Effect of casein phosphopeptide-amorphous calcium phosphate (CPP-ACP) topical agents on enamel demineralization around orthodontic braces: An in vitro study

Introduction Orthodontic treatment has many benefits including the improvement of patient’s dental and facial esthetics. Aim The aim of this study was to evaluate the in vitro effects of a casein phosphopeptide-amorphous calcium phosphate (CPP-ACP) topical agent in reducing enamel demineralization around orthodontic braces. Material and method This study comprised 60 healthy extracted premolars without any clinical sign of decalcification. The teeth (40) were divided in two groups according to the period of monitoring, after 1 and 6 months. Then they were coated with a topical gel - GC Tooth Mousse for 5 minutes thrice daily, and they have been stored in artificial saliva until analyzing. We measured the percentage of Ca and Mg in the enamel by using the method of flame atomic absorption spectrometry (FAAS). SEM was used to observe the acid-etched enamel surfaces treated with or without CPP-ACP. Twenty premolars were used for ultrastructural examination of the etched enamel surfaces by SEM. The two examined groups were coated with a topical gel - GC Tooth Mousse for 5 minutes thrice daily, and they were stored in artificial saliva for one and six months. After that, the samples were prepared for SEM analysis (JEOL JSM 5300). Results The mean values of Ca content in enamel were increased in the examined group, compared to the control group of teeth one month after the braces were bonded and topical application with casein phosphopeptide-amorphous calcium phosphate (CPP-ACP). The results indicated that SEM analysis detected the highest remineralization potential in the tooth samples exposed daily to topical gel - GC Tooth Mousse. Conclusions CPP-ACP has the potential to remineralize areas of enamel decalcification in orthodontic patients. The application of CPPACP can improve the mineralization of the teeth during orthodontic treatment with fixed appliance.


Introduction
Orthodontic treatment has many benefits in cluding the improvement of patient's dental and facial esthetics. However, the ideal esthetic out come can be compromised by development of demineralization around the appliances during orthodontic treatment. Enamel demineraliza tion associated with fixed orthodontic applianc es has been observed for years and continues to be a problem in spite of the advances in mate rials and techniques. While using fixed appli ances for orthodontic treatments, the formation of white spot lesions is an unaesthetic and com mon side effect. Bands and braces increase the retention of plaque and food on smooth tooth surfaces that would otherwise tend to have a low prevalence of caries 1 .
Published literature shows wide variation in the prevalence of demineralization and white spot development during fixed orthodontic treat ment. Gorelick et al. 2 observed that, at debon ing, 49.6% of patients showed white spot for mation on at least one tooth, while Sudjalim et al. 3 reported a wide variation within their study, with overall prevalence of white spot lesions ranging between 2% and 96%. Irrespective of prevalence, it is agreed upon that demineraliza tion and development of white spot lesions is a problem during orthodontic treatment. More over, it is established that white spot lesions may persist for years. In the light of the present state of knowledge, it has been shown that CPP-ACP can prevent enamel demineralization and promote remineralization of enamel subsurface lesions. Casein phosphopeptide-amorphous calcium phosphate (CPP-ACP) complex, a milk protein derivative, has been proven to have an ticariogenic effects 4,5,6 . It prevents enamel de mineralization and promotes remineralization through buffering free calcium and phosphate ion activities, 7,8 thereby incorporating ACP into plaque and onto tooth surface and main taining a state of supersaturation with respect to tooth enamel 9 . Remineralized enamel has an in creased resistance to subsequent acid challenge than untreated enamel. The use of CPP-ACP in orthodontic clinical practice has been proposed for white spots prevention and caries prophy laxis before braces bonding procedure 10 . The CPP-ACP complexes act as calcium and phos phate reservoir helping to maintain a state of super saturation of these minerals, which can enhance enamel remineralization. Presently, CPP-ACP complexes are available in a variety of gels, creams, and mousses and may also be incorporated into chewing gums 11 .
The aim of this study was to evaluate the in vitro effects of a casein phosphopeptide-amor phous calcium phosphate (CPP-ACP) topi cal agent in reducing enamel demineralization around orthodontic braces.

Material and method
This study comprised 60 healthy extracted premolars without any clinical sign of decalci fication. All teeth were cleaned and cut in half buccolingually with a diamond disc. Thus, the control and test specimens were obtained from the same teeth. The adhesive used for bonding braces was Con Tec LC (Dentaurum, Germa ny). The teeth (40) were divided in two groups according to the period of monitoring, after 1 and 6 months. Then they were coated with a topical gel -GC Tooth Mousse for 5 minutes thrice daily, and they have been stored in ar tificial saliva (20 mmol/l NaHCO 3 , 3 mmol/l NaH 2 PO 4 and 1 mmol/l CaCl 2 , neutral pH) un til analyzing. We measured the percentage of Ca and Mg in the enamel by using the meth od of flame atomic absorption spectrometry (FAAS), with a Varian Spectra AA 55 B atomic absorption spectrometer 12,13 . Before the analy sis, instrumental parameters for better precision and sensibility were optimized ( Table 1). The content of the investigated elements in the teeth enamel was determined by FAAS after miner alization in a microwave digestion system from Milestone, model Ethos Touch Control (Table  2).

Construction on calibration diagram
Calibration diagram was constructed by us ing of the method of standard solutions. Using regression analysis, functional relationsiphis between mass concetration and absorbance of Ca and Mg were obtained. Standard solutions of Ca and Mg with concetration of 1 mg/L were used. Analytical dependence on concentration absorbance on Ca is given by the equation: A = 0, 0078• γ(Ca)/μg/ml. Correlation coefficient was 0,992.

SEM Observations
SEM was used to observe the acid-etched enamel surfaces treated with or without CPP-ACP. Twenty premolars were used for ultra structural examination of the etched enamel surfaces by SEM. The crowns were sectioned from the roots with a diamond bur at the buc cal cement enamel junction, and each crown was cut longitudinally in an occlusogingival direction to obtain two buccal enamel surfaces. Each surface obtained from the same tooth was randomly allocated to one of two experimental groups. The two examined groups were coated with a topical gel -GC Tooth Mousse for 5 min utes thrice daily, and stored in artificial saliva for one and six months. After that, the samples were prepared for SEM analysis (JEOL JSM 5300), using sputter technique in a vacuum evaporator. The appropriate area of enamel surface was analyzed in order to determine mi cro morphology changes in the structure of the enamel, on the place of previous braces fixa tion.
For statistical evaluation, one-way analysis of variance (ANOVA) followed by Tukey's test were initially used to see if there was a signifi cant difference between the groups.

Results
In the examined group of teeth (Table 3) there were statistically significant differences of Ca content mean values in enamel after treat ment at one and six months (Analysis of vari ance F=3,710; p=0,0284).
The mean values of Ca content in enamel of the examined and control group of teeth one month after the braces were bonded and topi cal application with casein phosphopeptideamorphous calcium phosphate (CPP-ACP) are shown in Table 4.
These values were increased in the exam ined group, compared to the control group of teeth. After one month, a statistically significant difference of Ca values in enamel between the examined and control groups was noticed. Af ter six-month treatment were differences, but not statistically significant of the Ca mean val ues in enamel between the examined and con trol group.
In the examined group of teeth (Table 5), there were statistically significant differences of Mg content mean values in enamel after treatment at one and six months (Analysis of variance F=5,031; p=0,0085). The mean val ues of Mg content in enamel of the examined and control group of teeth one month after the braces were bonded and topical application with casein phosphopeptide-amorphous calci um phosphate (CPP-ACP) are shown in Table  6. After one month, no statistically significant difference of the Mg mean values in enamel between the examined and control group was found. After six-month treatment, there were statistically significant differences of the Mg mean values in enamel between the examined and control group.   U kontrolnoj grupi (veštačka pljuvačka), konfiguracija topografije gleđi očigledno je imala porozne defekte (Slika 1a; uvećanje 1500x). Pri još jačem uvećanju (3500x), po roznost je bila još evidentnija, a oko poroznih polja mogle su se uočiti linije mineralizacije (Slika 1b).
Representative SEM images of the etched enamel specimens pretreated with and without CPP-ACP are shown in Figures 1 -3.
In the control group (artificial saliva), the configuration of enamel topography was pre sented apparent with certain porous defects (Figure 1a; 1000× magnification). At a higher magnification of 2000×, the porosities were more evident and faint lines of mineralization can be seen in the same group and around the porosities (Figure 1b).
In the examined group (after the six-month treatment with CPP-ACP), areas of calcified deposits concentrated along the porous defects were evident (Figure 3a; 3500× magnification). At a higher magnification of 5000×, discern able areas of mineralized deposits and certain areas of calcifications along the porosities were seen in the same group (Figure 3b).

Discussion
It is generally accepted that the insertion of fixed orthodontic appliances creates stagna tion areas for plaque and makes tooth cleaning more difficult. The irregular surfaces of braces, bands, wires, and other attachments also limit naturally occurring self-cleansing mechanisms, such as the movement of the oral musculature and saliva. This in turn encourages a lower plaque pH in the presence of carbohydrates and accelerates the rate of plaque accumulation and plaque maturation. These changes in the local environment appear to favor colonization of aciduric bacteria such as Streptococcus mu tans and Lactobacilli. However, such microbial levels were found to decrease significantly to levels comparable with age matched controls, 6 to 15 weeks into a retention phase, after the removal of fixed appliances.
In one of the first clinical trials of CPP-ACP cream used specifically for treatment of postorthodontic WSLs, Bailey et al. 14 reported that use of CPP-ACP cream enhanced the regression of WSLs compared with placebo. Howev er, more recent clinical investigations show less promising results. In a clinical study, Beerens et al. 15 compared a fluoride-containing CPP-ACP paste with a control paste in 54 subjects. After a three-month treatment period, they found no advantage for use of the fluoridated CPP-ACP paste over regular oral hygiene in WSLs re gression as measured by QLF. The CPP-ACP complex acts as a vehicle for calcium and phosphate, transporting it to the tooth surface and localizing it in plaque and salivary pellicles. A reduction in pH results in a release of calcium and phosphate ions, which act to promote remineralization 16 .
Casein phosphopeptides (CPP) contain mul tiphosphoseryl sequences and these have the ability to stabilize calcium phosphate in nanocomplexes in solution in the form of amorphous calcium phosphate (ACP). These nano com plexes (CPP-ACP) remain very small and they have two effective functions. They can localize at the tooth surface and prevent enamel demin eralization and they are also small enough to be able to penetrate deep into the porous enam el and remineralize the surface of the enamel crystals. At the same time, the CPP-ACP will interact with available fluoride ions to produce an amorphous calcium fluoride phosphate and this can be stabilized by the CPP on the tooth surface. The result from the application of CPP-ACP is remineralization in depth, thus eliminat ing the porosity in the enamel and restoring the light properties and translucency. This study was undertaken to determine the remineraliza tion potential of CPP-ACP. The CPP molecules contain a cluster of phosphoseryl residues that markedly increase the apparent solubility of calcium phosphate by stabilizing amorphous calcium phosphate under neutral and alkaline conditions. CPP can stabilize over 100 times more calcium phosphate than is normally pos sible in aqueous solution at neutral or alkaline pH before spontaneous precipitation occurs. CPP-ACPF has, in addition, fluoride. Studies have shown that remineralization of small le sions with low-dose fluoride therapy is more efficient 17 .
In this in vitro study we examined the per centage of calcium, its effect on enamel de mineralization, and the alterations that were observed on the enamel surface after the use orthodontic bonding system (Con Tec LC Den taurum) and topical application of dental cream GC Tooth Mousse. The results obtained in this study showed a remarkably high percentage of Ca in enamel in the examined group for the first examined period (at one month). The SEM images of the three groups suggest remineral ization with the CPP-ACP showing the great est amount of mineral deposits, after the sixmonths treatment with CPP-ACP.

Conclusions
CPP-ACP has the potential to remineralize areas of enamel decalcification in orthodontic patients. However, since patients with enamel decalcification are generally those with poor oral hygiene, good oral hygiene instruction is essential in preventing enamel decalcification in orthodontic patients, with CPP-ACP used as an adjunct therapy.