The influence of chlorhexidine on the remineralization of demineralized dentine

The influence of chlorhexidine on the remineralization of demineralized dentine

Journal of Dentistry, 2011-12-01, Volume 39, Issue 12, Pages 855-862, Copyright © 2011 Elsevier Ltd

Abstract

Objectives

To examine the differences in the amounts of bound chlorhexidine (CHX) on demineralized dentine blocks and to investigate the different aspects of remineralization of demineralized dentine according to different concentrations of CHX.

Methods

Dentine blocks (2 mm × 7 mm × 0.9 mm) were demineralized in 0.2 M formic acid solution. Amount of bound CHX on the dentine blocks was measured on a spectrophotometer after the dentine block was soaked in 0.02%, 0.2%, or 2% CHX solutions for 1 min. The change in elastic modulus of dentine block stored in simulated body fluids was measured at 0 (baseline), 2, 4, and 6 weeks after storage. The micromorphological aspects of the samples were observed using a field emission scanning electron microscope after 6 weeks of storage.

Results

Higher concentrations of CHX caused a greater amount of CHX to bind to the dentine blocks ( p < 0.05). The group treated with the higher concentration of CHX had a smaller decrease in the elastic modulus at 2 weeks and a greater increase at 4 and 6 weeks. Dentine specimens with the 0.2% and 2% CHX had a greater deposition of granular minerals along the collagen fibrils compared to the 0.02% CHX-treated group.

Conclusion

The application of the 0.2% and 2% CHX seemed to be effective in promoting the remineralization of demineralized dentine.

Clinical significance

The application of the 0.2% and 2% CHX positively influences on the dentine remineralization

Introduction

Chlorhexidine (CHX) was introduced as an antiseptic for skin wounds in the 1940s and has been widely used in dentistry due to its broad spectrum antibacterial effects and substantivity. CHX is used as the most effective antiplaque and antigingivitis agent in periodontal treatment and is used as an effective antiseptic root canal irrigant in endodontic treatment. CHX varnish has also been reported to have an effect on the arrestment of dental caries when applied on carious dentine. It has recently been reported that CHX not only inhibits the activity of bacterial proteolytic enzymes but also effectively reduces collagen degradation by inhibiting host-derived proteases in demineralized dentine.

Many studies have reported that application of CHX for a certain amount of time in a prepared tooth cavity during the bonding procedure improved the stability of the hybrid layer in the resin–dentine bond and inhibited the reduction in bond strength due to ageing. This improvement in the durability of the resin–dentine bond is thought to happen because CHX inhibits the matrix metalloproteinase (MMP) activity, which is responsible for the self-degradation of incomplete resin-infiltrated collagen at the bottom of the hybrid layer. Various concentrations of CHX from low concentrations of 0.01–0.02% to relatively high concentration of 2% have been used in the durability studies of the resin–dentine bond and it was reported that the MMP inhibition effect was observed at all these concentrations. However, it was recently reported that CHX was not able to prevent collagen degradation completely but to only delay collagen degradation for some time because of limited substantivity. Conceptually, if the amount of bound CHX on the demineralized dentine was high, it might possibly better inhibit MMP more effectively or for a longer time. Except for one study which measured the binding amount of CHX using ground dentine powder, until now, there have been no reports on the differences in the amounts of bound CHX on demineralized dentine blocks that are more similar to a clinical environment compared to ground dentine powder according to concentrations of 0.02–0.2%, which have been mostly used in resin–dentine bond durability studies. Moreover, it is not clear if the exposed collagen fibril layer at the bottom of the hybrid layer keeps its original structure without changing, or is weakened or strengthened by any change when the endogenous MMP of the demineralized dentine is inhibited by CHX in a clinical environment.

Many studies on tooth remineralization have been done targeting mainly the enamel remineralization of incipient caries. The sites, which need a remineralization strategy in dentine, could be a root caries that occurs mainly in older patients with gingival recession and saliva flow reduction, a cervical area in which dentine is exposed by chemical erosion, each cavity that has remaining affected dentine at its walls/floor, and an exposed collagen fibril layer incompletely infiltrated by the adhesive resin after acid etching in the resin–dentine bond interface. Those lesions have similar characteristics because they all originated from demineralization and thus ideal restoration procedures should remineralize the affected areas. Theoretically, it would be possible to remineralize those lesions through a supply of calcium and phosphate ions from the saliva or dentinal tubular fluid if the collagen fibril scaffold maintains its sound structure. It is well known that the effect of MMP inhibition increases as the concentration of CHX becomes higher ; however, little is known how different concentrations of CHX influence the remineralization of collagen scaffolds in demineralized dentine when there are constant mineral elements.

The aims of this study were to examine the differences in the amounts of bound CHX on demineralized dentine blocks when different concentrations of CHX were used to treat the blocks and to investigate the different aspects of remineralization of demineralized dentine according to different concentrations of CHX when constant mineral elements were supplied.

The remineralization aspects of demineralized dentine were examined by the change in elastic modulus and by the micromorphological appearance of the samples using a field emission scanning electron microscope (FE-SEM).

Methods and materials

Specimen preparation

Forty extracted caries-free human third molars were collected and stored in 1% chloramine T solution with the patients’ informed consent. The study protocol was approved by the Institutional Review Board (IRB) committee of Kyung Hee University Medical Centre (KHB IRB 003-2). The enamel portion of each tooth was removed using a high-speed diamond bur with water-cooling and the mid-coronal dentine was exposed. A dentine block (2 mm × 7 mm × 0.9 mm) was obtained from each tooth using a slow-speed diamond saw (Isomet, Buehler Ltd., Lake Bluff, IL, USA) with water-cooling. Dentine blocks were demineralized in 0.2 M formic acid solution for 10 days. After demineralization, they were thoroughly washed with distilled water and dried with anhydrous calcium sulphate for 3 days. Then, the mass of each dried specimen was measured and rehydrated with distilled water.

Measuring the amount of bound CHX

The amount of bound CHX was measured using a modified protocol based on Kim et al.’s method. After the full absorbance spectrum (200–800 nm) of CHX was recorded using a spectrophotometer (Synergy HT, Bio Tek instruments, USA), the 225 nm wave length was selected as the specific peak for measuring the amount of bound CHX. A standard curve for CHX absorbance was established by measuring the absorbance peak at 225 nm for 11 CHX concentrations (ranging from 0.012% to 2%).

After each dentine block was inserted into a centrifuge tube, 1 mL of 0.02%, 0.2%, or 2% CHX was applied to the inside of the tube. Dentine block was completely soaked with CHX solution in the tube. The tube was then capped and tumbled for 1 min at 37 °C. The supernatant was placed in a 96-well plate and the absorbance at 225 nm was measured using a spectrophotometer. From the standard CHX curve, the amount of bound CHX was calculated using the difference in absorbance before and after the dentine block was soaked in the CHX solution. The reduced amount of CHX corresponds to the amount of bound CHX on the dentine block. This was expressed as mg CHX/g demineralized dentine block. This procedure was done for 10 dentine block specimens for each concentration of CHX except for the control group without any CHX treatment ( n = 10).

Measuring the elastic modulus

A simulated body fluid (SBF) was prepared by dissolving 136.8 mM NaCl, 4.2 mM NaHCO 3 , 3.0 mM KCl, 1.0 mM K 2 HPO 4 ⋅3H 2 O, 1.5 mM MgCl 2 ⋅6H 2 O, 2.5 mM CaCl 2 , and 0.5 mM Na 2 SO 4 in deionized water. The SBF was buffered to pH 7.4 with 0.1 M Tris base and 0.1 M HCl. All dentine block specimens were stored in the SBF for 6 weeks. A three-point bending test for each specimen was done with a universal testing machine (Vitrodyne V1000, John Chatillong & Sons, Greensboro, NC, USA) at 0 (baseline), 2, 4, and 6 weeks after storage. Each specimen was tested in a wet state immediately after being removed from the SBF. The measurement was done with a 2.5 mm support span and a 0.5 mm/min crosshead speed. The elastic modulus of each dentine block was calculated using the following formula:

E=mL34bh3 E = m L 3 4 b h 3
where E is the elastic modulus (MPa), m is the steepest slope (N/mm), L is the support span length (2.5 mm), b is the width of the dentine block (mm), and h is the thickness (mm). The elastic modulus at baseline was measured immediately after the dried demineralized specimen was rehydrated. The SBF was changed every two weeks.

FE-SEM evaluation

Dentine blocks were selected from each group after storage for 6 weeks and irrigated thoroughly with distilled water. The specimens were treated according to the protocol of Perdigao et al. to avoid any artefacts from dehydration shrinkage. They were fixed with 2.5% glutaraldehyde (0.1 M sodium cacodylate buffer, pH 7.2) for 4 h and irrigated thoroughly with distilled water. They were dehydrated in ascending order with 50%, 60%, 70%, 80%, 90%, and 100% ethanol and dried with hexamethyldisilazane (Sigma–Aldrich, St. Louis, USA). The specimens were Pt-sputter-coated and fixed in an aluminium stub. Micromorphological structures of the specimens were evaluated using FE-SEM (Leo Supra 55, Carl Zeiss, Germany).

Statistical analysis

The differences in the amounts of bound CHX according to the concentrations of CHX were analysed by one-way ANOVA. The differences in elastic modulus according to the CHX concentrations and the various lengths of the storage periods were analysed by repeated measure ANOVA. Tukey’s test was done as a post hoc analysis to compare the effects of the different CHX concentrations. Paired t -tests were conducted to compare the elastic modulus for the various lengths of the storage periods from the baseline within the group. The level of significance was set at α = 0.05. SPSS version 18.0 (SPSS Inc., Chicago, IL, USA) was used for all statistical analyses.

Results

The amounts of bound CHX according to the CHX concentrations treated in the dentine block are shown in Fig. 1 . There was a statistically significant difference in the amounts of bound CHX according to the concentrations of CHX ( p < 0.05). Higher concentrations of CHX caused a greater amount of CHX to bind to the dentine blocks. However, the difference in the amounts of bound CHX between the 0.02% and 0.2% groups was much greater than that between the 0.2% and 2% groups.

Amounts of bound CHX according to the CHX concentrations ( n = 10). The difference in the amounts of bound CHX between the 0.02% and 0.2% groups is much greater than that between the 0.2% and 2% groups.
Fig. 1
Amounts of bound CHX according to the CHX concentrations ( n = 10). The difference in the amounts of bound CHX between the 0.02% and 0.2% groups is much greater than that between the 0.2% and 2% groups.

The results of the elastic modulus test according to the different CHX concentrations and the various lengths of storage periods are presented in Table 1 . There was a significant difference between the groups of different CHX concentrations in the change in elastic modulus according to the various lengths of storage periods ( p < 0.05). However, there was no statistically significant difference between the control group and the 0.02% CHX-treated group and between the 0.2% and 2% CHX-treated groups ( p > 0.05). The elastic modulus of the control group with no CHX treatment decreased significantly after 2 weeks ( p < 0.05). In the 0.02% CHX-treated group, the elastic modulus after 2 weeks of storage was significantly lower than that of the baseline ( p < 0.05), but the elastic modulus for 4 and 6 weeks of storage did not have any significant differences from that of the baseline ( p > 0.05). In the 0.2% CHX-treated group, the value of the elastic modulus was significantly lower at 2 weeks, similar at 4 weeks and higher at 6 weeks compared to the baseline. In the 2% CHX-treated group, the value of the elastic modulus showed no statistically significant changes at 2 weeks but significantly increased at 4 weeks and 6 weeks from the baseline ( p < 0.05).

Table 1
Elastic modulus of the dentine blocks according to each CHX concentration and length of storage period in SBF (unit: MPa, n = 10).
Group Baseline 2 weeks 4 weeks 6 weeks
Control A 3.08 ± 0.44 b 2.18 ± 0.24 a 2.14 ± 0.34 a 2.07 ± 0.42 a
0.02% A 3.11 ± 0.59 b 2.17 ± 0.60 a 2.73 ± 0.64 b 2.85 ± 0.82 b
0.2% B 3.11 ± 0.34 b 2.68 ± 0.34 a 3.36 ± 0.38 b 3.66 ± 0.42 c
2% B 3.14 ± 0.40 a 2.94 ± 0.23 a 3.52 ± 0.41 b 3.94 ± 0.44 c
Groups with the same superscript show no statistically significant differences.
Same superscript amongst the time points within the group indicates no statistically significant differences.

In the control group with no CHX treatment, the exposed reticular nanostructure of the collagen fibrils showing the effect of demineralization was clearly observed. The diameter of the collagen fibrils was thin and the interfibrillar spaces were relatively wide and clear ( Fig. 2 ). In the 0.02% CHX-treated group, with a small quantity of particles deposited around the demineralized collagen fibrils, the diameter of the collagen fibrils appeared to become slightly thicker compared to the control group ( Fig. 3 ). In the 0.2% CHX-treated group, many clustered granular structures of spherical grains were observed to form around the collagen fibrils and, as a consequence, there was only a little remaining amount of interfibrillar space ( Fig. 4 ). The apparent deposition of irregular nanoparticles along the collagen fibrils was also found in the intratubular area. The 2% CHX-treated group showed a denser granular deposition of nanoparticles on the surface of specimen. Since there was not much interfibrillar space, it was difficult to find a trace of the collagen fibrils on the intertubular dentine area ( Fig. 5 ).

FE-SEM images of non-CHX-treated dentine: (a) 30,000× magnification view; (b) 100,000× magnification view. The exposed reticular nanostructures of the collagen fibrils are clearly observed showing the effect of demineralization. The diameter of the collagen fibrils is thin and the interfibrillar distance is about 50–100 nm.
Fig. 2
FE-SEM images of non-CHX-treated dentine: (a) 30,000× magnification view; (b) 100,000× magnification view. The exposed reticular nanostructures of the collagen fibrils are clearly observed showing the effect of demineralization. The diameter of the collagen fibrils is thin and the interfibrillar distance is about 50–100 nm.
FE-SEM images of the 0.02% CHX-treated dentine: (a) 30,000× magnification view; (b) 100,000× magnification view. The interfibrillar distance decreases and the diameter of the collagen fibrils become thicker compared to the non-CHX-treated group as a small quantity of particles is deposited around the demineralized collagen fibrils.
Fig. 3
FE-SEM images of the 0.02% CHX-treated dentine: (a) 30,000× magnification view; (b) 100,000× magnification view. The interfibrillar distance decreases and the diameter of the collagen fibrils become thicker compared to the non-CHX-treated group as a small quantity of particles is deposited around the demineralized collagen fibrils.
FE-SEM images of the 0.2% CHX-treated dentine: (a) 30,000× magnification view; (b) 100,000× magnification view. Many clustered granular structures of spherical grains around the collagen fibrils are formed so that there is little remaining interfibrillar space on the surface. The apparent deposition of the irregular nanoparticles along the collagen fibrils is also found in the intratubular area.
Fig. 4
FE-SEM images of the 0.2% CHX-treated dentine: (a) 30,000× magnification view; (b) 100,000× magnification view. Many clustered granular structures of spherical grains around the collagen fibrils are formed so that there is little remaining interfibrillar space on the surface. The apparent deposition of the irregular nanoparticles along the collagen fibrils is also found in the intratubular area.
FE-SEM images of the 2% CHX-treated dentine: (a) 30,000× magnification view; (b) 100,000× magnification view. Dense granular deposition of nanoparticles is shown on the surface. Since there is little interfibrillar space, it is difficult to find a trace of collagen fibrils on the intertubular dentine area.
Fig. 5
FE-SEM images of the 2% CHX-treated dentine: (a) 30,000× magnification view; (b) 100,000× magnification view. Dense granular deposition of nanoparticles is shown on the surface. Since there is little interfibrillar space, it is difficult to find a trace of collagen fibrils on the intertubular dentine area.

Discussion

A number of studies have shown in a direct way that the activity of endogenous MMP in dentine was inhibited according to the application of certain concentrations of CHX or in an indirect way such that the resin–dentine bond strength did not drop after ageing according to the application of certain concentrations of CHX. However, it is difficult to find a report on the direct effects of CHX on the remineralization of demineralized dentine. To our knowledge, the present study is the first that shows in an integrated way through mechanical and micromorphological changes in the aspects of remineralization in demineralized dentine according to concentrations of CHX. Moreover, this study presents a guide for the appropriate concentration of CHX that exerts the best effect through the results of the differences in the amounts of bound CHX on demineralized dentine blocks according to 0.02–2% concentrations of CHX, which have been mostly used in past studies on durable resin–dentine bonds. Based on the results of the present study, the application of 0.2% and 2% CHX for 1 min seemed to be more effective in promoting the remineralization of the demineralized dentine than that of 0.02% CHX.

Dentine demineralization could arise from an acid attack by bacteria and/or a food source, and from acid etching for resin–dentine bonding. Several conditions are required to remineralize the demineralized dentine. First, the collagen structure should be sound as a scaffold for the mineral crystals to grow. Second, there should be residual mineral crystals as a growth centre, or there should be at least a newly formed nucleation site in case of complete demineralization. Last, mineral sources containing calcium and phosphorous should be supplied to the lesion. Many studies have tried to remineralize an initial caries and root caries by application of various agents such as calcium phosphate, bioglass, and fluoride. Recent several studies have also tried a new remineralization strategy creating a mineral nucleus through the application of a biomimetic analogue on completely demineralized dentine. In the present study, we showed that the application of CHX, which is an effective MMP inhibitor, promoted demineralized dentine to remineralize. CHX could keep the collagen cross-linkage sound by inhibiting the MMP activity. Remineralization would then happen around the remaining mineral crystals by obtaining mineral sources from the SBF. A previous study reported that an incorporated MMP inhibited the remineralization process of demineralized dentine and remineralization proceeded when the MMP was inhibited by a tissue inhibitor of the metalloproteinase. Mukai and ten Cate showed that remineralization could also occur in demineralized root dentine simulating advanced root caries under in vitro conditions. Therefore, it is not surprising that remineralization happened under SBF, which contains calcium and phosphorous, as long as a sound collagen scaffold remains in the demineralized dentine. Nevertheless, the results of the present study show that the level of remineralization was different according to the different concentrations of CHX and could be considered as an important step in establishing a relationship between the amount of bound CHX on dentine, the level of MMP inhibition, and the level of remineralization.

Remineralization of dentine could occur by simple mineral precipitation between the collagen fibrils, which would be mainly extrafibrillar mineral formation. This type of remineralization could show an increase in mineral content but not have a tight association with the collagen matrix. Another type of remineralization is that the collagen fibrils of the dentine could be reinforced by intrafibrillar mineral formation so that the remineralized dentine would show an increase in mechanical properties, the so-called functional remineralization. In the present study, the elastic modulus of the remineralized dentine was shown to increase after storage in SBF for several weeks prominently in the cases of 0.2% and 2% CHX ( Table 1 ). Therefore, the remineralization in the present study might have occurred in an intrafibrillar manner. As a possible explanation for the functional remineralization, dentine specimens of the present study might be incompletely demineralized despite 10 days of storage in 0.2 M formic acid solution, which would leave nucleating mineral crystals in the collagen fibrils. Partially demineralized dentine was considered to have a capacity to restore the original mineralized state because it contains remnant mineral crystals and non-collagenous phosphoproteins, which act as a nucleation site of remineralization. That the aspect of mineral clogging in the dentinal tubule was not shown in the FE-SEM images might also suggest that remineralization of the dentine blocks in the present study was not a simple precipitation of minerals through the supply of calcium and phosphate irrespective of the sites but rather is closer to the optimal incorporation of minerals into the collagen fibrils. Despite that, further study needs to clarify the characteristics of the minerals formed through the remineralization process of this study. In order to verify functional remineralization through the application of CHX on demineralized dentine, the crystal structure of the calcium phosphate minerals formed in this study need to be examined using X-ray diffraction or infrared spectroscopy. Evaluation using transmission electron microscopy would also better help to understand the structural evidence of the remineralization in the collagen fibril scaffold rather than SEM, which is a convenient way to examine the surface of the remineralized dentine blocks.

In the present study, 0.2% and 2% concentrations of CHX had a significantly higher elastic modulus after storage in SBF and a higher level of remineralization according to the morphological aspect than that of the relatively low concentration of 0.02% CHX. This might be because higher concentrations of CHX inhibited MMP activity more strongly and subsequently had a more positive influence on mineral formation. The results of the present study might suggest that the capability of keeping collagen fibril structures sound would be dose-dependent on the amount of CHX applied onto the dentine surface. As another possible explanation for causing higher levels of remineralization due to higher concentrations of CHX, the bound CHX itself at greater amounts in the demineralized dentine collagen might attract the mineral phosphate strongly and promote mineral growth by an electrostatic attraction between the protonated amine groups of CHX and the mineral phosphates.

As for the control group without any CHX treatment, only the collagen fibre mesh was observed and the elastic modulus significantly decreased when measured every two weeks, which suggests that the endogenous MMP of the dentine hydrolyzed the collagen fibrils. This hydrolysis might cause the partial collapse of the collagen scaffold. Although the collagen fibril structure is sustained, the hydrolysis of specific amino acids in the collagen fibrils might weaken the structure or inhibit the growth of the minerals despite the supplied mineral elements through the SBF. In the present study, 0.02% CHX group did not show noticeable differences in the change in elastic modulus and morphologic aspect when compared to the control group. This reflects that the application of too low concentrations of CHX does not seem to be effective for inhibiting the MMP activity in the demineralized dentine. The present study is believed to support the previous studies that CHX cannot preserve the resin–dentine bond as long as the concentration of CHX is very low (0.05%).

If the exposed collagen fibril layer of the demineralized dentine through acid etching for the resin–dentine bond had the ideal environmental conditions for remineralization, the nanoleakage at the bottom of the hybrid layer might be a temporary phenomenon, which could be solved by remineralization. An old animal study reporting that demineralized dentine from acid etching underwent remineralization in 4 months when demineralized dentine was restored with an adhesive system is believed to support this possibility. The results of the present study that CHX application could promote the remineralization process could be extended to the possibility that the durability of the resin–dentine bond can be improved in a practical way by remineralization of the exposed collagen fibrils in the hybrid layer and that postoperative hypersensitivity can also be arrested by mineral sealing of the nanoleakage. Nanoleakage at the bottom of the hybrid layer is reported to happen not only in the case of the etch-and-rinse adhesive system but also in the case of the self-etch adhesive system due to the continuous etching effect of the acidic monomers. Considering that self-etch adhesives could leave remnant minerals as nucleating sites for remineralization in the hybrid layer due to a weaker etching effect than the etch-and-rinse adhesives, the self-etch adhesive system appears to have a greater possibility of reducing nanoleakage through remineralization with the adjunctive use of CHX. Although the substantivity of CHX is limited according to the concentrations, if the remineralization occurred before the MMP inhibition effect of CHX ended, the limitation of CHX could be overcome. This perspective requires more research on optimizing the remineralization period of the demineralized dentine in the future.

Conclusion

With 0.02–2% CHX, demineralized dentine blocks absorbed greater amounts of CHX at higher concentrations of CHX. When mineral sources were supplied, the application of 0.2% and 2% CHX for 1 min seemed to be more effective in remineralizing the demineralized dentine than that of the 0.02% CHX based on the results of the elastic modulus and FE-SEM in the present study.

Acknowledgement

This study was supported by a grant from the Kyung Hee University in 2011 (KHU-20110668).

References

  • 1. Lindhe J., Lang N.P., Karring T.: Rosengarten Family Fund. Clinical periodontology and implant dentistry.2008.Blackwell Munksgaard
  • 2. Cosyn J., Wyn I., De Rouck T., Sabzevar M.M.: Long-term clinical effects of a chlorhexidine varnish implemented treatment strategy for chronic periodontitis. Journal of Periodontology 2006; 77: pp. 406-415.
  • 3. Siqueira J.F., Paiva S.S., Rocas I.N.: Reduction in the cultivable bacterial populations in infected root canals by a chlorhexidine-based antimicrobial protocol. Journal of Endodontics 2007; 33: pp. 541-547.
  • 4. Bretz W.A., Djahjah C.A., Almeida R.S., Villar do Valle E., Fonseca C., Valente I., et. al.: Effect of a chlorhexidine varnish on caries lesions. Oral Health & Preventive Dentistry 1995; 85: pp. 29-30.
  • 5. Garcia M.B., Nör J.E., Schneider L.G., Bretz W.A.: A model for clinical evaluation of the effect of antimicrobial agents on carious dentin. American Journal of Dentistry 2001; 14: pp. 119-122.
  • 6. Radford J.R., Homer K.A., Naylor M.N., Beighton D.: Inhibition of human subgingival plaque protease activity by chlorhexidine. Archives of Oral Biology 1992; 37: pp. 245-248.
  • 7. Grenier D.: Reduction of proteolytic degradation by chlorhexidine. Journal of Dental Research 1993; 72: pp. 630-633.
  • 8. Hebling J., Pashley D.H., Tjaderhane L., Tay F.R.: Chlorhexidine arrests subclinical degradation of dentin hybrid layers in vivo. Journal of Dental Research 2005; 84: pp. 741-746.
  • 9. De Munck J., Van den Steen P.E., Mine A., Van Landuyt K.L., Poitevin A., Opdenakker G., et. al.: Inhibition of enzymatic degradation of adhesive–dentin interfaces. Journal of Dental Research 2009; 88: pp. 1101-1106.
  • 10. Brackett W.W., Tay F.R., Brackett M.G., Dib A., Sword R.J., Pashley D.H.: The effect of chlorhexidine on dentin hybrid layers in vivo. Operative Dentistry 2007; 32: pp. 107-111.
  • 11. Carrilho M.R., Geraldeli S., Tay F., de Goes M.F., Carvalho R.M., Tjaderhane L., et. al.: In vivo preservation of the hybrid layer by chlorhexidine. Journal of Dental Research 2007; 86: pp. 529-533.
  • 12. Campos E.A., Correr G.M., Leonardi D.P., Barato-Filho F., Gonzaga C.C., Zielak J.C.: Chlorhexidine diminishes the loss of bond strength over time under simulated pulpal pressure and thermo-mechanical stressing. Journal of Dentistry 2009; 37: pp. 108-114.
  • 13. Toledano M., Nieto-Aguilar R., Osorio R., Campos A., Osorio E., Tay F., et. al.: Differential expression of matrix metalloproteinase-2 in human coronal and radicular sound and carious dentine. Journal of Dentistry 2010; 38: pp. 635-640.
  • 14. Hiraishi N., Yiu C., King N., Tay F.: Effect of 2% chlorhexidine on dentin microtensile bond strengths and nanoleakage of luting cements. Journal of Dentistry 2009; 37: pp. 440-448.
  • 15. Loguercio A.D., Stanislawczuk R., Polli L.G., Costa J.A., Michel M.D., Reis A.: Influence of chlorhexidine digluconate concentration and application time on resin–dentin bond strength durability. European Journal of Oral Sciences 2009; 117: pp. 587-596.
  • 16. Gendron R., Grenier D., Sorsa T., Mayrand D.: Inhibition of the activities of matrix metalloproteinases 2, 8, and 9 by chlorhexidine. Clinical and Diagnostic Laboratory Immunology 1999; 6: pp. 437-439.
  • 17. Tay F.R., Pashley D.H.: Biomimetic remineralization of resin-bonded acid-etched dentin. Journal of Dental Research 2009; 88: pp. 719-724.
  • 18. Osorio R., Yamauti M., Osorio E., Ruiz-Requena M.E., Pashley D., Tay F., et. al.: Effect of dentin etching and chlorhexidine application on metalloproteinase-mediated collagen degradation. European Journal of Oral Sciences 2011; 119: pp. 79-85.
  • 19. Kim J., Uchiyama T., Carrilho M., Agee K.A., Mazzoni A., Breschi L., et. al.: Chlorhexidine binding to mineralized versus demineralized dentin powder. Dental Materials 2010; 26: pp. 771-778.
  • 20. Gonzalez-Cabezas C.: The chemistry of caries: remineralization and demineralization events with direct clinical relevance. Dental Clinics of North America 2010; 54: pp. 469-478.
  • 21. Gu Ls , Huffman B.P., Arola D.D., Kim Y.K., Mai S., Elsalanty M.E., et. al.: Changes in stiffness of resin-infiltrated demineralized dentin after remineralization by a bottom-up biomimetic approach. Acta Biomaterialia 2010; 6: pp. 1453-1461.
  • 22. Blackburn R.S., Harvey A., Kettle L.L., Manian A.P., Payne J.D., Russell S.J.: Sorption of chlorhexidine on cellulose: mechanism of binding and molecular recognition. The Journal of Physical Chemistry B 2007; 111: pp. 8775-8784.
  • 23. Kokubo T., Kushitani H., Sakka S., Kitsugi T., Yamamuro T.: Solutions able to reproduce in vivo surface-structure changes in bioactive glass–ceramic A–W. Journal of Biomedical Materials Research 1990; 24: pp. 721-734.
  • 24. Carrilho M.R., Tay F.R., Donnelly A.M., Agee K.A., Tjäderhane L., Mazzoni A., et. al.: Host-derived loss of dentin matrix stiffness associated with solubilization of collagen. Journal of Biomedical Materials Research – Part B Applied Biomaterials 2009; 90: pp. 373-380.
  • 25. Perdigao J., Lambrechts P., Van Meerbeek B., Vanherle G., Lopes A.L.: Field emission SEM comparison of four postfixation drying techniques for human dentin. Journal of Biomedical Materials Research 1995; 29: pp. 1111-1120.
  • 26. Breschi L., Mazzoni A., Nato F., Carrilho M., Visintini E., Tjaderhane L., et. al.: Chlorhexidine stabilizes the adhesive interface: a 2-year in vitro study. Dental Materials 2010; 26: pp. 320-325.
  • 27. Kuboki Y., Ohgushi K., Fusayama T.: Collagen biochemistry of the two layers of carious dentin. Journal of Dental Research 1977; 56: pp. 1233-1237.
  • 28. Xu Z., Neoh K.G., Kishen A.: A biomimetic strategy to form calcium phosphate crystals on type i collagen substrate. Materials Science and Engineering C 2010; 30: pp. 822-826.
  • 29. Peters M.C., Bresciani E., Barata T.J.E., Fagundes T.C., Navarro R.L., Navarro M.F.L., et. al.: In vivo dentin remineralization by calcium–phosphate cement. Journal of Dental Research 2010; 89: pp. 286-291.
  • 30. Peters M.C.: Strategies for noninvasive demineralized tissue repair. Dental Clinics of North America 2010; 54: pp. 507-525.
  • 31. Ten Cate J.M.: Remineralization of deep enamel dentine caries lesions. Australian Dental Journal 2008; 53: pp. 281-285.
  • 32. Reynolds E.C., Cai F., Cochrane N.J., Shen P., Walker G.D., Morgan M.V., et. al.: Fluoride and casein phosphopeptide-amorphous calcium phosphate. Journal of Dental Research 2008; 87: pp. 344-348.
  • 33. Vollenweider M., Brunner T.J., Knecht S., Grass R.N., Zehnder M., Imfeld T., et. al.: Remineralization of human dentin using ultrafine bioactive glass particles. Acta Biomaterialia 2007; 3: pp. 936-943.
  • 34. Tschoppe P., Zandim D.L., Sampaio J.E.C., Kielbassa A.M.: Saliva substitute in combination with high-concentrated fluoride toothpaste: effects on demineralised dentin in vitro. Journal of Dentistry 2010; 38: pp. 207-213.
  • 35. Kim J., Arola D.D., Gu L., Kim Y.K., Mai S., Liu Y., et. al.: Functional biomimetic analogs help remineralize apatite-depleted demineralized resin-infiltrated dentin via a bottom-up approach. Acta Biomaterialia 2010; 6: pp. 2740-2750.
  • 36. Nordbø H., Leirskar J., Ngo H., Mount G.J., Wahlgren J.: The influence of a matrix metalloproteinase on the remineralization of artificially demineralized dentin. Oral Health & Preventive Dentistry 2003; 1: pp. 267-272.
  • 37. Mukai Y., ten Cate J.M.: Remineralization of advanced root dentin lesions in vitro. Caries Research 2002; 36: pp. 275-280.
  • 38. Bertassoni L.E., Habelitz S., Kinney J.H., Marshall S.J., Marshall G.W.: Biomechanical perspective on the remineralization of dentin. Caries Research 2009; 43: pp. 70-77.
  • 39. Kinney J.H., Habelitz S., Marshall S.J., Marshall G.W.: The importance of intrafibrillar mineralization of collagen on the mechanical properties of dentin. Journal of Dental Research 2003; 82: pp. 957-961.
  • 40. Clarkson B.H., Feagin F.F., McCurdy S.P., Sheetz J.H., Speirs R.: Effects of phosphoprotein moieties on the remineralization of human root caries. Caries Research 1991; 25: pp. 166-173.
  • 41. Bertassoni L.E., Habelitz S., Marshall S.J., Marshall G.W.: Mechanical recovery of dentin following remineralization in vitro – an indentation study. Journal of Biomechanics 2011; 44: pp. 176-181.
  • 42. Carrilho M.R., Carvalho R.M., Sousa E.N., Nicolau J., Breschi L., Mazzoni A., et. al.: Substantivity of chlorhexidine to human dentin. Dental Materials 2010; 26: pp. 779-785.
  • 43. De Munck J., Mine A., Van den Steen P., Van Landuyt K.L., Poitevin A., Opdenakker G., et. al.: Enzymatic degradation of adhesive–dentin interfaces produced by mild self-etch adhesives. European Journal of Oral Sciences 2010; 118: pp. 494-501.
  • 44. Zhou J., Tan J., Chen L., Li D., Tan Y.: The incorporation of chlorhexidine in a two-step self-etching adhesive preserves dentin bond in vitro. Journal of Dentistry 2009; 37: pp. 807-812.
  • 45. Tatsumi T., Inokoshi S., Yamada T., Hosoda H.: Remineralization of etched dentin. The Journal of Prosthetic Dentistry 1992; 67: pp. 617-620.
  • 46. Oliveira S.S., Marshall S.J., Habelitz S., Gansky S.A., Wilson R.S., Marshall G.W.: The effect of a self-etching primer on the continuous demineralization of dentin. European Journal of Oral Sciences 2004; 112: pp. 376-383.
  • 47. Carvalho R.M., Chersoni S., Frankenberger R., Pashley D.H., Prati C., Tay F.R.: A challenge to the conventional wisdom that simultaneous etching and resin infiltration always occurs in self-etch adhesives. Biomaterials 2005; 26: pp. 1035-1042.
  • 48. Margvelashvili M., Goracci C., Beloica M., Papacchini F., Ferrari M.: In vitro evaluation of bonding effectiveness to dentin of all-in-one adhesives. Journal of Dentistry 2010; 38: pp. 106-112.

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The influence of chlorhexidine on the remineralization of demineralized dentine Duck-Su Kim , Jongryul Kim , Kyoung-Kyu Choi and Sun-Young Kim Journal of Dentistry, 2011-12-01, Volume 39, Issue 12, Pages 855-862, Copyright © 2011 Elsevier Ltd Abstract Objectives To examine the differences in the amounts of bound chlorhexidine (CHX) on demineralized dentine blocks and to investigate the different aspects of remineralization of demineralized dentine according to different concentrations of CHX. Methods Dentine blocks (2 mm × 7 mm × 0.9 mm) were demineralized in 0.2 M formic acid solution. Amount of bound CHX on the dentine blocks was measured on a spectrophotometer after the dentine block was soaked in 0.02%, 0.2%, or 2% CHX solutions for 1 min. The change in elastic modulus of dentine block stored in simulated body fluids was measured at 0 (baseline), 2, 4, and 6 weeks after storage. The micromorphological aspects of the samples were observed using a field emission scanning electron microscope after 6 weeks of storage. Results Higher concentrations of CHX caused a greater amount of CHX to bind to the dentine blocks ( p < 0.05). The group treated with the higher concentration of CHX had a smaller decrease in the elastic modulus at 2 weeks and a greater increase at 4 and 6 weeks. Dentine specimens with the 0.2% and 2% CHX had a greater deposition of granular minerals along the collagen fibrils compared to the 0.02% CHX-treated group. Conclusion The application of the 0.2% and 2% CHX seemed to be effective in promoting the remineralization of demineralized dentine. Clinical significance The application of the 0.2% and 2% CHX positively influences on the dentine remineralization 1 Introduction Chlorhexidine (CHX) was introduced as an antiseptic for skin wounds in the 1940s and has been widely used in dentistry due to its broad spectrum antibacterial effects and substantivity. CHX is used as the most effective antiplaque and antigingivitis agent in periodontal treatment and is used as an effective antiseptic root canal irrigant in endodontic treatment. CHX varnish has also been reported to have an effect on the arrestment of dental caries when applied on carious dentine. It has recently been reported that CHX not only inhibits the activity of bacterial proteolytic enzymes but also effectively reduces collagen degradation by inhibiting host-derived proteases in demineralized dentine. Many studies have reported that application of CHX for a certain amount of time in a prepared tooth cavity during the bonding procedure improved the stability of the hybrid layer in the resin–dentine bond and inhibited the reduction in bond strength due to ageing. This improvement in the durability of the resin–dentine bond is thought to happen because CHX inhibits the matrix metalloproteinase (MMP) activity, which is responsible for the self-degradation of incomplete resin-infiltrated collagen at the bottom of the hybrid layer. Various concentrations of CHX from low concentrations of 0.01–0.02% to relatively high concentration of 2% have been used in the durability studies of the resin–dentine bond and it was reported that the MMP inhibition effect was observed at all these concentrations. However, it was recently reported that CHX was not able to prevent collagen degradation completely but to only delay collagen degradation for some time because of limited substantivity. Conceptually, if the amount of bound CHX on the demineralized dentine was high, it might possibly better inhibit MMP more effectively or for a longer time. Except for one study which measured the binding amount of CHX using ground dentine powder, until now, there have been no reports on the differences in the amounts of bound CHX on demineralized dentine blocks that are more similar to a clinical environment compared to ground dentine powder according to concentrations of 0.02–0.2%, which have been mostly used in resin–dentine bond durability studies. Moreover, it is not clear if the exposed collagen fibril layer at the bottom of the hybrid layer keeps its original structure without changing, or is weakened or strengthened by any change when the endogenous MMP of the demineralized dentine is inhibited by CHX in a clinical environment. Many studies on tooth remineralization have been done targeting mainly the enamel remineralization of incipient caries. The sites, which need a remineralization strategy in dentine, could be a root caries that occurs mainly in older patients with gingival recession and saliva flow reduction, a cervical area in which dentine is exposed by chemical erosion, each cavity that has remaining affected dentine at its walls/floor, and an exposed collagen fibril layer incompletely infiltrated by the adhesive resin after acid etching in the resin–dentine bond interface. Those lesions have similar characteristics because they all originated from demineralization and thus ideal restoration procedures should remineralize the affected areas. Theoretically, it would be possible to remineralize those lesions through a supply of calcium and phosphate ions from the saliva or dentinal tubular fluid if the collagen fibril scaffold maintains its sound structure. It is well known that the effect of MMP inhibition increases as the concentration of CHX becomes higher ; however, little is known how different concentrations of CHX influence the remineralization of collagen scaffolds in demineralized dentine when there are constant mineral elements. The aims of this study were to examine the differences in the amounts of bound CHX on demineralized dentine blocks when different concentrations of CHX were used to treat the blocks and to investigate the different aspects of remineralization of demineralized dentine according to different concentrations of CHX when constant mineral elements were supplied. The remineralization aspects of demineralized dentine were examined by the change in elastic modulus and by the micromorphological appearance of the samples using a field emission scanning electron microscope (FE-SEM). 2 Methods and materials 2.1 Specimen preparation Forty extracted caries-free human third molars were collected and stored in 1% chloramine T solution with the patients’ informed consent. The study protocol was approved by the Institutional Review Board (IRB) committee of Kyung Hee University Medical Centre (KHB IRB 003-2). The enamel portion of each tooth was removed using a high-speed diamond bur with water-cooling and the mid-coronal dentine was exposed. A dentine block (2 mm × 7 mm × 0.9 mm) was obtained from each tooth using a slow-speed diamond saw (Isomet, Buehler Ltd., Lake Bluff, IL, USA) with water-cooling. Dentine blocks were demineralized in 0.2 M formic acid solution for 10 days. After demineralization, they were thoroughly washed with distilled water and dried with anhydrous calcium sulphate for 3 days. Then, the mass of each dried specimen was measured and rehydrated with distilled water. 2.2 Measuring the amount of bound CHX The amount of bound CHX was measured using a modified protocol based on Kim et al.’s method. After the full absorbance spectrum (200–800 nm) of CHX was recorded using a spectrophotometer (Synergy HT, Bio Tek instruments, USA), the 225 nm wave length was selected as the specific peak for measuring the amount of bound CHX. A standard curve for CHX absorbance was established by measuring the absorbance peak at 225 nm for 11 CHX concentrations (ranging from 0.012% to 2%). After each dentine block was inserted into a centrifuge tube, 1 mL of 0.02%, 0.2%, or 2% CHX was applied to the inside of the tube. Dentine block was completely soaked with CHX solution in the tube. The tube was then capped and tumbled for 1 min at 37 °C. The supernatant was placed in a 96-well plate and the absorbance at 225 nm was measured using a spectrophotometer. From the standard CHX curve, the amount of bound CHX was calculated using the difference in absorbance before and after the dentine block was soaked in the CHX solution. The reduced amount of CHX corresponds to the amount of bound CHX on the dentine block. This was expressed as mg CHX/g demineralized dentine block. This procedure was done for 10 dentine block specimens for each concentration of CHX except for the control group without any CHX treatment ( n = 10). 2.3 Measuring the elastic modulus A simulated body fluid (SBF) was prepared by dissolving 136.8 mM NaCl, 4.2 mM NaHCO 3 , 3.0 mM KCl, 1.0 mM K 2 HPO 4 ⋅3H 2 O, 1.5 mM MgCl 2 ⋅6H 2 O, 2.5 mM CaCl 2 , and 0.5 mM Na 2 SO 4 in deionized water. The SBF was buffered to pH 7.4 with 0.1 M Tris base and 0.1 M HCl. All dentine block specimens were stored in the SBF for 6 weeks. A three-point bending test for each specimen was done with a universal testing machine (Vitrodyne V1000, John Chatillong & Sons, Greensboro, NC, USA) at 0 (baseline), 2, 4, and 6 weeks after storage. Each specimen was tested in a wet state immediately after being removed from the SBF. The measurement was done with a 2.5 mm support span and a 0.5 mm/min crosshead speed. The elastic modulus of each dentine block was calculated using the following formula: E=mL34bh3 E = m L 3 4 b h 3 where E is the elastic modulus (MPa), m is the steepest slope (N/mm), L is the support span length (2.5 mm), b is the width of the dentine block (mm), and h is the thickness (mm). The elastic modulus at baseline was measured immediately after the dried demineralized specimen was rehydrated. The SBF was changed every two weeks. 2.4 FE-SEM evaluation Dentine blocks were selected from each group after storage for 6 weeks and irrigated thoroughly with distilled water. The specimens were treated according to the protocol of Perdigao et al. to avoid any artefacts from dehydration shrinkage. They were fixed with 2.5% glutaraldehyde (0.1 M sodium cacodylate buffer, pH 7.2) for 4 h and irrigated thoroughly with distilled water. They were dehydrated in ascending order with 50%, 60%, 70%, 80%, 90%, and 100% ethanol and dried with hexamethyldisilazane (Sigma–Aldrich, St. Louis, USA). The specimens were Pt-sputter-coated and fixed in an aluminium stub. Micromorphological structures of the specimens were evaluated using FE-SEM (Leo Supra 55, Carl Zeiss, Germany). 2.5 Statistical analysis The differences in the amounts of bound CHX according to the concentrations of CHX were analysed by one-way ANOVA. The differences in elastic modulus according to the CHX concentrations and the various lengths of the storage periods were analysed by repeated measure ANOVA. Tukey's test was done as a post hoc analysis to compare the effects of the different CHX concentrations. Paired t -tests were conducted to compare the elastic modulus for the various lengths of the storage periods from the baseline within the group. The level of significance was set at α = 0.05. SPSS version 18.0 (SPSS Inc., Chicago, IL, USA) was used for all statistical analyses. 3 Results The amounts of bound CHX according to the CHX concentrations treated in the dentine block are shown in Fig. 1 . There was a statistically significant difference in the amounts of bound CHX according to the concentrations of CHX ( p < 0.05). Higher concentrations of CHX caused a greater amount of CHX to bind to the dentine blocks. However, the difference in the amounts of bound CHX between the 0.02% and 0.2% groups was much greater than that between the 0.2% and 2% groups. Fig. 1 Amounts of bound CHX according to the CHX concentrations ( n = 10). The difference in the amounts of bound CHX between the 0.02% and 0.2% groups is much greater than that between the 0.2% and 2% groups. The results of the elastic modulus test according to the different CHX concentrations and the various lengths of storage periods are presented in Table 1 . There was a significant difference between the groups of different CHX concentrations in the change in elastic modulus according to the various lengths of storage periods ( p < 0.05). However, there was no statistically significant difference between the control group and the 0.02% CHX-treated group and between the 0.2% and 2% CHX-treated groups ( p > 0.05). The elastic modulus of the control group with no CHX treatment decreased significantly after 2 weeks ( p < 0.05). In the 0.02% CHX-treated group, the elastic modulus after 2 weeks of storage was significantly lower than that of the baseline ( p < 0.05), but the elastic modulus for 4 and 6 weeks of storage did not have any significant differences from that of the baseline ( p > 0.05). In the 0.2% CHX-treated group, the value of the elastic modulus was significantly lower at 2 weeks, similar at 4 weeks and higher at 6 weeks compared to the baseline. In the 2% CHX-treated group, the value of the elastic modulus showed no statistically significant changes at 2 weeks but significantly increased at 4 weeks and 6 weeks from the baseline ( p < 0.05). Table 1 Elastic modulus of the dentine blocks according to each CHX concentration and length of storage period in SBF (unit: MPa, n = 10). Group Baseline 2 weeks 4 weeks 6 weeks Control A 3.08 ± 0.44 b 2.18 ± 0.24 a 2.14 ± 0.34 a 2.07 ± 0.42 a 0.02% A 3.11 ± 0.59 b 2.17 ± 0.60 a 2.73 ± 0.64 b 2.85 ± 0.82 b 0.2% B 3.11 ± 0.34 b 2.68 ± 0.34 a 3.36 ± 0.38 b 3.66 ± 0.42 c 2% B 3.14 ± 0.40 a 2.94 ± 0.23 a 3.52 ± 0.41 b 3.94 ± 0.44 c Groups with the same superscript show no statistically significant differences. Same superscript amongst the time points within the group indicates no statistically significant differences. In the control group with no CHX treatment, the exposed reticular nanostructure of the collagen fibrils showing the effect of demineralization was clearly observed. The diameter of the collagen fibrils was thin and the interfibrillar spaces were relatively wide and clear ( Fig. 2 ). In the 0.02% CHX-treated group, with a small quantity of particles deposited around the demineralized collagen fibrils, the diameter of the collagen fibrils appeared to become slightly thicker compared to the control group ( Fig. 3 ). In the 0.2% CHX-treated group, many clustered granular structures of spherical grains were observed to form around the collagen fibrils and, as a consequence, there was only a little remaining amount of interfibrillar space ( Fig. 4 ). The apparent deposition of irregular nanoparticles along the collagen fibrils was also found in the intratubular area. The 2% CHX-treated group showed a denser granular deposition of nanoparticles on the surface of specimen. Since there was not much interfibrillar space, it was difficult to find a trace of the collagen fibrils on the intertubular dentine area ( Fig. 5 ). Fig. 2 FE-SEM images of non-CHX-treated dentine: (a) 30,000× magnification view; (b) 100,000× magnification view. The exposed reticular nanostructures of the collagen fibrils are clearly observed showing the effect of demineralization. The diameter of the collagen fibrils is thin and the interfibrillar distance is about 50–100 nm. Fig. 3 FE-SEM images of the 0.02% CHX-treated dentine: (a) 30,000× magnification view; (b) 100,000× magnification view. The interfibrillar distance decreases and the diameter of the collagen fibrils become thicker compared to the non-CHX-treated group as a small quantity of particles is deposited around the demineralized collagen fibrils. Fig. 4 FE-SEM images of the 0.2% CHX-treated dentine: (a) 30,000× magnification view; (b) 100,000× magnification view. Many clustered granular structures of spherical grains around the collagen fibrils are formed so that there is little remaining interfibrillar space on the surface. The apparent deposition of the irregular nanoparticles along the collagen fibrils is also found in the intratubular area. Fig. 5 FE-SEM images of the 2% CHX-treated dentine: (a) 30,000× magnification view; (b) 100,000× magnification view. Dense granular deposition of nanoparticles is shown on the surface. Since there is little interfibrillar space, it is difficult to find a trace of collagen fibrils on the intertubular dentine area. 4 Discussion A number of studies have shown in a direct way that the activity of endogenous MMP in dentine was inhibited according to the application of certain concentrations of CHX or in an indirect way such that the resin–dentine bond strength did not drop after ageing according to the application of certain concentrations of CHX. However, it is difficult to find a report on the direct effects of CHX on the remineralization of demineralized dentine. To our knowledge, the present study is the first that shows in an integrated way through mechanical and micromorphological changes in the aspects of remineralization in demineralized dentine according to concentrations of CHX. Moreover, this study presents a guide for the appropriate concentration of CHX that exerts the best effect through the results of the differences in the amounts of bound CHX on demineralized dentine blocks according to 0.02–2% concentrations of CHX, which have been mostly used in past studies on durable resin–dentine bonds. Based on the results of the present study, the application of 0.2% and 2% CHX for 1 min seemed to be more effective in promoting the remineralization of the demineralized dentine than that of 0.02% CHX. Dentine demineralization could arise from an acid attack by bacteria and/or a food source, and from acid etching for resin–dentine bonding. Several conditions are required to remineralize the demineralized dentine. First, the collagen structure should be sound as a scaffold for the mineral crystals to grow. Second, there should be residual mineral crystals as a growth centre, or there should be at least a newly formed nucleation site in case of complete demineralization. Last, mineral sources containing calcium and phosphorous should be supplied to the lesion. Many studies have tried to remineralize an initial caries and root caries by application of various agents such as calcium phosphate, bioglass, and fluoride. Recent several studies have also tried a new remineralization strategy creating a mineral nucleus through the application of a biomimetic analogue on completely demineralized dentine. In the present study, we showed that the application of CHX, which is an effective MMP inhibitor, promoted demineralized dentine to remineralize. CHX could keep the collagen cross-linkage sound by inhibiting the MMP activity. Remineralization would then happen around the remaining mineral crystals by obtaining mineral sources from the SBF. A previous study reported that an incorporated MMP inhibited the remineralization process of demineralized dentine and remineralization proceeded when the MMP was inhibited by a tissue inhibitor of the metalloproteinase. Mukai and ten Cate showed that remineralization could also occur in demineralized root dentine simulating advanced root caries under in vitro conditions. Therefore, it is not surprising that remineralization happened under SBF, which contains calcium and phosphorous, as long as a sound collagen scaffold remains in the demineralized dentine. Nevertheless, the results of the present study show that the level of remineralization was different according to the different concentrations of CHX and could be considered as an important step in establishing a relationship between the amount of bound CHX on dentine, the level of MMP inhibition, and the level of remineralization. Remineralization of dentine could occur by simple mineral precipitation between the collagen fibrils, which would be mainly extrafibrillar mineral formation. This type of remineralization could show an increase in mineral content but not have a tight association with the collagen matrix. Another type of remineralization is that the collagen fibrils of the dentine could be reinforced by intrafibrillar mineral formation so that the remineralized dentine would show an increase in mechanical properties, the so-called functional remineralization. In the present study, the elastic modulus of the remineralized dentine was shown to increase after storage in SBF for several weeks prominently in the cases of 0.2% and 2% CHX ( Table 1 ). Therefore, the remineralization in the present study might have occurred in an intrafibrillar manner. As a possible explanation for the functional remineralization, dentine specimens of the present study might be incompletely demineralized despite 10 days of storage in 0.2 M formic acid solution, which would leave nucleating mineral crystals in the collagen fibrils. Partially demineralized dentine was considered to have a capacity to restore the original mineralized state because it contains remnant mineral crystals and non-collagenous phosphoproteins, which act as a nucleation site of remineralization. That the aspect of mineral clogging in the dentinal tubule was not shown in the FE-SEM images might also suggest that remineralization of the dentine blocks in the present study was not a simple precipitation of minerals through the supply of calcium and phosphate irrespective of the sites but rather is closer to the optimal incorporation of minerals into the collagen fibrils. Despite that, further study needs to clarify the characteristics of the minerals formed through the remineralization process of this study. In order to verify functional remineralization through the application of CHX on demineralized dentine, the crystal structure of the calcium phosphate minerals formed in this study need to be examined using X-ray diffraction or infrared spectroscopy. Evaluation using transmission electron microscopy would also better help to understand the structural evidence of the remineralization in the collagen fibril scaffold rather than SEM, which is a convenient way to examine the surface of the remineralized dentine blocks. In the present study, 0.2% and 2% concentrations of CHX had a significantly higher elastic modulus after storage in SBF and a higher level of remineralization according to the morphological aspect than that of the relatively low concentration of 0.02% CHX. This might be because higher concentrations of CHX inhibited MMP activity more strongly and subsequently had a more positive influence on mineral formation. The results of the present study might suggest that the capability of keeping collagen fibril structures sound would be dose-dependent on the amount of CHX applied onto the dentine surface. As another possible explanation for causing higher levels of remineralization due to higher concentrations of CHX, the bound CHX itself at greater amounts in the demineralized dentine collagen might attract the mineral phosphate strongly and promote mineral growth by an electrostatic attraction between the protonated amine groups of CHX and the mineral phosphates. As for the control group without any CHX treatment, only the collagen fibre mesh was observed and the elastic modulus significantly decreased when measured every two weeks, which suggests that the endogenous MMP of the dentine hydrolyzed the collagen fibrils. This hydrolysis might cause the partial collapse of the collagen scaffold. Although the collagen fibril structure is sustained, the hydrolysis of specific amino acids in the collagen fibrils might weaken the structure or inhibit the growth of the minerals despite the supplied mineral elements through the SBF. In the present study, 0.02% CHX group did not show noticeable differences in the change in elastic modulus and morphologic aspect when compared to the control group. This reflects that the application of too low concentrations of CHX does not seem to be effective for inhibiting the MMP activity in the demineralized dentine. The present study is believed to support the previous studies that CHX cannot preserve the resin–dentine bond as long as the concentration of CHX is very low (0.05%). If the exposed collagen fibril layer of the demineralized dentine through acid etching for the resin–dentine bond had the ideal environmental conditions for remineralization, the nanoleakage at the bottom of the hybrid layer might be a temporary phenomenon, which could be solved by remineralization. An old animal study reporting that demineralized dentine from acid etching underwent remineralization in 4 months when demineralized dentine was restored with an adhesive system is believed to support this possibility. The results of the present study that CHX application could promote the remineralization process could be extended to the possibility that the durability of the resin–dentine bond can be improved in a practical way by remineralization of the exposed collagen fibrils in the hybrid layer and that postoperative hypersensitivity can also be arrested by mineral sealing of the nanoleakage. Nanoleakage at the bottom of the hybrid layer is reported to happen not only in the case of the etch-and-rinse adhesive system but also in the case of the self-etch adhesive system due to the continuous etching effect of the acidic monomers. Considering that self-etch adhesives could leave remnant minerals as nucleating sites for remineralization in the hybrid layer due to a weaker etching effect than the etch-and-rinse adhesives, the self-etch adhesive system appears to have a greater possibility of reducing nanoleakage through remineralization with the adjunctive use of CHX. Although the substantivity of CHX is limited according to the concentrations, if the remineralization occurred before the MMP inhibition effect of CHX ended, the limitation of CHX could be overcome. This perspective requires more research on optimizing the remineralization period of the demineralized dentine in the future. 5 Conclusion With 0.02–2% CHX, demineralized dentine blocks absorbed greater amounts of CHX at higher concentrations of CHX. When mineral sources were supplied, the application of 0.2% and 2% CHX for 1 min seemed to be more effective in remineralizing the demineralized dentine than that of the 0.02% CHX based on the results of the elastic modulus and FE-SEM in the present study. Acknowledgement This study was supported by a grant from the Kyung Hee University in 2011 (KHU-20110668). References 1. Lindhe J., Lang N.P., Karring T.: Rosengarten Family Fund. Clinical periodontology and implant dentistry.2008.Blackwell Munksgaard 2. Cosyn J., Wyn I., De Rouck T., Sabzevar M.M.: Long-term clinical effects of a chlorhexidine varnish implemented treatment strategy for chronic periodontitis. Journal of Periodontology 2006; 77: pp. 406-415. 3. Siqueira J.F., Paiva S.S., Rocas I.N.: Reduction in the cultivable bacterial populations in infected root canals by a chlorhexidine-based antimicrobial protocol. Journal of Endodontics 2007; 33: pp. 541-547. 4. Bretz W.A., Djahjah C.A., Almeida R.S., Villar do Valle E., Fonseca C., Valente I., et. al.: Effect of a chlorhexidine varnish on caries lesions. Oral Health & Preventive Dentistry 1995; 85: pp. 29-30. 5. Garcia M.B., Nör J.E., Schneider L.G., Bretz W.A.: A model for clinical evaluation of the effect of antimicrobial agents on carious dentin. American Journal of Dentistry 2001; 14: pp. 119-122. 6. Radford J.R., Homer K.A., Naylor M.N., Beighton D.: Inhibition of human subgingival plaque protease activity by chlorhexidine. Archives of Oral Biology 1992; 37: pp. 245-248. 7. Grenier D.: Reduction of proteolytic degradation by chlorhexidine. Journal of Dental Research 1993; 72: pp. 630-633. 8. Hebling J., Pashley D.H., Tjaderhane L., Tay F.R.: Chlorhexidine arrests subclinical degradation of dentin hybrid layers in vivo. Journal of Dental Research 2005; 84: pp. 741-746. 9. De Munck J., Van den Steen P.E., Mine A., Van Landuyt K.L., Poitevin A., Opdenakker G., et. al.: Inhibition of enzymatic degradation of adhesive–dentin interfaces. Journal of Dental Research 2009; 88: pp. 1101-1106. 10. Brackett W.W., Tay F.R., Brackett M.G., Dib A., Sword R.J., Pashley D.H.: The effect of chlorhexidine on dentin hybrid layers in vivo. Operative Dentistry 2007; 32: pp. 107-111. 11. Carrilho M.R., Geraldeli S., Tay F., de Goes M.F., Carvalho R.M., Tjaderhane L., et. al.: In vivo preservation of the hybrid layer by chlorhexidine. Journal of Dental Research 2007; 86: pp. 529-533. 12. Campos E.A., Correr G.M., Leonardi D.P., Barato-Filho F., Gonzaga C.C., Zielak J.C.: Chlorhexidine diminishes the loss of bond strength over time under simulated pulpal pressure and thermo-mechanical stressing. Journal of Dentistry 2009; 37: pp. 108-114. 13. Toledano M., Nieto-Aguilar R., Osorio R., Campos A., Osorio E., Tay F., et. al.: Differential expression of matrix metalloproteinase-2 in human coronal and radicular sound and carious dentine. Journal of Dentistry 2010; 38: pp. 635-640. 14. Hiraishi N., Yiu C., King N., Tay F.: Effect of 2% chlorhexidine on dentin microtensile bond strengths and nanoleakage of luting cements. Journal of Dentistry 2009; 37: pp. 440-448. 15. Loguercio A.D., Stanislawczuk R., Polli L.G., Costa J.A., Michel M.D., Reis A.: Influence of chlorhexidine digluconate concentration and application time on resin–dentin bond strength durability. European Journal of Oral Sciences 2009; 117: pp. 587-596. 16. Gendron R., Grenier D., Sorsa T., Mayrand D.: Inhibition of the activities of matrix metalloproteinases 2, 8, and 9 by chlorhexidine. Clinical and Diagnostic Laboratory Immunology 1999; 6: pp. 437-439. 17. Tay F.R., Pashley D.H.: Biomimetic remineralization of resin-bonded acid-etched dentin. Journal of Dental Research 2009; 88: pp. 719-724. 18. Osorio R., Yamauti M., Osorio E., Ruiz-Requena M.E., Pashley D., Tay F., et. al.: Effect of dentin etching and chlorhexidine application on metalloproteinase-mediated collagen degradation. European Journal of Oral Sciences 2011; 119: pp. 79-85. 19. Kim J., Uchiyama T., Carrilho M., Agee K.A., Mazzoni A., Breschi L., et. al.: Chlorhexidine binding to mineralized versus demineralized dentin powder. Dental Materials 2010; 26: pp. 771-778. 20. Gonzalez-Cabezas C.: The chemistry of caries: remineralization and demineralization events with direct clinical relevance. Dental Clinics of North America 2010; 54: pp. 469-478. 21. Gu Ls , Huffman B.P., Arola D.D., Kim Y.K., Mai S., Elsalanty M.E., et. al.: Changes in stiffness of resin-infiltrated demineralized dentin after remineralization by a bottom-up biomimetic approach. Acta Biomaterialia 2010; 6: pp. 1453-1461. 22. Blackburn R.S., Harvey A., Kettle L.L., Manian A.P., Payne J.D., Russell S.J.: Sorption of chlorhexidine on cellulose: mechanism of binding and molecular recognition. The Journal of Physical Chemistry B 2007; 111: pp. 8775-8784. 23. Kokubo T., Kushitani H., Sakka S., Kitsugi T., Yamamuro T.: Solutions able to reproduce in vivo surface-structure changes in bioactive glass–ceramic A–W. Journal of Biomedical Materials Research 1990; 24: pp. 721-734. 24. Carrilho M.R., Tay F.R., Donnelly A.M., Agee K.A., Tjäderhane L., Mazzoni A., et. al.: Host-derived loss of dentin matrix stiffness associated with solubilization of collagen. Journal of Biomedical Materials Research – Part B Applied Biomaterials 2009; 90: pp. 373-380. 25. Perdigao J., Lambrechts P., Van Meerbeek B., Vanherle G., Lopes A.L.: Field emission SEM comparison of four postfixation drying techniques for human dentin. Journal of Biomedical Materials Research 1995; 29: pp. 1111-1120. 26. Breschi L., Mazzoni A., Nato F., Carrilho M., Visintini E., Tjaderhane L., et. al.: Chlorhexidine stabilizes the adhesive interface: a 2-year in vitro study. Dental Materials 2010; 26: pp. 320-325. 27. Kuboki Y., Ohgushi K., Fusayama T.: Collagen biochemistry of the two layers of carious dentin. Journal of Dental Research 1977; 56: pp. 1233-1237. 28. Xu Z., Neoh K.G., Kishen A.: A biomimetic strategy to form calcium phosphate crystals on type i collagen substrate. Materials Science and Engineering C 2010; 30: pp. 822-826. 29. Peters M.C., Bresciani E., Barata T.J.E., Fagundes T.C., Navarro R.L., Navarro M.F.L., et. al.: In vivo dentin remineralization by calcium–phosphate cement. Journal of Dental Research 2010; 89: pp. 286-291. 30. Peters M.C.: Strategies for noninvasive demineralized tissue repair. Dental Clinics of North America 2010; 54: pp. 507-525. 31. Ten Cate J.M.: Remineralization of deep enamel dentine caries lesions. Australian Dental Journal 2008; 53: pp. 281-285. 32. Reynolds E.C., Cai F., Cochrane N.J., Shen P., Walker G.D., Morgan M.V., et. al.: Fluoride and casein phosphopeptide-amorphous calcium phosphate. Journal of Dental Research 2008; 87: pp. 344-348. 33. Vollenweider M., Brunner T.J., Knecht S., Grass R.N., Zehnder M., Imfeld T., et. al.: Remineralization of human dentin using ultrafine bioactive glass particles. Acta Biomaterialia 2007; 3: pp. 936-943. 34. Tschoppe P., Zandim D.L., Sampaio J.E.C., Kielbassa A.M.: Saliva substitute in combination with high-concentrated fluoride toothpaste: effects on demineralised dentin in vitro. Journal of Dentistry 2010; 38: pp. 207-213. 35. Kim J., Arola D.D., Gu L., Kim Y.K., Mai S., Liu Y., et. al.: Functional biomimetic analogs help remineralize apatite-depleted demineralized resin-infiltrated dentin via a bottom-up approach. Acta Biomaterialia 2010; 6: pp. 2740-2750. 36. Nordbø H., Leirskar J., Ngo H., Mount G.J., Wahlgren J.: The influence of a matrix metalloproteinase on the remineralization of artificially demineralized dentin. Oral Health & Preventive Dentistry 2003; 1: pp. 267-272. 37. Mukai Y., ten Cate J.M.: Remineralization of advanced root dentin lesions in vitro. Caries Research 2002; 36: pp. 275-280. 38. Bertassoni L.E., Habelitz S., Kinney J.H., Marshall S.J., Marshall G.W.: Biomechanical perspective on the remineralization of dentin. Caries Research 2009; 43: pp. 70-77. 39. Kinney J.H., Habelitz S., Marshall S.J., Marshall G.W.: The importance of intrafibrillar mineralization of collagen on the mechanical properties of dentin. Journal of Dental Research 2003; 82: pp. 957-961. 40. Clarkson B.H., Feagin F.F., McCurdy S.P., Sheetz J.H., Speirs R.: Effects of phosphoprotein moieties on the remineralization of human root caries. Caries Research 1991; 25: pp. 166-173. 41. Bertassoni L.E., Habelitz S., Marshall S.J., Marshall G.W.: Mechanical recovery of dentin following remineralization in vitro – an indentation study. Journal of Biomechanics 2011; 44: pp. 176-181. 42. Carrilho M.R., Carvalho R.M., Sousa E.N., Nicolau J., Breschi L., Mazzoni A., et. al.: Substantivity of chlorhexidine to human dentin. Dental Materials 2010; 26: pp. 779-785. 43. De Munck J., Mine A., Van den Steen P., Van Landuyt K.L., Poitevin A., Opdenakker G., et. al.: Enzymatic degradation of adhesive–dentin interfaces produced by mild self-etch adhesives. European Journal of Oral Sciences 2010; 118: pp. 494-501. 44. Zhou J., Tan J., Chen L., Li D., Tan Y.: The incorporation of chlorhexidine in a two-step self-etching adhesive preserves dentin bond in vitro. Journal of Dentistry 2009; 37: pp. 807-812. 45. Tatsumi T., Inokoshi S., Yamada T., Hosoda H.: Remineralization of etched dentin. The Journal of Prosthetic Dentistry 1992; 67: pp. 617-620. 46. Oliveira S.S., Marshall S.J., Habelitz S., Gansky S.A., Wilson R.S., Marshall G.W.: The effect of a self-etching primer on the continuous demineralization of dentin. European Journal of Oral Sciences 2004; 112: pp. 376-383. 47. Carvalho R.M., Chersoni S., Frankenberger R., Pashley D.H., Prati C., Tay F.R.: A challenge to the conventional wisdom that simultaneous etching and resin infiltration always occurs in self-etch adhesives. Biomaterials 2005; 26: pp. 1035-1042. 48. Margvelashvili M., Goracci C., Beloica M., Papacchini F., Ferrari M.: In vitro evaluation of bonding effectiveness to dentin of all-in-one adhesives. Journal of Dentistry 2010; 38: pp. 106-112.

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