Substantivity of chlorhexidine to human dentin

Abstract

Objectives

To better comprehend the role of CHX in the preservation of resin–dentin bonds, this study investigated the substantivity of CHX to human dentin.

Material and methods

Dentin disks ( n = 45) were obtained from the mid-coronal portion of human third molars. One-third of dentin disks were kept mineralized (MD), while the other two-thirds had one of the surfaces partially demineralized with 37% phosphoric acid for 15 s (PDD) or they were totally demineralized with 10% phosphoric acid (TDD). Disks of hydroxyapatite (HA) were also prepared. Specimens were treated with: (1) 10 μL of distilled water (controls), (2) 10 μL of 0.2% chlorhexidine diacetate (0.2% CHX) or (3) 10 μL of 2% chlorhexidine diacetate (2% CHX). Then, they were incubated in 1 mL of PBS (pH 7.4, 37 °C). Substantivity was evaluated as a function of the CHX-applied dose after: 0.5 h, 1 h, 3 h, 6 h, 24 h, 168 h (1 week), 672 h (4 weeks) and 1344 h (8 weeks) of incubation. CHX concentration in eluates was spectrophotometrically analyzed at 260 nm.

Results

Significant amounts of CHX remained retained in dentin substrates (MD, PPD or TDD), independent on the CHX-applied dose or time of incubation ( p < 0.05). High amounts of retained CHX onto HA were observed only for specimens treated with the highest concentration of CHX (2%) ( p < 0.05).

Conclusion

The outstanding substantivity of CHX to dentin and its reported effect on the inhibition of dentinal proteases may explain why CHX can prolong the durability of resin–dentin bonds.

Introduction

Chlorhexidine (1,1′-hexamethylene-bis-5(4-chlorophenyl)bisguanide CHX, Fig. 1 ) is a symmetric molecule with two ionizable guanidine moieties. Its pKa values are 2.2 and 10.3, thereby making it cationic over the entire range of physiological pH values . CHX has been shown to be effective against various oral bacteria. Due to its broad antimicrobial spectrum (i.e. against Gram positive/negative bacteria and fungi), CHX has been used to adjunctively treat either endodontic or periodontal diseases and to arrest/prevent caries progression .

Fig. 1
Chemical structure of chlorhexidine.

The recent finding that CHX also has potent anti-MMP-2, -8 and -9 activity encouraged some researchers to determine whether CHX could stabilize the organic matrix of resin–dentin bonds. This led to numerous in vitro and in vivo studies that demonstrated that CHX has beneficial effects on the preservation of resin–dentin bonds, thereby offering a valuable alternative to clinicians who seek to delay the degradation process of adhesive restorations.

The effectiveness of CHX, as an antimicrobial or an antiproteolytic agent, has been reported to be related with its substantivity to oral/dental structures . Substantivity is the prolonged association between a material (e.g. CHX) and a substrate (e.g. oral mucosa, oral proteins, dental plaque, dental surface), an association that can be greater and more extended than would be expected from a simple deposition mechanism. It is considered that the delivery of an agent to its site of action, in a biologically active form, and in effective doses, increases this agent effects for prolonged periods of time .

Substantivity of CHX, or its ability to be retained in dentin matrices, could be the reason why CHX-treated acid-etched dentin may form hybrid layers that are more stable over time . The success of CHX in increasing the durability of resin–dentin bonds requires that more efforts be made toward understanding the mechanisms responsible for CHX binding to mineralized and demineralized dentin, in an attempt to optimize how CHX should be used clinically to maximize its retention and effectiveness.

Studies on substantivity of CHX to oral structures began in the early seventies . Most of these studies investigated the retention of CHX in oral surfaces in order to determine its capacity to inhibit or decrease the bacterial growth/activity. Accordingly, as long as the number and growth of bacteria were controlled, CHX would be considered retained and effective. However, prolonged inhibition of bacteria growth/activity is merely an indirect way to estimate the substantivity of CHX to oral structures, and it may not be appropriate to explain its long-term antiproteolytic function.

The purpose of this study was to investigate the in vitro substantivity of CHX to human mineralized vs demineralized dentin. The tested null hypotheses were that: (1) CHX substantivity to mineralized and demineralized dentin is not different; (2) CHX substantivity to dentin substrates is independent on the concentration of applied CHX.

Material and methods

Chlorhexidine diacetate monohydrate salt (CHX) was purchased from Fluka (St Louis, MO, USA). Reagent grade hydroxyapatite and other chemicals were purchased from Sigma–Aldrich (St Louis, MO, USA) if not otherwise specified.

Specimen preparation

Forty-five extracted non-carious human third molars were collected from young patients (20–28 years old) after their informed consent had been obtained under a protocol reviewed and approved by the Human Assurance Committee of the University of São Paulo, São Paulo, Brazil. Teeth were stored in 0.9% NaCl containing 0.02% sodium azide at 4 °C for less than 1 month. After organic debris/calculus removal, teeth were sectioned at the cementum–enamel junction to remove the roots. The pulp tissue was scraped off with sterile spoon excavators. Enamel and cementum were completely removed from the crown segment with high-speed diamond burs under copious water cooling. Dentin disks (0.5 ± 0.02 mm thick and 6.0 ± 0.01 mm diameter) were obtained from the mid-coronal portion of each tooth using a slow speed diamond saw (Labcut 1010, Extec Corp., Enfield, CT, USA) under water cooling followed by the use of a diamond-incrusted coring drill with a 6 mm internal diameter (Continental Diamond Tool Corp., New Haven, IN, USA). Dentin disks were ultra-sonicated in distilled-water bath for 15 min. Then, they were pre-dried in a sealed desiccator containing fresh silica gel at 37 °C, until a constant dry mass was obtained in analytical balance (Mettler Toledo Inc., Columbus, OH, USA) (i.e. 0.057 ± 0.009 g). Afterwards, they were individually immersed in distilled water at 37 °C for 1 week, gently wiped with absorbent paper and weighed in an analytical balance for determination of their wet mass (i.e. 0.062 ± 0.01 g). The inner, exposed surface of dentin disks (i.e. those that previously were in close contact with the pulp chamber) was completely covered with two layers of a nail varnish (Risqué, Niasi S/A, São Paulo, SP, Brazil), left to dry for 30 min (at 25 °C, 56% relative humidity) and had their dry mass re-measured (i.e. 0.063 ± 0.008 g).

Specimens were assigned for one of the following groups ( n = 15): (1) dentin disks were kept mineralized (MD); (2) the outer, uncovered dentin surface of dentin disks was partially demineralized with liquid 37% phosphoric acid for 15 s and thoroughly rinsed with distilled water for 60 s (PDD); (3) dentin disks were totally demineralized with liquid 10% phosphoric acid for 12 h and washed for 24 h (TDD). Although a pilot study showed that the selected nail varnish is apparently acid-resistant (data not shown), care was taken to avoid direct contact between the varnish-covered dentin surface and the phosphoric acid solution. Disks of synthetic hydroxyapatite were also prepared . One of the surfaces of these disks was completely covered with two layers of the same nail varnish while the other was kept exposed, as previously described for the dentin disks. Additionally, films of nail varnish (0.5 ± 0.01 mm thick and 6.0 ± 0.01 mm diameter) were also prepared.

Treatment of specimen surface

The exposed, dried, uncovered surface of each substrate (i.e. MD, PDD, TDD and HA) was treated with: (1) 10 μL of distilled water (controls); (2) 10 μL of 0.2% CHX (3.2 mM) or (3) 10 μL of 2% CHX (32.0 mM). The test solutions were applied uniformly on the specimens’ surface using a micro-syringe and they were left undisturbed for 30 s. No excess solution was removed from the specimens’ surface. With a pair of small forceps, the specimens were carefully transferred to 2-mL plastic centrifuge tubes containing 1 mL of PBS (pH 6.8) and incubated at 37 °C. Film-like specimens of nail varnish were also individually incubated under same conditions.

Analysis of eluates

The 1 mL PBS contents of each tube containing any eluted CHX were spectrophotometrically analyzed in quartz cuvettes at 260 nm after the following periods of incubation: 0.5 h, 1 h, 3, 6, 24, 168, 672 and 1344 h. The eluates were never discarded so that after measuring their absorbance values, at each determined period, the eluates were returned to the same plastic centrifuge tubes for additional incubation of specimens. The cumulative concentration of CHX in the eluates was estimated as a function of their UV-absorbances plotted against a CHX standard curve. Substantivity of CHX to tested substrates was expressed (in percentage) based on the CHX-applied dose. In theory, if CHX was not able to bind to the tested substrates, one should expect to detect the entire amount of applied CHX (10 μL of 0.2% or 2%) to be released into 1000 μL of PBS. This theoretical amount is 32 nmol (in 1000 μL) for specimens treated with 0.2% CHX and 320 nmol (in 1000 μL) for specimens treated with 2% CHX.

Data were analyzed by a three-way repeated measures ANOVA with substrate (MD, PDD, TDD and HA), specimen treatment mode (without, with 0.2% CHX or with 2% CHX) and time of incubation (0.5–1344 h) as main factors, and with the eluate as factor of repetition. Post hoc multiple comparisons were performed using Holm-Sidak test. Statistical significance was preset at α = 0.05.

Material and methods

Chlorhexidine diacetate monohydrate salt (CHX) was purchased from Fluka (St Louis, MO, USA). Reagent grade hydroxyapatite and other chemicals were purchased from Sigma–Aldrich (St Louis, MO, USA) if not otherwise specified.

Specimen preparation

Forty-five extracted non-carious human third molars were collected from young patients (20–28 years old) after their informed consent had been obtained under a protocol reviewed and approved by the Human Assurance Committee of the University of São Paulo, São Paulo, Brazil. Teeth were stored in 0.9% NaCl containing 0.02% sodium azide at 4 °C for less than 1 month. After organic debris/calculus removal, teeth were sectioned at the cementum–enamel junction to remove the roots. The pulp tissue was scraped off with sterile spoon excavators. Enamel and cementum were completely removed from the crown segment with high-speed diamond burs under copious water cooling. Dentin disks (0.5 ± 0.02 mm thick and 6.0 ± 0.01 mm diameter) were obtained from the mid-coronal portion of each tooth using a slow speed diamond saw (Labcut 1010, Extec Corp., Enfield, CT, USA) under water cooling followed by the use of a diamond-incrusted coring drill with a 6 mm internal diameter (Continental Diamond Tool Corp., New Haven, IN, USA). Dentin disks were ultra-sonicated in distilled-water bath for 15 min. Then, they were pre-dried in a sealed desiccator containing fresh silica gel at 37 °C, until a constant dry mass was obtained in analytical balance (Mettler Toledo Inc., Columbus, OH, USA) (i.e. 0.057 ± 0.009 g). Afterwards, they were individually immersed in distilled water at 37 °C for 1 week, gently wiped with absorbent paper and weighed in an analytical balance for determination of their wet mass (i.e. 0.062 ± 0.01 g). The inner, exposed surface of dentin disks (i.e. those that previously were in close contact with the pulp chamber) was completely covered with two layers of a nail varnish (Risqué, Niasi S/A, São Paulo, SP, Brazil), left to dry for 30 min (at 25 °C, 56% relative humidity) and had their dry mass re-measured (i.e. 0.063 ± 0.008 g).

Specimens were assigned for one of the following groups ( n = 15): (1) dentin disks were kept mineralized (MD); (2) the outer, uncovered dentin surface of dentin disks was partially demineralized with liquid 37% phosphoric acid for 15 s and thoroughly rinsed with distilled water for 60 s (PDD); (3) dentin disks were totally demineralized with liquid 10% phosphoric acid for 12 h and washed for 24 h (TDD). Although a pilot study showed that the selected nail varnish is apparently acid-resistant (data not shown), care was taken to avoid direct contact between the varnish-covered dentin surface and the phosphoric acid solution. Disks of synthetic hydroxyapatite were also prepared . One of the surfaces of these disks was completely covered with two layers of the same nail varnish while the other was kept exposed, as previously described for the dentin disks. Additionally, films of nail varnish (0.5 ± 0.01 mm thick and 6.0 ± 0.01 mm diameter) were also prepared.

Treatment of specimen surface

The exposed, dried, uncovered surface of each substrate (i.e. MD, PDD, TDD and HA) was treated with: (1) 10 μL of distilled water (controls); (2) 10 μL of 0.2% CHX (3.2 mM) or (3) 10 μL of 2% CHX (32.0 mM). The test solutions were applied uniformly on the specimens’ surface using a micro-syringe and they were left undisturbed for 30 s. No excess solution was removed from the specimens’ surface. With a pair of small forceps, the specimens were carefully transferred to 2-mL plastic centrifuge tubes containing 1 mL of PBS (pH 6.8) and incubated at 37 °C. Film-like specimens of nail varnish were also individually incubated under same conditions.

Analysis of eluates

The 1 mL PBS contents of each tube containing any eluted CHX were spectrophotometrically analyzed in quartz cuvettes at 260 nm after the following periods of incubation: 0.5 h, 1 h, 3, 6, 24, 168, 672 and 1344 h. The eluates were never discarded so that after measuring their absorbance values, at each determined period, the eluates were returned to the same plastic centrifuge tubes for additional incubation of specimens. The cumulative concentration of CHX in the eluates was estimated as a function of their UV-absorbances plotted against a CHX standard curve. Substantivity of CHX to tested substrates was expressed (in percentage) based on the CHX-applied dose. In theory, if CHX was not able to bind to the tested substrates, one should expect to detect the entire amount of applied CHX (10 μL of 0.2% or 2%) to be released into 1000 μL of PBS. This theoretical amount is 32 nmol (in 1000 μL) for specimens treated with 0.2% CHX and 320 nmol (in 1000 μL) for specimens treated with 2% CHX.

Data were analyzed by a three-way repeated measures ANOVA with substrate (MD, PDD, TDD and HA), specimen treatment mode (without, with 0.2% CHX or with 2% CHX) and time of incubation (0.5–1344 h) as main factors, and with the eluate as factor of repetition. Post hoc multiple comparisons were performed using Holm-Sidak test. Statistical significance was preset at α = 0.05.

Results

UV-absorbances (A) for control specimens (i.e. not treated with CHX) were low (0.2 ± 0.05 A) and significantly different from the CHX-treated specimens, regardless of the tested substrate, CHX concentration or time of incubation ( p < 0.05) (data not shown). Such low values were coincident with the mean UV-absorbance (0.2 ± 0.1 A) of film-like specimens of pure varnish (data not shown), indicating that the low positive UV-absorbances of control groups were likely due the presence of leaching species from the varnish-covered surface. To confirm it, specimens of each substrate were prepared and stored in PBS without being covered with varnish and treated with CHX. As expected, these specimens exhibited null or negative UV-absorbance (data not shown). Thus, since all experimental groups had their own control (i.e. specimens that were covered with nail varnish and were not treated with CHX), the mean UV-absorbance of each control group was subtracted from the mean UV-absorbance of the corresponding CHX-treated group.

Substantivity of CHX for all treated substrates expressed as a percentage the CHX-applied dose is summarized in Table 1 , while Fig. 2 shows graphically the percent of bound CHX, separately, to specimens treated with 0.2% ( Fig. 2 A) and 2.0% CHX ( Fig. 2 B).

Table 1
Percentage of CHX remaining bound (i.e. substantivity) over time as a function of the CHX-applied dose.
0.5 h 1 h 3 h 6 h 24 h 168 h (1 week) 672 h (4 weeks) 1344 h (8 weeks)
Substantivity of chlorhexidine
MD 0.2% CHX 86.3 ± 5.1 a,A 87.3 ± 5.0 a,A 85.6 ± 4.0 a,AB 85.6 ± 3.7 a,A 88.7 ± 4.1 a,AB 86.3 ± 3.2 a,A 86.2 ± 3.9 a,A 86.9 ± 3.2 a,A
MD 2.0% CHX 68.9 ± 3.8 a,B 68.8 ± 5.0 a,B 68.8 ± 3.5 a,B 68.7 ± 4.6 a,B 68.6 ± 3.2 a,C 68.2 ± 4.0 a,C 68.3 ± 3.1 a,D 68.2 ± 2.8 a,C
HA 0.2% CHX 7.9 ± 1.0 a,D 7.5 ± 1.8 a,D 7.4 ± 2.2 a,E 6.8 ± 1.5 a,D 9.9 ± 3.1 a,E 6.6 ± 1.2 ab,E 3.7 ± 0.9 b,F 3.1 ± 1.2 b,E
HA 2.0% CHX 48.3 ± 2.1 a,C 49.1 ± 1.8 a,C 48.0 ± 3.3 a,D 48.3 ± 1.2 a,C 49.5 ± 1.5 a,D 49.4 ± 1.9 a,D 50.5 ± 3.0 a,E 49.0 ± 2.7 a,D
PDD 0.2% CHX 92.6 ± 3.3 ab,A 91.3 ± 3.1 b,A 90.8 ± 3.6 b,A 90.9 ± 2.5 b,A 90.9 ± 1.5 b,A 97.1 ± 2.0 a,A 97.5 ± 1.6 a,A 97.9 ± 3.1 a,A
PDD 2.0% CHX 45.7 ± 2.7 b,C 44.6 ± 2.0 b,C 46.3 ± 1.5 b,D 48.9 ± 2.6 ab,C 47.6 ± 3.0 b,D 49.9 ± 2.4 ab,D 55.2 ± 2.8 a,E 54.1 ± 2.9 a,D
TDD 0.2% CHX 74.4 ± 3.3 a,B 75.9 ± 2.1 a,B 72.3 ± 1.9 a,C 71.2 ± 2.8 a,B 75.1 ± 3.0 a,C 75.0 ± 2.5 a,C 76.1 ± 3.0 a,C 74.2 ± 2.7 a,C
TDD 2.0% CHX 85.8 ± 4.5 a,A 85.4 ± 3.5 a,A 83.0 ± 2.0 a,B 84.2 ± 3.1 a,A 84.8 ± 1.6 a,B 83.6 ± 4.1 a,B 83.6 ± 2.3 a,B 83.0 ± 4.7 a,B
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Nov 30, 2017 | Posted by in Dental Materials | Comments Off on Substantivity of chlorhexidine to human dentin
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