Dental Gum

Casein Phosphopeptide-Amorphous Calcium Phosphate and the Remineralization of Enamel

a report by

Eric C Reynolds, AO
Professor of Dental Science and Head, School of Dental Science, The University of Melbourne

Casein phosphopeptide-amorphous calcium phosphate (CPP-ACP) nanocomplexes are derived from bovine milk protein, casein and calcium and phosphate. The concept of CPP-ACP as a remineralizing agent was first postulated in 1998.1 A number of subsequent studies have demonstrated CPP-ACP to have anticariogenic activity in laboratory, animal, and human in situ experiments.2-4 This has led to the incorporation of CPP-ACP into food products and dental products as a new tool in the fight against caries.
CPP-ACPA New Adjunctive Agent for Caries Control

Reynolds and colleagues, using an in situ animal caries model, showed that exposure of enamel plaque to solutions of tryptic peptides of casein significantly reduced enamel subsurface demineralization. Casein peptides were incorporated into enamel plaque and were associated with increases in the plaques content of calcium and phosphate.The investigators concluded that the tryptic peptides responsible for caseinates anticariogenic activity were the calcium phosphatestabilizing CPPs (casein phosphopeptides).1 These CPPs contain a specific sequence, which markedly increases the apparent solubility of calcium phosphate by stabilizing ACP, forming solutions that are supersaturated with respect to the calcium phosphates. In an animal caries study, a 0.5% weight per volume (w/v) solution of CPP-ACP nanocomplexes was demonstrated to be comparable with a 500ppm fluoride solution in reducing caries activity.10 CPPACP solutions, applied twice daily to the teeth of specific-pathogen-free rats orally infected with Streptococcus sobrinus, a bacterium that causes tooth decay in humans, significantly reduced caries activity, with 0.1% w/v CPP-ACP producing a 14% reduction, and 1.0% w/v CPP-ACP producing a 55% reduction relative to the distilled water control. The anticariogenicity of CPP-ACP and fluoride was

Caries initiation is associated with demineralization of subsurface tooth enamel. Calcium and phosphate are lost from the subsurface enamel, resulting in the formation of a subsurface lesion. At this early stage, the caries lesion is reversible via a remineralization process involving the diffusion of calcium and phosphate ions into the subsurface lesion to restore lost structure. Since several studies had demonstrated that milk-based products appeared to have anticariogenic properties in animal models,5-8 attention was focused on identifying the specific milk-based agents that were responsible for the anticaries effect.

1. Reynolds EC,Anticariogenic complexes of amorphous calcium phosphate stabilized by casein phosphopeptides: A review, Spec Care Dentist (1998);8: pp. 816. 2. Reynolds EC, et al., Advances in enamel remineralization: Anticariogenic casein phosphopeptide - amorphous calcium phosphate, J Clin Dent (1999);10: pp. 8688. 3. Reynolds EC, et al.,Retention in plaque and remineralization of enamel lesions by various forms of calcium in a mouthrinse or sugar-free chewing gum, J Dent Res (2003);82: pp. 206211. 4. Shen F, et al., Remineralization of enamel subsurface lesions by sugar-free chewing gum containing casein phosphopeptideamorphous calcium phosphate, J Dent Res (2001);80: pp. 20662070. 5. Reynolds EC, Black CL, Confectionery composition and rat caries, Caries Res (1987);21: pp. 538545. 6. Navia JM, Lopez H,Rat caries assay of reference foods and sugar-containing snacks, J Dent Res (1983); 62: pp. 893898. 7. Bowen WH, et al., A method to assess cariogenic potential of food-stuffs, J Am Dent Assoc (1980); 100: pp. 677681. 8. Harper DS, et al., Cariostatic evaluation of cheeses with diverse physical and compositional characteristics, Caries Res (1986);20: pp. 123130. 9. Reynolds EC, et al.,Anticariogenicity of calcium phosphate complexes of tryptic casein phosphopeptides in the rat, J Dent Res (1995)74: pp. 12721279. 10. Moller IJ, Poulsen S, The effect of sorbitol containing chewing gum on the incidence of dental caries, plaque and gingivitis in Danish schoolchildren, Community Dent Oral Epidemiol (1973);1: pp. 5867.

Eric C Reynolds, AO is Professor of Dental Science and Head of the School of Dental Science at The University of Melbourne. Professor Reynolds is also Associate Dean of the Faculty of Medicine, Dentistry and Health Sciences at The University of Melbourne and Chief Executive Officer of the Cooperative Research Centre for Oral Health Science. He has been researching and teaching for over 20 years on the etiology and prevention of oral diseases. He has over 200 scientific publications and 16 patents. Professor Reynolds is on the editorial board for the Australian Dental Journal and has also been a member of the editorial board for the Journal of Dental Research . He has received numerous awards and distinctions, with the most significant being the Clunies Ross National Science and Technology Award in 2002 , which honours those who make important contributions to science for the economic, social and environmental benefit of Australia, and the Victoria Prize in 2005, which is awarded to an individual whose scientific discovery or technological innovation has significantly advanced the State of Victorias knowledge base and has potential to lead to a commercial outcome. In 2005, he was appointed an Officer of the Order of Australia (AO) for service to community dental health through research and development of preventive and restorative products, to dental education through curriculum development, and as an administrator and teacher.

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additive, as animals receiving 0.5% CPP-ACP plus 500ppm fluoride had significantly lower caries activity than those animals receiving either CPP-ACP or fluoride alone.10 A human in situ caries model study evaluated the ability of a 1.0% CPP-ACP solution as a mouthrinse, twice daily, to prevent enamel demineralization. The enamel plaque that developed in this model was bacteriologically similar to normal supragingival plaque on frequent exposure to sucrose solutions. The twicedaily use of the 1.0% CPP-ACP solution resulted in a 144% increase in calcium level and a 160% increase in inorganic phosphate level in the interenamel plaque recovered from the removable intraoral appliance used in the study. Moreover, CPP-ACP produced a 51 19% reduction in enamel mineral loss caused by frequent sugar-solution exposure.1 The probable anticariogenic mechanism for CPPACP is the localization of calcium and phosphate ions at the tooth surface, which buffers the free calcium and phosphate ion activities, thereby helping to maintain a state of supersaturation with respect to tooth enamel, depressing demineralization, and enhancing remineralization.1-2
CPP-ACP in Chewing Gum

For each study, all subjects crossed over to each randomly assigned treatment, with at least one week between treatments. All subjects used standard fluoride dentifrice for the duration of each study and chewed the gums at their natural chewing frequency for 20 minutes, four times daily for 14 days.The appliances were worn for the 20 minutes of gum chewing and for 20 minutes following. In the no-treatment control, the appliances were worn for 40 minutes. Subjects were instructed not to eat or drink while wearing the appliance and to rinse and clean their appliances using the fluoride-free denture cleanser plate and toothbrush provided. They were informed not to brush the area containing the enamel blocks. No alterations were made to the subjects diet and oral hygiene procedures for the duration of each study. After completion of each treatment period, the enamel blocks were removed from the appliances for processing. Microradiographic analyses of the enamel lesions were evaluated using appropriate computerized imaging software. The chewing of sugar-free gum produced a four- to sevenfold increase in salivary flow rate over the two-minute period in all subjects. No significant difference was observed between the sorbitol-based and xylitol-based gums in their ability to stimulate saliva or remineralize subsurface enamel lesions. The addition of CPP-ACP to either the sorbitol- or xylitol-based gums at 10.0, 18.8, or 56.4mg produced a significant (p>0.001) increase in enamel remineralization, with a 63%, 102%, and 152% average increase, respectively, relative to the sugar-free gum not containing CPP-ACP. No significant correlation was found between individual unstimulated or stimulated salivary flow rates and percentage enamel remineralization values obtained for any of the treatments. These results indicated that the addition of CPP-ACP to sugar-free chewing gum significantly enhanced remineralization of enamel subsurface lesions in a dose-related manner, independent of gum weight or type. The ability of CPP-ACP to be retained in supragingival plaque and remineralize enamel subsurface lesions in situ when delivered in a mouthrinse or sugar-free gum was studied in randomized, double-blind trials involving 30 adult subjects.13 The mouthrinse study was a randomized, double-blind, cross-over design involving four coded

Clinical trials of sugar-free chewing gum have shown that the gum is non-cariogenic and, in fact, can have an anticariogenic effect through the stimulation of saliva.1113 Therefore, it was proposed that sugar-free gum may be an excellent delivery vehicle for CPP-APP to remineralize enamel subsurface lesions. The effect of incorporating CPP-ACP into sugar-free gum on enamel remineralization was evaluated in three randomized, double-blind, crossover studies in 30 patients.4 The subjects in the studies wore removable palatal acrylic appliances with six humanenamel half-slabs inset containing subsurface demineralized lesions. The protocols of the three studies were identical except for the specific sweetener (sorbitol or xylitol), weight, and type (slab or pellet) of sugar-free gum, CPP-ACP dose, and number of treatments.The gums were provided as coded products and were stored at room temperature. All chewing gum treatments were double-blinded and randomized.

11. Kandelman D, Gagnon G,A 24-month clinical study of the incidence and progression of dental caries in relation to consumption of chewing gum containing xylitol in school preventive programs, J Dent Res (1990);69: pp. 17711775. 12. Beiswanger BB, et al., The effect of chewing sugar-free gum after meals on clinical caries incidence, J Am Dent Assoc (1998);129: pp. 16231626. 13. Reynolds EC, et al.,Retention in plaque and remineralization of enamel lesions by various forms of calcium in a mouthrinse or sugar-free chewing gum, J Dent Res (2003);82(3): pp. 206211. 52

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REFERENCES: 1. Data on file. 2. Shen P, Cai F, Nowicki A, Vincent J, Reynolds EC. Remineralization of enamel subsurface lesions by sugar-free chewing gum containing casein phosphopeptide-amorphous calcium phosphate. J Dent Res. 2001; 80: 2066-2070. 3. Reynolds EC, Cai F, Shen P, Walker GD. Retention in plaque and remineralization of enamel lesions by various forms of calcium in a mouthrinse or sugar-free chewing gum. J Dent Res. 2003; 82: 206-211. RECALDENT is milk derived and, therefore, not recommended for your patients with milk allergies. RECALDENT is digestible by patients with lactose intolerance. RECALDENT is a registered trademark of Recaldent Pty. Ltd.

Dental Gum

mouthrinses.Two of the mouthrinses contained CPPACP (2% w/v and 6% w/v, respectively), the third mouthrinse contained an unstabilized slurry of 60mM CaCl2 and 40mM sodium phosphate. The fourth mouthrinse was de-ionized water and acted as the control. In the mouthrinse study, only the CPP-ACPcontaining mouthrinses significantly increased plaque calcium and inorganic phosphate levels, and the CPP were immunolocalized to the surfaces of bacterial cells, as well as to the intercellular plaque matrix. In the chewing gum studies, two randomized, double-blind cross over remineralization trials were conducted with three pellet and three slab sugar-free gums containing different forms of calcium, including CPP-ACP. According to the results, the gum containing the CPP-ACP, although not containing the most calcium per piece of gum, produced the highest level of enamel remineralization independent of gum-chewing frequency and duration. The CPP could be detected in plaque extracts three hours after subjects chewed the CPP-ACP-containing gum. These results highlight the importance of CPP in delivering ACP to the tooth surface. Moreover, the results also demonstrate the importance of the CPP in stabilizing ACP and producing a highly water-soluble calcium phosphate phase. The CPP-ACP gums were superior to the other sugar-free gums in remineralizing enamel subsurface lesions in situ, even though the other gums contained from five to 13 times the level of total calcium. In a recent double-blind, randomized, cross-over study, designed to investigate the acid resistance of enamel lesions remineralized in situ by a sugar-free chewing gum containing CPP-ACP, the active treatment group demonstrated superior resistance to a subsequent acid challenge.14 The study compared sugar-free gum containing 18.8mg of CPP-ACP and sugar-free gum not containing CPP-ACP. Subjects wore removable palatal appliances with insets of human enamel containing demineralized subsurface lesions, and chewed the gum for 20 minutes four times daily for 14 days. After each treatment, the enamel slabs were removed and half of each lesion challenged with acid in vitro for eight or 16 hours.The level of remineralization was determined using microradiography. The gum containing CPP-ACP produced approximately twice the level of remineralization as the control sugar-free gum. The eight- and 16-hour acid challenge of the lesions remineralized with the control gum resulted in

65.4% and 88.0% reductions, respectively, of deposited mineral, while for the CPP-ACP-remineralized lesions the corresponding reductions were 30.5% and 41.8%. The acid challenge after in situ remineralization for both control and CPP-ACP-treated lesions resulted in demineralization underneath the remineralized zone, indicating that the remineralized mineral was more resistant to subsequent acid challenge. CPP-ACP gum has also been shown to slow the progression and enhance the regression of dental caries in adolescents. 2,720 subjects were randomly assigned to either a test or control group. All subjects received accepted preventive procedures and optimum systemic fluoride exposure. The test group received a sugar-free gum containing 54.4mg CPP-ACP, while the control group received an identical gum without CPP-ACP. Subjects were instructed to chew their assigned gum for 10 minutes, three times per day, with one session supervised on school days, over the two-year study period. Standardized digital bitewing radiographs were taken at the baseline and at the completion of the clinical trial. Analysis of caries progression or regression was undertaken using a transition matrix. There was a statistically significant difference in the distributions of the transition scores between the two groups (p<0.001). The CPP-ACP gum slowed progression of caries compared with the control gum. Nineteen percent more of the approximal surfaces experienced caries progression in the control gum group compared to the same surfaces in the CPP-ACP gum group.The CPP-ACP gum also enhanced regression of caries compared with the control gum. Fifty-six percent more of the approximal surfaces experienced caries regression in the CPP-ACP gum group compared to those in the control gum group.15
Summary

The enamel remineralizing properties of CPP-ACP have been determined from many years of basic and evidencebased clinical research. The incorporation of CPP-ACP into chewing gum has been shown to increase enamel remineralization.This represents a significant advance in treatment of early carious lesions and prevention in the 21st century. Dental practitioners should recommend CPP-ACP-containing products to their patients, particularly children, as well as adults who are at high risk for caries. Remineralization of early carious lesions may continue to emerge in importance as fluoride did in the past for caries prevention and reduction.

14. Iijima Y, et al., Acid resistance of enamel subsurface lesions remineralized by a sugar-free chewing gum containing casein phosphopeptide-amorphous calcium phosphate, Caries Res (2004);38(6): pp. 551556. 15. Morgan MV , et al.,CPP-ACP gum slows progression and enhances regression of dental caries, 84th IADR General Session and Exhibition, (2006) June 28 July 1: Abstract 2445. 54

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