Abstract
Objective
Biomodification of dentin by a natural crosslinker has been recommended to improve a mechanical property of demineralized dentin. This study investigated the effect of various plant-derived agents (hesperidin, proanthocyanidin, epigallocatechin gallate and genipin) on the stability of dentin collagen matrix to resist collagenase degradation.
Methods
The dentin specimens were treated with glutaraldehyde (0.5% and 5.0%) and each plant-derived test solution (0.5%). They were subjected to ultimate tensile strength (UTS) and swelling ratio measurements. Demineralized human dentin powder was incubated with 0.02%, 0.1% and 0.5% of each test agent and followed by bacterial collagenase digestion. The extent of collagen degradation was investigated using hydroxyproline assay.
Results
The UTS and swelling ratio measurements revealed that the mechanical property of dentin was improved by the use of these natural agents. The greatest reduction in collagen degradation was shown following the use of hesperidin, proanthocyanidin, and epigallocatechin gallate at 0.5%.
Significance
The use of hesperidin, proanthocyanidin, and epigallocatechin gallate could improve the mechanical properties of collagen and resist enzymatic degradation, leading to functional repair of pathological dentin lesion.
1
Introduction
Dentin caries process cannot be explained solely by terms of physicochemical dissolution of mineral components by bacterial acids. Organic components of dentin are type I collagen fibrils, and non-collagenous proteins, which are embedded with mineral crystallites . The mineral deposition and dispersion within the collagen matrix may be governed by the condition of collagen matrix . The collagen matrix can be degraded mechanically and biochemically, which may be responsible for the progression of pathological lesion in dentin . The modification of collagen matrix by potential therapeutic agents should be considered to enhance the stability of collagen matrix.
In the attempts to stabilize collagen matrix, glutaraldehyde (GA) and natural cross-linkers, such as proanthocyanidin (PA) and genipin (GNP) have been used in human dentin to demonstrate their effects on demineralized lesion . PA, a grape seed polyphenol, is well known as a natural collagen cross-linker. The cross-linking effect of PA is attributed to its interaction with proline-rich proteins such as collagen . Intermolecular cross-links within collagen matrix may enhance the mechanical stability of collagen matrix and resist caries progression . GNP is the active compound found in the gardenia fruit extract. GNP has also been reported as an excellent natural cross-linker for proteins, collagen, gelatin, and chitosan cross-linking with a low toxicity .
Endogenous metalloproteinases (MMPs), such as a collagenase (MMP-8) and gelatinases (MMP-2 and -9), are a group of proteolytic enzymes that participate in collagen breakdown in dentin and involved to biodegradation of organic matrix of dentin collagens . Those enzymes incorporated into mineralized dentin may be uncovered and activated during dentin caries progression . Cysteine cathepsins are also proteolytic enzymes in dentin. Cathepsin B is a major cysteine cathepsin in carious and sound dentin, while cathepsin K is found in resorption of dentin . Since a positive correlation between cysteine cathepsins and MMPs activities has recently been shown in dentin , MMPs and cysteine cathepsins may be major proteases responsible for collagen breakdown. Hence, another approach to stabilize collagen matrix should be to inhibit the proteolytic activity in sound and carious dentin. Epigallocatechin gallate (EGCG), a green tea polyphenol, has been proven to have such an inhibitory effect against MMPs . In a recent in vitro study, EGCG has been shown to reduce the MMPs activity in dentin, which arrested dentin erosions .
Hesperidin (HPN), hesperetin-7-O-rutinoside, is a flavonoid extracted from citrus fruits. The novel medical benefits of this citrus flavonoid include antioxidant, anti-inflammatory and anti-carcinogenic effects . We first attempted the use of HPN in dental tissue using an in vitro caries model and provided evidence that treatment with HPN may resist collagenase degradation and arrest demineralization of bovine root dentin .
Thus, there has been a growing interest in the use of plant-derived natural agents that have potential to stabilize dentin collagen matrix. In the present study, we examined the effect of natural plant-derived agents, HPN, PA, EGCG and GNP, on the mechanical properties of dentin collagen matrix and stability of collagen matrix against collagenase digestion. We compared the effects of these natural agents with that of GA, which is one of the most effective synthetic protein cross-linking agents. The null hypotheses were that there would be no differences: (i) in mechanical properties (the UTS and swelling ratio) of demineralized dentin after the use of each cross-linking agent, and (ii) in proteolytic biodegradation of collagen following bio-modification by each agent.
2
Materials and methods
2.1
Preparation of dentin specimen and tested chemicals
Thirty-six freshly extracted 36 intact human third molars were obtained from 20- to 41-yr-old patients and used according to the guidelines of the Ethics Committee of the Graduate School and Hospital, Tokyo Medical and Dental University. Teeth were sliced parallel to their long axis, with a thickness of 0.5 ± 0.1 mm, by means of a low-speed diamond saw (Isomet, Buehler Ltd., Lake Bluff, IL, USA) under water cooling. Two slabs were chosen from the center of 23 teeth for the UTS test, and of 13 teeth for the swelling test. For the UTS test, hourglass-shaped specimens were prepared with neck area of 0.5 ± 0.1 × 0.5 ± 0.1 mm at middle dentin, 2 mm above the pulp chamber using a cylindrical diamond bur . For the swelling ratio test, a few I-shaped beam specimens (0.5 ± 0.1 × 1.7 ± 0.1 × 7.0 ± 0.5 mm size) were further sectioned from each slab . Prepared specimens were immersed in 10% phosphoric acid solution for 5 h at room temperature to complete demineralization and thoroughly rinsed with distilled water for 10 min.
Another 10 extracted teeth were obtained from unerupted, human third molars obtained from 19- to 29-yr-old patients and were used for collagenase degradation by means of hydroxyproline assay test. Following manual removal of pulp, periodontal ligament, cementum and enamel, the remaining dentinal fragments were pulverized under liquid N 2 in a cryogenic mill (CertiPrep 6750, SPEX CertiPrep, Metuchen, USA) into particles with diameters <75 μm.
The cross-linking agents used in the study were GA (Sigma–Aldrich, St. Louis, MO, USA), HPN (Wako Pure Chemical Industries, Ltd., Tokyo, Japan), PA (Kikkoman Biochemifa Company, Tokyo, Japan), EGCG (Sigma–Aldrich, St. Louis, MO, USA) and GNP (Sigma–Aldrich, St. Louis, MO, USA). The chemical structures of these plant-derived agents are shown in Fig. 1 .
2.2
Ultimate tensile strength (UTS) testing
The test agents were first dissolved in 100% dimethyl sulfoxide (DMSO) and diluted by distilled water to be 0.5 wt% in 10% DMSO as the final concentration. Low (0.5 wt%) and high (5.0 wt%) concentrations of GA were similarly prepared in 10% DMSO. Demineralized hourglass-shaped samples ( n = 6) were treated in each test solution or distilled water (the control) at 37 °C for 12 h. After this treatment, specimens were subjected to UTS evaluation. The specimens were glued to a jig using a cyanoacrylate adhesive (Zapit, Dental Ventures of America, Corona, CA), which was mounted on a universal testing machine (EZ Test, Shimazu, Kyoto, Japan) subjected to a tension force at a crosshead speed of 1 mm/min until failure.
2.3
Swelling ratio testing
The test solutions were prepared in the same manner as described for the UTS testing. Demineralized I-shaped specimens ( n = 8) were treated for 12 h either in the test solution or in distilled water, which was used as a control. After treatment, the specimens were swollen in water and equilibrated overnight in phosphate buffered saline (pH 7.4) at room temperature. The specimens were removed and blotted with filter paper to remove excess surface water and weighed immediately. Dentin samples were then placed in distilled water for 10 min to remove the buffer salts. They were dried in a desiccator to a constant weight and weighted. The swelling ratio was calculated as the ratio of the weight of swollen sample to that of dry sample .
2.4
Collagenase degradation and hydroxyproline assay
The test solutions of cross-linking agents were prepared at 0.02 wt%, 0.1 wt% and 0.5 wt% concentrations in 10% DMSO. The dentin samples were demineralized with 0.5 M EDTA (pH 7.4) for 10 days at 4 °C, extensively washed with distilled water and lyophilized. Six 2 mg aliquot of demineralized dentin samples were allocated to each test solution ( n = 6). The samples were incubated in 2 ml of each test solution for 12 h at 37 °C with stirring. After incubation, the samples were centrifuged and extensively washed with distilled water three times. Each residue was digested with 2 ml of bacterial collagenase derived from Clostridium histolyticum (7.5 U/1 ml) for 24 h in artificial saliva (50 mmol/L HEPES, 1.5 mmol/L CaCl 2 ·2H 2 O, 150 mmol/L NaCl, and 3 mmol/L NaN 3 ; pH 7.2) with stirring at 37 °C. The samples that were not incubated with any test agents were further divided into the positive and negative control, in which collagenase challenging was performed or not, respectively. Degradation of collagen was determined by calculating collagen content that was dissolved during collagenase digestion. After centrifuging, the supernatants were subjected to chemical analysis of collagen degradation by estimating the amount of hydroxyproline (an amino acid characteristic of collagen) using the simplified chloramine T method . In brief, the sample aliquot was hydrolyzed with 2 N sodium hydroxide by autoclaving at 120 °C for 20 min. Chloramine T was added to the hydrolyzate to allow oxidation of hydroxyproline, followed by adding of Ehrlich’s aldehyde reagent. When chromophore was developed, the absorbance of each sample was read at 550 nm using a spectrophotometer (VICTOR X Multilabel Plate Readers; PerkinElmer Inc., Boston, MA) and was converted to concentration of hydroxyproline. The amount of collagen degraded was calculated assuming that 12.5% of collagen is hydroxyproline .
2.5
Statistical analysis
The statistical analysis was performed using a statistical package (SPSS Ver.11 for Windows, SPSS Inc., Chicago, IL, USA). The UTS and swelling ratio data were analyzed by one-way ANOVA. The amount of collagen degradation was analyzed by two-way ANOVA using the tested agents and their concentration as two factors, further by one-way ANOVA. In case of significance, statistical analyses were performed by Tukey multiple comparisons test ( p = 0.05).
2
Materials and methods
2.1
Preparation of dentin specimen and tested chemicals
Thirty-six freshly extracted 36 intact human third molars were obtained from 20- to 41-yr-old patients and used according to the guidelines of the Ethics Committee of the Graduate School and Hospital, Tokyo Medical and Dental University. Teeth were sliced parallel to their long axis, with a thickness of 0.5 ± 0.1 mm, by means of a low-speed diamond saw (Isomet, Buehler Ltd., Lake Bluff, IL, USA) under water cooling. Two slabs were chosen from the center of 23 teeth for the UTS test, and of 13 teeth for the swelling test. For the UTS test, hourglass-shaped specimens were prepared with neck area of 0.5 ± 0.1 × 0.5 ± 0.1 mm at middle dentin, 2 mm above the pulp chamber using a cylindrical diamond bur . For the swelling ratio test, a few I-shaped beam specimens (0.5 ± 0.1 × 1.7 ± 0.1 × 7.0 ± 0.5 mm size) were further sectioned from each slab . Prepared specimens were immersed in 10% phosphoric acid solution for 5 h at room temperature to complete demineralization and thoroughly rinsed with distilled water for 10 min.
Another 10 extracted teeth were obtained from unerupted, human third molars obtained from 19- to 29-yr-old patients and were used for collagenase degradation by means of hydroxyproline assay test. Following manual removal of pulp, periodontal ligament, cementum and enamel, the remaining dentinal fragments were pulverized under liquid N 2 in a cryogenic mill (CertiPrep 6750, SPEX CertiPrep, Metuchen, USA) into particles with diameters <75 μm.
The cross-linking agents used in the study were GA (Sigma–Aldrich, St. Louis, MO, USA), HPN (Wako Pure Chemical Industries, Ltd., Tokyo, Japan), PA (Kikkoman Biochemifa Company, Tokyo, Japan), EGCG (Sigma–Aldrich, St. Louis, MO, USA) and GNP (Sigma–Aldrich, St. Louis, MO, USA). The chemical structures of these plant-derived agents are shown in Fig. 1 .
2.2
Ultimate tensile strength (UTS) testing
The test agents were first dissolved in 100% dimethyl sulfoxide (DMSO) and diluted by distilled water to be 0.5 wt% in 10% DMSO as the final concentration. Low (0.5 wt%) and high (5.0 wt%) concentrations of GA were similarly prepared in 10% DMSO. Demineralized hourglass-shaped samples ( n = 6) were treated in each test solution or distilled water (the control) at 37 °C for 12 h. After this treatment, specimens were subjected to UTS evaluation. The specimens were glued to a jig using a cyanoacrylate adhesive (Zapit, Dental Ventures of America, Corona, CA), which was mounted on a universal testing machine (EZ Test, Shimazu, Kyoto, Japan) subjected to a tension force at a crosshead speed of 1 mm/min until failure.
2.3
Swelling ratio testing
The test solutions were prepared in the same manner as described for the UTS testing. Demineralized I-shaped specimens ( n = 8) were treated for 12 h either in the test solution or in distilled water, which was used as a control. After treatment, the specimens were swollen in water and equilibrated overnight in phosphate buffered saline (pH 7.4) at room temperature. The specimens were removed and blotted with filter paper to remove excess surface water and weighed immediately. Dentin samples were then placed in distilled water for 10 min to remove the buffer salts. They were dried in a desiccator to a constant weight and weighted. The swelling ratio was calculated as the ratio of the weight of swollen sample to that of dry sample .
2.4
Collagenase degradation and hydroxyproline assay
The test solutions of cross-linking agents were prepared at 0.02 wt%, 0.1 wt% and 0.5 wt% concentrations in 10% DMSO. The dentin samples were demineralized with 0.5 M EDTA (pH 7.4) for 10 days at 4 °C, extensively washed with distilled water and lyophilized. Six 2 mg aliquot of demineralized dentin samples were allocated to each test solution ( n = 6). The samples were incubated in 2 ml of each test solution for 12 h at 37 °C with stirring. After incubation, the samples were centrifuged and extensively washed with distilled water three times. Each residue was digested with 2 ml of bacterial collagenase derived from Clostridium histolyticum (7.5 U/1 ml) for 24 h in artificial saliva (50 mmol/L HEPES, 1.5 mmol/L CaCl 2 ·2H 2 O, 150 mmol/L NaCl, and 3 mmol/L NaN 3 ; pH 7.2) with stirring at 37 °C. The samples that were not incubated with any test agents were further divided into the positive and negative control, in which collagenase challenging was performed or not, respectively. Degradation of collagen was determined by calculating collagen content that was dissolved during collagenase digestion. After centrifuging, the supernatants were subjected to chemical analysis of collagen degradation by estimating the amount of hydroxyproline (an amino acid characteristic of collagen) using the simplified chloramine T method . In brief, the sample aliquot was hydrolyzed with 2 N sodium hydroxide by autoclaving at 120 °C for 20 min. Chloramine T was added to the hydrolyzate to allow oxidation of hydroxyproline, followed by adding of Ehrlich’s aldehyde reagent. When chromophore was developed, the absorbance of each sample was read at 550 nm using a spectrophotometer (VICTOR X Multilabel Plate Readers; PerkinElmer Inc., Boston, MA) and was converted to concentration of hydroxyproline. The amount of collagen degraded was calculated assuming that 12.5% of collagen is hydroxyproline .
2.5
Statistical analysis
The statistical analysis was performed using a statistical package (SPSS Ver.11 for Windows, SPSS Inc., Chicago, IL, USA). The UTS and swelling ratio data were analyzed by one-way ANOVA. The amount of collagen degradation was analyzed by two-way ANOVA using the tested agents and their concentration as two factors, further by one-way ANOVA. In case of significance, statistical analyses were performed by Tukey multiple comparisons test ( p = 0.05).
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