Comparison of Remineralization Effect of Casein Phosphopeptide Amorphous Calcium Phosphate and Sodium Fluoride on Enamel Surface after Orthodontic Debonding: An In vitro study



Hafiza Asma Jawaid1           BDS, MDS
Asmi Shaheen2                    BDS, M.Phil, FCPS
Muhammad Ilyas3                BDS, FCPS
Waheed-ul-Hamid4               BDS, M.Orth RCSED, MS (Ortho)
Ahmad Shamim5                  BDS, MDS
Asif Shakoor6                       BDS, M.Phil



OBJECTIVE: The present study was under taken to determine the anti-fungal effect of Silver Nano partial coating in
concentrations of 0.1%, 0.2%, 0.5% and 1% on heat cure acrylic denture through diffusion disc method. Poly methyl methacrylate
(PMMA) or simply acrylic is most commonly used material for construction of complete dentures. Denture stomatitis is an
inflammatory disorder of oral mucosa, frequently observed in denture wearers. The unpolished intaglio surface of PMMA base
dentures coupled with adverse conditions such as poor hygiene, dry mouth and compromised immune system leads to denture
related stomatitis in 50-70% of complete denture wearers. Antifungal agents such as silver have been added to acrylic denture
bases to in part self-disinfect property.
METHODOLOGY: The supplied modeling wax sheet was cut in to 25 specimens of dimensions 10x10x 2mm with help of
wax knife. The wax sheet was invested in stone plaster with in metal flask using open flasking method for mould formation.
Heat cured acrylic resin polymer and monomer (Meadway Royale Heat Cure, MR. Dental, and UK) was mixed according to
manufacturer's instructions of 2.5gm powder to 1ml monomer. The mixed acrylic was packed in dough stage followed by
pressure packing in hydraulic bench press for 30 minutes under 9.8 MPa. Curing was done by placing the flask in water at room
temperature until boiling. It remained in boiling water for 45 minutes and then allowed to cool down in water bath. A total of
25 acrylic plates were recovered from the flask and divided into five groups. Group A have no coating , group B coated with
0.1% sliver nano particles, group C coated with 0.2% silver nano particles, group D coated with 0.5% silver nano particles and
group E coated with 1% silver nano particles solution. Each specimen was cut in to 6mm disc by Laser Engraving Machine.
These discs were utilized for calculating zone of inhibition through diffusion disc method in agar media.
RESULTS: The diameter of zone of inhibition increased with the increasing concentrations of silver nano particles. When the
concentration of silver nano particles was 1%, the zone of inhibition size was maximum (20.48mm). When the concentration was
0.1%, the size of zone of inhibition was minimum (10.02mm) .This difference was statistically found to be highly significant (0.005).
CONCLUSION: This study results demonstrate that silver nano-particles have good antifungal activity against Candida Albicans
when used as surface coating. This antifungal property is directly influenced by the concentration of silver nano particles used.
KEY WORDS: Antifungal property, Silver nano particles (AgNPs), Candida Albicans (CA).
HOW TO CITE: Haider B, Imran M, Raza M, Riaz Z, Hanif A, Akram S. Antifungal effect of silver nano particles coating
on denture base specimens made of acrylic resin. J Pak Dent Assoc 2022;31(2):59-64.
Received: 17 June 2021, Accepted: 07 January 2022

Fixed appliances are mostly the appliance of choice for orthodontic treatment.1 The advantages of fixed orthodontic treatment are patient comfort and conservative method of bonding.2 Disadvantages include surface roughness, scratches, discoloration, demineralization
and caries.3 After fixed orthodontic treatment, the most difficult problem is the control of enamel demineralization.4 The rough surfaces of bands, wires and brackets confine the cleaning activity of cheeks, tongue and lips.5 Thus, plaque accumulation and cariogenic micro-organisms increase widely during orthodontic treatment.6 Clinically visible
demineralized lesions arise as quickly as 4 weeks following placement of orthodontic appliances with the incidence of 24.9% to 96%.7,8
Demineralization is the removal of enamel’s inorganic constituents by acids that are produced by bacteria existing
in the plaque.9 These acids soften calcium phosphate content of dentine and enamel, and result in demineralization.10 Few
teeth are extra prone to demineralization such as maxillary lateral incisors, canines, especially in the gingival third and
first molars in the middle third.9,11 Demineralization presents an aesthetic concern because it compromises the aesthetics of smile.12 Regular remineralization via mineral ions existing in saliva occurs simply in the shallow layers of demineralized
Though, different managements are mentioned to support remineralization. The scientific base of fluoride use is that fluoride be able to enter into crystal-like arrangement of enamel, reduces its solubility plus increase resistance for acids. Fluoride ions substitute hydroxyl assembly of hydroxyapatite, then form fluorapatite.13 Thus low dose fluoride application is advised for sub surface remineralization.11
A new remineralizing agent CPP-ACP, is a derivative of milk casein, has ability to absorb through enamel surface.14,15 It preserves supersaturated position of enamel mineral, phosphate and calcium in plaque and encourage remineralization,16 delays the growth of biofilm plus hinders the adhesion of bacteria to tooth surface.17,18 It works like buffering agent that stop a fall of pH and regulate acid-base
balance.17 So, CPP-ACP has been included in various manufactured goods for example, chewing gums, mouth rinses, sports drinks, glass ionomer cements, topical creams and water based mousse.19,5
Therefore, the rationale of this research was to determine the most effective method for remineralization of demineralized enamel
which facilitates the clinicians to overcome the demineralization of enamel after orthodontic debonding.

    This experimental study of twelve months was performed in Orthodontics department of de’Montmorency College of Dentistry, Lahore. The sample size was calculated by convenient sampling technique followed by randomization, keeping power of study equal to 90% and level of significance equal to 5% using formula.
(no caries, no cracks, no exposure to chemicals, no filling) are collected from department of Oral Surgery, Punjab Dental Hospital, Lahore and divided into 3 groups A, B and C, each of 15 teeth. The labial surface of teeth was “ground wet” by using 200, 400, plus 600 grit paper of silicon carbide (Automata A, JearWirtz, West Germany) and then buffed by alumina suspension (BUEHLER, IL, USA) to representUSA) was poured in Customized molds of plastic. The roots of teeth were detached 2 mm beneath the cemento-enamel joint with a diamond disk (3M UnitekCorp. Monrovia, Calif) and coronal portion were fixed horizontally into self-cured acrylic resin. Then teeth were burnished with non-fluoridated pumice, washed with water and dried with oil free air.
The baseline Microhardness of samples was determined using Microvickers Hardness Tester (Wilson Wolpert Microvickers, 402 MVD, Japan) with a force of 100 grams for 15 seconds. 5 indentations, at a distance of 120 micrometer were made and baseline MH of every sample was determined by averaging the value of all five indentations. The objective of baseline MH determination was to evaluate the alterations
that occur following demineralization and remineralization.
Then teeth were etched by 37% phosphoric acid gel “(3M/ESPE, St. Paul, MN, USA)” for 30 sec, showered by water, then dry with air for 10 sec. to obtain frosty white appearance. Light cure primer “(Transbond XT of 3M Unitek Monrovia, CA, USA)” was applied on enamel surface. Then resin composite (Transbond XT of 3M Unitek) was coated on bracket base, the bracket was bonded and resin was light
cured using high intensity blue light (intensity 5 W, wavelength 420 nm~480 nm, Woodpecker Medical Instrument) for 20 sec. All samples were saved in deionized water for 1 week. The brackets were deboned with a debonding plier (UnitekCorp. Monrovia, Calif) and remaining
composite was removed by Sof Lex aluminum oxide finishing disks (3M UnitekCorp. Monrovia, Calif) in low speed hand piece (NSK, Pana Air, Japan) operated at 10,000 rpm. Then the teeth of all groups were stored in separate beakers containing deionized water. The artificial caries solution/demineralizing solution was prepared with the pH of 4.4 [2.2mM/L CaCl2, 2.2 Mm/L NaH2PO4, 50Mm/L acetic acid, 100 Mm/L NaCl (Merck, USA)][20] . In order to create artificial demineralization, all samples were immersed in 100ml demineralizing solution at room temperature for 1 week. The demineralizing solution was replaced every day to avoid accumulation of substances formed through demineralization. After 1 week, they were kept in separate bottles that contain deionized water. After verification of demineralized lesion by visual inspection, SMH of each sample was re-measured for demineralization as described above. After that, surface treatments were carried out.
“CPP-ACP” was applied on the teeth of gp B and NaF  was applied on the teeth of gp C two times daily for 3 minutes for remineralization. All teeth were cycled between deionized water and surface treatment with remineralizing agent for 12 weeks. Group A was considered as control gp for assessment of remineralization. The MH of gp B and gp C samples was re-measured for remineralization. The result of study shows that here is substantial enhancement in microhardness after using CPP ACP and NaF. But group B shows more significant improvement in microhardness than group C. This shows that CPP-ACP is extra efficient remineralizing agent as compared to NaF.

   The collected information was assessed by IBM SPSS Statistics 20 (Statistical Package for Social Sciences). Mean ± SD were given for quantitative variable (MH). One way ANOVA was applied to compare the mean differences in MH and following demineralization among the groups. Paired t-test was practiced to match the MH prior to and following demineralization. Independent t-test was applied to compare MH among gp B and gp C. P value of < 0.05 was measured statistically significant.

The MH of every group A (Deionized water), B (CPP ACP) and C (NaF) was measured for demineralization and remineralization. The outcome of CPP-ACP and NaF on the alterations in MH of enamel (%age of microhardness retrieval, %VHNR) was concluded by following formula: % VHNR = (remineralized enamel microhardness – demineralized enamel microhardness) / (baseline enamel microhardness – demineralized enamel microhardness) x 100.
Results proved that here was a considerable decrease in enamel micohardness among three groups after demineralization (P < 0.001).
The results of the current study shows that CPP-ACP is statistically significant than NaF in increasing MH of demineralized enamel. Thus CPP-ACP is helpful in our clinical practice if used daily during orthodontic treatment as it increases the rate of remineralization.

Demineralization is one of the causes of tooth loss that causes molecular alterations in apatite minerals of tooth, to an obvious demineralized area, association of dentin, and ultimate cavitation.15 Following the end of fixed orthodontic treatment, the most difficult problem is the control of enamel demineralization. Usually, surface treatment of demineralization was concerned about the mineral loss and
mineral gain to the tooth. Thus objective is the facilitation of recovering new minerals.21 Hence, demineralization persist as primary apprehension for both patients and orthodontists. Since the increased incidence of demineralization in orthodontic patients and the importance of esthetics, occurrence of demineralization must be prohibited. Therefore, we assessed the efficiency of NaF and CPP ACP for inhibition of demineralization to find the best effective agent for this goal.13
Techniques for delivering calcium and phosphorous to assist remineralization have been backbone of this kind of study.21 Fluoride becomes incorporated into the tooth, and replace the hydroxyl group of the hydroxyapatite.15 By the use of low-concentration of calcium and phosphate solution, no remineralization occur because this is not effective in localizing the ions on surface of tooth in large concentrations.22
The CPP contains multiple phosphoryl sequences with ability to stabilize calcium phosphate complexes in solutions such as ACP.15 Previously, the researchers had focused on primary prevention of demineralization, but in current study, we have emphasised on secondary prevention, chiefly, management of persisting demineralized lesions. In previous studies, numerous techniques have been practiced to evaluate the amount of remineralization, comprising quantitative light-induced fluorescence (QLF), DIAGNOdent, x-ray spectrophotometer, polarized light microscope and microcomputed tomography.15,23,24 In our study, we have used Vickers indenter for measurement of MH of enamel and baseline SMH value range from 290.0 to 289.8 VHN that was related to preceding study by Lussi et al. in 2000. The sharp decrease in SMH occurred after demineralization. The test materials (NaF and CPP ACP) were applied to enamel for remineralization. After that, mean SMH of samples raised by 230.2 VHN and 207.5 VHN in both groups. The percentage of SMHR show maximum improvement of 41.2% in CPP-ACP and 19.1% in NaF group. Thus, NaF encourage remineralization of enamel, but not as efficiently as CPP-ACP.
This study is in line with Kumar et al. Qiong et al. Guçlu et al. and Lopatiene. Kumar described that CPP-ACP is more efficient as compared to fluoride in decreasing lesion depth. Though fluoride causes an increase in enamel resistance, but resulting remineralization, is a self-restricting phenomenon. This explain the increased efficacy of CPP-ACP in increasing MH than that of fluoride.25 Qiong et al, conducted an in vitro study to compare CPP-ACP crème with 500 ppm NaF solution for prevention of primary caries in childhood. The result showed that CPP-ACP cream is useful for remineralization of premature enamel lesions of deciduous teeth, extra efficient as compared to 500 ppm NaF.26 Guçlu et al, told that application of CPP-ACP paste can manage demineralized lesions in permanent teeth, and additional application of 5% NaF varnish had no useful results. They alleged that fluoride causes hyper mineralization of superficial layer, which blocks entry of mineralizing ions into subsurface area.27 In a systematic analysis, Lopatiene proved that ACP as well as fluoride are equally efficient in
treatment of demineralized lesions throughout and afterward fixed orthodontic treatment, but CPP-ACP was extra useful in improving lesions.28
Farzanegan et al. compared the efficiency of amorphous calcium phosphate (ACP) and fluoride on MH recovery of damaged enamel. Results showed that MH of samples in the ACP and fluoride groups had considerably enhanced afterward treatment as compared to control gp. Conferring to the above study, both 0.05% ACP as well as 0.05% fluoride solutions improved micro-hardness of enamel in the management of demineralized lesions.29
Actually, CPP stop the quick conversion of calcium phosphate phase and thus, ions become stable and retained at tooth surface. So, CPP has ability to transport phosphate, fluoride and calcium ions deeply  into the subsurface lesions.30 Nano-sized crystals were organized on body of lesions after application of CPP-ACP with less inter crystalline gaps.26 The efficiency of CPP-ACP could be improved in mouth
when a biofilm is present, that could attach to CPP and used as storage for phosphate and calcium ions.25 Thus CPP-ACP may be helpful in our clinical practice if used on regular basis during the period of orthodontic treatment.

After this study, it is concluded that, a remineralizing cream having CPP-ACP has promising results in remineralization of demineralized lesions as compared to NaF. CPP-ACP increases MH of enamel more efficiently as compared to NaF with the addition of few minerals and has aesthetic benefits for patients.

None declared.


1. Harry DR, Sandy J. Orthodontics. Part 5: Appliance choices. Br Dent J 2004;196:9-18.

2. Miksic M, Slaj M, Mestrovic S. Qualitative analysis of the enamel surface after removal of remnant composite. Acta Stomat Croat 2003;

3. Trakyali G, Ozdemir FI, Arun T. Enamel colour changes at debonding and after finishing procedures using five different adhesives. Eur J
Orthod 2009;31:397-01.

4. Elkabbany SMH, Mosleh AA, Metwally NI. Remineralization effect of diode laser, Nanoseal®, and Zamzam water on initial enamel carious lesions induced around orthodontic brackets. J Nat Sci Med 2021; 4:50-7.

5. Jena A, Duggal R. Enamel scars in orthodontics. Am J Orthod Dentofac Orthop 2004;125:36-41.

6. Akin M, Basciftci FA. Can white spot lesions be treated effectively? Angle Orthod 2012; 82:770-75.

7. Bebber LV, Campbell PM, Honeyman AL, Spears R, Buschang PH. Does the amount of filler content in sealants used to prevent
decalcification on smooth enamel surfaces really matter? Angle Orthod 2011;81:134-40.

8. Kaua CH, Wangb J, Palombinic A, Abou-Kheirb N, Christou T. Effect of fluoride dentifrices on white spot lesions during orthodontic
treatment:A randomized trial. Angle Orthod 2019;89:365-71.

9. Alsubhi H, Mohammad Gabbani M, Alsolami A, Alosaimi M, Abuljadayel J, Taju W, Bukhari O. A comparison between two different
remineralizing agents against white spot lesions: an in vitro study. Int J Dent 2021;1-5.

10. Uysal T, Amasyali M, Ozcan S, Koyuturk AE, Akyol M, Sagdic D. In vivo effect of amorphous calcium phosphate-containing
orthodontic composite on enamel demineralization around orthodontic brackets. Aust Dent J 2010;55:285-91.

11. Willmot DR. White spot lesions after orthodontic treatment. Sem in Orthod 2008;14:209-19.

12. Bailey DL, Adams GG, Tsao CE, Hyslop A, Escobar K, Manton DJ, Reynolds EC, Morgan MV. Regression of post orthodontic lesions
by a remineralizing cream. J Dent Res 2009;88:1148-53.

13. Tahmasbi S, Mousavi S, Behroozibakhsh M, Badiee M. Prevention of white spot lesions using three remineralizing agents: An in vitro
comparative study. J Dent Res Dent Clin Dent Prospect. 2019;13:36-42.

14. Bhadoria N, Gunwal MK, Kukreja R, Maran S, Devendrappa SN, Singla S. An in vitro evaluation of remineralization potential of
functionalized tricalcium phosphate paste and cpp-acpf on artificial white spot lesion in primary and permanent enamel. Int J Clin Pediatr
Dent 2020;13:579-84.

15. Geeta RD, Vallabhaneni S, Fatima K. Comparative evaluation of remineralization potential of nanohydroxyapatite crystals, bioactive
glass, casein phosphopeptide-amorphous calcium phosphate, and fluoride on initial enamel lesion (scanning electron microscope analysis)-
An in vitro study. J Conserv Dent. 2020;23:275-9.

16. Siddika F, Khan MSR, Bao RJ, Sheng MW. Managing white spot lesion during and after the orthodontic treatment. J Pak Dent Assoc
2018; 27:1-8.

17. Beerens MW. White spot lesions after orthodontic fixed appliance treatment, the effectiveness of MI Paste Plus® as a remineralizing
agent: A randomized controlled trial. Thesis 2018; University of Amsterdam and the VU University Amsterdam, the Netherlands.
Corpus ID: 80324168.

18. Cochrane NJ, Cai F, Huq NL, Burrow MF, Reynolds EC. New approaches to enhanced remineralization of tooth enamel. J Dent Res

19.amorphous calcium phosphate and a cola soft drink on in vitro enamel hardness. J Am Dent Assoc. 2009;140:455-60.

20. Paschos E, Kleinschrodt T, Luedemann TC, Huth KC, Hickel R, Kunzelmann KH, Janson IR. Effect of different bonding agents on
prevention of enamel demineralization around orthodontic brackets. Am J Orthod Dentofac Orthop. 2009; 135(5):603-12.

21. Sammel SA. In vitro remineralization of human enamel with bioactive glass containing dentifrice using conofocal microscopy and
nanoindentation analysis for early caries defence. Thesis 2004; University of Florida, USA.  Reynolds EC. Calcium phosphate-based remineralization systems: scientific evidence? Aust Dent J 2008;53:268-73.

22. Behroozibakhsh M, Shafiei F, Hooshmand T, Moztarzadeh F, Tahriri M, Bagheri Gorgani H. Effect of a synthetic nanocrystalline
fluorohydroxyapatite on the eroded enamel lesions. Dent Mater. 2014; 30(Supplement 1): e117-e118.

24. Featherstone JD, Lussi, A. Understanding the chemistry of dental erosion. Monogr Oral Sci 2006;20:66-76.

25. Kiana SE, Romina M, Leila P. Effect of treatment with various remineralizing agents on the microhardness of demineralized enamel
surface. J Dent Res Dent Clin Dent Prospect. 2015;9:239-45.

26. Qiong Z, Jing Z, Ran Y, Xuedong Z. Remineralization effects of casein phosphopeptide-amorphous calcium phosphate crème on artificial
early enamel lesions of primary teeth. Int J Paediatr Dent. 2011;21: 374-81.

27. Guclu ZA, Alacam A, Coleman NJ. A 12-Week Assessment of the Treatment of White Spot Lesions with CPP-ACP paste and/or fluoride
varnish. biomed Res Int 2016; 8357621.

28. Lopatiene K, Borisovaite M, Lapenaite E. Prevention and Treatment of White Spot Lesions during and after treatment with fixed orthodontic appliances: a systematic literature review. J Oral Maxillofac Res 2016;7(2):e1.

29. Farzanegan F, Mostafavi SMS, Ameri H, Khaki H. Effects of fluoride versus amorphous calcium phosphate solutions on enamel
microhardness of white spot lesions: An in-vitro study. J Clin Exp Dent. 2019;11:219-24.

30. Cross KJ, Huq NL, Palamara JE, Perich JW, Reynolds EC. Physicochemical characterization of casein phosphopeptide-amorphous
calcium phosphate nanocomplexes. J Biol Chem. 2005;280:15362-69.