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Evaluation of the Effect of Casein Phosphopeptide – Amorphous Calcium Phosphate Mousse versus Natural Raw Fresh Milk on Enamel Surface Roughness After a pH Challenge

Aims: The aim of this study is to evaluate the effect of CPP−ACP, raw, fresh buffalo milk and cow milk on surface roughness of artificial initial caries lesions. Materials and Methods: 100 sound maxillary first premolar extracted for the purpose of orthodontic treatment had been collected and randomly divided into four groups, in all groups the teeth subjected to pH cycle procedure then treated with: Group1: n(25) deionized water, group2: n(25) CPP−ACP tooth mousse, group3: n(25) raw cow milk, group4: n(25) raw buffalo milk. Enamel surface was assessed by a profilometer device at a baseline and after demineralization and after remineralization. Results: In all groups, there was a high statistically significant increase in surface roughness after demineralization. And there was a high statistically significant decrease in surface roughness in all groups except deionized water after remineralization, the highest remineralization effect was found in CPP−ACP tooth mousse group followed by buffalo milk and then cow milk.Conclusions: Within the limits of the current study, it was concluded that CPP−ACP tooth mousse, raw fresh buffalo milk and cow milk were effective remineralizing agents which was reflected by decreasing surface roughness of artificial initial caries lesion, but with different potentials, CPP−ACP tooth mousse was the best followed by fresh raw buffalo milk which showed superior results in reducing surface roughness in comparison with fresh raw cow milk.

In general milk is considered as a protecting agent against dental caries, due to its high level of calcium and phosphorous, and moreover it has protein buffering capacity, especially if consumed immediately after acid intake (5,6) .
Also, it has been proposed that the fat of milk plays an important role in prevention of dental caries, either by its physical effect by decreasing the adhesion between the food and tooth surface, or by microbiological effect by having a bacteriostatic property (7) .
Roughness is a fundamental property of teeth, which affects the attachment of exogenous materials to enamel surfaces and caries progression (2,8) .
The aim of the current study is to evaluate the effect of casein phosphopeptideamorphous calcium phosphate versus natural raw fresh buffalo and cow milk on enamel surface roughness after a pH challenge.

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 specific 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. 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 surface roughness testing (9) .

Milk:
Fresh raw buffalo and cow milk were obtained from local farm dealer (local breed) in Mosul City by single milking dose, the milk was kept cooled in the refrigerator.

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 surface roughness 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 was repeated two times daily for 7 days (10) . 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 (11) . Then the teeth were stored in deionized water until subjected to surface roughness 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 (11) . Then the teeth were stored in deionized water until subjected to surface roughness test. All the groups can be seen in Figure

The pH Cycling Procedure:
Each group was immersed in 250 ml of the demineralization solution and retained for 6 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 (12,13) .  Figure (2) and the resulted value was expressed in µm (14,15) .

Surface Roughness Test:
The cutoff value (reference length) was adjusted at 0.8 mm. Three values of surface roughness for each specimen were taken and the average of these values was considered.     In remineralization there was a statistically significant difference between the groups, the lowest SR mean value was found in CPP−ACP followed by fresh raw buffalo milk followed by fresh raw cow milk group, while the highest value was found in the deionized water group.

RESULTS
However none of the mentioned groups decreased the SR mean values to the baseline. leads to an increase in susceptibility to bacterial adhesion (17,18) (19,20) . In addition, CPP−ACP reduce the lesion depth, improve surface roughness and promote enamel remineralization by deposition of mineral into the porous zone surface of the enamel due to its ability to be as reservoir of bioavailable calcium and phosphate ions (21,22) .

DISCUSSION
The results of CPP−ACP group is in agreement with Piątek-Jakubek et al. (16) (24) who concluded that, milk and CPP−ACP were not significantly effective in decreasing enamel loss after specimens were extra orally eroded.
Moreover, Table ( and cow milk, the calcium content is higher in buffalo milk than in milk from cow, and it contains more colloidal calcium and phosphorus, beside that it is richer in proteins, mineral and fat (25) . In addition to its protective effect we should not forget that milk in all its types is the arch criminal in development of nursing bottle syndrome which is due to bad feeding habits and is always correlated to improper and prolonged bottle use or breast feeding, particularly at bedtime (26) . So the use of milk whether it has a beneficial or harmful effect will depend on the way that it is used.