Evaluation of the Effect of Casein Phos-phopeptide – Amorphous Calcium Phosphate Mousse versus Natural Raw Fresh Milk on Enamel Hardness After a pH Challenge

Aims: The aim of this study was to evaluate the effect of CPP−ACP, raw fresh buffalo milk and cow milk on the microhardness of artificial initial caries lesions. Materials and Methods: 100 sound maxillary first premolars were collected and randomly divided into four groups, the teeth in all groups were subjected to pH cycling procedure then treated with: Group1: n (25) deionized water, group2: n (25) CPP−ACP tooth mousse, group3: n (25) fresh raw cow milk, group4: n (25) fresh raw buffalo milk. Enamel surface was assessed by Vickers microhardness test device at a baseline and after demineralization and after remineralization. Results: In all groups, there were high statistically significant reduction of surface microhardness after demineralization. There was high statistically significant increase in surface microhardness in all groups except deionized water after remineralization, the highest remineralization effect was found in CPP−ACP tooth mousse group followed by the milk groups that showed encouraging results with fresh raw buffalo milk having superior results then fresh raw cow milk. Conclusions: CPP−ACP tooth mousse, raw fresh buffalo milk and cow milk were effective remineralizing agents which were reflected by increasing the surface microhardness of artificial initial caries lesion, but with different abilities, CPP−ACP tooth mousse was the best followed by buffalo milk then cow milk.


INTRODUCION
Dental caries is considered as a major public oral health problem worldwide (1) . To control this problem, management must start with the first sign of the disease which appears as white spot lesion also known as initial carious lesion and is defined as a non-cavitated carious lesion restricted to visual change in color of enamel and texture (2) .
The principles of minimum invasive technique in dentistry dictate the need for clinical effective measures to remineralize early caries lesions. While fluoride-based remineralization, agents are the cornerstone of caries management techniques, a number of new remineralization procedures commercially available that concentrate or enhance deeper remineralization of caries lesions and decrease the potential risks related with high-fluoride products, and control caries disease over a lifetime (3) .
Casein phosphopeptide − amorphous calcium phosphate is considered a bioactive agent, milk base products. Casein phosphopeptide (CPP) is capable of stabilizing amorphous calcium phosphate (ACP) and also localizes it in dental plaque. This nanocomplex represents as a reservoir for bioavailable calcium and phos-phate, so facilitates remineralization by maintaining the solution in supersaturated level (4,5) .
Milk has a high ability in supporting enamel remineralization. Milk can be obtained from different sources. Casein of the milk which is the main protein of milk, high concentration of phosphate and calcium. All these play an important role in preventing demineralization and promoting remineralization (6,7) .
The aim of the current study is to evaluate the effect of casein phosphopeptide-amorphous calcium phosphate versus natural raw fresh buffalo and cow milk on enamel hardness after a pH challenge.

MATERIALS AND METHODS
The study was approved by Research Ethics Committee board (University of Mosul, College of Dentistry, REC reference No. POP/S.14/6/20).

Sample Collection
The sample of this study consisted of (100) human permanent first premolars extracted for the purpose of orthodontic treatment. After extraction, the teeth were cleaned with tap water and examined with 10X magnifying lens. The selection of the teeth followed specif-Al-Ani SA. Al-Naimi RJ ic criteria: the teeth must be sound, free from enamel defects, decay, stain, cracks, demineralization, fluorosis and restorations. The teeth were stored in 0.1% thymol solution.

Sample Preparation
The teeth were cleaned with rubber cup and non-fluoridated pumice by using low speed hand piece, then the crowns of the teeth were separated from the roots at cemento-enamel junction by using diamond disc in hand piece under copious of water, then the teeth were adapted in a cold cure acrylic mold with specific dimensions (20mm diameter,15mm depth) in a technique that the buccal surfaces of crowns were exposed and paralleled to the floor as seen in Figure (1). The buccal surface of each tooth was grounded and polished using grit paper (grit 400, 600) ten times in one direction, in order to get a flat surface of each specimen of tooth for microhardness testing (8) .

Groups Design and Methods:
Group 1: (control negative) n=25 the teeth were subjected to pH cycling procedure for ten days and then were stored in deionized water until subjected to microhardness test.
Group 2: n=25 the teeth were subjected to pH cycling procedure for ten days and then CPP−ACP tooth mousse was applied. The protocol of application GC tooth mousse in this study was followed the manufacturer's instructions by application thin layer of the cream on each tooth specimen by fine brush for 3minutes, then again the cream was distributed by the brush and left for 30 minutes, this procedure repeated two times daily for 7 days (9) .
Between sessions the teeth were stored in deionized water.
Group 3: n=25 the teeth were subjected to pH cycling procedure for ten days and then, the teeth were immersed in 250 ml of raw fresh cow milk for 50 hours continuously. The milk was replaced every 2 hours (10) . Then the teeth were stored in deionized water until subjected to microhardness test.
Group 4: n=25 the teeth were subjected to pH cycling procedure for ten days and then, the teeth were immersed in 250 ml of raw fresh buffalo milk for 50 hours continuously, the milk was replaced every 2 hours (10) . Then the teeth were stored in deionized water until subjected to microhardness test. All the groups can be seen in Figure (

Formation of Initial Like Caries Lesion in Enamel Surface of Specimens
The formation of initial like caries lesion followed the protocol of demineralizing and remineralizing solutions preparation, and adjustment of the pH (11,12) . hours. Then the teeth were removed and rinsed with running deionized water for one minute.
After that, each group was immersed in 250 ml of remineralizing solution for 17 hours .This procedure was done one time each day and repeated for a period of 10 days (11,12) .

Microhardness Test:
The enamel hardness of the specimens was measured by Vickers microhardness tester device (Wolpert, Germany). The measurements were conducted using 500 gm load for 15 seconds to the occlusal third of the enamel surface of the specimens. The time and load were constant for all the specimens for the whole study.
For each specimen three indentations were measured and the average value of them was calculated.
The Vickers hardness number (VHN) was calculated after measuring the two diagonal d1,d2 length of the square impression which was formed by the indenter using scaled microscope (70X lens) in the device (13,14) and then

DISCUSSION
Microhardness studies provide a relatively simple, easy nondestructive, and rapid method in mineralization studies. Therefore, in the present study, the microhardness values for each specimen are measured three times at the baseline, and after pH cycling procedures that caused the induction of carious lesion (demineralization), and after the remineralization process. It also allows repeated measurements taken over a period of time of the same specimen, and needs tiny area of specimen surface for assessment (15,16,17) .
In the past fluoride was the corner stone of most remineralization studies (18,19) but due to the increased risk of dental fluorosis especially in children which has led to recommendation of the need to assess the amount of exposure of the population before introducing any additional fluoridation for caries prevention (20) due to fear of increasing dental fluorosis. Therefore, there is a need for development of other novel enamel remineralization systems other than fluoride. The most promising of these remineralizing agents is the CPP−ACP (3) .
Our study showed that the results of CPP−ACP, fresh raw buffalo and cow milk application resulted in remineralization, the results were highly promising considering the two types of milk used and is considered the first study that has used fresh raw buffalo and cow milk, and there was an increase in hardness values of the initial caries lesion that was created.
In Table ( within groups due to the differences in remineralizing ability of each treatment regime. In Table ( (21,22) .  (25) . Also, milk has bioactive peptides, casein phosphor proteins that are strongly absorbed in the surface of enamel and able to reduce the activity and adsorption of glucosyltransferase enzymes, also preventing or reducing the dissolution of the enamel (26) . The results of remineralization effect of milk are in agreement with Vieira et al. (27) that concluded that whole milk showed significant higher gain of enamel microhardness of eroded enamel as compared to filtered water.

Al-Ani SA. Al-Naimi RJ
Our study revealed that CPP−ACP exhibited the highest remineralization , this could be due to the concentrated amount of calcium and phosphate and casein in the synthetic product of CPP−ACP tooth mousse in comparison to milk. Although the increase in the microhardness was significant in both types of milk used, but it was more and showed superior results in buffalo milk than cow milk which might be attributed to that the calcium content in buffalo milk which is more than the cow milk and the protein (80% of it casein) was higher in buffalo milk than cow milk also it might be attributed to the reason that the whey protein is greater in buffalo compared to cow milk (28) .
This result was in agreement with Vongsavan et al. (29) who concluded that the mean microhardness value increased in both buffalo and cow milk in dry milk powder formula, but was more in buffalo milk than cow milk.

CONCLUSION
Within the limitations of the current study, the demineralization process by pH cycling procedure decreased the microhardness and created initial like caries lesion on enamel surface. CPP−ACP tooth mousse, raw fresh buffalo milk and cow milk showed promising remineralizing effect which were reflected by increasing surface microhardness of artificial initial caries lesion, but with different abilities, CPP−ACP tooth mousse had the superior effect followed by fresh raw buffalo milk which was superior to fresh raw cow milk in remineraliza-tion abilities expressed by increased hardness scores.