|Year : 2014 | Volume
| Issue : 2 | Page : 33-38
A comparative study of the preventive effect of chlorhexidine o. 12% and nano zinc oxide particles on the distraction of collagen scaffolding of the hybrid layer by two immunohistochemistry and microleakage tests
Homayoon Alaghemand1, Behnaz Esmaeili1, Pouralibaba Firouz2, Mohammad Hossein Soltani1, Marzyeh Rouhaninasab1, Ali Dehghani nazhvani3, Ali Bijani4
1 Department of Oral Medicine, Dental materials Research Center, Babol University of Medical Science, Babol, Iran
2 Department of Oral Medicine, Tabriz University of Medical Science, Tabriz, Iran
3 Department of Esthetic and Restorative Dentistry, Babol University of Medical Science, Babol, Iran
4 Department of Oral Pathology, Shiraz University of Medical Science, Shiraz, Social Determinant of Health Research Center, Babol University of Medical Science, Babol, Iran
|Date of Web Publication||21-Oct-2014|
Mohammad Hossein Soltani
Department of Esthetic and Restorative Dentistry, Babol University of Medical Science, Babol
Source of Support: None, Conflict of Interest: None
Introduction: Hybrid layer degradation is mainly attributed to matrix metalo proteinases (MMP) activity. In this study, we compare inhibitory effect of nano zinc oxide with chlorhexidine (0.12%) on distraction of collagen scaffolding of the hybrid layer by immunohistochemistry (IHC) and microleakage tests. Materials and Methods: A total of 60 extracted molars and premolar teeth with no caries and visible anatomic defects were selected. Class 5 cavities with dimensions of 2 × 2 × 2 mm was performed for each tooth on the buccal surface 1 mm above cemento enamel junction. Results: In microleakage test, there was no significant difference in the result for groups pretreated with nano zinc oxide mixed with single bond (C), pretreated with chlorhexidine (0.12%) applied before bonding (B) and not pretreated with these agents (A) whether in 24 h or 1-week. in IHC test, there was a significant difference (P < 0.05) between all groups except in group B and group C. Conclusion: (1) The inhibitory effect of chlorhexidine and nano zinc oxide on MMPs activity is different. (2) Microleakage amount is different in composite resin restoration used in a cavity pretreated with nano zinc oxide contained bonding or chlorhexidine 24 h and 1-week later.
Keywords: Anatomic defects, immunohistochemistry, matrix metalo proteinases
|How to cite this article:|
Alaghemand H, Esmaeili B, Firouz P, Soltani MH, Rouhaninasab M, nazhvani AD, Bijani A. A comparative study of the preventive effect of chlorhexidine o. 12% and nano zinc oxide particles on the distraction of collagen scaffolding of the hybrid layer by two immunohistochemistry and microleakage tests. Dent Med Res 2014;2:33-8
|How to cite this URL:|
Alaghemand H, Esmaeili B, Firouz P, Soltani MH, Rouhaninasab M, nazhvani AD, Bijani A. A comparative study of the preventive effect of chlorhexidine o. 12% and nano zinc oxide particles on the distraction of collagen scaffolding of the hybrid layer by two immunohistochemistry and microleakage tests. Dent Med Res [serial online] 2014 [cited 2020 Aug 4];2:33-8. Available from: http://www.dmrjournal.org/text.asp?2014/2/2/33/143327
| Introduction|| |
Dentinal adhesive systems were prone to show failure primarily. Despite now-a-days improvement in dentinal adhesive's quality and dentin-resin bond strength, there is still high demand to enhance this system. 
In recent studies, it has been shown that some enzymes exist in dentinal matrix which are released during bonding process by environmental pH modification due to acid etching and gradually distract dentin-resin bond; which consequently causes secondary caries, marginal discoloration and marginal leakage and finally the failure of restorations. Matrix metalo proteinases (MMPs) are a 26 membered family proteolytic enzymes.  Some of these have been detected in a carious lesion including MMP2 (gelatinase), MMP8 (collagenase), MMP9 (gelatinase) and MMP20 (enamelysin). Dentinal proteins contain 90% collagen (mostly type 1) and 10% non-collagen. By releasing of MMPs; collagen proteins will be impacted primarily by MMP8 and after low pH induced-dentin demineralization within carious lesion and etching process, degradation of collagen shall be completed by MMP2 and MMP9. These MMPs can exist in saliva and pulp or also they can settle in dentin and release during caries lesion. 
These enzymes can be activated at pH 4.5 or less, and this activation phase can even remain if pH returns to its normal level. It has been suggested that organic acids and also some other acids such as phosphoric acid can have a special role in activation of these enzymes. Activation of MMPs is done by linking Zn and Ca ions to N-terminal branch. 
This activation would be disturbed in two circumstances and with some inhibitors; (1) That other enzymes and ions compete with Ca and Zn ions and hence deactivation occurs or (2) by increase in Zn ions, occupying other linking sites with less activation affinity by zinc will happen and inhibition of MMPs activation will be done by changes in its configuration.
Till now the inhibitory effect of chlorehexidine, zinc, galardin and others on MMPs activity has been studied. Osorio et al. evaluated zinc's effect on MMPs and concluded that zinc oxide nano particles when combined with etch and rinse adhesive resin like single bond; has the best inhibitory effect on MMPs activity.  Hebling et al. and Carrilho et al. reported successful use of chlorhexidine adjunctively with conventional etch and rinse adhesives.  Even though more research is needed in this field.
Since hybrid layer degeneration and collagen scaffolding degradation is one reason of microleakage among others , and also MMPs releasing can cause this damage to collagen framework, in this study we compared the inhibitory effect of chlorhexidine (0.12%) and Nano zinc oxide by two tests of immunohistochemistry (IHC) and also microleakage, 24 h and 1-week after (under thermocycling process) restoration preparation.
| Materials and methods|| |
Sixty extracted molars and premolar teeth with no caries and visible anatomic defects were selected. Teeth were restored in thymol (0.2%) for 24 h after extraction and then were preserved in normal saline till beginning of the study. Class 5 cavities with dimensions of 2 mm × 2 mm × 2 mm was prepared for each tooth on the buccal surface 1 mm above cemento enamel junction (CEJ). Sixty teeth were restored with three different methods in three groups (n = 20). In group A, teeth were 15 s etched with phosphoric acid 38% (Etch-Rite, Pulpdent, USA) then rinsed and dried off. Single bond (3M, ESPE, USA) as a bonding agent was applied on cavity surfaces with microbrush, after 10 s gentle air drying, 20 s light curing (LED VALO, Ultradent, USA) with intensity of 600 m W/cm 2 was performed and then cavity was restored incrementally with layer of 1 mm composite (Filtek Z250, shade A2, 3M, USA) and cured for 40 s. In group B, after etching the cavity was pretreated by chlorhexidine 0.12% for 60 s using microbrushes, blot-dried to remove the excess chlorhexidine and then bonding agent and composite were applied similar to group A. In group C, essential amount of nano zinc oxide powder was added to single bond binding and manually mixed for 10 min (single bond + 10% weight nano zinc oxide) and applied to the cavity after etching, cured and then composite filling similarly to the later groups, was done. Teeth were then polished for 30 s under running [Table 1] water with 800 and 600 grits silicon carbide papers. Teeth were then divided to two groups. In the half of them, 2 layers of nail polish varnish were applied on coronal and radicular teeth surfaces except the restoration and the surrounding 1mm and also 4 layers of nail polish varnish was used to cover the apex to prevent the color infiltration. Then they were immersed in silver nitrate solution (50% weight) in a dark room for 2 h, rinsed under water and restored in emerge solution for 4 h under fluorescent lamp then rinsed and restored in normal saline till 24 h after restoration preparation.  Then they were cut bucco-lingually with a chisel blade and the score* of microleakage was evaluated at gingival dentin-resin interfaces under stereomicroscope (MEIJI, Japan) (group A1 n = 10, group B1 n = 10, group C1 n = 10). For the second half all the above steps were done after a week under aging process (NEMO Thermocycling Machine, 5°C-55°C, each for 30 s with dwelling time of 30 s, 250 cycles) (group A2 n = 10, group B2 n = 10, group C2 n = 10).
- Specimens were classified due to these scores:
- Gingival interface 0: Without dye infiltration in tooth composite interface 1: Dye infiltration to first half of gingival wall 2: Dye infiltration beyond first half of dentin without reaching axio-cervical line angel 3: Dye infiltration to axio-cervical line angel and on axial wall [Figure 1].
|Figure 1: ×100 graphs (a) Microleakage score (0) (b) Microleakage score (3) (c) Microleakage score (2)|
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Teeth preparation before immunohistochemistry
10 teeth without obvious caries and anatomic defect were selected. Boxes with dimensions of 1 mm depth 3 mm height and 3 mm length were prepared just above CEJ at buccal, lingual, mesial and distal surfaces of teeth. Not any treatment was done for distal boxes (positive control group) (group A n = 10). 10 mesial boxes were etched with phosphoric acid (38%) for 15 s (MMPs activation) rinsed for 10 s and dried off (group B n = 10). Buccal boxes were etched, pretreated with chlorehexidine (0.12%) for 60 s and then blot-dried to remove the excess CHX (group C n = 10). For the remaining 10 buccal boxes after etching process, pretreatment with nano zinc oxide solution (10% weight) for 20 s was done and then were blot-dried group D n = 10).
Teeth were immersed in ethylenediaminetetraacetic acid (EDTA) solution with neutral PH for almost a week to chelate the mineral phase (1750 ml distilled water + disodium salt, EDTA 250 g). Color of solution is a marker of neutral pH (change of color from milky to clear by adding 25 g NaOH means pH is neutral).
Forty sections of dentin with 4 μm thickness was cut by microtome machine (Leitz 1600, Ernst Leitz, Germany). Sections were then deparaffinized and re-hydrated with graded ethanol series. These sections were treated in an autoclave with an EDTA (0.005 M) for 15 min. When buffer solution reached room temperature they were rinsed, and the endogenous peroxidase activity was quenched by peroxide hydrogen in methanol, (0.01 M) in a darkroom for 10 min. Then after covering the sections with type I collagen antibody (Nb600-408, novus biological, USA) for 30 min negative control group were covered with tris-buffered saline and then tissue surfaces were covered with peroxidase labeled polymer (envision code k8000_DAKO LTD) in a 37°C temperature for 30 min. After rinsing in buffer solution, tissue surfaces were covered with di amino benzidine chromogen solution (DAB-Code k8004_DAKO LTD) at room temperature for 10 min. After rinsing in buffer solution, all sections were under color reaction with hematoxylin and then mounted. Slides were examined by a calibrated observer using olympus (BX51, Olympus Optical, Tokyo, Japan). Staining intensity was classified as weakly positive (+), moderately positive (++) or strongly positive (+++) due to DAB impregnation [Figure 2].
|Figure 2: ×400 olympus BX51 microscope graphs (a) Score +++ for acid etched group B (b) Score ++ for CHX pretreated group C (c) Score + for nano zinc oxide pretreated group D|
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| Results|| |
All data were analyzed by nonparametric tests. Chi-square and Kruskal-Wallis were used for comparison of group A, B and C whether 24 h or 1 week after beginning of the test. There was no statistically difference between all three groups whether CHX (0.12)% or nano zinc oxide were used or not, both in the period of 24 h and 1-week in microleakage scores (P > 0.05).
Wilcoxon test was used to analyze the difference in microleakage score in periods of 24 h and 1-week for each group individually. Group A and B showed significant difference scores when had been thermocycled and analyzed 1-week later (P < 0.05), whereas in group C, no significantly different was shown (P > 0.05) [Table 2].
Using Chi-square and Wilcoxon tests, for two by two comparisons of all the four groups, there was significant difference (P < 0.05) between all groups except in group b and group c. There was no statistical difference between these groups (P > 0.05) [Table 3].
| Discussion|| |
In this study, IHC scores in group D (nano zinc oxide) was significantly different from group C (chlorhexidine 0.12%) and group B (acid only pretreated), but not different comparing group B and group C with each other. In microleakage test, scores were not significantly different in 3 groups tested. Comparing each group 24 h and 1-week later, group C was the only one which didn't show significant difference. Our null hypothesis, which the inhibitory effect of chlorehexidine and nano zinc oxide powders on MMPs activity is different, was not rejected. In IHC test, we used phosphoric acid 38% to activate MMPs activity and also in two different groups we pretreated the cavity surfaces with nano zinc oxide powders and chlorhexidine as inhibitors of MMPs activity. We simulated most of specimen preparation steps for both IHC and microleakage tests to attain two goals: (1) To simulate the clinical situation and (2) to simulate IHC test to the microleakage one in order to compare the results with each other. Inhibitory Effect of zinc and chlorhexidine had been studied in previous researches, ,,,, but simultaneous comparison of chlorhexidine and nano zinc oxide particles by both microleakage and IHC test had not been done before. In IHC test, the median intensity of type I collagen fibrils staining was moderately positive (++) in nano zinc oxide group (D), while in chlorehexidine (C) and acid only used groups (B) the median were strongly positive (+++), whereas the positive control group (A) showed weakly positive score (+). Staining intensity was classified as weakly positive (+), moderately positive (++) or strongly positive (+++) according to DAB impregnation.  It may mean that when we prepare cavity in dentin, whether followed by acid etching or not, the occurring thermal changes will cause mild coagulation of collagen proteins, which disturb fibril's integrity and hence mild positive score (+) is seen under microscope. Group B and C showed the same score, which can imply that the inhibitory effect of chlorehexidine (0.12%) on MMPs activity is not significant. Staining intensity may elevate with increase in collagen disturbance. On the other hand, disturbance caused by acid etching is inhibited more by nano zinc oxide powders than chlorhexidine 0.12%. IHC analyzing of hybrid layer degradation is not a very routine way on detecting MMPs activity; whereas zymography, scanning electron microscopy and transmission electron microscopy analyze, bond strength and microleakage tests are used more often. ,,, In microleakage test there was no significant difference in the result for groups pretreated with nano zinc oxide mixed with single bond (C), pretreated with chlorhexidine (0.12%) applied before bonding (B) and not pretreated with these agents (A), whether in 24 h or 1-week. It may be due to the short term followup considered, or even low chlorhexidine concentration (0.12%) or low amount of zinc infiltration to dentin that is in contrast with Toledano et al. or Osorio et al. who considered 8 h immersion of dentin beams in zinc contained resin bonding. , To simulate all clinical steps we applied nano zinc oxide contained bonding for 10 s and cured it for 20 s. Since we used neutral pH decalcification solution (weak aqueous EDTA solution) in order not to adversly effect the specimens due to low PH and it took 1-week to complete; we consider two period of 24 h and 1-week for microleakage test, although comparing each group individually showed significant difference in microleakage scores of group A and B, but not in group C. It means that when we used nano zinc oxide contained single bond as bonding agent the integrity of hybrid layer stabilized and hence the miroleakage score didn't differ significantly after 1-week. Osorio et al. evaluated the collagen degradation and bond strength using nano zinc oxide contained single bond (etch and rinse) versus clearfil self-etching primer and bond (self-etch adhesive) for period of 24 h, 1 and 4 week and concluded that the only resin able to stabilize collagen degradation over time (within the study period) was zinc oxide contained single bond and bond strength had not been affected. ZnO is an amphoteric oxide, although it normally shows basic properties. It is nearly insoluble in water and alcohol, but it is soluble in and degraded by acids. Small size particles of ZnO were selected to propitiate the highest interfibrillar infiltration. The high solubility of ZnO when combined with acid could also account for the effective release of zinc ions at the resin-dentin interface.  It has already been published that zinc may not only act as an inhibitor of MMPs, but may also influence signaling pathways and stimulate a metabolic effect in hard tissue mineralization.  Zinc has also been shown to inhibit dentin demineralization.  All these characters make zinc attractive as a therapeutic agent and adhesive products. We used chlorhexidine 0.12% as an inhibitor for MMPs activity. Although there was the difference in microleakage scores of chlorhexidine pretreated group (B) and no pretreatment group (A) but it was not statistically meaningful. Chlorhexidine has been shown to inhibit MMP2, 8, and 9 activities directly at extremely low concentrations.  Although Carrilho et al. concluded that chlorhexidine 2% pretreated dentin exhibit normal structure integrity,  Vicente Casta showed that short application of chlorhexidine (2%) at low concentration prevent hybrid layer degradation and positively affect bond strength over time, ,,,,,, we didn't find any statistically difference between microleakage scores of group A and B. It may be due to the chlorhexidine concentration we used (0.12%), while others have used more concentration. ,, The influence of concentration and application time of CHX have been evaluated to show that times <1-min and concentration less than 2% may prevent degradation in vitro by Loguercio et al.  The use of chlorhexidine (0.12%) mouthwash to rub on the preparation might seem to be the most immediate way to treat self-etch dentin bonds. Since chlorhexidine (0.12%) (Peridex, 3M, USA) has a routine use in dental offices and for dentistry patients, we decided to use this chlorehexidine concentration applying it before bonding in etch and rinse adhesives. Our reason to select etch and rinse system was the fact that adding inhibitor to their bonding have showed better result than self-etch ones. 
| Conclusion|| |
Within the limitation of this study, we report that nano zinc oxide pretreatment protect hybrid layer by inhibiting collagen degradation and hence marginal integrity is less affected by MMPs activity, and also chlorhexidine may act as an inhibitor but it is not statistically meaningful. Inhibitory effect of nano zinc oxide particles is more than chlorhexidine 0.12% although knowledge perfection in this field is still needed. We suggest future researches about accumulative effect of zinc and chlorhexidine and also evaluating other properties affected by these two factors such as bond strength and bond durability in even longer periods than 1-week.
| Acknowledgments|| |
This project had been done with the support of Babol University of Medical Sciences (No: 10374985).
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[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3]