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 Table of Contents  
ORIGINAL ARTICLE
Year : 2019  |  Volume : 16  |  Issue : 3  |  Page : 163-168

Effect of two tissue inhibitors on the antimicrobial activity of chitosan nanoparticles and chlorhexidine


1 Department of Endodontic, Faculty of Dentistry, Tanta University, Tanta, Egypt
2 Department of Pharmaceutical Microbiology, Faculty of Pharmacy, Tanta University, Tanta, Egypt

Date of Submission29-Jun-2019
Date of Acceptance29-Sep-2019
Date of Web Publication14-Jan-2020

Correspondence Address:
Muhammad S. Anwar
Department of Endodontic, Faculty of Dentistry, Tanta University, Tanta, Algharbeia
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/tdj.tdj_26_19

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  Abstract 

Aim: To assess the effect of dentin powder (DB) and human serum albumin (HSA) as tissue inhibitors on the antibacterial activity of chitosan nanoparticles (CSnps) and chlorhexidine (CHX).
Materials and methods: The antibacterial effect of CSnps and CHX in presence or absence of inhibitors was tested on planktonic Enterococcus faecalis OG1RF standard strain. Study groups were group 1: DB and CSnps, group 2: DB and CHX, group 3: HSA and CSnps, and group 4: HSA and CHX, group 5: CSnps suspension (positive control for groups 1, 3), group 6: CHX solution (positive control for groups 2, 4), and bacteria suspension as a negative control (group 7). The assigned medications were added to tissue inhibitors for an hour before bacterial suspension addition. After 1 min, 24, and 72 h of bacterial suspension addition a sample was taken. Bacterial culturing was done on brain heart infusion plates at 37°C. The plates were incubated for 24 h and were inspected for growth by naked eye and the colonies the number of surviving bacteria was calculated. Data were collected and the mean and SD of log10 colony forming units was calculated and statistically analyzed using one-way analysis of variance and pairwise comparison between groups was performed whenever significant difference was found. Level of significance was set at P value less than or equal to 0.05.
Results: Group 3and group 7 were statistically significant than all tested groups at all times. Also, significant difference between group 4 versus group 2 and group 6 recording P value of 0.048 at 1 min. However, there was no significant difference between groups 1, 2, 4, 5, 6 at 24 and 72 h.
Conclusions: HSA had significant inhibitory effect on antibacterial activity of CHX and CSnps than DB at 1 min. At 24 and 72 h, neither HSA nor DB had significant inhibitory effect on antibacterial activity of CHX while HSA had a significant effect on antibacterial activity of CSnps.

Keywords: chitosan nanoparticles, chlorhexidine, dentin powder, human serum albumin, tissue inhibitors


How to cite this article:
Anwar MS, Darrag AM, Al-Madboly LA, Ghoneima WM. Effect of two tissue inhibitors on the antimicrobial activity of chitosan nanoparticles and chlorhexidine. Tanta Dent J 2019;16:163-8

How to cite this URL:
Anwar MS, Darrag AM, Al-Madboly LA, Ghoneima WM. Effect of two tissue inhibitors on the antimicrobial activity of chitosan nanoparticles and chlorhexidine. Tanta Dent J [serial online] 2019 [cited 2020 Sep 24];16:163-8. Available from: http://www.tmj.eg.net/text.asp?2019/16/3/163/275936


  Introduction Top


It has been accepted for many years that microorganisms are the main etiological agent of pulpal and periapical pathosis. Despite best efforts in decontaminating root canal systems, persistence of intracanal bacteria and reinfection via coronal leakage are the most common causes of endodontic treatment failure [1]. Enterococcus faecalis is a facultative anaerobic gram-positive coccus that is present in 24–74% of asymptomatic and persistent endodontic infections [2].

Elimination of microorganisms from the main canals, as well as from the accessory canals, links between canals, dentinal tubules or apical deltas is a big challenge for endodontists as the mechanical preparation of the root canals is not effective alone. Therefore, the additional chemical disinfection of root canals is an essential part of a successful root canal treatment[3],[4].

Ideally, chemical disinfectants should be antibacterial, nontoxic, minimally destructive to tooth structure, provide dissolution of organic and inorganic materials, not affected by presence of organic matter and be relatively easy to use [5]. Chlorhexidine gluconate (CHX) has gained considerable popularity in endodontics as an irrigating solution and as an intracanal medicament because of its substantivity and antimicrobial effect via prolonged binding to hydroxyapatite [6].

Chitosan is a nontoxic biopolymer derived by the deacetylation of chitin. Chitin is a natural polymer occurring in the exoskeleton of the crustaceans. It is a bioadhesive that readily binds to negatively charged surfaces and has excellent antimicrobial and antifungal activities. Chitosan nanoparticles (CSnps) are found to have higher antibacterial activity, this is due to the higher surface area and charge density of nanoparticles, which enable them to achieve a greater degree of interaction with the negatively charged surface of bacterial cells [7].

Many root canal disinfecting agents with excellent antimicrobial activity against endodontic bacteria and yeast in vitro often fail to completely disinfect the root canal infected by the same species in vivo [8]. The infected root canal system is known to contain bacteria and its byproducts, inflammatory exudate, entering the root canal apically, which is rich in proteins, such as albumin, dentin, dentin matrix, and pulpal tissue remnants. These root canal constituents have been shown to reduce or completely inhibit the antibacterial activity of CHX, iodine potassium iodide, and calcium hydroxide[9],[10]. However, no specific mechanism has been suggested for the inhibitory effect of different organic components. So, it is important that all the newly developed disinfection strategies are tested in the presence of these tissue inhibitors to evaluate their intracanal antibacterial efficacy [11].

Based on the above evidence this study aimed to assess the effect of dentin and human serum albumin (HSA) as tissue inhibitors on the antibacterial activity of CSnps and CHX.


  Materials and Methods Top


Preparation of bacterial suspension

E. faecalis OG1RF standard strain was used as a test organism in the present study. E. faecalis was grown on bile esculin agar specific media (Oxoid, Basingstoke, Hampshire, England). A sterilized tooth pick was used to pick up one colony of the pure E. faecalis which was inserted carefully into sterile test tube containing 7 ml sterile normal saline solution (Pharmaceutical Solutions industries Co., 10th of Ramadan, Egypt) and mixed well. This was repeated several times, observed by naked eye until the turbidity of this suspension was nearly equal to the turbidity of 0.5 McFarland standard solution [12–14]. The turbidity of E. faecalis suspension was confirmed spectrophotometrically (UNICO Instruments Co., Shanghai, China)[11] to give cell density of 1.5 × 108 colony forming units (CFU) per ml for the stock solution.

Preparation of tissue inhibitors

The substances tested for the inhibition of the antibacterial activity of the medicaments were dentin powder (DB) and HAS (Sigma Chemical Co., St. Louis, Missouri, USA). All compounds were used in powder form. The DB was prepared by using different sizes of gates glidden (MANI Inc., Tochigi, Japan) ranging from size two to five in up and down motion inside the root canal against the root canal walls and collecting it in Eppendorf tube. Of each powder, 28 mg was suspended in 50 μl sterilized thioglycolate broth. All procedures were performed complete aseptic condition in a laminar air flow chamber (American Middle East Marketing Co., USA).

Preparation of tested medicaments

The medicaments tested were 10 mg/ml CSnps suspension (Nanotech, 6-October City, Egypt) and 2% CHX (Cerkamed, Kwiatkowskiego, Poland) solution.

Groups assignment

Group 1: DB and CSnps.

Group 2: DB and 2% CHX.

Group 3: HSA and CSnps.

Group 4: HSA and 2% CHX.

Group 5: CSnps suspension (positive control for groups 1, 3).

Group 6: 2% CHX solution (positive control for groups 2, 4).

Group 7: bacterial suspensions (negative control).

For experimental groups (groups 1–4) 50 μl of the assigned medicaments was added to aliquots of 50 μl of DB or HSA suspension in thioglycolate broth and thoroughly mixed and incubated in sealed Eppendorf tubes at 37°C[4],[15]. After 1 h, 50 μl of bacterial suspension was added to the mixture of the inhibitors and assigned medicament. For positive control groups (groups 5, 6) 50 μl of thioglycolate broth without adding DB or HSA was thoroughly mixed and incubated with 50 μl of the assigned medicaments in sealed Eppendorf tube at 37°C for 1 h before adding 50 μl of bacterial suspension. For negative control group (group 7) 50 μl bacterial suspension was added and mixed with 100 μl thioglycolate broth. Serial 10-fold dilutions were made. A sample of 10 μl was taken for bacterial culturing after 1 min, 24 and 72 h of bacterial suspension addition. Bacterial culturing was done on brain heart infusion plates ± at 37°C. After that, the plates were put in the incubator for 24 h[11],[15]. The plates were inspected for growth by naked eye and the colonies were counted and the total number of surviving bacteria was calculated. These processes were repeated ten times for each group.

Statistical analysis

Data were collected and the mean and SD of log10 CFU in each group was calculated and statistically analyzed using one-way analysis of variance (ANOVA) and pairwise comparison between groups was performed whenever significant difference was found. The significance level was set at P value less than or equal to 0.05. Statistical analysis was performed with SPSS, version 17.0 (SPSS Inc., Chicago, Illinois, USA.).


  Results Top


Regarding the antimicrobial effect of the assigned medicaments in the presence and absence of tissue inhibitor on E. faecalis after 1 min of bacterial suspension addition; the lowest mean value of log10 CFU/ml was recorded with CHX in the presence of DB (group 2) and in group 6 where CHX was used without inhibitor. More colonies were found in group 4 where CHX was used in the presence of HSA, followed by group 3 in which CSnps was used in the presence of HSA. While the highest mean value was recorded in negative control group as shown in [Table 1].
Table 1 The means and SDs of the numbers of the log10 colony forming unit/ml for each group after 1 min of bacterial suspension addition and their statistical analysis

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One-way ANOVA revealed that there was a statistical significant difference among the tested groups (P > 0.001). So, Tukey's pairwise comparison test was performed and revealed that, there were statistical significant difference between group 3 versus all tested groups and between group 7 versus all tested groups. There were also significant difference between group 4 versus group 2 and between group 4 versus group 6.

Similarly, the antimicrobial effect of the assigned medications in the presence and absence of tissue inhibitors on E. faecalis after 24 h of bacterial suspension addition was considered and recorded in [Table 2]. One-way ANOVA revealed that there was a statistically significant difference among the tested groups (P < 0.001). Therefore, Tukey's pairwise comparison test was done, and it revealed that, there were a statistically significant differences between group 3 versus all other tested groups and between negative control group versus other tested groups.
Table 2 The means and SDs of the numbers of the log10 colony forming unit/ml for each group after 24 h of bacterial suspension addition and their statistical analysis

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At 72 h of bacterial suspension addition; mean values of CFU/ml were recorded in [Table 3]. A statistically significant difference among the tested groups (P < 0.001) was recorded using one-way ANOVA. Tukey's test revealed that groups 3 and 7 have statistically significant difference versus all other tested groups.
Table 3 The means and SDs of the numbers of the log10 colony forming unit/ml for each group after 72 h of bacterial suspension addition and their statistical analysis

Click here to view



  Discussion Top


The use of antimicrobial agents in conjunction with mechanical canal preparation has been shown to reduce bacterial load in the root canals[16],[17]. Despite the high antimicrobial efficacy of conventional disinfectants in vitro, clinical studies have shown bacterial persistence within the root canal system[18],[19] due to presence of various tissue inhibitors that are known to impede the efficacy of conventional disinfection strategies[9],[20],[21]. So, it is important that all the newly developed disinfection strategies are tested in the presence of these tissue inhibitors [11]. Therefore, this study was conducted to evaluate and compare the effect of DB and HSA as tissue inhibitors on the antimicrobial activity of a commonly used disinfecting agent which is CHX solution and a newly developed disinfecting agent, CSnps suspension.

E. faecalis was chosen as a mono-infection bacterium in root canals [22] in this study for several reasons; it is probably the key species in many persistent endodontic infections and most frequently found in nonhealed cases of apical periodontitis requiring retreatment [22–27], it is considered more resistant to some local endodontic medicaments than most other microbes[28],[29], it is easy to grow and identify, nonfastidious[30],[31], it can efficiently and rapidly invade and colonizes dentinal tubules in vitro and has the ability to withstand long period of starvation and high pH levels [32–35].

The DB model appears to be an adequate and efficient tool for the study of interactions between the local endodontic medicaments, bacteria and dentin[15] as it has a small particle size and can easily be delivered in standardized quantities, which allows controlled mixing with a variety of disinfecting agents as well as microorganisms [36].

In the present study the autoclaving was done before powdering because when roots were autoclaved before gates glidden grinding, the powder was relatively easy to handle. However, if autoclaving was done after powdering, the physical properties, possibly cohesive forces, of the dentin were changed in such a way that made it difficult to divide it into equal portions [15].

Previous studies used BSA as an organic tissue inhibitor but in the present study HSA was used to simulate the clinical environment. Despite there are no data available about the amount of albumin and other proteins entering the root canal in inflammatory exudate or the amount of DB formed during chemomechanical preparation of the root canal [36], 28 mg DB and HSA was added to simulate the organic load content inside the root canal that could influence the antimicrobial capability of intracanal medication according to previous studies[4],[8],[9],[13],[15],[37],[38].

In our modified model we tried to adapt the parameters more relevant to the endodontic clinical practice. We have used 1 min, 24 and 72 h time measuring points instead of 10, 30 s as described by others[15],[37]. Seventy-two hours measuring time point was chosen to resemble the duration needed for intracanal medication in the in vivo condition and to avoid inaccurate results due to short experimental times. On the other hand, short measuring time points were chosen in order to test the bactericidal activity of the assigned antimicrobial agents, as these agents could be used as irrigating agents.

The present study showed that, 2% CHX was effective on E. faecalis even in the presence of inhibitor at 24 and 72 h as no significant results were found between groups 2, 4, and 6. These finding may be explained by the fact that CHX has a wide antimicrobial spectrum and it is effective against both gram-positive and gram-negative bacteria [39]. This was supported by Athanassiadis et al. [40] who proved that CHX has much greater activity against gram-positive than gram-negative organisms and this finding explaining the great reduction of E. faecalis count in the present study which are gram-positive bacteria.

Furthermore, these findings may be attributed to electrostatic interaction between CHX and bacterial cell wall as it is positively charged, and the bacterial wall is negatively charged[41],[42]. Moreover, CHX solution was used in the present study in 2% concentration which could be a possible reason of great inhibition of tested microorganisms. Furthermore, CHX can bind to surrounding tissues and then be released again slowly at therapeutic levels over extended periods of time, a phenomenon known as substantivity[30],[43]. Interestingly, the ability of CHX to efficiently inhibit the initial adherence, further accumulation and biofilm formation of fungi and other microorganisms may played an important role for its best antimicrobial effect [44].

The results of the present study at 24 and 72 h were in agreement with Haapasalo et al. [15] and Portenier et al. [8],[9],[45] who found CHX was effective on E. faecalis in the presence of DB and BSA. They found that binding to DB has resulted in a reduced, but not abolished, activity which can be seen as delayed killing of the bacteria [15].

In contrary, another lower concentration of CHX (0.05%) was tested by Haapasalo et al. [15] who observed that even after 24 h. E. faecalis was reduced but not totally eliminated by CHX in the presence of DB. This could be attributable to the lower CHX concentration used in that study and they used bovine dentin, whose chemical composition has been poorly investigated and different than human dentin which may influence the intensity of the dentin buffering effect, responsible for the inhibition of irrigants. Portenier et al. [8],[45] also found that when BSA was incubated, together with CHX 0.02 and 0.05% and bacteria, much of the antibacterial effect of CHX was lost even after 24 h of incubation and more than 20% of E. faecalis cells were viable. Such controversial results may be explained by the lower CHX concentrations used in that study.

The efficacy of CHX at 1 min was lower than that after 24 and 72 h and the inhibitory effect of HSA was significantly higher than that of DB. However no specific mechanism has been suggested for the inhibitory effect of different organic components, it is generally known that organic material can inactivate or weaken the effect of CHX [46]. Additionally, the inorganic fraction of dentin is also known to play an important role as an inhibitor because of its buffering action [47].

These results were in agreement with Morgental et al. [37] and Herczegh et al. [13]. These results were also supported by the finding of Quintana et al. [38] who observed that 2% CHX was unable to eliminate E. faecalis within 2 min in the presence of BSA. However, the same study [38] showed that 2% CHX was able to eliminate E. faecalis within 2 min in the presence of DB. These discrepancies may be explained by the difference in the methodologies. Also, Sassone et al. [48] and Sassone et al. [49] noted that CHX are able to eliminate E. faecalis immediately in the presence of BSA. Lower BSA concentrations used in those studies could explain the complete bacterial eradication.

Results of this study showed that, CSnp possess strong antibacterial effect against E. faecalis as shown by group 5 at 1 min, 24 and 72 h. This antibacterial effect was not affected by presence of the DB but it was significantly inhibited by HSA at all time intervals.

The antibacterial mechanism of chitosan has not yet been fully clarified. Several hypotheses have been postulated, based on its cationic nature [50]. Chitosan with low molecular weight was considered to have the ability to penetrate the bacterial cell membrane and then to bind to the DNA, inhibiting its transcription and mRNA synthesis, while chitosan with high molecular weight was surmised to bind to the negatively charged components of the bacterial cell wall, forming an impermeable layer and blocking transportation into the cell [51].

Another alternative hypothesis for the antibacterial mechanism of chitosan is thought to be as a result of its ability to bind to the negatively charged bacterial cell membrane, increasing its permeability and ultimately resulting in leaking of the cytoplasmic contents and bacterial cell death [52]. Others postulated that as chitosan has the ability to chelate metals, microbial growth was inhibited by reducing enzyme activity through metal chelation [53].

The cationic CSnp is highly reactive and interacts physicochemically with other charged particles. The components of pulp and BSA could also interact physicochemically with CSnps, compromising their antibacterial efficacy [11] and this could explain its compromised antibacterial action against E. faecalis in the presence of HSA.

However, dentin and dentin matrix showed a lesser inhibitory effect. The weaker chemical interaction of CSnps with dentin matrix components could have resulted in the lack of inhibition of its antibacterial efficacy [11] and this could explain its efficient antibacterial action against E. faecalis in the presence of DB.

The results of the present study were in agreement with Shrestha et al. [54], who revealed that 10 mg/ml CSnps eliminated planktonic E. faecalis after 8 h in contrast to E. faecalis biofilm which survived even after 72 h. Also, this was supported by Kishen et al. [55] who showed that when dentin was treated with CSnps, the bacterial adherence was reduced significantly. Furthermore, the results of the present study were in agreement with Shrestha and Kishen [11] where CSnps was effective on E. faecalis in the presence of DB and strongly inhibited by BSA at 24 h.

Within the limitations of this study, it can be concluded that two percent CHX and CSnps 10 mg/ml had good antibacterial activity against E. faecalis. HSA had significant inhibitory effect on antibacterial activity of CHX and CSnps than DB in a short period of time (1 min). At longer periods of time (24 and 72 h), neither HSA nor DB had significant inhibitory effect on antibacterial activity of CHX while HSA had a significant effect on antibacterial activity of CSnps.



Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

 
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