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Received: 15 May 2018    Accepted: 3 December 2018

DOI: 10.1111/jicd.12400

ORIGINAL ARTICLE
Conservative Dentistry

Preventive effects of carbon dioxide laser and casein

phosphopeptide amorphous calcium phosphate fluoride varnish
on enamel demineralization: A comparative, in vitro study

Moufida Abufarwa1  | Amal Noureldin2 | Taha Azimaie3 | Phillip M. Campbell4 | 

Peter H. Buschang4

1
Department of Biomedical Science, Texas
A&M University College of Dentistry, Dallas,
Texas
2
Department of Public Health
Sciences, Texas A&M University College of
Dentistry, Dallas, Texas
3
Rutgers School of Dental Medicine,
Newark, New Jersey
4
Department of Orthodontics, Texas A&M
University College of Dentistry, Dallas, Texas
Correspondence
Moufida Abufarwa, Department of
Biomedical Science, Texas A&M University
College of Dentistry, Dallas, TX.
Email: mofify@gmail.com
Abstract
Aim: The aim of the present study was to compare the effects of carbon dioxide
(CO2) laser and casein phosphopeptide amorphous calcium phosphate (CPP-ACP)
fluoride varnish on enamel demineralization.
Methods: Human teeth were randomly assigned to three groups. The enamel was
treated with fluoride varnish, 10.6 μm CO2 laser, or no treatment (control), followed
by 9 days of pH cycling. Baseline and final FluoreCam images were used to quantify
the area, intensity, and impact of demineralization; cross-­sectional microhardness
was used to measure the mechanical properties of the enamel.
Results: There were statistically-­significant changes in the area, intensity and impact of
demineralization in the control and laser groups (P < 0.05), but not in the fluoride group.
The control group showed a significantly greater area and impact of enamel deminerali-
zation compared to the fluoride group. The area of demineralization in the laser group
was significantly greater than that of the fluoride group. Enamel demineralization of the
laser and control groups was comparable. The fluoride group showed statistically-­
significant harder enamel than the control at 20, 40, and 60 μm depths; the laser group
enamel was significantly harder than the control at 20 and 40 μm depths. The fluoride
group showed statistically-­significant harder enamel than the laser group at 20 μm depth.
Conclusions: CPP-­ACP fluoride varnish is more effective than CO2 in preventing
enamel demineralization.

KEYWORDS
carbon dioxide laser, casein phosphopeptide amorphous calcium phosphate, enamel
demineralization, FluoreCam, fluoride varnish

1 |  I NTRO D U C TI O N
The development of white spot lesions (WSL) is a common risk associ-
ated with orthodontic treatment using fixed appliances. WSL, which
appear as chalky white patches on the buccal and labial surface of
1
the teeth, jeopardize the esthetic benefits of treatment. The prev-
alence of visually-­assessed WSL among orthodontic patients ranges
from 25% to 28% in university and private practice settings. 2,3 The
development of WSL has been attributed to prolonged plaque accu-
mulation around the brackets, as well as shifts in the bacterial flora.4
While caries development usually takes at least 6 months, WSL in
orthodontic patients have been reported as early as 4 weeks after
appliance insertion.1
Fluoride therapy is the gold standard for caries prevention.
Among the various fluoride vehicles, varnish has proven to be
among the most successful methods for reducing the incidence

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https://doi.org/10.1111/jicd.12400
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of WSL in orthodontic patients. 5 Recently, an enhanced form of

dental varnish, MI Varnish (GC America, Alsip, IL, USA), has been
developed. It combines the preventive effects of fluoride and the
casein phosphopeptide amorphous calcium phosphate (CPP-ACP)
complex. CPP-­ACP provides bioavailable calcium and phosphate
to the tooth surface when oral pH falls below the critical level
(pH = 5.0), thus enhancing enamel remineralization. 6 The anti-
cariogenic activity of CPP-­ACP,7 as well as its synergistic effect
with fluoride, 6 have been demonstrated. MI Varnish enhances
enamel's resistance against acidity 8,9 and rehardens early cari-
ous lesions.10 Furthermore, it releases phosphate11 and greater
amounts of fluoride11,12 and calcium ions than other varnishes.11
Lasers also potentially inhibit enamel demineralization.12,13
Carbon dioxide (CO2) lasers are the most promising.13 Because
CO2 laser wavelengths can be absorbed by enamel, higher pre-
ventive effects with minimum harm to dental tissues have been
reported.13 The 10.6 μm CO2 laser wavelength appears to have
the greatest preventative effect.12,14 While various CO2 laser set-
tings have been reported, those used by Kato et al have achieved
the most promising clinical results.15 Lasers also have a synergis-
tic effect with fluorides.16 The mechanisms of lasers’ cariostatic
effect include purification of enamel hydroxyapatite, reduction
of enamel permeability, and increased enamel fluoride deposition
and uptake.16
No previous studies have compared the anticariogenic ef-
fect of fluoride varnish containing CPP-­ACP and 10.6 μm CO2
laser using the FluoreCam (DARZA, Noblesville, IN, USA). The
FluoreCam system is a new optical detection method designed for
early WSL detection and is one of the most advanced and reliable
devices. 17
Based on the autofluorescence phenomenon of enamel,
the FluoreCam system depicts demineralized areas as being
darker than the surrounding healthy enamel and provides three
outputs: area of demineralized enamel, intensity (light-­intensity
loss), and impact of demineralization (product of intensity and
area). Therefore, the aim of the present study was to compare
the effect of a single application of each treatment on enamel
demineralization.
2 |  M ATE R I A L S A N D M E TH O DS
2.1 | Sample size and power analysis
Sound human molars and premolars were collected and stored in
0.1% (weight/volume) thymol solution at 4°C (Texas A&M University
College of Dentistry, Dallas, Texas, USA; IRB no. 2015-­0 413-­BCD).
Because the entire tooth's surface was not needed, the teeth were
sectioned mesiodistally into buccal and lingual halves using a low-­
speed (0.56 g-force) diamond-­saw (Buehler1000; Isomet, Lake Bluff,
IL, USA). They were then randomly assigned to three groups accord-
ing to the surface treatment (Figure 1): fluoride varnish group, laser
group, or untreated control group. Based on an effect size of 1.3,18
21 specimens per group were necessary to ensure a type I error rate
of 0.02 and 90% power.
F I G U R E   1   Flowchart illustrating the design of the experiment
2.2 | FluoreCam baseline record
Baseline FluoreCam images were obtained under standardized
conditions.19 The FluoreCam was held at a fixed distance from a
mounting table. For each specimen, a mold was made using an im-
pression material (Exaflex Putty; GC America, Alsip, IL, USA). The
FluoreCam was positioned to contact the enamel surface of the
specimen and the impression material was molded around the tip,
providing a reference indentation for later FluoreCam reposition-
ing. After image capture, the FluoreCam computer software re-
corded the area (mm2), intensity (pixel), and impact (pixel.mm2) of
demineralization.
2.3 | Surface treatment
The specimens were covered with two layers of an acid-­resistant nail
polish (cherry color; Revlon, New York, NY, USA), leaving a 2 × 4 mm
window of exposed enamel. Photographs were taken to demarcate
the windows. The windows of the fluoride group were treated with
one layer of MI Varnish (GC America, Alsip, Ill, USA) and immedi-
ately immersed in 5 mL artificial saliva for 24 hours (1.5 mmol L−1 Ca,
0.9 mmol L−1 P, 150 mmol L−1 KCl, 0.05 μg F mL−1 in 0.1 mol L−1 Tris
buffer, pH 7.0).19 After 24 hours, the specimens were washed and
the varnish was removed, exposing the treated enamel.
The enamel windows of the laser group were irradiated with
10.6 μm CO2 laser (2-­watt pulsed mode with 0.2 second irradia-
tion time) using a 0.8-­mm diameter ceramic tip (LX-­20SP Novapulse
CO2 laser; Luxar, Bothell, WA, USA). The parameters were set to
an average power of 0.3 watts, pulse width of 10 m seconds, fre-
quency of 15 Hz, and energy density of 15 J cm−2.15 The distance
from the ceramic tip to the enamel surface was approximately
2 mm. Following irradiation, the specimens where soaked in arti-
ficial saliva for 24 hours.
2.4 | pH cycling
The specimens were subjected to 9 days of pH cycling.19 Each
specimen was suspended in a Falcon tube (VWR, Radnor, PA,
USA) and immersed in 50 mL demineralizing solution for 4 hours
(1.28 mmol L−1 calcium nitrate, 0.74 mmol L−1 sodium dihydrogen
ABUFARWA et al. |
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F I G U R E   3   Baseline between-­group differences in (A) area, (B)

intensity, (C) impact of enamel demineralization. , control; ,
fluoride; , laser

F I G U R E   2   Cross-­sections of tooth section showing Cross-

sectional microhardness indentations within (3 bands) and outside
(2 bands) of the exposed enamel window

phosphate, 0.05 mol L−1 acetate buffer, 0.03 g F mL−1 , pH 5.0).

They were then rinsed with distilled water, dried with absor-
bent paper, and immersed in 25 mL remineralizing solution for
20 hours (1.5 mmol L−1 Ca, 0.9 mmol L−1 P, 150 mmol L−1 KCl,
0.05 μg F mL−1 in 0.1 mol L−1 Tris buffer, pH 7.0.). During the
cycling procedure, the Falcon tubes were kept in an incubator
at 37°C under constant agitation (0.112 g-force). The pH was
checked daily. Both solutions were replaced every fourth day.
On day 9, the specimens were kept in the remineralizing solution
for 24 hours. The proportion of solutions per exposed enamel
area was 6.25 and 3.12 mL mm−2 for the demineralizing and rem-
ineralizing solutions, respectively.19
F I G U R E   4   Between-­group differences in changes
(Δ = final − baseline) in (A) area, (B) intensity, and (C) impact of
2.5 | FluoreCam system assessment enamel demineralization. , control; , fluoride; , laser

Following pH cycling, the nail polish was removed because it can

affect the FluoreCam readings. The specimens were reinserted in
their prefabricated molds. Using the same baseline settings, final
FluoreCam images were captured.
2.6 | Cross-­sectional microhardness assessment
Ten specimens from each group were randomly selected for
microhardness testing. The crowns were cross-­
s ectioned per-
pendicular to the enamel windows using a low-­speed (100 rpm)
diamond saw (IsoMet 1000; Buehler, USA). One of the halves was
randomly selected and embedded in self-­curing epoxy resin, leav-
ing the cross-­s ectional surfaces exposed. The tooth's surface was
polished (Ecomet, Buehler Lake Bluff, IL, USA) with silicon carbide
discs of diminishing grit area (250, 320, 400, 600, 800, and 1200).
The mechanical properties of enamel were assessed using a mi-
crohardness tester (FM-­le Digital Microhardness Tester; Future-­
Tech Corp, Novi, MI, USA), with a Knoop indenter (load: 50 g,
dwell time: 5 seconds, room temperature: 23± 1°C). 20 Three sets
of six indentations (20, 40, 60, 80, 100, and 120 μm from the outer
enamel surface) were made; one set at the central region of the
(exposed) enamel window, and another two sets approximately
100 μm distant from the central row (Figure 2). The first indenta-
tions were made 20 μm from the outer enamel surface to avoid
surface cracking. 20 To evaluate the (unexposed) sound enamel,
two additional sets were made 1 mm from each side of the window
(Figure 2). The Knoop hardness number (KNH) was obtained from
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4 of 6       ABUFARWA et al.

TA B L E   1   Differences and probabilities

∆ Area ∆ Intensity ∆ Impact
of the FluoreCam ∆ area, ∆ intensity and
Groups Difference P-­value Difference P-­value Difference P-­value ∆ impact of the fluoride, laser, and control
groups
Fluoride −1.7 0.002* 1.87 0.08 21.98 0.002*
vs
control
Fluoride −1.91 0.001* 0.58 0.99 14.17 0.07
vs laser
Laser vs 0.20 0.99 1.28 0.36 7.81 0.63
control

*Significant P < 0.05.

3 | R E S U LT S

At baseline, the FluoreCam data showed no statistically-­significant

F I G U R E   5   Between-­group differences in enamel microhardness
(exposed–unexposed enamel) 20-­120 μm from the enamel surface.
, control; , fluoride; , laser and * Significant P < 0.05
−2 −1 2
the relationship KNH (kg mm ) = 14 230 K L , where K was the
applied force (g), and L was the observed indentation length (μm).
between-­
group differences (Figure 3). There were statistically-­
significant between-­group differences in the changes that occurred
for area and impact, but not for intensity (Figure 4). The fluoride
group showed a significantly smaller area and less impact of enamel
demineralization than the control group. Compared to the laser
group, the area of enamel demineralization in the fluoride group was
significantly smaller (Table 1). Enamel demineralization of the laser
group and the control were comparable.There were statistically-­
significant between-­group differences in enamel hardness at all six
depths (Figure 5). The fluoride group had significantly harder enamel
than the control group at 20, 40, and 60 μm; they had significantly
harder enamel than the laser group at 20 μm. The laser group had
significantly harder enamel than the control group at 20 and 40 μm
(Table 2; Figure 6).

2.7 | Statistical analyses
All data were normally distributed (version 22; SPSS, Chicago, IL,
USA). The baseline data were evaluated using analysis of variance
(ANOVA). Within-­group differences were analyzed using one-­sample
t tests. Between-­group differences in the changes that occurred
were compared using ANOVA, followed by a set of Bonferroni post-­
hoc tests. Differences in enamel hardness (Δ hardness = exposed–
unexposed) were evaluated using analysis of covariance controlling
for unexposed hardness.
4 | D I S CU S S I O N
Fluoride varnish containing CPP-­ACP prevents enamel deminer-
alization. Enamel treated with MI Varnish showed no significant
enamel demineralization when challenged by pH cycling, whereas
the laser and control groups did. In vitro studies have shown
that MI Varnish releases significant levels of phosphate ions and
greater cumulative calcium ions.11 Furthermore, it has the highest

TA B L E   2   Differences and probabilities

Fluoride vs control Laser vs control Fluoride vs laser
of the microhardness differences of the
Depth (μm) Difference P-­value Difference P-­value Difference P-­value fluoride, laser, and control groups in
contrast to each other
20 268.6 <0.001** 160.8 <0.001** 107.8 0.001*
40 132.7 <0.001** 85 0.001** 47.7 0.06
60 76.4 0.007* 39.3 0.17 37.1 0.16
80 48.7 0.15 30.5 0.39 18.3 0.63
100 51.4 0.11 23.3 0.50 28.2 0.42
120 18.4 0.57 14.1 0.69 4.4 0.91

*Significant P < 0.05.
**Significant P < 0.001.
ABUFARWA et al.
(A) (B)
(C)
F I G U R E   6   Cross-­sectional microhardness indentations. (A)
Untreated control, (B) casein phosphopeptide amorphous calcium
phosphate fluoride varnish, (C) carbon dioxide laser
−1
cumulative fluoride release (303 μg mL ) compared to other var-
nishes.10,12,13 MI Varnish, which has 22 000 ppm F, could provide
better protection than MI Paste Plus (GC America, Alsip, Ill, USA),
21
which has only 900 ppm F. Fluoride varnish containing CCP-­ACP
has been shown to be superior to other varnishes in increasing the
acid resistance of enamel, 8,9 and was therefore chosen for the pre-
sent study.
Fluoride varnish containing CPP-­ACP increases enamel micro-
hardness. In the present study, enamel treated with MI Varnish was
significantly harder than control enamel, suggesting an increase in
22
mineral content. MI Varnish has previously been shown to increase
10
enamel hardness of early carious lesions. Increased hardness can
be attributed to the synergistic effect of the fluoride and CPP-­ACP
6
complex. During pH cycling, the fluoride ions interact with partially
demineralized enamel and create a less soluble and harder form of
6
the enamel crystals (fluorapatite).
Importantly, the present study showed that MI Varnish increases
enamel hardness to a depth of at least 60 μm, indicating that the rem-
ineralizing ions penetrated deep into the enamel. Previous studies have
reported fluoride uptake localized to the outermost (10 μm) surface
layer.23 The CPP-­ACP complex could be responsible for fluoride pen-
etration into the deeper layers.24 Fluoride incorporation is significantly
higher in enamel treated with CPP-­ACP than with fluoride alone, indi-
24
cating that the fluoride incorporation is calcium phosphate limited. At
low pH, fluoride with the CPP-­ACP complex develops a neutral form
of fluoride, calcium, and phosphate that does not precipitate onto the
enamel surface. This inactive form allows for the diffusion of fluoride,
together with calcium and phosphate ions, deep into the enamel, and
thus accelerates remineralization of enamel subsurface lesions.24
CO2 lasers potentially enhance enamel's resistance to demineral-
ization. There were significant increases in enamel microhardness to a
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depth of 40 μm. Although not statistically significant, the laser group
showed less demineralization than the control group. CO2 lasers have
previously been shown to decrease demineralization and increase
enamel hardness.25 Post-­hoc analysis revealed that there was insuffi-
cient power to detect between-­group differences. The smaller effect
in the present study could have been due to the laser parameters used.
Various laser settings have been used for increasing enamel resis-
tance, but no standards have been established. Esteves-­Oliveira et al.
suggested optimal laser parameters for caries prevention using an
industrial laser that irradiates at a distance 19.8 cm, which might be
impractical clinically.12 The present study adopted laser settings used
in a clinical trial that prevented occlusal caries for 3 years in high-­risk
patients.15
The mechanism by which lasers increase enamel resistance to de-
mineralization remains poorly understood. Lasers might increase the
temperature of the enamel surface, altering its mineral phase com-
position and decreasing its permeability and solubility.14 CO2 lasers
can melt enamel, leading to surface damage.26 To prevent enamel de-
mineralization, the laser must decrease the solubility of enamel, and
the energy produced must be absorbed and efficiently converted to
heat, without damaging the enamel or surrounding tissues.27 Based
on previous research,15 the laser parameters used in the present
study might be expected to have been minimal. More studies are re-
quired to determine safe and appropriate laser parameters for WSL
prevention.
CPP-­ACP fluoride varnish is more effective than CO2 laser in re-
ducing enamel demineralization. In the present study, laser-­treated
enamel showed significant enamel demineralization over time,
whereas MI Varnish-­treated enamel did not. The varnish also pro-
duced harder enamel surfaces than CO2 laser irradiation. Previous in-
vestigators have reported that fluoride is superior to CO2 in reducing
enamel demineralization.28,29 Fluoride gels (1.2% NaF) provide higher
surface microhardness than the CO2 laser alone, which was compara-
ble to the untreated control.28,29 Others have shown that CO2 laser is
superior to fluoride varnish (5% NaF).30,31 These contradictory results
could, once again, be due to the different laser parameters used. The
varnish in the present study could be more effective than varnishes
previously used, because it contains CPP-­ACP, which has a synergis-
tic effect with fluoride.6 MI Varnish also has been shown to have a
higher fluoride, calcium, and phosphate ions release,10,11 and prevents
enamel demineralization more than other varnishes.8
In vitro studies are important because they provide standardized
and more controlled conditions than clinical settings. In vivo studies
might yield different results, which are difficult to predict due to the
complexity of the oral environment (composition/flow rate of saliva,
food, and microbiota). Thus, future clinical studies are recommended
to confirm these findings.
5 | CO N C LU S I O N S
With the limitations of this in vitro study CPP-­ACP fluoride varnish
has a preventive effect on enamel WSL formation, and CO2 laser
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6 of 6       ABUFARWA et al.
could have the potential to increase acid resistance; however, its ap-
plication solely for WSL prevention would not appear to be practical
under the present set of parameters.
C O N FL I C T O F I N T E R E S T
The authors declare no potential conflicts of interest or sources of
funding related to the authorship and/or publication of this article.
ORCID
Moufida Abufarwa  https://orcid.org/0000-0002-9633-6742
REFERENCES
1. Benham AW, Campbell PM, oBuschang PH. Effectiveness of pit and
fissure sealants in reducing white spot lesions during orthodontic
treatment. A pilot study. Angle Orthod. 2009;79:338‐345.
2. Brown MD, Campbell PM, Schneiderman ED, Buschang PH. A
practice-­b ased evaluation of the prevalence and predisposing
etiology of white spot lesions. Angle Orthod. 2016;86:181‐186.
3. Julien KC, Buschang PH, Campbell PM. Prevalence of white spot
lesion formation during orthodontic treatment. Angle Orthod.
2013;83:641‐647.
4. Opsahl Vital S, Haignere-Rubinstein C, Lasfargues JJ, Chaussain
C. Caries risk and orthodontic treatment. Int Orthod. 2010;8:
28‐45.
5. Benson PE, Parkin N, Dyer F, et al. Fluorides for the prevention of
early tooth decay (demineralised white lesions) during fixed brace
treatment. Cochrane Database Syst Rev. 2013;12:Cd003809.
6. Reynolds EC, Cai F, Cochrane NJ, et al. Fluoride and casein
phosphopeptide-­ amorphous calcium phosphate. J Dent Res.
2008;87:344‐348.
7. Reynolds EC, Cain CJ, Webber FL, et al. Anticariogenicity of calcium
phosphate complexes of tryptic casein phosphopeptides in the rat.
J Dent Res. 1995;74:1272‐1279.
8. Tuloglu N, Bayrak S, Tunc ES, Ozer F. Effect of fluoride varnish with
added casein phosphopeptide-­amorphous calcium phosphate on the
acid resistance of the primary enamel. BMC Oral Health. 2016;16:103.
9. Bayrak S, Tuloglu N, Bicer H, Sen Tunc E. Effect of fluoride var-
nish containing CPP-­ACP on preventing enamel erosion. Scanning.
2017;2017:1897825.
10. Al Dehailan L, Martinez-Mier EA, Lippert F. The effect of fluo-
ride varnishes on caries lesions: an in vitro investigation. Clin Oral
Investig. 2016;20:1655‐1662.
11. Cochrane NJ, Shen P, Yuan Y, Reynolds EC. Ion release from
calcium and fluoride containing dental varnishes. Aust Dent J.
2014;59:100‐105.
12. Esteves-Oliveira M, Zezell DM, Meister J, et al. CO2 Laser (10.6 mi-
crom) parameters for caries prevention in dental enamel. Caries Res.
2009;43:261‐268.
13. Featherstone JD, Barrett-Vespone NA, Fried D, Kantorowitz Z,
Seka W. CO2 laser inhibitor of artificial caries-­like lesion progres-
sion in dental enamel. J Dent Res. 1998;77:1397‐1403.
14. Zuerlein MJ, Fried D, Featherstone JD. Modeling the modification
depth of carbon dioxide laser-­treated dental enamel. Lasers Surg
Med. 1999;25:335‐347.
15. Kato J, Moriya K, Jayawardena JA, Wijeyeweera RL, Awazu K.
Prevention of dental caries in partially erupted permanent teeth
with CO2 laser. J Clin Laser Med Surg. 2003;21:6.
16. Liu Y, Hsu CY, Teo CM, Teoh SH. Potential mechanism for the
laser-­
fluoride effect on enamel demineralization. J Dent Res.
2013;92:71‐75.
17. Abufarwa M, Noureldin A, Campbell PM, Buschang PH. Reliability
and validity of FluoreCam for white-­spot lesion detection: an in vitro
study. J Investig Clin Dent. 2017; https://doi.org/10.1111/jicd.12277
18. Abufarwa M, Noureldin A, Campbell PM, Buschang PH.
Comparative study of two chemical protocols for creating white
spot lesions: an in vitro FluoreCam evaluation. J Investig Clin Dent.
2017; https://doi.org/10.1111/jicd.12274
19. Queiroz CS, Hara AT, Paes Leme AF, Cury JA. pH-­c ycling models
to evaluate the effect of low fluoride dentifrice on enamel de-­and
remineralization. Braz Dent J. 2008;19:21‐27.
20. Delbem A, Sassaki K, Vieira A, et  al. Comparison of methods for
evaluating mineral loss: hardness versus synchrotron microcom-
puted tomography. Caries Res. 2008;43:359‐365.
21. Llena C, Leyda AM, Forner L. CPP-­ACP and CPP-­ACFP versus flu-
oride varnish in remineralisation of early caries lesions. A prospec-
tive study. Eur J Paediatr Dent. 2015;16:181‐186.
22. Featherstone J, Ten Cate J, Shariati M, Arends J. Comparison of
artificial caries-­like lesions by quantitative microradiography and
microhardness profiles. Caries Res. 1983;17:385‐391.
23. Petersson LG, Odelius H, Lodding A, Larsson SJ, Frostell G. Ion
probe study of fluorine gradients in outermost layers of human
enamel. J Dent Res. 1976;55:980‐990.
24. Cochrane NJ, Saranathan S, Cai F, Cross KJ, Reynolds EC. Enamel
subsurface lesion remineralisation with casein phosphopeptide
stabilised solutions of calcium, phosphate and fluoride. Caries Res.
2008;42:88‐97.
25. Esteves-Oliveira M, Pasaporti C, Heussen N, et al. Rehardening of
acid-­softened enamel and prevention of enamel softening through
CO2 laser irradiation. J Dent. 2011;39:414‐421.
26. Featherstone JD, Barrett-Vespone NA, Fried D, et  al. Rational
choice of laser conditions for inhibition of caries progression. Paper
presented at: Photonics West'95. 1995.
27. Rodrigues LK, Nobre dos Santos M, Pereira D, Assaf AV, Pardi
V. Carbon dioxide laser in dental caries prevention. J Dent.
2004;32:531‐540.
28. Mirhashemi AH, Hakimi S, Ahmad Akhoundi MS, Chiniforush N.
Prevention of enamel adjacent to bracket demineralization follow-
ing carbon dioxide laser radiation and titanium tetra fluoride solu-
tion treatment: an in vitro study. J Lasers Med Sci. 2016;7:192‐196.
29. Ramos-Oliveira TM, Ramos TM, Esteves-Oliveira M, et al. Potential
of CO2 lasers (10.6 microm) associated with fluorides in inhibiting
human enamel erosion. Braz Oral Res. 2014;28:1‐6.
3 0. Souza-Gabriel AE, Colucci V, Turssi CP, Serra MC, Corona SA.
Microhardness and SEM after CO(2) laser irradiation or fluo-
ride treatment in human and bovine enamel. Microsc Res Tech.
2010;73:1030‐1035.
31. Rechmann P, Charland DA, Rechmann BM, Le CQ, Featherstone
JD. In-­vivo occlusal caries prevention by pulsed CO2 -­laser and
fluoride varnish treatment–a clinical pilot study. Lasers Surg Med.
2013;45:302‐310.
How to cite this article: Abufarwa M, Noureldin A, Azimaie T,
Campbell PM, Buschang PH. Preventive effects of carbon
dioxide laser and casein phosphopeptide amorphous calcium
phosphate fluoride varnish on enamel demineralization: A
comparative, in vitro study. J Invest Clin Dent. 2019;e12400.
https://doi.org/10.1111/jicd.12400