo-halide. A benefit of this method is that the reaction products are generated at virtually quantitative amounts and require minimal purification . Briefly, 10 mmol of 1-(dimethylamino)docecane (Sigma, St. Louis, MO) and 10 mM of 2-bromoethyl methacrylate (BEMA, Monomer-Polymer and Dajec Labs, Trevose, PA) were combined with 3 g of ethanol in a 20 mL scintillation vial. The vial was stirred at 70 °C for 24 h for the reaction to be completed. Then the solvent was removed via evaporation, yielding DMADDM as a clear, colorless and viscous liquid . The reaction and products were verified via Fourier transform infrared spectroscopy (FTIR) in a previous study .

Inhibition of soluble recombinant human matrix metalloproteinases (rhMMPs)

Purified rhMMP-8 (catalog No. 72008), rhMMP-9 (catalog No. 55576-1) and the Sensolyte Generic MMP colorimetric assay kit (catalog No. 72095) were obtained from AnaSpec Inc. (San Jose, CA). MMP-8 is the major collagenase in human dentin . MMP-9 is the most studied gelatinase in dentin . The Sensolyte Generic MMP kit contains a thiopeptide that can be cleaved by MMPs to release a sulfhydryl group. The sulfhydryl group reacts with 5,5′-dithiobis(2-nitrobenzoic acid) to produce a colored reaction product 2-nitro-5-thiobenzoic acid, which can be detected spectrophotometrically at 412 nm .

DMADDM was dissolved in deionized water at DMADDM/(DMADDM + water) = 0.1%, 1%, 2.5%, 5%, 7.5% and 10% by mass. The thiopeptolide substrate solution was diluted to 0.2 mM with the supplied assay buffer in a 1:50 volume ratio. The assay was performed in a 96-well plate using four replicate wells for each DMADDM solution. For example, for rhMMP-9, each well contained 2 μL of rhMMP-9 (19.6 ng/well), 10 μL of a DMADDM solution at various concentrations (0.1, 1, 2.5, 5, 7.5 and 10 mg of DMADDM per well, respectively), and 50 μL of thiopeptolide substrate solution. Additional buffer was added to achieve a total of 100 μL for each well. As this resulted in a 10-fold dilution of the anti-MMP agent DMADDM, the DMADDM solutions at the aforementioned concentrations were prepared to 10 times their designated final concentrations. The DMADDM solutions were pre-incubated with each MMP enzyme for 20 min to prevent a burst of uninhibited enzyme activity, based on a previous study .

The control groups included: (1) a positive control containing rhMMP only without anti-MMP agent; (2) an inhibitor control containing rhMMP and 10 μL of the 20 μM GM6001, a known MMP inhibitor supplied in the assay kit by the manufacturer; (3) a test compound control containing assay buffer and the GM6001 or the DMADDM solution at each concentration to be tested; (4) a substrate control containing assay buffer. Additional assay buffer was added to bring the total volume of all control wells to 50 μL prior to adding 50 μL of the thiopeptolide substrate solution to obtain the 100 μL/well.

The reagents were mixed by shaking the plate gently for 30 s. Then they were read kinetically at every 10 min for 60 min by measuring the absorbance at 412 nm using a plate reader (SpectraMax M5, Molecular Devices, Sunnyvale, CA) . Background absorbance was determined using the mean absorbance readings from the “substrate control” wells and subtracted from the readings of other wells containing the thiopeptolide substrate .

The potency of MMP inhibition by the proprietary MMP inhibitor GM6001 (inhibitor control) and the DMADDM at six concentrations was calculated as the percentage of the adjusted absorbance of the positive control . Percentage of MMP inhibition = 1 − (absorbance of test compound group − absorbance of test compound control)/(absorbance of the positive control − absorbance of substrate control). The test compound control was used for subtraction of background absorbance from the compound group. The substrate control was used for subtraction of background absorbance from the positive control group.

For statistical analysis, as the normality and homoscedasticity assumptions of the data appeared to be valid, the percentages of inhibition in the six groups (for the aforementioned six DMADDM concentrations) were analyzed using one-way ANOVA on ranks and Tukey’s multiple comparison tests at α = 0.05.

Preparation and incubation of demineralized human dentin beams

Extracted human third molars were collected with approval by the University of Maryland. The teeth were stored at 4 °C in 0.9% NaCl supplemented with 0.02% sodium azide to prevent bacterial growth and used within 1 month after extraction. For each tooth, the enamel and superficial dentin were removed by horizontal sectioning at 1 mm below the central fissures using a water-cooled cutting saw (Isomet, Buehler, Lake Bluff, IL). A 1-mm thick dentin disk consisting of midcoronal dentin was prepared from each tooth. Two dentin beams of 6 × 2 × 1 mm were sectioned from the middle of each disk under water cooling . Prior to demineralization, for each dentin beam, a small dimple was made at the end of one of the two 6 × 2 mm surfaces to allow subsequent measurements to be performed on the same surface. The beams were submerged in 10% phosphoric acid for 18 h to completely demineralize the dentin . Absence of residual mineral was confirmed using digital radiography. Then, the elastic modulus of each demineralized dentin beam was determined using three-point flexure (Section 2.4 ), and this is termed the elastic modulus at 0 d.

Demineralized dentin beams were randomized into four groups for aging in four types of solutions ( n = 10). A calcium- and zinc-containing complete storage medium was used which contained 5 mM HEPES, 2.5 mM CaCl 2 ·H 2 O, 0.05 mM ZnCl 2 , and 0.3 mM NaN 3 (adjusted to pH 7.4 with 1 M HCl), and this is referred to as control medium. The four solutions were: control medium; control medium plus 5% DMADDM; control medium plus 0.2% CHX (Sigma); and control medium plus 5% MDPB. MDPB powder was kindly provided by Kuraray Medical Inc. (Tokyo, Japan), included here since it was a well-known and potent quaternary ammonium methacrylate. For calculation of the mass%, for example, DMADDM/(DMADDM + control medium) = 5%. The 5% DMADDM was based on Section 2.2 which showed that the inhibition of rhMMP-8 and rhMMP-9 in the 5% DMADDM group was excellent and exceeded 90%. Furthermore, the result of our previous study showed that an adhesive containing 5% DMADDM had strong antibacterial activity after being cured . Each demineralized dentin beam was placed in a polypropylene tube containing 1 mL of one of the four solutions. The sealed tubes were incubated in a shaker at 60 cycles/min and 37 °C for 30 d . Elastic modulus of dentin beams were determined using three-point flexure (Section 2.4 ), and this is termed the elastic modulus at 30 d.

Elastic modulus of demineralized dentin beams

Elastic modulus of demineralized dentin beams were measured using three-point flexure with a span of 4 mm, suitable for testing small-sized human tooth samples. A universal testing machine (model V1000, John Chatillon & Sons, Greensboro, NC) was used with a 1 N load-cell (Transducer Techniques, Temecula, CA). Each beam was loaded to a maximum displacement of 200 μm at a rate of 60 μm/min, which was determined in preliminary study to be suitable for measuring elastic modulus of demineralized dentin in the elastic region. After reaching maximum displacement, the load was reduced to 0 N within 15 s . The dentin beam was taken out of the immersion; it remained saturated with solution and wet during testing. Elastic modulus ( E ) was calculated as: E = mL 3 /4 bd 3 , where m is slope of load-displacement curve, L is span, b is dentin beam width, and d is dentin beam thickness . As this test used relatively small dentin beams, the elastic modulus was approximate, to determine the ranking of elastic modulus of dentin for the different treatment groups .

The data were analyzed using a two factor repeated measures analysis of variance to examine the effects of the four solutions and the repeated factor storage time (0 d vs. 30 d), and the interaction of the two variables on elastic modulus. Pair-wise multiple comparisons were performed using the Tukey’s test. Statistical significance was set at α = 0.05.

Loss of mass versus immersion time for demineralized dentin

Demineralized dentin beams at 0 d were placed in uncapped polypropylene tubes and placed in a container with anhydrous calcium sulfate (Drierite, Hammond Drierite, Xenio, OH), to be desiccated until a constant weight was reached. The dry mass of each beam was measured to an accuracy of 0.1 mg via an analytical balance (AB204, Mettler Toledo, Switzerland). After the measurement, each dried and shrunken dentin beam was placed in its original polypropylene tube with incubation medium. This completely rehydrated the dried beam, and reversibly recovered its original dimensions . After 30 d, the beam was dried again as described above, and the dry mass was again measured. Percentage of mass loss in dentin = (dry mass at 0 d − dry mass at 30 d)/dry mass at 0 d. The data were analyzed using Kruskal–Wallis one-way ANOVA and Dunn’s multiple comparison tests at α = 0.05.

Dissolution of collagen peptides from demineralized dentin beams

When the demineralized dentin beams were incubated in the calcium and zinc-containing medium, the activated matrix-bound MMPs slowly digested the demineralized collagen and released the solubilized peptide fragments . A hydroxyproline assay (Sigma) was used to evaluate the collagen degradation. At 30 d, 100 μL of the medium was collected from each vial and transferred to a labeled polypropylene tube. An aliquot of 100 μL concentrated hydrochloric acid (HCl, 12 M) was added into the tube and the mixture was hydrolyzed at 120 °C for 3 h. Then, 5 mg of activated charcoal was added, mixed and centrifuged at 13 kg for 2 min. An aliquot of 10 μL supernatant was transferred to a 96 well plate. At the same time, hydroxyproline standards for colorimetric detection were prepared by diluting 10 μL of the 1 mg/mL hydroxyproline standard solution with 90 μL of water to prepare a 0.1 mg/mL standard solution. An amount of 0.1 mg/mL hydroxyproline standard solution, at 0, 2, 4, 6, 8, or 10 μL, was added into a 96 well plate, generating corresponding standards of 0 (blank), 0.2, 0.4, 0.6, 0.8, and 1.0 μg/well. The plate was placed in a 60 °C oven to evaporate all the wells. Then, 100 μL Chloramine T/Oxidation Buffer Mixture was added to each sample well and standard well. After incubation at room temperature for 5 min, 100 μL 4-(dimethylamino) benzaldehyde (DMAB) reagent was added to each sample well and standard well, and incubated for 90 min at 60 °C. Then the absorbance was measured at 560 nm using the plate reader. The background for the assay was the value obtained for the 0 (blank) hydroxyproline standard. The blank value was subtracted from all the readings. The amount of hydroxyproline (μg/mL) was determined from a pre-established calibration curve derived from a linear regression equation of the absorbance of hydroxyproline against the known concentrations of hydroxyproline in the standards, following the manufacturer’s instructions for the hydroxyproline assay.

The resulting amount of hydroxyproline was used to estimate the percentage of solubilized collagen fragments in the medium. This estimate was based on the assumption that 90% of the dry mass of demineralized dentin consisted of type I collagen according to a previous study , and that the dentin collagen contained 9.6 mass% of hydroxyproline . Hence, the total hydroxyproline mass in the dentin beam at 0 d = dentin dry mass at 0 d × 0.9 × 0.096. Therefore, the percentage of solubilized collagen at 30 d = the measured mass of solubilized hydroxyproline at 30 d/the total hydroxyproline mass in the dentin beam at 0 d = the measured mass of solubilized hydroxyproline at 30 d/(dentin dry mass at 0 d × 0.9 × 0.096). However, in order to compare with previous study , the dissolved collagen from the demineralized dentin beam was expressed as μg of solubilized hydroxyproline per mg dry mass of demineralized dentin at 0 d. The amounts of dissolved collagen from the demineralized dentin beams in the four groups were evaluated using Kruskal–Wallis one-way ANOVA and Dunn’s multiple comparison tests at α = 0.05.

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Nov 23, 2017 | Posted by in Dental Materials | Comments Off on Inhibition of matrix metalloproteinase activity in human dentin via novel antibacterial monomer
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