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Table of Contents   
ORIGINAL RESEARCH  
Year : 2020  |  Volume : 31  |  Issue : 5  |  Page : 768-773
Esterase like activity of Enterococcus faecalis and Lactobacillus casei on microhardness and weight loss of resin luting cements


1 Department of Conservative Dentistry and Endodontics, Thai Moogambigai Dental College & Hospital, Dr. MGR Educational and Research Institute (Deemed to be University), Chennai, Tamil Nadu, India
2 Department of Biotechnology, Dr. A.P.J Abdul Kalam Centre for Excellence in innovation and entrepreneurship, Dr. M.G.R Educational and Research Institute, Maduravoyal, Chennai, Tamil Nadu, India

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Date of Submission26-Jul-2020
Date of Decision24-Oct-2020
Date of Acceptance27-Oct-2020
Date of Web Publication08-Jan-2021
 

   Abstract 


Introduction: Gap-free/continuous cement margins have been considered important for the longevity of indirect dental restorations. Bacterial species have demonstrated esterase-like activity that can cause biodegradation of resin composites. Aim: The aim of this study was to evaluate the effect of the esterase-like activity of E. faecalis and L. casei on three resin luting cements. Settings and Design: In-vitro study materials and three resin luting cements tested were: Variolink N, Rely X U200 and Panavia F2.0. E. faecalis and L. casei suspensions and supernatants were assessed for enzymatic activity by bacterial esterase activity assay. Circular samples of resin luting cements were exposed to suspensions of E. faecalis and L. casei for 7 and 28 days followed by testing for solubility, microhardness and bishydroxy propoxy phenyl propane (BisHPPP) release. Results: E. faecalis and L. casei both demonstrated esterase-like activity. Bacterial suspensions had significantly increased enzymatic activity than supernatant solutions (P < 0.05). There was no significant reduction in microhardness or increased weight loss in all three cements after incubation in E. faecalis and L. casei for 7 and 28 days. BisHPPP release signifying resin degradation was seen after 7 and 28 days of incubation in E. faecalis and L. casei. Conclusion: Within the limitations of this in vitro study, E. faecalis and L. casei demonstrated esterase-like activity. BisHPPP release was evident in all three cements after 7 and 28 days. However, the bacterial strains did not significantly reduce the microhardness or cause weight loss of the tested resin luting cements (Variolink N, Panavia F2.0 and Rely X U200) after 7 and 28 days of incubation.

Keywords: E. faecalis, esterase activity, L. casei, microhardness, resin luting cements

How to cite this article:
SreeVidya G, Archana D, Prithika U, Sivapriya E, Tejaswi B, PradeepKumar AR. Esterase like activity of Enterococcus faecalis and Lactobacillus casei on microhardness and weight loss of resin luting cements. Indian J Dent Res 2020;31:768-73

How to cite this URL:
SreeVidya G, Archana D, Prithika U, Sivapriya E, Tejaswi B, PradeepKumar AR. Esterase like activity of Enterococcus faecalis and Lactobacillus casei on microhardness and weight loss of resin luting cements. Indian J Dent Res [serial online] 2020 [cited 2021 Jan 28];31:768-73. Available from: https://www.ijdr.in/text.asp?2020/31/5/768/306460



   Introduction Top


Leakage of bacteria and bacterial elements along the margins of restorations and root canal fillings can lead to treatment failure.[1],[2] Though well prepared and filled root canals may resist bacterial penetration,[3] leaking coronal restorations can play an important role in treatment failure.[1]

Previous studies have shown that  Enterococcus faecalis Scientific Name Search aecalis),[4]  Streptococcus mutans Scientific Name Search ns)[5] and salivary derived enzymes[6] can degrade the dentin–methacrylate interface and increase the interfacial bacterial biofilm proliferation leading to restoration failure. Also, human salivary enzymes and bacterial esterases can readily catalyse the biodegradation of resin composites, adhesives, and adversely affect the physical properties of root canal sealers.[4],[7],[8] The degradation of resin composites and adhesives was assessed by the release of bishydroxy propoxy phenyl propane (BisHPPP), a BisGMA-derived product using high-performance liquid chromatography (HPLC).[4],[7]

Resin cements can be used for cementing ceramic restorations and prefabricated fibre posts.[9] However, adhesive restorations are less effective in preventing secondary caries which can form due to various factors such as high caries risk profile of the host, technique-sensitive placement procedure and degradation through marginal gaps in restoration-tooth interface caused by polymerisation shrinkage on curing.[6],[10] Also, the luting cements can be exposed to the oral environment at the margins which may lead to degradation.[11]

E. faecalis is a facultative anaerobic Gram-positive bacterium that can invade dentinal tubules and survive nutritional deprivation.[12],[13] E. faecalis has been reported to adapt to the habitat of filled root canals better than other microorganisms.[14] E. faecalis has been found to possess esterase-like activity that can degrade methacrylate resin.[4] Lactobacillus casei (L. casei) is a Gram-positive, facultative anaerobe identified in significant proportion in dentinal caries[15] and is also reported to have active esterases.[16]

While there have been studies investigating the effect of bacteria and salivary enzymes on methacrylate-based resin composites,[4],[8] the effect of bacteria on resin luting cements is yet to be explored.

Therefore, this in-vitro study was formulated to evaluate the effect of the esterase-like activity of E. faecalis and L. casei on microhardness, weight loss and BisHPPP release of different resin luting cements (Variolink N, RelyX U200 and Panavia F2.0). The null hypothesis was that the bacterial esterase-like activity evaluated does not affect the investigated resin luting cements.


   Materials and Methods Top


Preparation of samples

Three resin luting cements were selected: Variolink N (Ivoclar Vivadent, Schaan, Liechtenstein), Panavia F 2.0 (Kuraray, Okayama, Honshu, Japan) and RelyX U200 (3M EPSE, Seefeld, Bavaria, Germany). They were prepared according to the manufacturer's instructions, placed into a metal mould measuring 10 mm in diameter and 1 mm height.[8] A clear matrix sheet was placed on both sides of the mould[5] which was then light-cured for 20 seconds (LED Bluephase N, Ivoclar Vivadent, Schaan, Liechtenstein) on each side.

Two bacterial species used to test the resin luting cements were E. faecalis ATCC 29212 (American Type Culture Collection) and L. casei MTCC (Microbial Type Culture Collection) strains.

Three groups were tested: Group 1- Variolink N (n = 35), Group 2- Panavia F2.0 (n = 35) and Group 3- Rely X U200 (n = 35). Each group was further subdivided into two sub-groups based on the bacterial strain used: Subgroup 1A (n = 10) Variolink N in E. faecalis; 1B (n = 10) Variolink N in L. casei; 2A (n = 10) Panavia F2.0 in E. faecalis; 2B (n = 10) Panavia F2.0 in L. casei; 3A (n = 10)- RelyX U200 in E. faecalis; and 3B (n = 10) RelyX U200 in L. casei. Control samples were incubated in brain–heart infusion (BHI) (n = 6) and De Man, Rogosa and Sharpe Agar (MRS) (n = 6) for each cement group.

A total of 105 circular resin luting cement samples were prepared. The prepared samples were incubated to enable complete setting for 72 h at 37°C and 95–100% humidity.[8] Wet-polishing of sample surfaces was done sequentially with silicon carbide-based sandpapers 600, 800, 1000 and 1200 grit (Rhynowet, Indasa Redline, Secunderabad, Telangana, India).[8]

Preparation of bacterial suspension

Overnight cultures (10 ml) of E. faecalis in BHI and L. casei in MRS were raised. A portion (5 mL) of each was taken separately and heat inactivated (HIN) for 30 minutes at 80°C. The other half was diluted with saline (1:20) and incubated (37°C) until reaching the log phase. Bacterial suspensions with heat inactivation (HIN) and without heat inactivation were centrifuged at 13,000 rpm for 10 minutes (Eppendorf, Germany). A 0.22-mm pore size filter was used to filter the bacterial supernatant and phosphate-buffered saline (PBS) was used to resuspend the bacterial cells.[4]

Substrate preparation

The enzyme substrate was prepared by dissolving p-nitrophenyl acetate (Sigma Aldrich, St. Louis, Missouri, USA) in 1 mL methanol. 100 mL of 0.1 M sodium acetate with pH 5.0, was added to dilute the dissolved solution to obtain a final concentration of 1 mM. 1.0 mL of 0.05 M phosphate buffer (pH 7.0) was then added to 0.5 mL of the prepared p-nitrophenyl acetate solution (p-NPA).[7] Eight solutions were prepared; solution A (1 mL of E. faecalis suspension with 0.5 mL of p-NPA), solution B (1 mL of E. faecalis supernatant with 0.5 mL p-NPA), solution C (1 mL of HIN E. faecalis suspension with 0.5 mL p-NPA), and solution D (1 mL of HIN E. faecalis supernatant with 0.5 mL p-NPA), solution E (1 mL of L. casei suspension with 0.5 mL of p-NPA), solution F (1 mL of L. casei supernatant with 0.5 mL p-NPA), solution G (1 mL of HIN L. casei suspension with 0.5 mL p-NPA), and solution H (1 mL of HIN L. casei supernatant with 0.5 mL p-NPA).[4]

Esterase enzyme activity

Spectrophotometric measurements (Biorad, Hercules, California, U.S) were taken at 410 nm[7] for 1 mL of solutions A, B, C, D, E, F, G, and H to measure the nitrophenyl-dependent esterase-like activity of E. faecalis and L. casei [Figure 1] and [Figure 2]. Spectrometric measurements of 1 mL of BHI, PBS, p-NPA and MRS were tested as controls.
Figure 1: Enzymatic activity of Enterococcus faecalis

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Figure 2: Enzymatic activity of Lactobacillus casei

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Exposure to E. faecalis and L. casei

A total of 96 resin cement samples were incubated in sterile vials containing 2 mL of solution A (experimental n = 10 per subgroup 1A, 2A and 3A), solution E (experimental n = 10 per subgroup 1B, 2B and 3B) and controls (BHI (n = 6) and MRS (n = 6) per cement group) for 7 and 28 days[4],[8] resulting in A, B, C, D, E, F, G and H to measure the nitrophenyl-dependent esterase-like activity of E. faecalis and L. casei. Spectrometric measurements of 1 mL of BHI, PBS, p-NPA and MRS were a total of 32 samples per cement group. The broth was replaced every 48 hours.[4] Solutions (1 mL) from each subgroup (n = 1) were collected from the vials after 7 days and 28 days. Methanol (100%) was added to these collected solutions to halt the enzymatic activity.[4] The solutions were then filtered and refrigerated for further HPLC analysis.

Pre-experimental weight evaluation

A total of 48 samples (five from each subgroups 1A, 1B, 2A, 2B, 3A, and 3B and 18 controls including three in BHI and three in MRS for each cement group) were kept in a desiccator at 37 ± 1°C for 22 h. Thereafter, all samples were stored in a second desiccator at 25 ± 1°C for 2 h and the samples were weighed using a precision analytical balance (Shimadzu, Kyoto, Japan) until a constant weight for each sample (W1) was obtained.[17]

Solubility evaluation by measuring post-experimental weight

After a 7-day incubation period, the samples were stored in a desiccator as described above and weighed again (W2). After 28 days, the same 48 samples (30 experimental and 18 controls) were weighed after (W3) storing in the desiccator. The difference between the pre-incubation weight (W1) and post-incubation weights (W2 and W3) was calculated.[8],[17]

Microhardness test

A total of 48 samples (five from each sub-group 1A, 1B, 2A, 2B, 3A and 3B and 18 controls including three in BHI and three in MRS for each cement group) were retrieved after 7 days and then washed, dried and subjected to the microhardness test with a Vickers hardness tester (HDNS Kelly, Shanghai, China). The same 48 samples (30 experimental and 18 controls) were retrieved after 28 days and washed, dried and then again subjected to a microhardness test.[8],[18] Nine samples (Variolink-3; Rely X U200-3; and Panavia F2.0-3) were tested as controls.

HPLC analysis

A Varian HPLC system (Palo Alto, CA, USA) was used to assess the release of BisHPPP, a Bis-GMA derived product.[4] A Diamonsil C18 column (4.6 × 100 mm, 5 μm particle size) was used to achieve the chromatographic separation. One sample from each cement subgroup at 7 days and 28 days (totally 12 samples) was assessed.

Statistical analysis

The statistical analysis of the data was done using the Statistical Package for Social Sciences, IBM Corporation, SPSS Inc., Chicago, IL, USA version 21 software package (SPSS). Descriptive statistics including mean, standard deviation and frequencies were computed for various parameters. Normality of the data was assessed using the Shapiro–Wilk test. Further analysis was done using non-parametric tests since the data did not follow a normal distribution. Friedman test was used to compare the mean rank differences between the groups at different time intervals. Wilcoxon signed-rank test was used for comparison between paired groups. The level of significance in the present study was kept at P < 0.05.


   Results Top


Enzymatic activity of E. faecalis and L. casei

Solution A (E. faecalis suspension in BHI with 0.5 mL of p-NPA) significantly increased the esterase-like enzymatic activity than solution B (E. faecalis supernatant in BHI with 0.5 mL of p-NPA), C (HIN E. faecalis suspension in BHI with 0.5 mL of p-NPA), D (HIN E. faecalis supernatant in BHI with 0.5 mL of p-NPA) and controls (P < 0.05). Solution E (L. casei suspension in MRS with 0.5 mL of p-NPA) significantly increased the esterase-like enzymatic activity (P < 0.05) compared to solution F (L. casei supernatant in MRS with 0.5 mL of p-NPA), G (HIN L. casei suspension in MRS with 0.5 mL of p-NPA), H (HIN L. casei supernatant in MRS with 0.5 mL of p-NPA) and controls.

Microhardness

Rely X U200 exhibited the highest initial microhardness values followed by Variolink N and Panavia F 2.0 which was not statistically significant. There was no significant reduction in microhardness for all the three cements after incubation in E. faecalis and L. casei for 7 days (P > 0.05) and 28 days (P > 0.05) [Table 1].
Table 1: Microhardness values of tested cements (HV)

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Solubility

None of the tested cements showed significant weight loss [Table 2] after incubation for 7 days (P > 0.05) and 28 days (P > 0.05) in E. faecalis and L. casei.
Table 2: Weight of tested cements (gms)

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BisHPPP release

Bis HPPP release was [Table 3] evident in all three cements after 7 and 28 days of incubation.
Table 3: BisHPPP release from tested cements (μg/cm2)

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   Discussion Top


Resin luting cements can be used for luting indirect restorations and posts.[9] However, resin cements may become exposed to saliva and bacteria which can lead to degradation over time.[11]

The cholesterol esterase (CE)-like and pseudocholinesterase (PCE)-like activity of saliva[7] and CE-like activity of bacteria[4] can hydrolyse the synthetic matrix component of composite resin systems resulting in the following products BisHPPP,[4] triethylene glycol methacrylate (TEGMA) and methacrylic acid.[19] In this study, the impact of the esterase-like activity of E. faecalis and L. casei on resin cements was investigated. Variolink N is a dual-cure luting composite system. It is suitable for the cementation of indirect restorations made of composite resins, leucite-reinforced glass ceramics and lithium disilicate glass-ceramics.[20] Panavia F2.0 is a dual-cure, self-etch resin cement[21] which contains 10-methacryloyloxydecyl dihydrogen phosphate (10- MDP).[22] MDP has demonstrated a moisture-resistant chemical bonding potential to zirconia.[23] RelyX U200 is a dual-cure self-adhesive resin cement which had demonstrated adequate clinical success compared to conventional resin cement.[24]

E. faecalis was selected in the present study as it is the most common bacteria to be found in infected root canal–treated teeth.[25] The present study evaluated whether E. faecalis has an effect on resin cements used to lute fibre posts. L. casei was chosen as it is one of the most prevalent microorganisms in advanced caries.[15]

Bacteria in a culture media demonstrates different phases of growth namely lag, stationary and log phases.[4],[26] The esterase-like activity was found to increase in the log phase of bacteria.[4] Therefore, in the current study, E. faecalis and L. casei were cultured in the log phase.

The amount of filler content affected resin composite surface degradation after exposure to CE and more surface degradation was reported with higher filler content.[27] Also, adequate polymerization is essential for the stability and biocompatibility of the resin composite.[28] Dual-cure resin cements demonstrated a better degree of conversion from monomer to polymer than self-cure resin cements.[29] The filler contents of RelyX U200 and Panavia F2.0 are 43% vol[30] and 59% vol,[31] respectively. The filler content of Variolink N base is 46.7% vol and catalyst is 43.6% vol.[32] As the resin luting cements evaluated in this study have a lower filler content of 30%–66%,[33] and all the three resin cements used were dual cure resin cements, they may be more resistant to CE-mediated surface degradation.

In the current study, heat-inactivated E. faecalis and L. casei suspensions were used as negative bacterial controls and they showed a significantly reduced esterase-like activity compared with the bacterial suspensions without HIN. The bacterial suspension showed higher esterase-like activity than bacterial supernatant which was similar to the results reported by Marashdeh et al.[4] Therefore, in the current study, bacterial suspensions were used to incubate the resin samples.

All the three cements tested showed a decrease in microhardness values when incubated in E. faecalis and L. casei which was not significant. Increased filler content of the resin cement is associated with the better microhardness of the material.[29] However, there was no significant difference in the initial microhardness values of RelyX U200, Panavia F2.0 and Variolink N.

A study done by Marashdeh et al. reported a significant decrease in microhardness values of methacrylate-based resin composite after incubation in simulated human salivary esterase for 28 days.[8] The present study has reported no significant difference in microhardness values for all three tested resin cements after 28 days incubation in bacterial suspensions demonstrating esterase-like activity. This could be due to the dual polymerization ability of the resin cements used which can provide a better conversion of free monomers and maximize their mechanical properties.[34]

There was no significant weight loss seen in the resin luting cements tested when they were incubated in E. faecalis and L. casei for 28 days. Similar results were observed in previous studies that evaluated resin composites over a period of 28 days.[8]

10-MDP present in Panavia F2.0 can interact with calcium and form a nanolayering of 10-MDP_Ca salts in the adhesive and hybrid layers. The water resistance property of these calcium salts contributes to the long-term stability of 10-MDP–based adhesives.[35]

Previous studies have indicated that E. faecalis and S. mutans possess CE-like activity that can degrade resin composites.[4],[5] Simulated human salivary esterases were also found to affect resin composites and root canal sealers.[7],[8] In the current study, all the tested cements showed BisHPPP release after 7 days and 28 days of incubation in E. faecalis and L. casei which demonstrate that these bacterial strains can degrade the tested resin luting cements. Variolink N showed a lesser amount of BisHPPP release than Panavia F2.0 and RelyX U200. E. faecalis has been reported to possess the esterase-like activity that could significantly degrade resin composites and adhesives after 30 days of incubation with a significant increase in the release of the by-product BisHPPP.[4] Thus, the null hypothesis was accepted.

Limitations of the present study include the short incubation period of 28 days. An increased incubation period could give a different perspective on the effect of the esterase-like activity on resin cements since resin degradation can increase with time.[6]


   Conclusion Top


Within the limitations of this in vitro study, E. faecalis and L. casei demonstrated the esterase-like activity. Bis HPPP release was evident in all three cements after 7 and 28 days. However, the bacterial strains did not significantly reduce the microhardness or cause weight loss of the tested resin luting cements (Variolink N, Panavia F2.0 and Rely X U200) after 7 and 28 days of incubation.

Acknowledgements

The authors thank Dr. Kesavan MDS for statistical analysis.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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Correspondence Address:
Dr. Angambakkam Rajasekaran PradeepKumar
Professor and Head of the Department, Department of Conservative Dentistry and Endodontics, Thai Moogambigai Dental College & Hospital, Dr. MGR Educational and Research Institute (Deemed to be University), Chennai - 600095, Tamil Nadu
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijdr.IJDR_747_20

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