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. 2015 Jun;49(2):104–111. doi: 10.15644/asc49/2/3

Antimicrobial Capacity of Casein Phosphopeptide/Amorphous Calcium Phosphate and Enzymes in Glass Ionomer Cement in Dentin Carious Lesions

SÉRGIO LUIZ PINHEIRO 1, GIULIANA RODRIGUES AZENHA 1, FLÁVIA DE MILITO 1, YASMIN MARIALVA DEMOCH 1
PMCID: PMC4988828  PMID: 27688392

Abstract

Objective

To evaluate the ability of casein phosphopeptide/amorphous calcium phosphate (CPP/ACP) and lysozyme, lactoferrin, and lactoperoxidase (LLL) added to glass ionomer cement (GIC) to inhibit the growth of S. mutans in a caries model.

Material and methods

Eighty permanent third molars were selected. The dentin of these teeth was exposed and flattened. Except for the coronal dentin, the specimens were waterproofed, autoclaved, and submitted to cariogenic challenge with standard strain of S. mutans. The carious lesions were sealed as follows: group 1 (n=20): GIC without additives; group 2 (n=20): GIC + CPP/ACP; group 3 (n=20): GIC + LLL; group 4 (n=20): GIC + CPP/ACP + LLL. S. mutans counts were performed before the caries were sealed (n=5), after 24 hours (n=5), at 1 month (n=5), and at 6 months (n=5). The results were analyzed using descriptive statistical analysis and the Kruskal-Wallis test (Student-Newman-Keuls test).

Results

GIC + LLL caused a significant reduction of S. mutans 1 month after sealing (p<0.01); however, there was a significant growth of S. mutans 6 months after sealing. GIC, GIC + CPP/ACP, and GIC + CPP/ACP + LLL showed similar behavior with significant reduction of S. mutans after 24 hours (p<0.05) and increase after 1 and 6 months.

Conclusion

The addition of LLL to GIC increases the antimicrobial action of GIC on S. mutans. This leads to control of bacterial biofilm for 1 month, thus stopping the progression of carious lesions.

Key words: Caries, Streptococcus mutans, casein phosphopeptide-amorphous calcium phosphate nanocomplex, Glass Ionomer Cements, Anti-Bacterial Agents, Muramidase, Lactoferrin, Lactoperoxidase

Introduction

Minimally invasive dentistry recommends the removal of the infected dentin and the preservation of the caries-affected dentin. The infected dentin is characterized by severe softening and degeneration of its collagen fibers, whereas the caries-affected dentin is characterized by moderate softening with repairable collagen fibers (1, 2). Six different layers have been observed in carious lesions: 1- outer irreversibly demineralized layer; 2- translucent layer; 3- subtransparent layer; 4- sclerotic layer; 5- healthy dentin; 6- predentin. Layers 2, 3, and 4 are the repairable caries-affected tissues (3).

The microbial nutrition is restricted by the sealing of the dentin-pulp complex, thus stopping the progression of carious lesions (1-4). Demineralization and remineralization of the dentin are dynamic processes involved in the onset, progression, and reversal of the carious lesion. The balance between these processes is essential for the prevention and treatment of this dental disease (5, 6).

Different treatment options to promote the remineralization process have been evaluated, including the use of casein phosphopeptide/amorphous calcium phosphate (CPP/ACP). CPP/ACP is a bioactive compound that stimulates the repair of tooth structure by first releasing calcium and phosphate ions (7, 8) and then attracting them to the tooth surface, thus increasing the concentration gradient on the enamel subsurface and promoting in situ remineralization (5, 9). CPP/ACP is commercially available as toothpaste and induces the mineralization of the intrafibrillar and interfibrillar dentin on the collagen surface by attracting calcium ions through electrostatic force (10). A nucleation site is formed to trigger the remineralization needed for the growth of hydroxyapatite crystals (10). After enamel is remineralized using CPP/ACP, it becomes more resistant to acid changes when compared with normal carbonated enamel (11). This compound has osteoconductivity, biodegradability, bioactivity, high cell adherence, no cytotoxicity, and kinetic stabilization (7). There are reports in the literature on the use of CPP/ACP as an active component of many products and materials, especially mouthwashes, toothpastes, some dental bleaching, abrasive pastes, varnishes, and glass ionomer cement (GIC) (5, 7).

The addition of antibacterial substances to GIC has been shown to increase the antimicrobial potential of this material (12-14). According to the literature, the most commonly used antimicrobials added to GIC are antibiotics (12), propolis (15), chlorhexidine (13, 16, 17), triclosan (14, 18), cetrimide (19), and cetylpyridinium chloride (19). The results reported in these studies show that the addition of these antimicrobials is able to reduce the number of viable bacteria and may contribute to the successful treatment of caries using minimal intervention. Moreover, according to Tüzüner & Ulusu and Mazzaoui et al. (20, 21), the addition of antimicrobial agents does not affect the physical and mechanical properties of GIC.

Another therapeutic possibility is the use of enzymes such as lysozyme, lactoferrin, and lactoperoxidase (LLL) (22). These enzymes are present in saliva and exert an antimicrobial effect against bacterial, viral, and fungal pathogens (23, 24). The interaction of bacteria with the salivary components that form the biofilm on the enamel surface is associated with the selective adherence of S. mutans to the surface. The salivary components that interact with S. mutans are mucin, lysozyme, lactoperoxidase, agglutinin, proline, and secretory immunoglobulin. Goodman et al. (25) demonstrated that, when lysozyme and other substances found in saliva interact, they may be effective in the lysis of microorganisms. Lactoferrin, an iron-binding glycoprotein present in saliva, is produced by neutrophils and glandular epithelial cells (26). This enzyme is a multifunctional bioactive molecule playing an important role in the physiological system (26). Salivary lactoperoxidase catalyzes the conversion of thiocyanate into hypothiocyanite, which regulates the incidence of caries (27).

The objective of the present study was to evaluate the ability of CPP/ACP and LLL added to GIC to inhibit the growth of S. mutans in a caries model.

Materials

The GIC used throughout this study was the Ketac™ Cem Easymix (3M ESPE, Seefeld, Germany), which comprises powder/liquid components and is commercially available for manual mixing. This GIC was chosen for this series of experiments because it has the potential for serving as a cavity liner; thus, in case the procedure of adding LLL and CPP/ACP weakened the mechanical strength of GIC, the cavity would be protected by a filling layer. The following enzymes were used: lysozyme (Sigma, Sao Paulo, SP, Brazil), lactoferrin (Sigma), and lactoperoxidase (Sigma). Using a precision scale (Uni Bloc, Shimadzu Auy220, Kyoto, Japan), 1% of each of the enzymes was added to the powder of GIC and mixed in a sterile porcelain mortar and pestle set. To prepare CPP/ACP, 1 g of GC Tooth Mousse Plus paste (GC Corporation, Tokyo, Japan) was weighed on a precision scale (Uni Bloc) and diluted in 4 ml of distilled water to minimize possible changes in the setting chemistry of GIC. Then, 3% of this solution was added to the liquid of GIC and mixed until a homogeneous solution was obtained. The materials used in the study along with the manufacturer’s name and batch number are described in Table 1.

Table 1. Materials, manufacturers, and batch numbers.

Product Manufacturer Lot
Glass Ionomer Cement
(Ketac Cem Easymix)
 3M ESPE.
Germany.
 494562
Lysozyme
 SIGMA- Aldrich Brasil Ltda.
Săo Paulo, Brazil.
 SLBF1341V
Lactoferrin
 SIGMA- Aldrich Brasil Ltda.
Săo Paulo, Brazil.
 SLBB2642V
Lactoperoxidase
 SIGMA- Aldrich Brasil Ltda.
Săo Paulo, Brazil.
 SLBB5243V
Tooth Mousse Plus paste GC Corporation.
Tokyo, Japan.		 120816S
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Methods

This study was approved by the Research Ethics Committee of PUC-Campinas (Protocol: 0345/11).

Selection of samples

Eighty permanent third molars were selected at the Dental Clinic of PUC-Campinas. All donor patients signed an Informed Consent Form.

  • The inclusion criteria were:

    • Permanent third molars;

    • No fissures or fractures detected by a 10x magnifying glass (Stemi DV4 Carl Zeiss, Săo Paulo, Brazil).

Procedures The teeth were stored in sodium chloride 0.9% containing sodium azide (0.02%) (LabCenter, Săo Paulo, Brazil) at 4°C (28). The occlusal third was removed from the specimens using double-sided diamond disc (KG Sorensen Indústria e Comercio LTDA, Săo Paulo, Brazil) at low speed under water-cooling for dentin exposure. The dentin surfaces were polished with wet silicon carbide sandpaper sheets, P600 grit (Água T223 advance, Norton, Indústria Brasileira, Săo Paulo, Brazil). The specimens were sealed using epoxy resin (Araldite, Săo Paulo, Brazil) and nail polish (Colorama, Sao Paulo, Brazil), except for the coronal dentin in the laminar flow (Veco, Campinas, SP, Brazil). After sealing the teeth, the specimens were sterilized in an autoclave (Sercon, Săo Paulo, Brazil) for 20 minutes at 121°C and 1 atm (29) and submitted to cariogenic challenge.

With the purpose of simulating caries-affected dentin, the teeth were placed in sterile test tubes with brain heart infusion (BHI) broth (LabCenter, Săo Paulo, Brazil) plus 0.5% yeast extract (LabCenter, Sao Paulo, Brazil), 1% glucose (LabCenter, Săo Paulo, Brazil), and 1% sucrose (LabCenter, Săo Paulo, Brazil). The standard strain of S. mutans (ATCC 25175) (Fundaçăo André Tosello, Campinas - SP, Brazil) standardized to 0.5 McFarland standard was added to the BHI (LabCenter, Săo Paulo, Brazil). The specimens were incubated at 37°C for 1 month in anaerobic jars with gas-generating envelopes (LabCenter, Săo Paulo, Brazil) in an atmosphere containing 85% nitrogen (N2), 10% carbon dioxide (CO2), and 5% hydrogen (H2). After that, the teeth were stored in a bacteriological incubator (Fanem Ltda, Săo Paulo, SP, Brazil). During this period, the BHI broth (LabCenter, Săo Paulo, Brazil) was replaced every 2 days (adapted from de Carvalho et al.) (30). Then, the teeth were randomly assigned to one of four groups of materials for sealing the carious lesion (n​​=20 per group):

Group 1: GIC (powder/liquid components) without any additives.

Group 2: 3% of CPP/ACP added to the liquid of GIC.

Group 3: 1% of lysozyme, 1% of lactoferrin, and 1% of lactoperoxidase added to the powder of GIC .

Group 4: 3% of CPP/ACP added to the liquid of GIC + 1% of lysozyme, 1% of lactoferrin, and 1% of lactoperoxidase added to the powder of GIC.

In each group (n=20), S. mutans counts were performed before sealing the carious tissue (n=5), after 24 hours (n=5), 1 month (n=5), and 6 months (n=5). The GIC restorations were removed using sterile spherical diamond burs (KG Sorensen, Săo Paulo, Brazil) at high speed under saline cooling. The collections of caries-affected dentin were performed using a sterile #20 spoon excavator (SSWhite Duflex, Rio de Janeiro, Brazil). The specimens were immediately immersed in BHI broth (LabCenter, Săo Paulo, Brazil). This material was manipulated within a period of 24 hours and homogenized for 3 minutes in a tube shaker (Phoenix, Araraquara, SP, Brazil). Immediately after, 5 decimal dilutions were performed. Three aliquots of 25 uL of these dilutions were seeded in the surface of the mitis salivarius bacitracin (MSB) medium. All plates were incubated in jars (Oxoid Ltd., Basingstoke, Hampshire, England) for 5 days at 37°C in 85% nitrogen (N2), 10% carbon dioxide (CO2), and 5% hydrogen (H2). Such atmosphere was obtained using anaerobic jars with gas-generating envelopes and anaerobic indicators (Oxoid Ltd., Basingstoke, Hampshire, England). After incubation, we counted the total number of viable bacteria (31).

Statistical Analysis

We compared the total amount of viable S. mutans before sealing the carious tissue, after 24 hours, 1 month, and 6 months using Bioestat 4.0. The results were analyzed using descriptive statistical analysis and the Kruskal-Wallis test followed by Student-Newman-Keuls test. The level of significance was set at 5% (p<0.05).

Results

After comparing the counts of S. mutans in the carious lesions before sealing and after 24 hours, 1 month, and 6 months, we found that GIC without additives, GIC with the addition of CPP/ACP, and GIC with the addition of CPP/ACP and LLL showed similar behavior with significant reduction of S. mutans after 24 hours (p<0.05) and increased count after 1 and 6 months. GIC with the addition of LLL resulted in a significant reduction of S. mutans after 1 month (p<0.01). However, at 6 months, carious lesions sealed with GIC with the addition of LLL showed a significant growth of S. mutans (Table 2 and Figure 1).

Table 2. Comparison of S. mutans counts in carious lesions before and after sealing with different glass ionomer cement (GIC)-based materials.

SEALING MATERIAL BEFORE 24 HOURS 1 MONTH 6 MONTHS p*
GIC
 3.44
(0.20)a
 0.00
(0.00)b
 3.11
(4.77)a
 4.98
(0.17)a
 0.0085
GIC+CPP/ACP
 3.71
(0.94)a
 2.90
(0.06)b
 3.12
(0.30)a
 3.86
(0.11)a
 0.0052
GIC+LLL
 3.10
(0.30)a
 1.60
(0.00)a
 0.00
(0.00)b
 3.50
(0.36)a
 0.0004
GIC+CPP/ACP+LLL 3.58
(0.50)a 		2.82
(0.39)b 		3.60
(0.26)a 		3.61
(0.04)a 		0.0186
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GIC: GIC without additives. GIC+CPP/ACP: GIC with addition of casein phosphopeptide/amorphous calcium phosphate. GIC+LLL: GIC with addition of lysozyme, lactoferrin, and lactoperoxidase. GIC+CPP/ACP+LLL: GIC with addition of casein phosphopeptide/amorphous calcium phosphate and addition of lysozyme, lactoferrin, and lactoperoxidase. 
Values are expressed as median (interquartile deviation). Different letters in the same row indicate significant difference (p<0.05).
* Kruskal-Wallis test followed by Student-Newman-Keuls test.

Figure 1.

Figure 1

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Medians of S. mutans counts before and 24 hours, 1 month, and 6 months after sealing of caries with different glass ionomer cement (GIC)-based materials

Discussion

Our study is different from other studies from the literature that stored the specimens in distilled water (16, 17, 20) and saline (32). By storing our specimens in BHI broth, we could preserve microbial viability during the experimental period if there were marginal fissures in the interface between GIC and the tooth.

Based on our results, we found that conventional GIC showed a significant reduction of S. mutans in the first 24 hours after sealing; however, after 1 and 6 months there was bacterial growth when compared with the count performed after 24 hours. This finding is in agreement with the literature (13, 17, 21), since there are reports that the antibacterial action of this material is more prevalent in the first hours after sealing. This may be explained by the higher fluoride release that occurs (17) during GIC setting. Such release displaces the ionically active elements (including fluoride) in the early stages of gelation. In addition, the effective sealing of the dentin-pulp complex is another possible explanation for the antibacterial action, because it promotes chemical adherence through the interaction between the carboxyl groups of the polyalkenoic acid of GIC and the calcium ions of hydroxyapatite, thus restricting microbial nutrition and stopping the progression of carious lesions (1-4).

The addition of LLL to GIC in this study resulted in reduction of S. mutans 24 hours and 1 month after sealing. However, after just 6 months of sealing, there was growth of S. mutans. This is in agreement with Jyoti et al. (33) because these authors considered that the action and addition of lactoperoxidase in oral hygiene products were effective. LLL have bacteriostatic and bactericidal effect against oral cavity pathogens, including S. mutans. Lysozyme causes bacterial lysis by breaking the binding between the N-acetyl muramic acid and the N-acetyl-glucosamine acid. Therefore, it degrades the peptidoglycan of the bacterial cell wall by muramidase activity and shows non-muramidase activity caused by the rupture of the membrane (34). Lysozyme has bactericidal activity even after heat inactivation, probably because of its cationic characteristic (35). Lactoferrin has bacteriostatic antimicrobial properties because it reduces the amount of viable iron provided to the bacteria (36). Human saliva also includes the antibacterial system of lactoperoxidase, hydrogen peroxide, and thiocyanate ion. The hydrogen peroxide present in the oral cavity is produced by microorganisms. Lactoperoxidase catalyzes the oxidation of thiocyanate ions by means of hydrogen peroxide, thus generating hypothiocianous acid or hypothiocyanite anion, both with antibacterial action. Hypothiocyanite acts as bacterial inhibitor interfering in cell metabolism (27). The action of hypothiocyanite inhibiting glucose metabolism of S. mutans becomes more effective as the pH of the medium is reduced (37).

The addition of CPP/ACP and CPP/ACP + LLL to GIC showed a significant reduction of S. mutans 24 hours after sealing of the caries lesions when compared with the S. mutans count before sealing. However, after 1 and 6 months, there was growth of S. mutans. Thus, we found that the addition of LLL enzymes to GIC improves the antibacterial properties of the material. Nevertheless, when CPP/ACP is added, the material begins to have the same behavior as pure conventional GIC, thus demonstrating that CPP/ACP probably inhibited the antimicrobial effect of the enzymes because of antagonist chemical action. This may be explained by the fact that CPP/ACP, in addition to promoting remineralization, increases the pH of the medium (38, 39) by increasing the concentration of calcium and phosphate ions (5, 7, 8), thus the medium becomes more alkaline in caries-affected dentin. Therefore, in the present study, changes in pH may have inhibited the activity of LLL, which have an antimicrobial effect between pH 5 and 6 (26, 37, 40, 41).

The growth of S. mutans in all groups after 6 months is explained by the loss and/or wear of the seal during storage. Czarnecka et al. (32) found marginal fissures in GIC in teeth with preexisting carious lesions after 21 days of storage of specimens in saline. GIC incorporates air bubbles during handling, which cause porosities that are exposed during the acid erosion or abrasion, thus contributing to increased surface roughness. GIC roughness may decrease the wear resistance and make this surface more prone to increased deposition of bacterial biofilm with consequent surface degradation and marginal leakage (42).

In conclusion, the addition of lysozyme, lactoferrin, and lactoperoxidase to the tested GIC enhanced its short-term antimicrobial properties and it may be used for microbial reduction of S. mutans in caries-affected dentin. The addition of CPP/ACP did not add antimicrobial properties to GIC. After 6 months, all groups showed bacterial viability and increase of S. mutans. This may be related to the aging process of the restoration and the presence of marginal fissures, thus enabling the nutrition of the remaining bacteria.

Acknowledgments

The authors would like to thank Fundaçăo de Amparo ŕ Pesquisa do Estado de Săo Paulo (FAPESP - process no. 2012/10350-6).

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