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J Dent Res 82(11): 914-918, 2003
© 2003 International and American Associations for Dental Research

RESEARCH REPORT
Biomaterials & Bioengineering

Incorporation of Casein Phosphopeptide-Amorphous Calcium Phosphate into a Glass-ionomer Cement

S.A. Mazzaoui1,2, M.F. Burrow1, M.J. Tyas1, S.G. Dashper1, D. Eakins1, and E.C. Reynolds1,*

1 School of Dental Science, The University of Melbourne, 711 Elizabeth Street, Melbourne, Victoria 3000, Australia; and
2 Dental Research Institute, The University of Zulia, Maracaibo, Venezuela;

* corresponding author, e.reynolds{at}unimelb.edu.au


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Casein phosphopeptide-amorphous calcium phosphate (CPP-ACP) nanocomplexes have been shown to prevent demineralization and promote remineralization of enamel subsurface lesions in animal and in situ caries models. The aim of this study was to determine the effect of incorporating CPP-ACP into a self-cured glass-ionomer cement (GIC). Incorporation of 1.56% w/w CPP-ACP into the GIC significantly increased microtensile bond strength (33%) and compressive strength (23%) and significantly enhanced the release of calcium, phosphate, and fluoride ions at neutral and acidic pH. MALDI mass spectrometry also showed casein phosphopeptides from the CPP-ACP nanocomplexes to be released. The release of CPP-ACP and fluoride from the CPP-ACP-containing GIC was associated with enhanced protection of the adjacent dentin during acid challenge in vitro.

KEY WORDS: glass-ionomer cement • CPP-ACP • calcium • inorganic phosphate and fluoride release • microtensile bond strength


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Casein phosphopeptide-amorphous calcium phosphate nanocomplexes (CPP-ACP) have been shown to prevent enamel demineralization and promote remineralization of enamel subsurface lesions in animal and human in situ caries models (Reynolds et al., 1995, 1999, 2003). The anticariogenic potential of CPP-ACP has been attributed to the ability of the CPP to localize amorphous calcium phosphate at the tooth surface, thereby helping to maintain a state of supersaturation with respect to tooth mineral (Reynolds et al., 2003). Recently, the CPP-ACP have been shown to interact with fluoride ions to produce an additive anticariogenic effect through the formation of a stabilized amorphous calcium fluoride phosphate phase (Reynolds et al., 1995; Reynolds, 1998).

Glass-ionomer cements (GICs) are water-based, tooth-colored, and chemically adhesive materials used in dentistry as bases and restorations. Microleakage around restorations remains a significant problem which can lead to caries of the underlying tooth tissues (Pachuta and Meiers, 1995). However, GICs are ion-releasing materials, and the incorporation into, and slow release of fluoride ions from, the cement provides a significant anticariogenic property (Forss, 1993).

In an approach to enhance the anticariogenic potential of a GIC, we incorporated 1.56% w/w CPP-ACP into a selected commercial GIC and investigated the cement’s physical and chemical properties. Compressive strength, net setting time, and microtensile bond strength to dentin, as well as calcium, phosphate, and fluoride release at neutral and acidic pH values, were determined.


   MATERIALS & METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Compressive Strength and Net Setting Time
Cylinders, 4 mm diam x 6 mm long, were made from the GICs for the compressive strength test and discs, 10 mm diam x 5 mm thick, for the net setting time test. The control GIC was prepared with a self-curing glass-ionomer cement (Fuji IX GP, liquid batch No. 080561 and powder batch No. 061051, GC International, Tokyo, Japan). The GIC containing CPP-ACP (RecaldentTM) was prepared from the same batch, with 1.56% w/w CPP-ACP incorporated. The CPP-ACP and GIC powder were manually mixed, and the powder:liquid ratio used was as recommended by the manufacturer. Compressive strength and net setting time tests were performed following ISO methods (ISO, 1991).

Microtensile Bond Strength to Dentin
Non-carious human molars stored in saline solution containing thymol were used within 2 mos following extraction. The use of human teeth for the study was approved by The University of Melbourne Human Research Ethics Committee. Bar-shaped specimens, half GIC and half dentin, were prepared with GIC (Fuji IX GP, batch No.9909021, GC International, Japan) and the same GIC containing 1.56% w/w CPP-ACP. Microtensile bond strength tests were performed as previously described (Phrukkanon et al., 1998; Tanumiharja et al., 2000), and the specimens were stressed in tension at a cross-head speed of 1 mm/min until failure. Mean bond strength values were calculated according to the standard formula (ISO, 1991), and we observed the fractured specimens in a scanning electron microscope (SEM 515; Phillips, Eindhoven, The Netherlands) to assess the mode of failure (Phrukkanon et al., 1998).

Ion Measurements and CPP Detection
We prepared discs, 6 mm diam x 2 mm thick, using the GIC with and without 1.56% w/w CPP-ACP. The GIC was mixed, as described above, injected into the molds, condensed, and allowed to set at 37°C and 100% RH for 1 hr. During setting, the bottom and top of the filled molds were covered by mylar strips and microscope slides under hand pressure. The discs were removed from the molds, placed in individual sealed plastic tubes, and incubated at 37°C in either 2 mL of de-ionized water (pH 6.9) (Milli-Q Water, Millipore Corporation, Melbourne, Victoria, Australia), or 2 mL of 50 mM sodium lactate buffer at pH 5.0. The solutions were changed every 24 hrs for 3 days, and the release of calcium, inorganic phosphate, and fluoride ions was measured in each solution. Calcium concentrations were determined by atomic absorption spectrophotometry (Adamson and Reynolds, 1995), inorganic phosphate colorimetrically (Itaya and Ui, 1966), and fluoride ion with the use of an ion-selective electrode (Ion 85 Radiometer, Copenhagen, Denmark). The release of the ions was expressed as µmol/mm2 surface area of the GIC exposed.

The presence of CPP in the solutions was determined by Matrix Assisted Laser Desorption/Ionisation-Mass Spectrometry (MALDI-MS) (Voyager-DE, Perseptive Biosystems; Framingham, MA, USA) with a matrix of 2,5-dehydroxybenzoic acid in 66% water, 33% CH3CN, and 1% formic acid.

Acid Demineralization Test
Box-shaped cavities (6 mm x 2 mm x 1.5 mm) along the cemento-enamel junction of human molars were prepared. The cavity margins were finished with a slow-speed cylindrical bur, giving a cavo-surface angle as close as possible to 90°. The cavities were filled with the GIC with and without the incorporated 1.56% w/w CPP-ACP. The teeth were then stored at 37°C and 100% relative humidity for 24 hrs. The GIC surfaces were finished and polished, and the integrity of the cavo-surface margins was confirmed under a light microscope. Two coats of an acid-resistant varnish were then applied to the tooth surface, leaving a 1-mm window around the cavity margins. The teeth were then placed in 25 mL of acid buffer containing 2.2 mM CaCl2, 2.2 mM NaH2PO4, and 50 mM acetic acid (pH 5.0) (ten Cate and Duijsters, 1982) for 4 days at 37°C. The acid solution was replaced every 24 hrs. After 4 days, longitudinal sections (100 µm) were obtained through the center of each restoration as described previously (Reynolds, 1997). The sections were observed under polarized light with quinoline as the imbibing medium (Wefel et al., 1985). A first-order red wavelength plate was inserted between the crossed polarizers to highlight the changes in the demineralized dentin, and a 103-µm-length graticle was used to measure the areas of the lesions. The area of the body of the lesion was measured from the margin of the restoration to 568 ± 3 µm from the restoration. Five measurements were made of each lesion.

Statistical Analysis
Data from compressive strength, net setting time, areas of demineralized lesions, and microtensile bond strength were compared by Student’s t test. Chi-squared distribution was used to compare the mode of failure of fractured specimens. Data from the ion release analyses were compared by a one-way classification analysis of variance, with the least significant difference test.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Physical Properties
Incorporation of 1.56% w/w CPP-ACP into the GIC resulted in a 23% increase in compressive strength, a 33% increase in microtensile bond strength, and a 40-second increase in mean setting time (Table 1Go). The increase in setting time did not exceed the acceptable standard for a self-cured GIC (ISO, 1991). The distribution in the mode of failure was analyzed by SEM. Type 2 fracture (partial adhesive and partial cohesive failure) was more frequent with the CPP-ACP-containing GIC, whereas Type 4 (cohesive failure) was more frequent with the control (Table 1Go). The microstructures of the 2 cements examined by SEM at a 360X magnification appeared similar, although a more porous and roughened fracture surface was observed with the control GIG when compared with the CPP-ACP-containing cement (Fig. 1Go).


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  		Table 1. Physical Properties and Mode of Failure of CPP-ACP-containing GIC and Control
 


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  		Figure 1. Representative scanning electron micrographs of fractured cement surfaces of the GIC containing 1.56% w/w CPP-ACP (a) and the control GIC (b).

 
Ion and CPP Release
Mean values for the release of fluoride, calcium, and phosphate ions in water (pH 6.9) and sodium lactate buffer (pH 5.0) from the GIC containing 1.56% w/w CPP-ACP and the control GIC are shown in Table 2Go. The pattern of fluoride release in water was similar between samples and controls, with the highest release occurring during the first 24 hrs and a slower but continued release during the next two 24-hour periods. Fluoride release was significantly higher in the pH 5.0 sodium lactate buffer than in water for both materials. Significantly higher fluoride release was found with the CPP-ACP-containing GIC when compared with the control at both pH values. No calcium release was detected in water from either material. However, calcium ion release was detected with the CPP-ACP-containing GIC in the pH 5.0 sodium lactate buffer. The release was low relative to fluoride but continuous during the three 24-hour periods. At both pH values, inorganic phosphate ion release was significantly higher from the CPP-ACP-containing GIC than from the control material during the first 24-hour period. The release of inorganic phosphate was significantly higher in sodium lactate buffer at pH 5.0 than in water for both materials. By MALDI-MS analysis, CPP could be detected in the water and pH 5.0 buffer after 24 hrs’ incubation with the GIC containing CPP-ACP. The mass spectrum obtained was the same as that for standard CPP-ACP.


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  		Table 2. Fluoride, Calcium, and Inorganic Phosphate Release from CPP-ACP-containing GIC and Control at Neutral and Acid pH
 
Incorporation of the CPP-ACP into the GIC significantly enhanced protection against acid demineralization of the adjacent dentin (Fig. 2Go). The mean area of demineralized lesion adjacent to the control GIC was 40,060 ± 4870 µm2 (n = 12), which was significantly larger (p < 0.05) than the area of the lesion (22,686 ± 8075 µm2, n = 12) adjacent to the GIC containing 1.56% w/w CPP-ACP.



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  		Figure 2. Representative photomicrographs of demineralized lesions in dentin adjacent to the control GIC (a) and the GIC containing 1.5% w/w CPP-ACP (b).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Incorporation of 1.56% w/w CPP-ACP into a GIC was shown to increase compressive strength and microtensile bond strength, enhance the release of calcium, phosphate, and fluoride ions, and enhance protection of the adjacent dentin to acid demineralization.

A major reason for the use of GICs in a variety of clinical applications is their capacity to bond chemically to different surfaces such as enamel, dentin, and resin composite (Akinmade and Nicholson, 1993). GICs are used routinely in conjunction with resin composites (Li et al., 1996), in Atraumatic Restorative Treatment (Frencken et al., 1996), in tunnel restorations (Svanberg, 1992), and in the restoration of primary teeth (Frankenberger et al., 1997). Bond strength, therefore, is an important property of the GIC. The mean microtensile bond strength of the CPP-ACP-containing GIC was 33% higher than that of the control GIC. The testing method used has been successfully used on specimens with different dentin thicknesses and dentinal tubule orientations and with disease-affected dentin specimens (Phrukkanon et al., 1998). Therefore, factors such as the quality, depth, and moisture of the dentin substrate (Burrow et al., 1994) were believed not to affect the results of this study.

The most common mode of failure in the adhesion between a GIC and the dentin during microtensile bond strength tests is cohesive failure within the GIC (Tanumiharja et al., 2000). This was the predominant mode of failure of the control GIC found in this study. The predominant mode of failure for the CPP-ACP-containing GIC was partial cohesive failure in the GIC and partial adhesive failure between the GIC and the dentin. It has not been clearly established whether the mode of failure for a particular material is associated with its bond strength or elastic modulus. It is known that the pores in a solid body act as stress-concentration points where fracture can initiate (Griffith, 1920), and it has been speculated that this explains the frequency of cohesive failure within a GIC (Tanumiharja et al., 2000). SEM analyses of the fractured surfaces of the different GICs did suggest a greater porosity of the control GIC relative to the CPP-ACP containing GIC. However, whether this was responsible for the difference in bond strength is unknown. The CPP-ACP in the GIC may have also directly increased microtensile bond strength by the incorporation of the CPP-ACP nanoparticles into the cross-linked matrix of the GIC.

With respect to the release of ions from the GICs, it was shown that the fluoride release in sodium lactate buffer (pH 5.0) was significantly higher than that in water (pH 6.9). This finding has been previously reported for normal GICs (Forss, 1993). However, in the current study, fluoride release was significantly higher from the CPP-ACP-containing GIC than from the control GIC at both pH values. It is possible that the CPP-ACP promoted the release of fluoride ions from the GIC by forming casein phosphopeptide-amorphous calcium fluoride phosphate (CPP-ACFP) nanocomplexes (Reynolds, 1998), which were released from the cement matrix.

Significantly more inorganic phosphate was also released from the CPP-ACP-containing GIC at both pH values (5.0 and 6.9) than from the control GIC, consistent with the addition of the stabilized amorphous calcium phosphate. Calcium ions were not detected at neutral pH with either GIC, and were detected with the CPP-ACP-containing GIC only at acid pH. It is likely that a low level of calcium ion release at neutral pH from the CPP-ACP-containing GIC did occur, since CPP, Pi, and F release at this pH was detected. However, this level of calcium release was below the detection limit of the analytical method. Fuji IX is a non-calcium-containing GIC in which calcium ions have been replaced by strontium ions (Wilson and McLean, 1988). Calcium is an ion not easily leachable from GIC once the cement has set (Matsuya et al., 1984), due to its rapid binding, in an insoluble form, to the polyacrylic acid matrix (Crisp et al., 1976). Elution of calcium, when it does occur, has been attributed to acid erosion of the cement matrix (Matsuya et al., 1984; Forss, 1993). The CPP-ACP nanoparticles may have been physically encapsulated into the set GIC, as has been found with unreacted glass particles (Matsuya et al., 1984), and therefore released as the acid eroded the cement in the acidic buffer. The acid-catalyzed release of the CPP-ACP nanoparticles from the GIC is consistent with the protection of the adjacent dentin observed during acid challenge. CPP-ACP nanocomplexes have been shown to prevent enamel demineralization and promote enamel subsurface lesion remineralization in situ when used in a mouthwash or sugar-free gum (Reynolds et al., 1999, 2003).

In conclusion, the results of this study suggest that the 1.56%-CPP-ACP-containing GIC might be a superior restorative/base with an improved anticariogenic potential.


   ACKNOWLEDGMENTS
 
Dr. S.A. Mazzaoui was a student receiving financial support from the Vice-Rectory of Academic Affairs of La Universidad del Zulia, Maracaibo, Venezuela. All other authors were employed by The University of Melbourne and received no consultant fees in relation to this work. All financial support for equipment, consumables (apart from the glass-ionomer cement), and non-author technical support was provided by The University of Melbourne. Thanks to GC International, Tokyo, Japan, for providing the Fuji IX GP glass-ionomer cement and to Dr. K. Hirota, Research Director of GC International, for his advice regarding the chemistry and composition of Fuji IX GP. The assistance of Joseph Palamara and Peter Riley is gratefully acknowledged.

Received July 30, 2001; Last revision July 23, 2003; Accepted July 30, 2003


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Adamson NJ, Reynolds EC (1995). Characterisation of tryptic casein phosphopeptides prepared under industrially-relevant conditions. Biotech Bioeng 45:196–204.

Akinmade AO, Nicholson JW (1993). Glass-ionomer cements as adhesives. Part I. Fundamental aspects and their clinical relevance. J Mater Sci-Mater M 4:93–101.

Burrow MF, Takakura H, Nakajima M, Inai N, Tagami J, Takatsu T (1994). The influence of age and depth of dentin on bonding. Dent Mater 10:241–246.[ISI][Medline]

Crisp S, Lewis BG, Wilson AD (1976). Glass ionomer cements: chemistry of erosion. J Dent Res 55:1032–1041.[Abstract/Free Full Text]

Forss H (1993). Release of fluoride and other elements from light-cured glass ionomers in neutral and acidic conditions. J Dent Res 72:1257–1262.[Abstract/Free Full Text]

Frankenberger R, Sindel J, Kramer N (1997). Viscous glass-ionomer cements: a new alternative to amalgam in the primary dentition? Quintessence Int 28:667–676.[Medline]

Frencken JE, Pilot T, Songpaisan Y, Phantumavanit P (1996). Atraumatic restorative treatment (ART): rationale, technique and development. J Public Health Dent 56:135–140.[ISI][Medline]

Griffith AA (1920). The phenomema of rupture and flow in solids. Philos Trans R Soc London A 221:163–198.

ISO (1991). International Organization for Standarization. ISO 9917. Dental water-based cements. Geneva.

Itaya K, Ui M (1966). A new micromethod for the colorimetric determination of inorganic phosphate. Clin Chim Acta 14:361–366.[ISI][Medline]

Li J, Liu Y, Söremark R, Sundström F (1996). Flexure strength of resin-modified glass ionomer cements and their bond strength to dental composites. Acta Odontol Scand 54:55–58.[ISI][Medline]

Matsuya S, Matsuya Y, Yamamoto Y, Yamane M (1984). Erosion process of glass ionomer cement in organic acids. Dent Mater J 3:210–219.[Medline]

Pachuta SM, Meiers JC (1995). Dentin surface treatment and glass ionomer microleakage. Am J Dent 8:187–190.[ISI][Medline]

Phrukkanon S, Burrow MF, Tyas MJ (1998). Effect of cross-sectional surface area on bond strengths between resin and dentin. Dent Mater 14:120–128.[ISI][Medline]

Reynolds EC (1997). Remineralization of enamel subsurface lesions by casein phosphopeptide-stabilized calcium phosphate solutions. J Dent Res 76:1587–1595.[Abstract/Free Full Text]

Reynolds EC (1998). Anticariogenic complexes of amorphous calcium phosphate stabilized by casein phosphopeptides: a review. Spec Care Dentist 18:8–16.[Medline]

Reynolds EC, Cain CJ, Weber FL, Black CL, Riley PF, Johnson IH, et al. (1995). Anticariogenicity of calcium phosphate complexes of tryptic casein phosphopeptides in the rat. J Dent Res 74:1272–1279.[Abstract/Free Full Text]

Reynolds EC, Black CL, Cai F, Cross KJ, Eakins D, Huq NL, et al. (1999). Advances in enamel remineralisation: casein phosphopeptide-amorphous calcium phosphate. J Clin Dent 10:86–88.

Reynolds EC, Cai F, Shen P, Walker GD (2003). Retention in plaque and remineralization of enamel lesions by various forms of calcium in a mouthrinse or sugar-free chewing gum. J Dent Res 82:206–211.[Abstract/Free Full Text]

Svanberg M (1992). Class II amalgam restorations, glass ionomer tunnel restorations, and caries development on adjacent tooth surfaces: a 3-year clinical study. Caries Res 26:315–318.[ISI][Medline]

Tanumiharja M, Burrow MF, Tyas MJ (2000). Microtensile bond strengths of glass ionomer (polyalkenoate) cements to dentine using four conditioners. J Dent 28:361–366.[ISI][Medline]

ten Cate JM, Duijsters PP (1982). Alternating demineralization and remineralization of artificial enamel lesions. Caries Res 16:201–210.[ISI][Medline]

Wefel JS, Clarkson BA, Heilman JR (1985). Natural root caries: a histologic and microradiographic evaluation. J Oral Pathol 14:615–623.[ISI][Medline]

Wilson AD, McLean JW (1988). Glass-ionomer cement. Chicago, IL: Quintessence Publishing Co., Inc.





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