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Table of Contents   
ORIGINAL RESEARCH  
Year : 2020  |  Volume : 31  |  Issue : 6  |  Page : 916-920
Antimicrobial Capacity of a Hydroxyapatite–Lysozyme–Lactoferrin–Lactoperoxidase Combination Against Streptococcus mutans for the Treatment of Dentinal Caries


1 Department of Dentistry, Master's Program in Health Sciences, Pontificia Universidade Catolica de Campinas (PUC-Campinas), Campinas, SP, Brazil
2 Department of Dentistry, PUC-Campinas, Campinas, SP, Brazil

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Date of Submission01-Jun-2018
Date of Decision28-Sep-2020
Date of Acceptance29-Sep-2020
Date of Web Publication22-Mar-2021
 

   Abstract 


Context: Hydroxyapatite has shown to regenerate the mineralized layer of dentin, whereas the combination of the enzymes lysozyme, lactoferrin, and lactoperoxidase may exhibit antimicrobial properties against oral pathogens. Aims: To evaluate a combination of hydroxyapatite and lysozyme, lactoferrin, and lactoperoxidase for the treatment of dentinal caries by measuring viable Streptococcus mutans. Settings and Design: Laboratory study with experimental groups. Methods and Material: Carious lesions in 20 permanent third molars were treated with a combination of hydroxyapatite and the enzymes lysozyme, lactoferrin, and lactoperoxidase. Carious dentin was collected and homogenized in a vortex shaker. After homogenization, five decimal dilutions were performed. Three aliquots of 25 μL of each dilution were seeded onto the surface of mitis salivarius bacitracin (MSB) medium. All plates were incubated in anaerobic jars. After incubation, the viable bacterial count was determined. S. mutans counts were obtained before and 24 h, 1 month, and 6 months after treatment. Statistical Analysis Used: Descriptive statistical analysis and the Kruskal–Wallis test, supplemented by the Student–Newman–Keuls test. Results: A significant reduction in S. mutans counts was observed 24 h after sealing with a combination of hydroxyapatite, lysozyme, lactoferrin, and lactoperoxidase as compared to counts after 1 month and after 6 months (P < 0.05). Conclusions: The combination of hydroxyapatite with lysozyme, lactoferrin, and lactoperoxidase may be an alternative for S. mutans control in dentinal caries.

Keywords: Dentinal caries, hydroxyapatite, lactoferrin, lactoperoxidase, lysozyme, S. mutans

How to cite this article:
Pinheiro SL, da Silva CC, da Silva LA, Cicotti MP. Antimicrobial Capacity of a Hydroxyapatite–Lysozyme–Lactoferrin–Lactoperoxidase Combination Against Streptococcus mutans for the Treatment of Dentinal Caries. Indian J Dent Res 2020;31:916-20

How to cite this URL:
Pinheiro SL, da Silva CC, da Silva LA, Cicotti MP. Antimicrobial Capacity of a Hydroxyapatite–Lysozyme–Lactoferrin–Lactoperoxidase Combination Against Streptococcus mutans for the Treatment of Dentinal Caries. Indian J Dent Res [serial online] 2020 [cited 2021 Apr 15];31:916-20. Available from: https://www.ijdr.in/text.asp?2020/31/6/916/311656



   Introduction Top


Histologically, healthy dentin is composed of 80% organic and 20% inorganic matter.[1] The inorganic mineral component is made up of calcium hydroxyapatite crystals. The organic matrix of dentin consists of type I collagen with fractional inclusions of glycoproteins, proteoglycans, phosphoproteins, and sialoproteins embedded in amorphous ground substance.[2],[3],[4] When visualized under an atomic force microscope, the collagen fibers of healthy intertubular dentin show a periodicity (cross-banding distance) of approximately 67 nm, and diameters ranging from 90 to 120 nm. The percentage of type I collagen, phosphoproteins, and sialoproteins in the dentin-pulp complex is higher in healthy teeth than in teeth with carious enamel and dentin. These three proteins play an important role in dentinogenesis, assisting in the formation of reparative and reactive dentin.[4]

From a histological standpoint, dentin caries can be classified as superficial (infected dentin) or deep (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 recalcification.[5],[6] Six different layers are visible in carious dentin: 1 - irreversibly demineralized outer layer; 2- translucent layer; 3 - subtransparent layer; 4 - sclerotic layer; 5 - healthy dentin; and 6 - predentin. Layers 2, 3, and 4 are the repairable caries-affected tissues.[7]

The tenets of minimally invasive dentistry recommend the removal of infected dentin and preservation of caries-affected dentin. Sealing of the dentin-pulp complex restricts microbial nutrition, thus stopping the progression of carious lesions.[2],[3],[5],[6],[7],[8],[9]

Carbonated hydroxyapatite nanocrystals are compatible in size, morphology, chemical composition, and crystal structure with native dentin and can thus be used to remineralize enamel.[10] The optimal concentration of nanohydroxyapatite for remineralization of early-stage carious lesions in enamel is 10%.[11] Hydroxyapatite has been used in toothpastes and to seal pits and fissures. Hydroxyapatite crystals are able to penetrate efficiently into dentin tubules and obliterate them within 10 min, thus regenerating the mineralized layer of dentin.[10],[11],[12]

In the same line, another agent that may help reorganize dentin affected by caries, due to its antimicrobial properties, is a combination of the enzymes lysozyme, lactoferrin, and lactoperoxidase. Lysozyme, lactoferrin, and peroxidases are among the various antimicrobial agents associated with the immune function of saliva.[13],[14] These agents may exert an inhibitory and even bactericidal effect on oral pathogens, including Streptococcus mutans. The interaction of bacteria with the salivary components that form the biofilm on the enamel surface is associated with the selective adherence of Streptococcus to the surface. The salivary components that interact with Streptococcus are mucin, lysozyme, lactoperoxidase, agglutinin, proline, and secretory immunoglobulin. According to Wilhelmus,[15] 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. Just as Wilhelmus,[15] Shimada et al.[16] noted that lysozyme is an antimicrobial compound capable of degrading peptidoglycans in the bacterial cell wall and found that lysozyme plays an important role in protection against pathogen invasion in the middle ear, particularly in the early stages of infection. These investigators suggested that exogenous lysozyme may be a useful adjunct therapy in the treatment of otitis media, particularly in light of growing microbial resistance to currently used antibiotics. Davis et al.[17] highlighted that in addition to its muramidase activity, which catalyzed hydrolysis of peptidoglycans, lysozyme also has non-muramidase activity, which causes bacterial membrane lysis through antimicrobial cationic peptides.

Within this context, the present study sought to evaluate a combination of hydroxyapatite and lysozyme, lactoferrin, and lactoperoxidase for the treatment of dentinal caries by measuring total viable S. mutans counts before carious tissue treatment and 24 h, 1 month, and 6 months after treatment. The null hypothesis was that no differences in S. mutans counts would be detected after application of hydroxyapatite with lysozyme, lactoferrin, and lactoperoxidase.


   Subjects and Methods Top


This study was approved by the Institutional Research Ethics Committee and was conducted in accordance with the Helsinki Declaration of 1975, as revised in 2000.

Sample selection

Twenty permanent third molars were selected at the dental clinic of our institution. All donor patients signed an Informed Consent Form.

The specimen inclusion criteria were Healthy permanent third molars with no cracks or fractures under visualization by a 10× magnifying glass (Stemi DV4, Carl Zeiss, São Paulo, Brazil). After sample selection, the occlusal third was removed from each specimen using a double-sided diamond disc (KG Sorensen Indústria e Comércio Ltda., São Paulo, Brazil) using a low-speed handpiece (KaVo Dental Excellence Ind. Com. Ltda., Joinville, Santa Catarina, Brazil), under refrigeration, to expose the dentin surface. The dentin surfaces were polished with wet silicon carbide sandpaper sheets, P600 grit (Água T223 advance, Norton, Indústria Brasileira, São Paulo, Brazil). A 4 × 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, SP, Brazil), the specimens were then sealed using epoxy resin (Araldite, São Paulo, Brazil) and nail polish (Colorama, São Paulo, Brazil), except for the coronal dentin. After sealing, the label was removed from the occlusal third of each specimen to enable generation of the carious lesion.

Microbiological processing

Cariogenic challenge

To simulate caries-affected dentin, teeth were then exposed to a cariogenic challenge in brain–heart infusion (BHI) medium (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). The samples were incubated in anaerobic jars at 37°C and subsequently stored in a bacteriological incubator (Fanem Ltda., São Paulo, Brazil) for 15 days. During this period, BHI broth (LabCenter) was replaced every 24 h.

S. mutans counts

S. mutans counts were obtained 24 h, 1 month, and 6 months after sealing (N = five specimens per time point). The number of specimens was based on sample size calculation performed by analysis of variance, with a minimum difference between treatment means = 0.02, standard error = 0,008, number of treatments = 4, statistical power = 0.80, and alpha = 0.05.

After the cariogenic challenge, caries-affected dentin was collected using sterile curettes (Golgran, São Caetano do Sul, São Paulo, Brazil) of a size consistent with the induced lesions.

Carious tissue was collected from five specimens. The remaining specimens underwent acid etching of enamel with 37% phosphoric acid (FGM Produtos Odontológicos LTDA, Joinville, Santa Catarina, Brazil) for 15 s and were rinsed and dried with sterile cotton points. Carious lesions in dentin were treated with a combination of hydroxyapatite and the enzymes, lactoferrin, and lactoperoxidase (Sigma-Aldrich, São Paulo, São Paulo, Brazil) in Tergenform medium (Fórmula and Ação, São Paulo, São Paulo, Brazil). Briefly, 0.018 mg of each enzyme, in powder, form, was added to 0.018 mg of hydroxyapatite powder (Sigma-Aldrich). This blend was then diluted in 1.8 mL Tergenform medium (Fórmula and Ação) to yield a 1% suspension of enzymes in vehicle. The mixture thus compounded was applied with a microbrush (KG Sorensen Indústria e Comércio Ltda., São Paulo, Brazil) for 1 min onto the carious lesions. Specimens were then sealed with an adhesive system (Dentsply, São Paulo, Brazil) and composite resin restorations were performed.

Before carious tissue could be collected, teeth had to be unsealed. For this procedure, the composite resin seal (Dentsply, São Paulo, Brazil) was removed with a round diamond bur (KG Sorensen Indústria e Comércio 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).

For microbiological processing, all samples were placed in BHI medium (LabCenter) and homogenized for 3 min in a vortex mixer (Prolab, São Paulo, Brazil). Immediately after homogenization, five decimal dilutions were performed, and three 25-μL aliquots from each dilution were seeded onto the surface of mitis-salivarius-bacitracin (MSB) medium (LabCenter, São Paulo, Brazil). All plates were incubated in anaerobic jars (Oxoid Ltd., Basingstoke, Hampshire, England) with gas-generating envelopes (Probac do Brasil Produtos Bacteriológicos Ltda., São Paulo, Brazil) for 2 days at 37°C. After incubation, the viable bacterial count was determined.

Statistical analysis

Statistical analyses were carried out in BioEstat 4.0. The results (in colony forming units [CFU]/mL) were log-transformed and analyzed using descriptive statistics and the Kruskal–Wallis test, supplemented by the Student–Newman–Keuls test.


   Results Top


A significant reduction in S. mutans counts was observed 24 h after sealing with a combination of hydroxyapatite, lysozyme, lactoferrin, and lactoperoxidase as compared to counts after 1 month (P = 0.0248) and after 6 months (P = 0.0001). There was no significant difference between counts before and after 24 h (P = 0.1995) or before and after 1 month (P = 0.3360). The highest microbial counts were seen at 6 months, with a significant difference from counts measured before and after the 24-hour time point (P < 0.05). There was no significant difference in counts at 1 and 6 months (P > 0.05) [Table 1], [Figure 1].
Table 1: Minimum values (MN), maximum values (MX), medians (MD), interquartile ranges (IR), and statistical analysis of Kruskal–Wallis test (Student–Newman–Keuls test) between groups (log10)

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Figure 1: Microbial count of each sample in each group (log10)

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   Discussion Top


Several alternatives have been proposed in the literature to assist in remineralization of dental caries and provide antimicrobial activity, including the use of lysozyme, lactoferrin, lactoperoxidase, and hydroxyapatite, which were combined for application in the present study. The combination of antimicrobial agents to broaden their spectrum of activity has been previously described as an alternative to inactivate bacteria in carious lesions.[18],[19],[20]

The results of the present study reject the null hypothesis, as we observed a significant reduction (P < 0.05) in S. mutans counts 24 h after sealing with a combination of hydroxyapatite, lysozyme, lactoferrin, and lactoperoxidase as compared with counts at 1 and 6 months. This is consistent with the explanation provided by Wilhelmus[15] that 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. Just as Aminlari et al.,[21] Shimada et al.,[16] and Wilhelmus[15] stated that lysozyme has antimicrobial properties and is capable of degrading peptidoglycans in the bacterial cell wall.

Many Gram-positive bacteria in the oral flora, including S. mutans, are resistant to direct lysis by lysozyme. As it is a low-molecular-weight cationic protein, lysozyme may have a bactericidal mechanism of action similar to that of other cationic peptides, which act on the cell membrane to produce loss of selective permeability and increase membrane permeability to electrolytes, which is followed by osmotic changes within the cell. Thus, lysozyme causes massive potassium loss in S. mutans, which may result in inactivation of potassium-dependent enzymes and a marked decline in membrane potential.[22],[23]

The antimicrobial effect of lactoferrin is attributed to the fact that this enzyme restricts microbial access to iron, an essential element for metabolic processes. Lactoferrin remains bound to iron even at low pH (4.5–5.0), which is the result of bacterial metabolic activity.[24],[25],[26] Lactoferrin can also inhibit S. mutans adhesion, modulating cluster formation and dental biofilm development.[26],[27] Finally, according to Nocerino et al.[25] and Drago-Serrano et al.[28] lactoferrin can interact with the cell membrane in some bacterial species, altering its permeability and, consequently, causing bacterial lysis.

The significant reduction in S. mutans counts after 24 h may also be attributable to the effects of lactoperoxidase, which catalyses oxidation of thiocyanate ions through hydrogen peroxide to generate hypothiocyanous acid or the hypothiocyanite anion, both of which have antimicrobial activity.[29],[30],[31],[32] Hydroxyapatite, which in the present study was combined with lysozyme, lactoferrin, and lactoperoxidase, is known to help remineralize dentin. Its crystals are able to penetrate efficiently into dentin tubules and obliterate them, thus regenerating the mineralized layer of dentin.[10],[11],[12],[33]

The significant increases in S. mutans counts observed at the 1-month and 6-month time points may be explained by degradation of the restoration interface, as well as by infiltration. According to Ribeiro,[34] the lack of adhesion of restorative materials to the underlying dental structure leads to marginal microleakage, which is the single greatest factor having a negative influence on the longevity of restorations. Leakage at the tooth-restoration interface may lead to marginal degradation of restorations.

Bonding to dentin, as in the present study, is more challenging than bonding to enamel, due to the organic nature of dentin and to the moisture contained in the dentin tubules.[35],[36] Dentin is made up of water-based components with a distinct, variable morphology. When bonding to dentin, micromechanical retention is considered one of the most important factors. It occurs when the hydrophilic monomers of the bonding agent penetrate into the exposed collagen-fiber meshwork, forming a mixed structure of fibers embedded in resin and hydroxyapatite crystals.[36],[37] As the optimal moisture level of dentin for this to occur is difficult to determine, excess water has been reported as an issue. Excess water on the dentin surface appears to cause phase separation between the hydrophobic and hydrophilic components of adhesive systems, causing a gap to form at the resin–dentin interface, which facilitates leakage and bacterial growth.[35] This phenomenon may explain the high S. mutans counts found in our sample at 6 months.

We conclude that the combination of hydroxyapatite with lysozyme, lactoferrin, and lactoperoxidase may be an alternative for S. mutans control in dentinal caries. The reduction in S. mutans counts 24 h after sealing with a combination of hydroxyapatite, lysozyme, lactoferrin, and lactoperoxidase as compared to counts obtained at 1 and 6 months may be explained by the antimicrobial properties of these enzymes, coupled with the remineralizing activity of hydroxyapatite.

Declaration of patient consent

The authors certify that they have obtained all appropriate patient consent forms. In the form, the patient(s) has/have given his/her/their consent for his/her/their images and other clinical information to be reported in the journal. The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed.

Key messages

The combination of hydroxyapatite with lysozyme, lactoferrin, and lactoperoxidase may be an alternative for S. mutans control in dentinal caries.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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Correspondence Address:
Prof. Sérgio L Pinheiro
Rua das Quaresmeiras, 54, Residencial Jardim das Palmeiras, Vinhedo - 13284-503, SP
Brazil
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijdr.IJDR_474_18

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