Journal of Conservative Dentistry
Home About us Editorial Board Instructions Submission Subscribe Advertise Contact e-Alerts Login 
Users Online: 1093
Print this page  Email this page Bookmark this page Small font sizeDefault font sizeIncrease font size
 


 
Table of Contents   
ORIGINAL RESEARCH ARTICLE  
Year : 2016  |  Volume : 19  |  Issue : 5  |  Page : 465-468
Capacity of a hydroxyapatite–lysozyme combination against Streptococcus mutans for the treatment of dentinal caries


Department of Restorative Dentistry, School of Dentistry, Pontifícia Universidade Católica de Campinas, Campinas, São Paulo, Brazil

Click here for correspondence address and email

Date of Submission01-Mar-2016
Date of Decision29-Aug-2016
Date of Acceptance09-Aug-2016
Date of Web Publication7-Sep-2016
 

   Abstract 


Background: One current strategy for the treatment of carious lesions is the use of biomaterials with antimicrobial activity.
Aims: The aim of this study was to evaluate a combination of hydroxyapatite and lysozyme for the treatment of dentinal caries by measuring Streptococcus mutans counts before carious tissue sealing, and 24 h, 1 month, and 6 months after treatment.
Materials and Methods: Forty permanent third molars were selected, and flat dentin surfaces were prepared. The teeth were exposed to a cariogenic challenge with S. mutans. After challenge, the dentinal caries were collected from five specimens. The remaining specimens were treated with a mixture of hydroxyapatite and lysozyme in sodium laureth sulfate and sealed with composite resin. S. mutans counts were obtained 24 h, 1 month, and 6 months after sealing.
Statistical Analysis: The results were evaluated by descriptive statistics and Wilcoxon signed-rank test.
Results: a significant reduction in S. mutans (CFU/mL) was observed in dentinal lesions 1 month after treatment with hydroxyapatite/lysozyme in sodium laureth sulfate (P = 0.0254). Comparison of S. mutans counts obtained 24 h, 1 month, and 6 months after treatment revealed reductions only at the 1-month time point (P = 0.0318).
Conclusions: the combination of hydroxyapatite and lysozyme may be an alternative for reducing the S. mutans burden in dentinal caries.

Keywords: Dental caries; enzyme; Streptococcus mutans

How to cite this article:
Pinheiro SL, da Rocha NN, Peres MH. Capacity of a hydroxyapatite–lysozyme combination against Streptococcus mutans for the treatment of dentinal caries. J Conserv Dent 2016;19:465-8

How to cite this URL:
Pinheiro SL, da Rocha NN, Peres MH. Capacity of a hydroxyapatite–lysozyme combination against Streptococcus mutans for the treatment of dentinal caries. J Conserv Dent [serial online] 2016 [cited 2020 Apr 8];19:465-8. Available from: http://www.jcd.org.in/text.asp?2016/19/5/465/190026



   Introduction Top


Dental caries are a very common condition caused by bacteria that form a biofilm on the surface of the teeth. In the presence of sugars or fermentable carbohydrates, the bacteria present in the biofilm produce acids, which demineralize the dental surface.[1] One current strategy for the treatment of carious lesions is the use of biomaterials with antimicrobial activity.[2],[3]

From a histological standpoint, dentin caries can be classified as infected dentin or affected dentin.[4] The first layer exhibits extensive decalcification and degeneration of collagen fibers. The second layer is characterized by intermediate decalcification, reversible alterations in collagen fibers, and odontoblasts undergoing active re-calcification. Minimally invasive dentistry recommends the removal of infected dentin and preservation of the caries-affected dentin.[4],[5]

Carious dentin can be regenerated by hydroxyapatite crystals, which penetrate and obliterate the dentinal tubules.[2],[3],[4],[5],[6] Carbonated hydroxyapatite nanocrystals are compatible in size, morphology, chemical composition, and crystal structure with native dentin, and can thus be used to re-mineralize enamel.[2]

In the same line, another agent that may help re-organize dentin affected by caries is lysozyme, which has antimicrobial properties. This agent has been reported to exert an inhibitory, bactericidal effect on oral pathogens, including Streptococcus mutans.[3]

Lysozyme induces bacterial lysis by breaking down linkages between N-acetylmuramic acid and N-acetylglucosamine in the cell wall, thus neutralizing pathogenicity in Gram-positive and Gram-negative bacteria by degrading peptidoglycans in the bacterial cell wall.[7]

S. mutans, a Gram-positive oral pathogen, is considered the primary etiologic agent of dental caries. Some oral Gram-positive bacteria, including S. mutans, are resistant to direct lysis by lysozyme.[8] Instead, lysozyme may act on S. mutans by increasing cell permeability.[9]

The aim of this study was to evaluate the antimicrobial activity of a combination of hydroxyapatite and lysozyme on viable S. mutans counts before the sealing of caries-affected tissue, and after 24 h, 1 month, and 6 months after the sealing.


   Materials and Methods Top


This study was approved by the PUC-Campinas Research Ethics Committee (protocol #388.376).

Sample selection

Forty permanent third molars were selected at the PUC-Campinas dental clinic. All donor patients signed an informed consent form. The criteria for inclusion were permanent third molars with no cracks or fractures. After sample selection, the occlusal third was removed from each specimen using a double-sided diamond disc (KG Sorensen Indústria e Comercio Ltda, São Paulo, Brazil) using a low-speed handpiece (KaVo Dental Excellence Ind. Com. Ltda, Joinville, Santa Catarina, Brazil) under water cooling for dentin exposure, and the dentin surfaces were polished with wet silicon carbide sandpaper sheets, P600 grit (Água T223 advance, Norton, São Paulo, Brazil). A 4 mm × 4 mm paper label (Kalunga, São Paulo, Brazil) was placed onto each specimen to standardize the location of the carious lesion.

Under a laminar flow hood (Veco, Campinas, São Paulo, Brazil), the specimens were sealed using epoxy resin (Araldite, São Paulo, Brazil) and nail polish (Colorama, São Paulo, Brazil), except on the coronal dentin. After sealing, the label was removed from the occlusal third of each specimen to enable generation of the carious lesion.

Teeth were then exposed to a cariogenic challenge in brain–heart infusion (BHI) broth (LabCenter, São Paulo, Brazil), supplemented with 0.5% yeast extract (LabCenter, São Paulo, Brazil), 1% glucose (LabCenter, São Paulo, Brazil), 1% sucrose (LabCenter, São Paulo, Brazil), and S. mutans type strain ATCC 25175 (Fundação André Tosello, Campinas, São Paulo, Brazil), standardized to 0.5 McFarland turbidity (Probac do Brasil Produtos Bacteriológicos Ltda., São Paulo, Brazil). Samples were incubated in anaerobic jars (Oxoid Ltd., Basingstoke, Hampshire, England) at 37°C and subsequently stored in a bacteriological incubator (Fanem Ltda, São Paulo, Brazil) for 14 days. During this period, BHI broth (LabCenter, São Paulo, Brazil) was replaced for every 48 h.

After the cariogenic challenge, dentinal carious lesions were collected from ten specimens, placed in BHI broth (LabCenter, São Paulo, Brazil) and homogenized for 3 min in a vortex mixer (Prolab, São Paulo, Brazil). Five decimal dilutions were performed, and three 25 μL aliquots from each dilution were seeded onto the surface of mitis-salivarius-bacitracin medium (LabCenter, São Paulo, Brazil). All plates were incubated in the anaerobic jars (Oxoid Ltd., Basingstoke, Hampshire, England) with gas-generating envelopes (Probac do Brasil Produtos Bacteriológicos Ltda., São Paulo, Brazil) for 5 days at 37°C. After incubation, the viable bacterial count was determined.

The remaining samples were treated with hydroxyapatite and lysozyme in sodium laureth sulfate, applied with a microbrush (KG Sorensen Indústria e Comercio Ltda, São Paulo, Brazil) for 1 min onto the carious lesion. Specimens were then sealed with a composite resin (Dentsply, São Paulo, Brazil). The mixture of lysozyme and hydroxyapatite in sodium laureth sulfate was compounded as follows: 0.018 mg of 1% lysozyme (Sigma-Aldrich, São Paulo, Brazil) and 0.045 mg of 3% hydroxyapatite (Sigma-Aldrich, São Paulo, Brazil), both in powder form, were mixed in 1.8 mL sodium laureth sulfate (Tergentol ®, Fórmula and Ação, São Paulo, Brazil) as vehicle. S. mutans counts were obtained 24 h, 1 month, and 6 months after sealing (n = 10 specimens per time point). For this procedure, the composite resin seal was removed with a round diamond bur (KG Sorensen Indústria e Comercio Ltda, São Paulo, Brazil) in a high-speed handpiece (Kavo Dental Excellence Ind. Com. Ltda, Joinville, Santa Catarina, Brazil), under saline cooling (Dauf, Fortaleza, Ceará, Brazil), to enable removal and collection of the carious lesions. All microbiological procedures (homogenization, dilution, and seeding) were described for counts obtained before sealing.

The results were evaluated by descriptive statistics and the Wilcoxon signed-rank test.


   Results Top


A significant reduction in S. mutans counts (CFU/mL) was observed in dentinal lesions 1 month after the treatment with hydroxyapatite/lysozyme in sodium laureth sulfate (P = 0.0254). S. mutans counts obtained 24 h after treatment were not significantly reduced as compared with baseline counts or those obtained at 6 months (P > 0.05). Comparison of S. mutans counts obtained 24 h, 1 month, and 6 months after the treatment revealed reductions at the 1-month time point (P < 0.05) [Table 1].
Table 1: Medians and interquartile ranges (Wilcoxon signed-rank test) of carious lesion samples before treatment and 24 h, 1 month, and 6 months after treatment with hydroxyapatite/lysozyme in sodium laureth sulfate (log10)

Click here to view



   Discussion Top


Several substances with antimicrobial activity that may be used as adjuncts in the re-mineralization process of caries-affected teeth have been described in literature, including lysozyme and hydroxyapatite, as used in the present study. Combining antimicrobial agents broadens their spectrum of activity to inactivate many of the bacterial species present in the carious lesion, thus halting disease progression.[10]

A significant reduction in S. mutans counts was observed 1 month after treatment. This may be explained by the antimicrobial activity of lysozyme and by the effect of cavity sealing. This significant reduction in S. mutans 1 month after sealing is consistent with previous reports in the literature,[7],[8],[9],[10],[11] which state that lysozyme has a beneficial effect on the re-organization of caries-affected dentin by exerting antimicrobial activity.

In this study, the bactericidal effect of lysozyme was observed only 1 month after treatment. There are three possible explanations for this finding. The first possible explanation for this late effect of lysozyme is that S. mutans can synthesize extracellular polysaccharides such as glucan, which may reduce the permeability to lysozyme of the biofilm present in the carious lesion. Several active enzymes have been identified among the components of the dental pellicle, including lysozyme itself,[12] bacterial glycosyltransferase, and fructosyltransferase.[13] The lysozyme component of the pellicle has bactericidal activity and reduces the adhesion of S. mutans to hydroxyapatite.[14] The presence of lysozyme affects the amount of glucan produced by glycosyltransferase B, but it has no effect on the glucan structure.[15] Another possible explanation for an effect of lysozyme being observed only at the 1-month time point could be the acidic pH of the caries environment; this hinders enzyme activity, which is immediate at neutral pH.[16] The last hypothesis is that lysozyme acts as a cationic, low-molecular weight protein that is capable of inducing bacterial lysis by catalyzing the breakage of glycosidic bonds [8] between N-acetylmuramic acid and N-acetyl-D-glucosamine in the polysaccharide backbone of the bacterial cell wall.[12] Curiously, many Gram-positive bacteria in the oral flora, including S. mutans, are resistant to direct lysis by lysozyme.[8] Therefore, lysis does not necessarily have to be the primary cause of cell death induced by lysozyme. Lysozyme may have a bactericidal mechanism of action similar to that of the other cationic peptides, which act on the cell membrane to produce a loss of selective permeability [17] and increasing membrane permeability to electrolytes, which is followed by osmotic changes within the cell.[9] Bacterial membranes work as proton barriers and are capable of maintaining a relatively basic cytoplasm even in an acidic environment by proton extrusion, generally through proton translocation driven by membrane-bound ATPase.[16] In S. mutans, lysozyme causes massive potassium loss, which may lead to a marked decline in membrane potential, as K+ is the major monovalent cation in bacterial cells; loss of the energy-dependent membrane transport function; inactivation of potassium-dependent cellular enzymes; loss of turgor pressure; cessation of growth; and cell death.[17] This is consistent with the findings of this study at the 1-month time point.

After 6 months, S. mutans counts had increased, possibly due to degradation of the tooth restoration interface and bacterial infiltration and growth, as the samples were submerged in BHI, a nutrient-rich medium.


   Conclusion Top


The results of this study show that incorporation of lysozyme and hydroxyapatite into composite resin reduced S. mutans counts 1 month after sealing, which is justified by the antimicrobial and re-mineralizing properties of these compounds. This combination may be a feasible alternative for the reduction of S. mutans burden in dentinal caries.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

 
   References Top

1.
Venault A, Yang HS, Chiang YC, Lee BS, Ruaan RC, Chang Y. Bacterial resistance control on mineral surfaces of hydroxyapatite and human teeth via surface charge-driven antifouling coatings. ACS Appl Mater Interfaces 2014;6:3201-10.  Back to cited text no. 1
    
2.
Huang SB, Gao SS, Yu HY. Effect of nano-hydroxyapatite concentration on remineralization of initial enamel lesion in vitro. Biomed Mater 2009;4:034104.  Back to cited text no. 2
    
3.
Felizardo KR, Gonçalves RB, Schwarcz WD, Poli-Frederico RC, Maciel SM, Andrade FB. An evaluation of the expression profiles of salivary proteins lactoferrin and lysozyme and their association with caries experience and activity. Science Dent J. (Online) 2010;25:344-9.  Back to cited text no. 3
    
4.
Banerjee A, Watson TF, Kidd EA. Dentine caries excavation: a review of current clinical techniques. Br Dent J 2000;188:476-82.  Back to cited text no. 4
    
5.
Rimondini L, Palazzo B, Iafisco M, Roveri LN. The remineralizing effect of carbonate-hydroxyapatite nanocrystals on dentine. Mater Sci Forum 2007;1:539-43.  Back to cited text no. 5
    
6.
Rao A, Malhotra N. The role of remineralizing agents in dentistry: a review. Compend Contin Educ Dent 2011;32:26-33.  Back to cited text no. 6
    
7.
Shimada J, Moon SK, Lee HY, Takeshita T, Pan H, Woo JI, et al. Lysozyme M deficiency leads to an increased susceptibility to Streptococcus pneumoniae-induced otitis media. BMC Infect Dis 2008;8:134.  Back to cited text no. 7
    
8.
Arnold RR, Brewen M, Gauthier JJ. Bactericidal activity of human lactoferrin: Sensitivity of a variety of microorganisms. Infect Immun 1980;28:893-8.  Back to cited text no. 8
    
9.
Kagan BL, Selsted ME, Ganz T, Lehrer RI. Antimicrobial defensin peptides form voltage-dependent ion-permeable channels in planar lipid bilayer membranes. Proc Natl Acad Sci U S A 1990;87:210-4.  Back to cited text no. 9
    
10.
Thomas EL, Aune TM. Lactoperoxidase, peroxide, thiocyanate antimicrobial system: correlation of sulfhydryl oxidation with antimicrobial action. Infect Immun 1978;20:456-63.  Back to cited text no. 10
    
11.
Terán WI. Actividad antiproliferativa de la lisozima en su forma nativa y modificada. Actual Nutr 2013;15:107-14.  Back to cited text no. 11
    
12.
Rölla G, Ciardi JE, Bowen WH. Identification of IgA, IgG, lysozyme, albumin, alpha-amylase and glucosyltransferase in the protein layer adsorbed to hydroxyapatite from whole saliva. Scand J Dent Res 1983;91:186-90.  Back to cited text no. 12
    
13.
Vacca-Smith AM, Venkitaraman AR, Schilling KM, Bowen WH. Characterization of glucosyltransferase of human saliva adsorbed onto hydroxyapatite surfaces. Caries Res 1996;30:354-60.  Back to cited text no. 13
    
14.
Roger V, Tenovuo J, Lenander-Lumikari M, Söderling E, Vilja P. Lysozyme and lactoperoxidase inhibit the adherence of Streptococcus mutans NCTC 10449 (serotype c) to saliva-treated hydroxyapatite in vitro. Caries Res 1994;28:421-8.  Back to cited text no. 14
    
15.
Kho HS, Vacca Smith AM, Koo H, Scott-Anne K, Bowen WH. Interactions of Streptococcus mutans glucosyltransferase B with lysozyme in solution and on the surface of hydroxyapatite. Caries Res 2005;39:411-6.  Back to cited text no. 15
    
16.
Kobayashi H. A proton-translocating ATPase regulates pH of the bacterial cytoplasm. J Biol Chem 1985;260:72-6.  Back to cited text no. 16
    
17.
Wang YB, Germaine GR. Effect of lysozyme on glucose fermentation, cytoplasmic pH, and intracellular potassium concentrations in Streptococcus mutans 10449. Infect Immun 1991;59:638-44.  Back to cited text no. 17
    

Top
Correspondence Address:
Sérgio Luiz Pinheiro
Rua Raul Gasparini, no 525 Bairro Panorama, Vinhedo, Sao Paulo 13280-000
Brazil
Login to access the Email id

Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0972-0707.190026

Rights and Permissions



 
 
    Tables

  [Table 1]



 

Top
 
 
 
  Search
 
 
    Similar in PUBMED
   Search Pubmed for
   Search in Google Scholar for
 Related articles
    Email Alert *
    Add to My List *
* Registration required (free)  
 


    Abstract
   Introduction
    Materials and Me...
   Results
   Discussion
   Conclusion
    References
    Article Tables

 Article Access Statistics
    Viewed1874    
    Printed16    
    Emailed0    
    PDF Downloaded151    
    Comments [Add]    

Recommend this journal