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Theses and Dissertations
Spring 2010
The efficacy of Novamin powered technology
Oravive and TopexRenew, Crest and Prevident
5000 Plus in preventing enamel demineralization
and white spot lesion formation
Andre Correia Jham
University of Iowa
Copyright 2010 Andre Correia Jham
This thesis is available at Iowa Research Online: http://ir.uiowa.edu/etd/522
Recommended Citation
Jham, Andre Correia. "The efficacy of Novamin powered technology Oravive and TopexRenew, Crest and Prevident 5000 Plus in
preventing enamel demineralization and white spot lesion formation." MS (Master of Science) thesis, University of Iowa, 2010.
http://ir.uiowa.edu/etd/522.
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Part of the Orthodontics and Orthodontology Commons
THE EFFICACY OF NOVAMIN POWERED TECHNOLOGY ORAVIVE AND
TOPEXRENEW, CREST AND PREVIDENT 5000 PLUS IN PREVENTING ENAMEL
DEMINERALIZATION AND WHITE SPOT LESION FORMATION
by
Andre Correia Jham
A thesis submitted in partial fulfillment
of the requirements for the Master of
Science degree in Orthodontics
in the Graduate College of
The University of Iowa
May 2010
Thesis Supervisor: Professor Robert N. Staley
Graduate College
The University of Iowa
Iowa City, Iowa
CERTIFICATE OF APPROVAL
_______________________
MASTER'S THESIS
_______________
This is to certify that the Master's thesis of
Andre Correia Jham
has been approved by the Examining Committee
for the thesis requirement for the Master of Science
degree in Orthodontics at the May 2010 graduation.
Thesis Committee: __________________________________
Robert. N. Staley, Thesis Supervisor
___________________________________
James Wefel
___________________________________
Lina Moreno
___________________________________
Fang Qian
To my dear wife Raquel, my loving parents Alice and Gulab, and my brother and best
friend Bruno for their unconditional love and support. To everyone one else who has, in
one way or the other, been a part of this great journey. You are way too many to
mention.
ii
.
Never look down on anybody unless you’re helping him up.
Jesse Jackson
.
iii
ACKNOWLEDGMENTS
I would like to thank Drs. Robert Staley, James Wefel, Fang Qian, and Lina
Moreno for all their valuable advice and insight. In addition, I am very thankful to Jeff
Harless and Maggie Hogan for their willingness to help at all times during this project. I
would also like to express my deepest gratitude to Jared Bolding and Molly Bremen for
all their help and dedication since the start of this project, and to Abby Kershner and
Treagan White for being available to help when needed.
I would also like to thank the staff and faculty of the Department of Orthodontics,
University of Iowa for providing me with a high quality education, in special to Dr. Tom
Southard for believing in me. And last but not least, a big thank you for my co-residents
for being this great group of people and putting up with me for the last two years. I will
always consider you amongst my closest friends.
iv
TABLE OF CONTENTS
LIST OF TABLES ............................................................................................................. vi
LIST OF FIGURES .................................................................................................... … vii
INTRODUCTION ...............................................................................................................1
Purpose of this Study...............................................................................................2
LITERATURE REVIEW……………………………………………………………... .....4
Demineralization Process/White Spot Lesion Formation .......................................4
White Spot Lesions in Orthodontic Patients…………………...…….......……...…….6
Methods to Decrease Demineralization during Orthodontic Treatment……….......7
Mechanism of Action of Fluoride…………………. ..............................................9
Fluoride Dentifrice………………………………...... ..........................................10
Mechanism of action of Novamin……………………………………………..................11
Polarized Light Microscopy…………………………………………………. .....13
MATERIALS AND METHODS………………………………………………….… ......17
Sample Preparation……………………………………………………………....17
Experimental Procedures………………………………… ...................................19
Statistical Analysis…………………………………………………………... ......22
RESULTS…………………………………………………………………….……... ......23
DISCUSSION…………………………………………………………………….… .......35
SUMMARY AND CONCLUSIONS……………………………………………….. ......40
REFERENCES…………………………………………………………………….... ......42
v
LIST OF TABLES
Table
1.
Descriptive statistics of mean lesion depth by treatment group ...............................31
2.
Mean lesion depth by treatment group .....................................................................32
vi
LIST OF FIGURES
Figure
1.
Clinical example of before and after treatment in a patient exhibiting severe
demineralization…………………………………………..........................................3
2.
Diagram representing the events of demineralization and
remineralization……………………………………………………………... ...........5
3.
Histologic zones of a carious lesion………………………………………… .........16
4.
Tooth painted with acid-resistant varnish to produce a 1mm x 6 mm window
of exposed enamel ………………………………………….. .................................17
5.
Cusp tips were ground flat to facilitate the sectioning
procedure……………………………………... .......................................................18
6.
Tooth following acidic challenge……..…………………………...…….................18
7.
Prevident 5000 Plus, Crest, Renew and
Oravive………………………………….………………….....................................19
8.
Polarized light microscopy image of representative lesion from the Control
group ………………....………………………………………... .............................24
9.
Polarized light microscopy image of representative lesion from the Oravive
group…………………………...…………………. .................................................25
10.
Polarized light microscopy image of representative lesion from the Renew
group……………………………………... ..............................................................26
11.
Polarized light microscopy image of representative lesion from the Crest
group ………………........ ........................................................................................27
12.
Polarized light microscopy image of representative lesion from the Prevident
5000 Plus group …………………………………………………. ..........................28
13.
Measuring technique used by Image Pro Plus computer software (Media
Cybernetics, Silver Spring, Maryland) to record mean lesion depth of
lesion.…………………….……………… ...............................................................29
14.
Measuring technique used for eroded lesions ...........................................................29
15.
Composite of polarized light microscopy images of representative lesions………....30
vii
16.
Mean lesion depth comparison between groups …………………………………………..33
17.
Percent (%) reduction of mean lesion depth compared to the Control group ……….34
viii
1
INTRODUCTION
There are several benefits to orthodontic treatment, such as significant
improvements in a patient’s esthetics, function and overall self-esteem. However, the
esthetic result of orthodontic treatment can be severely compromised by demineralization
of the tooth structure around the orthodontic appliances. This could result in white,
opaque areas of demineralization, also known as white spot lesions (Figure 1). An
opaque white spot lesion looks chalky and could result in cavitation of tooth structure if
mineral loss continues (Mitchell et al., 1992). Enamel demineralization associated with
fixed orthodontics has also been shown to be a very rapid process caused by a high and
continuous cariogenic challenge in the plaque developed around the brackets (Ogaard et
al., 1988).
Enamel demineralization associated with fixed orthodontic appliances has been
observed for years and continues to be a problem even with advances in materials and
techniques (Farhadian et al., 2008).
The most common method of preventing demineralization around orthodontic
appliances is application of topical fluoride. Topical fluoride application methods
include fluoride gels, rinses and dentifrice, which are all patient compliant - dependant
treatment. Strateman and Shannon (1974) found that when patients used stannous
fluoride gel, only 2% developed white spot lesions, compared with 58% of the patients
who did not use the gel. Geiger et al (1992) found that the use of a fluoride rinse caused a
25% reduction in the number of patients exhibiting white spot lesions. However, most
topical fluoride regimens rely on patient compliance. Patients who would benefit most
from supplemental fluoride due to their poor hygiene are also the least likely to comply.
A viable alternative in this situation would be the use of non-compliant treatment,
such as fluoride varnishes, fluoride containing bracket/band cements and light-cured
sealants. In an in vitro study by Frazier et al (1996), 80% of sealed teeth exhibited no
2
signs of demineralization whereas all untreated controls exhibited demineralization.
Resin modified glass ionomer cements are able to continuously release fluoride over time
after the initial topical fluoride application (Vorhies et al., 1998).
Novamin falls into a newer category of bioactive glass-ceramic material that has
been available since the 1960’s as materials to help in bone repair. The active ingredient
is a calcium sodium phosphosilicate that reacts when exposed to aqueous media, thus
providing calcium and phosphate ions to the applied surface. Novamin has received FDA
clearance for products aiming to reduce sensitivity by blockage of dentinal tubules, and
some of the products containing Novamin have also been used for reducing bleeding and
gingivitis, as well as preventing demineralization and enhancing remineralization.
Examples of Novamin powered technology include Oravive, a product from Natural
Health Organics, which is a non-fluoridated, non-prescription dentifrice containing 5%
Novamin. Another example is TopexRenew, a product of Sultan Health Care, a
prescription strength 5000 ppm fluoridated dentifrice, also containing 5% Novamin.
Purpose of this Study
The purpose of this study was to compare in vitro the effectiveness of Novamin
powered technology Oravive and TopexRenew, with Crest and Colgate’s PreviDent 5000
Plus in decreasing demineralization and preventing white spot lesion formation on
extracted human teeth. Teeth were cycled between demineralization and remineralization
solutions and polarized light microscopy was used to analyze and compare lesion
formation. Under our hypothesis, Oravive and TopexRenew would, respectively, be more
effective than Crest and Prevident 5000 Plus in the prevention of enamel
demineralization.
3
Figure 1. Clinical example of before and after treatment in a
patient exhibiting severe enamel demineralization.
4
LITERATURE REVIEW
Demineralization Process/White Spot Lesion Formation
Dental caries is a multi-factorial disease that involves the interaction between diet,
dental plaque containing bacteria, and host factors, such as tooth surface, saliva, and the
acquired pellicle (Zero, 1999). Dental caries is initiated via demineralization of tooth
mineral by organic acids. The organic acids are produced by plaque bacteria following
exposure to fermentable carbohydrates. When a critical pH is reached, the organic acids
are able to diffuse into the enamel surface through the acquired pellicle, commencing
demineralization. During demineralization, less soluble phases of dicalcium phosphate
dihydrate (CaHPO4.2H2O) and fluoridated hydroxyapatite (Ca5(PO4)3(OH)xF(1-x))
precipitate out of the enamel. This process continues until equilibrium is achieved
between the enamel and the oral environment. Demineralization can continue as long as
the oral pH remains acidic (Margolis and Moreno, 1990).
When the oral pH rises above the acidic level, the remineralization process of the
tooth surface can begin. Calcium and phosphate, which are present in the saliva, diffuse
into the enamel with the help of fluoride to remineralize crystalline structures in
demineralized areas. The rebuilt structures consist of fluoridated hydroxyapatite and
fluorapatite, which are much more resistant to acid attack than the original structure
(Selwitz et al., 2007). The processes of demineralization and remineralization occur
several times throughout the day and if balanced, will not result in carious lesions.
However, if the balance is tipped more towards demineralization rather than
remineralization, the lesion will progress and eventually become a frank cavitation
(Featherstone, 2000). A diagram of the demineralization and remineralization cycle is
shown in Figure 2.
5
A white spot lesion is the first clinical presentation of dental caries prior to
cavitation. The white opaque appearance is a result of the loss of subsurface enamel,
resulting in the loss of enamel translucency (Zero, 1999). At this stage, the lesion may
either progress to frank cavitation or may be arrested or reversed by modifying any of the
causative factors of increasing preventive measures (Margolis and Moreno, 1990).
Figure 1. Diagram representing the events of demineralization and
remineralization.
6
White spot lesions in Orthodontic Patients
Patients in fixed orthodontic appliances are quite susceptible to plaque
accumulation and, consequently, white spot lesion formation. In vivo experiments by
Ogaard et al. (1988) have shown that visible white spot lesions can develop in
orthodontic patients within four weeks in the absence of any fluoride supplementation.
Zachrisson and Zachrisson (1971) have shown almost a linear correlation between plaque
accumulation and development of carious lesions in orthodontic patients.
Visible plaque and the presence of mutans streptococci have been indicated to be
good predictors of white spot lesions (Øgaard, 1989).
Only at the time of removal of the appliances is the full extent of the damage
realized, with the clinical appearance of white rings on a tooth around the area where the
bracket once was. Once the condition is present, the patient is left with straight but
esthetically unappealing teeth, counteracting the beneficial effect of the treatment.
Although decalcification during orthodontic treatment is a widespread problem,
published reports of prevalence vary widely. Zachrisson et al. (1971) reported that 89%
of patients developed white spot lesions, while Strateman et al. (1974) observed 58%
prevalence. Boersma et al. (2005), observed an even higher prevalence of decalcification,
with 97% of their subjects displaying lesions after treatment. The differences in reported
prevalence may be attributed to geographical location, as well as variation in the
definition of a white spot lesion or the criteria applied for each individual study.
However prevalent they may be, it is established that white spot lesions may persist for
years, resulting in a permanent, unaesthetic result with the potential of worsening to the
point of requiring permanent restoration (Øgaard, 1989; Sudjalim et al., 2006).
7
Methods to Decrease Demineralization during Orthodontic Treatment
There are several methods to minimize or prevent demineralization and white spot
lesion formation in the orthodontic patient.
Patients should receive diet counseling that emphasizes a minimum intake of
fermentable carbohydrates. Frequent consumption of sugary foods or drinks causes the
pH in plaque to drop below the critical level of 5.5 (Mitchell, 1992). Especially in
children and adolescents, proper nutrition is essential for maintaining overall systemic
health and optimal oral health during and after treatment.
Thorough oral hygiene has been shown to be an effective way to prevent and/or
decrease demineralization in the orthodontic patient (Artun et al., 1986). Effective tooth
brushing, flossing, and routine prophylactic cleanings will minimize the amount of dental
plaque, thereby decreasing the probability of developing areas of decalcification (Øgaard,
1989).
Streptococcus Mutans is one of the main bacteria involved in the carious process
(Van Houte, 1992). Chlorhexidine is an antimicrobial agent that is effective at reducing
levels of streptococcus mutans (Ribeiro, 2007). Therefore, a protocol utilizing a
chlorhexidine rinse, gel, or varnish may assist in preventing demineralization.
Fluoride delivery in the oral cavity is also one of the most effective methods to
reduce demineralization. Fluoride can be delivered directly to the tooth structure in
multiple ways. Sources of fluoride include fluoridated dentifrice, rinses, foams, gels,
varnishes, and fluoride-releasing bonding agents and cements. Fluoridated dentifrices,
rinses, and gels are intended to be used by the patient at home and therefore rely on
patient compliance. It can be difficult to have good levels of patient compliance. Geiger
et al (1992) reported only 13% of compliance among patients participating in a fluoride
rinse protocol study.
8
Thus, the use of fluoride products that do not rely on patient compliance has
become increasingly popular. For instance, a fluoride varnish containing high amounts of
fluoride can be applied at office visits, brackets and bands can be cemented with fluoridereleasing materials, and sealants can be placed to cover the facial surface of each tooth
(Schmit et al., 2002; Todd et al., 1999; Ashcraft et al., 1997; Vorhies et al., 1998). Pro
Seal™ (Reliance Orthodontic Products, Itasca, IL) is an example of an enamel sealant
applied prior to bracket placement. This product has shown promise as a protective
barrier against demineralization in non-compliant patients (Buren et al., 2008)
MI Paste™ is a sugar-free, water-based cream containing RecaldentTM (GC
Corporation, Tokyo, Japan) and was introduced to the American market in October 2004.
The active ingredient of MI Paste™ is casein phosphopeptide-amorphous calcium
phosphate (CPP-ACP). RecaldentTM is the trademark applied to CPP-ACP technology.
MI Paste™ has been approved for treating patients with dentinal hypersensitivity, yet is
also marketed for helping prevent demineralization of tooth enamel and enhancing
remineralization. The proposed anticariogenic mechanism of CPP-ACP is to localize
amorphous calcium phosphate within dental plaque at the tooth surface, buffer the
acidogenic challenge, and maintain a state of supersaturation of calcium and phosphate
ions on the enamel surface. This process results in a decrease in demineralization during
a cariogenic challenge and an increase in the subsequent remineralization of the enamel
(Pulido et al., 2008). Eng (2009) found in an in vitro study that CPP-ACP showed a
statistically significant reduction in lesion depth, however he could not conclude that this
equates to a clinical reduction of visible demineralization.
9
Mechanism of Action of Fluoride
Fluoride has several mechanisms of action to aid in the prevention of dental
decay. It can incorporate into the enamel by combining with hydroxyapatite to form
fluoroapatite, resulting in a less soluble and more resistant enamel when faced with an
acidic challenge (Zipkin, 1970). It is also effective in inhibiting Enolase, an enzyme
necessary for glycolysis, which is sensitive to fluoride at low levels. Subsequently, oral
bacteria are unable to utilize fermentable carbohydrates (Levine, 1991). Besides, it can
be effective in inhibiting bacterial colonization of the tooth surface via competitive
binding. Due to its electronegative properties, fluoride competes with bacteria for these
binding sites and prevents adhesion (Levine, 1991).
However the main mechanism of action of fluoride is the remineralization of
affected enamel. After an acid attack, fluoride ions react with calcium and phosphate of
hydroxyapatite to form fluorapatite and can be incorporated into the partially damaged
enamel structure, resulting in remineralization of the surface. This incorporation will
slow, and may even stop, the progression of the lesion (Biestrock et al, 1998). Harris and
Cristen (1995) actually concluded that the resulting enamel is more resistant to
subsequent demineralization than the original one.
While persistent, low levels of topical fluoride exhibit many beneficial properties,
excessive amounts may demonstrate adverse effects. Research has indicated that
increased levels of topical fluoride will lead to rapid mineral precipitation on the enamel
surface and subsequent obliteration of surface enamel pores (García-Godoy et al., 2008).
This limits the ability to remineralize the subsurface areas. In essence, a hard enamel
shell is created with an area of demineralization remaining. For this reason, it is not
recommended to apply high concentrations of fluoride (such as fluoride varnish) to areas
of decalcification, as it will seal the outer enamel shell, and will prevent further
remineralization from occurring, allowing the white spot lesion to remain indefinitely.
10
High amounts of topical fluoride also have the potential to be ingested, increasing the
amount of systemic fluoride intake. In younger patients (under the age of six), this may
lead to an increased incidence of dental fluorosis or potentially fluoride toxicity (Zero,
2006; Warren et al., 2003).
Fluoride Dentifrice
The most commonly used fluoride delivery method worldwide is fluoridated
dentifrice. Burt and Eckland (2005) concluded fluoridated dentifrices have played a
significant role in decreasing caries in industrialized countries. Marinho et al (2003)
conducted a meta-analysis of seventy studies and came to the conclusion that fluoridated
toothpaste is effective in preventing dental caries.
Fluoride concentrations in OTC dentifrice range from 600 ppm to 1100 ppm.look it up The low and high concentrations of fluoride are intended for patients under the
age of six and those with high caries risk, respectively (Ammari, 2003). There are
different sources of fluoride that can be used in dentifrice, such as sodium fluoride (NaF),
monofluorophosphate (MFP) and stannous fluoride (SnF2)-SUBSCRIPT. In the United
States, MFP or NaF are the leading types used (Zero, 2006).
There are a few studies with prescription dentifrice such as Prevident 5000
(Colgate Oral Pharmaceuticals, Inc., Canton, Massachusetts). Baysan (2001) evaluated
root caries treated with Prevident 5000 Plus (5000 ppm) versus Crest (1100 ppm) and
found a greater reversal of primary root caries lesions among the group treated with
Prevident 5000 Plus. In a systematic review, Twetman (2003) found that dentifrice
containing 1500 ppm fluoride to have a “superior preventive effect” compared to that
containing 1000 ppm fluoride.
11
Mechanism of Action of Novamin
Bioactive glasses have been available since the late 1960’s. They contain oxides
of calcium, sodium, phosphorus, and silicon in a proportion providing the material with
surface activity and concomitantly with the property of forming a strong bond with bone.
Bioactive glasses have been tested under different clinical situations, such as having an
antibacterial effect (Allan 2001), assisting in the prevention of gingivitis, and having a
scaffolding effect for new bone growth (Loty et al., 2001).
Novamin is a calcium sodium phosphosilicate that belongs to a class of bioactive
glasses that react when exposed to aqueous media, providing calcium and phosphate ions.
It was originally developed as a bone regenerative material (Hench 1993) and contains
calcium, phosphorus, sodium and silica molecules.
In addition to the clinical uses mentioned above, Novamin has received approval
from the FDA to be used in products that decrease hypersensitivity by the occlusion of
exposed dentinal tubules. Marini et al conducted a pilot clinical study and concluded
that Novamin-containing dentifrice was statistically more effective than a placebo
dentifrice.
There is a wide array of products containing Novamin. Oravive, a product from
Natural Health Organics, is a type of non-fluoridated, non-prescription dentifrice
containing 5% Novamin. NuPro is a type of prophy paste marketed by Dentsply
containing 5000 ppm Fluoride and Novamin. TopexRenew, a product of SultanHealth
Care, is also powered by 5% Novamin, and is a prescription strength 5000 ppm
fluoridated dentifrice. Other products by Sultan Health Care include a root desensitizer
called Vitalmin (100% Novamin and water) and a fluoride varnish (Durashield, 10%
Novamin and 5% NaF). SootheRx is a prescription paste for hypersensitivity, containing
7.5% Novamin marketed by 3M/Omni in the United States. Dr. Collins Restore
Remineralizing Toothpaste is a fluoride-free toothpaste also containing Novamin. All of
12
the above products have undergone clinical studies that support claims of decreased
sensitivity through the occlusion of dentinal tubules. Burwell (2006) conducted an in
vitro study comparing DenShield (Novamin containing dentifrice) and GC Tooth Mousse
(Recaldent containing dentifrice) on bovine incisors following a 10-day demin/remin
cycling protocol. At the end of the study the samples were analyzed with SEM (Scanning
Electron Microscopy) that showed a greater number of occluded tubules with the
Novamin-containing Denshield dentifrice, compared to GC Tooth Mousse.
The key question to be answered is whether Novamin can prevent
demineralization and promote remineralization of the enamel surface. Laboratory studies
on Novamin have been released by the company. One of these studies was done by
Alaudin and Fontana (2007) and compared a dentifrice containing 5% Novamin and
Fluoride (MFP) to a commercially available dentifrice in remineralization of subsurface
carious lesions in human tooth enamel. It used confocal laser scanning microscopy
(CLSM), which is able to distinguish between sound enamel and demineralized enamel
using a fluorescent dye. The authors came to the conclusion that Novamin-containing
dentifrice was statistically more significant in decreasing the lesion area than the
commercial dentifrice containing MFP fluoride alone.
Although several studies have shown blockage of dentinal tubules when using
Novamin, one must understand that this fact supports its effectiveness against
hypersensitivity, not its capability to prevent demineralization and enhance
remineralization. It must be kept in mind that only a few studies on the effect of Novamin
on white spot lesions are available. Thus, further in vitro studies are needed to improve
the delivery systems and protocols used (Wefel, 2009).
13
Polarized Light Microscopy
Polarized light microscopy allows visualization of enamel demineralization and
remineralization and is often used to evaluate carious lesions. Demineralization can also
be studied by light microscopy, microradiography, surface hardness, electron
microscopy, and confocal laser scanning microscopy (Ogaard et al., 1996). Polarized
light microscopy is beneficial in that it permits qualitative and quantitative evaluation due
to its ease of visualization of the color spectrum (Hicks, 1981).
A polarized light microscope consists of a light microscope plus a polarizer and
analyzer set perpendicular to one another. Both are made of prisms of calcite or a sheet of
Polaroid, which transmit light oscillation in one plane (Weyrich, 1994). When a sample is
placed between the polarizer and the analyzer, the sample modifies the plane of light and
produces a series of interference colors.
A crystal is described as isotropic when it transmits light with equal velocity in all
directions. It is anisotropic when the crystal transmits light at different velocities in
different directions. Anisotropic crystals are subdivided into uniaxial and biaxial
depending on the number of axes present. Hydroxyapatite is a uniaxial anisotropic
crystal, meaning it has one optic axis that is coincident with its crystal axis.
Hydroxyapatite is also birefringent, splitting a light ray into two components,
which travel at different velocities and are polarized at right angle to one another,
releasing different color and light intensities (Weyrich, 1994). A material that consists of
a non-cubic crystal is given a sign of birefringence determined by the velocity of its
resultant rays. Slow rays are designated positive (+) and fast rays are designated negative
(-) (Theuns 1977). Enamel is composed mostly of inorganic hydroxyapatite (-) with a
small amount of organic material (+) interspersed.
Birefringence can be measured indirectly through polarized light microscopy.
Enamel is a uniaxial birefringent object. When enamel is oriented with its optic axis
14
parallel to the direction of plane polarized light propagation, it will act as an isotropic
crystal. If enamel is arranged with its optic axis in a plane perpendicular to the direction
of propagation, the light will split into two beams. These beams are referred to as the
extraordinary (ne) and ordinary (no) rays, which result in a series of interference colors
(Silverstone, 1967).
Birefringence is further categorized as intrinsic birefringence and form
birefringence. Intrinsic birefringence is the difference between the refractive indices of
the extraordinary ray (ne) and the ordinary ray (no), or (ne-no) (Hicks, 1981). Crstalline
mineral volume, orientation of crystallites towards the light beam, and crystallite
birefringence are all factors influencing intrinsic birefringence such that (ne – no)i = δ c
(ne –no)HAP where (ne – no)i = intrinsic birefringence, δ = pore volume occupied by
crystallites, c = crystallite orientation factor, and (ne – no)HAP = birefringence of
crystallites (Theuns and Groenveld, 1977).
During the demineralization process the space between the enamel crystals
becomes larger. Form birefringence occurs when a medium that fills the voids in carious
enamel has a different refractive index. The amount of form birefringence produced is
determined by the size of the spaces and the refractive index of the medium. If the
medium has the same refractive index as enamel, then no form birefringence will be
created. Thus, when using polarized microscopy, different media are used.
The total birefringence of enamel is the sum of 1) the negative intrinsic
birefringence of the inorganic material, 2) the positive intrinsic birefringence of the
organic material, 3) the positive form birefringence of the spaces in relation to the
mineral and possibly to the organic material, and 4) the positive form birefringence of the
organic versus the mineral material. The organic mineral content is so small that it can be
disregarded. Therefore, the observed total birefringence is made up almost entirely by the
intrinsic birefringence of the inorganic mineral and the form birefringence of the spaces
in relation to the inorganic mineral (Silverstone, 1967).
15
Enamel is divided into longitudinal sections so that the crystallites of apatite are
aligned along the length of the prism. Four zones of a carious lesion are visible with
polarized light microscopy (Figure 3). The amount of space or tissue lost defines the
zones, therefore different zones have different pore volumes. The surface zone is the
outermost zone, which appears relatively unchanged and intact. It is a zone of
remineralization and has a pore volume of 1-5 percent. The second zone is called the
body of the lesion and displays more tissue destruction than any of the other zones. The
pore volume in the body of the lesion is 5-25 percent. The third zone is the dark zone and
is caused by dissolution of enamel cross striations. The dark zone has 2-4% pore volume.
Like the surface zone, remineralization can occur in the dark zone. The dark zone also
exhibits loss of enamel rod core to a greater extent than the fourth zone, the translucent
zone. The translucent zone is the deepest of the four zones and exhibits a tenfold increase
in the amount of space compared to the normal enamel. It is created by loss of enamel rod
periphery and has a pore volume of 1 percent. (Silverstone 1967)
Different imbibing media have different refractive indices that correspond to the
zones of demineralization. Air, water, and potassium mercuric iodide dilutions (Thoulet’s
solution) are some of the media used in polarized light microscopy. Air (RI = 1.0)
corresponds with 1 percent pore volume, water (RI = 1.33) with 5 percent pore volume,
Thoulet’s 1.41 with 10 percent pore volume, and Thoulet’s 1.47 with a 25 percent pore
volume.
Different zones of a longitudinally sectioned lesion were ‘mapped’ by imbibing
with different media. The maps provided qualitative assessment of internal lesion pore
volume. Quantitative information can be easily obtained by measuring the positively
birefringent area of the lesion in the various imbibition media. Image Pro Plus (Media
Cybernetics, Silver Spring, Maryland) software is used to measure a polarized light
photomicrograph for statistical analysis. The polarized light microscope does allow for
lesions to be quantified indirectly (Hicks, 1981).
16
Figure 3. Histologic zones of a carious lesion
17
MATERIALS AND METHODS
Sample Preparation
Eighty-two recently extracted non-carious human third molar teeth without
observable white-spot lesions, decalcification, or dental fluorosis were selected for this in
vitro study. The teeth were disinfected in Streck Tissue Fixative for two weeks, washed
thoroughly with deionized water, and then stored in a 0.1% thymol solution. All
remaining soft tissue, calculus, and bone were removed from the teeth utilizing razor
blades and dental scalers. A small hole was drilled near the apex of the root of each tooth
and a piece of waxed dental floss was tied to the root to facilitate suspension of the tooth
in the experimental solutions. Each tooth was then coated with a thin layer of acidresistant varnish leaving a 1 mm x 6 mm window of exposed enamel. An example of the
prepared tooth is demonstrated in Figure 4. Cusp tips were ground flat to facilitate
sectioning as illustrated in Figure 5.
Figure 4. Tooth painted with acid-resistant varnish to produce a 1 mm x 6 mm window
of exposed enamel.
18
Figure 5. Cusp tips were ground flat to facilitate the sectioning procedure.
Figure 6. Tooth following acidic challenge.
19
Experimental Procedure
Procedures
This in vitro study compared the effectiveness of Novamin powered technology
Oravive and TopexRenew with Crest an
and
d Colgate’s PreviDent 5000 Plus in decreasing
demineralization and preventing white spot lesion formation on extracted human teeth.
teeth
Oravive.
Figure 7. Prevident 5000 Plus, Crest, Renew and Oravive
20
Following preparation, the teeth were randomly divided into five groups as
follows:
Group 1 - Control (deionized water)
Group 2 - Oravive
Group 3 - Renew
Group 4 - Crest
Group 5 - Prevident5000
Treatment slurries for Groups 2-5 were created at a ratio of 1:3 (w/v) with
deionized water. Oravive, Renew, Crest and Prevident 5000 Plus were measured and
placed in separate beakers. Each product was incorporated into water by hand spatulation
then stirred using a stir bar and stir plate (rpm = 400) to ensure even distribution of the
active ingredient, until the slurry was uniform in color and consistency. Fresh treatment
slurries were mixed twice daily for the total duration of the study (10 days).
The study commenced with the treatment application protocol. The teeth were
suspended fully submerged in their respective treatment slurry (Group 1 in deionized
water, Group 2 in Oravive, Group 3 in Renew, Group 4 in Crest, and Group 5 in
PreviDent 5000 Plus™) for two minutes. Following the treatment protocol, the teeth
were rinsed with distilled water and blotted dry. They were then placed in Parafilm® Mcovered beakers with room-temperature, constantly-circulating (rpm = 150) artificial
caries solution. The demineralizing solution consisted of of 2.2 mM Ca2+, 2.2 mM PO43-,
0.05 M acetic acid and 0.25 ppm Fluoride at pH = 4.4 (ten Cate and Duijsters, 1982).
Each group had separate beakers of demineralization solution.
Following 4 hours of demineralization, the teeth were removed from the acid
solution, rinsed thoroughly with distilled water, blotted dry, and placed in a constantlycirculating (rpm = 150), room temperature, artificial saliva (remineralization) solution
consisting of 20 mM NaHCO3, 3 mM NaH2PO4, 1 mM CaCl2·2H20, at pH = 7
21
(Birkeland, 1973). Similarly to the demineralization solution, each group had separate
beakers of remineralization solution.
After 1 hour of remineralization, the teeth were removed, rinsed thoroughly with
distilled water, blotted dry, and placed in the demineralization solution for another 4
hours. Following the second period of demineralization the treatment application
protocol was repeated, which included a two minute exposure in the respective treatment
slurry.
Following the second treatment application protocol the teeth were rinsed
thoroughly with distilled water, blotted dry, and placed in the remineralization solution
for 14 hours (overnight). The cycling process proceeded for ten continuous days until
significant white spot lesions were visible in the control teeth.
Both demineralization and remineralization solutions were changed at the end of
Day 5 to maximize the demineralization ability and the remineralization capacity of the
respective solutions. Teeth were monitored for chipped acid-resistant varnish during the
study and were repainted as necessary.
After ten days of cycling, one control tooth was sectioned and examined under the
polarized light microscope to evaluate lesion depth. Based on the depth of the lesions
observed in the control samples it was determined that the study had progressed
sufficiently and that cycling should be terminated. Subsequently, all teeth were removed
from solution, rinsed thoroughly with distilled water, and blotted dry.
Longitudinal sections were made using a Series 1000 Deluxe hard tissue
microtome (Scientific Fabrication, Littleton, Colorado) to yield sections 100-140 µm
thick. Sections were imbibed with deionized water (refractive index = 1.33) and
photographed under a polarized light microscope (Olympus model BX-50, Olympus
Corporation, Lake Success, New York). Three representative sections of every tooth
were photographed and measured using Image Pro Plus computer software (Media
Cybernetics, Silver Spring, Maryland). The software allowed for average depth (µm) to
22
be recorded across the length of the lesion. For most of the control teeth, the enamel
surface had to be recreated due to erosion of the outer enamel surface. The resulting
depths of each of the three sections were averaged together to produce an overall mean
lesion depth (µm) for the tooth.
Statistical Analysis
Descriptive statistics were conducted. Because of lack of normality, the white
spot lesion depths were analyzed using one-way ANOVA to the ranked data, followed by
post-hoc Bonferroni multiple comparison test, an equivalent test statistics to the
nonparametric Kruskal-Wallis test.
An underlying assumption in order to use the ANOVA procedure is the normality
of the residuals. Normality of residuals was checked with a non significant Shapiro-Wilk
test and normal probability plots.
All tests employed a 0.05 level of statistical significance. Statistical analyses
were carried out with the statistical package SAS® System version 9.1 (SAS Institute Inc,
Cary, NC, USA).
23
RESULTS
The teeth in the study were cycled for ten continuous days. Following the cycling
protocol the teeth were sectioned using the hard tissue microtome producing three to five
sections per tooth. This procedure led to the loss of six teeth (3 from the Control group, 1
from the Crest group and 2 from the Prevident group) due to damaged enamel and/or
unusable sections. Of the remaining 76 teeth, three representative sections of each tooth
were selected to be photographed and measured. Erosion of the outer enamel surface was
observed in most of the teeth in the Control (Group 1) and Oravive (Group 2) groups.
Representative sample lesions from each group were photographed and are displayed in
Figures 8-12. In addition, a sample of the measuring technique using the Image Pro Plus
computer software to determine lesion boundaries and average lesion depth is displayed
in Figure 13.
When measuring the teeth that displayed cavitation of the enamel surface, the
surface was approximated by connecting intact enamel borders (Figure 14). This allowed
for an estimated measurement of lesion depth for these teeth. A composite of all five
treatment groups is depicted in Figure 15. In order to ensure the independence of
samples for performing the appropriate statistical analysis, the average of three
measurements was used for the data analysis.
Descriptive statistics for the comparison of lesion depths between treatment
groups are summarized in Table 1.
24
Figure 8. Polarized light microscopy image of representative lesion from the Control
group.
25
Figure 9. Polarized light microscopy image of representative lesion from the Oravive
group.
26
Figure 10. Polarized light microscopy image of representative lesion from the Renew
group.
27
Figure 11. Polarized light microscopy image of representative lesion from the Crest
group.
28
Figure 12. Polarized light microscopy image of representative lesion from the Prevident
5000 Plus group.
29
Figure 13. Measuring technique used by Image Pro Plus computer software (Media
Cybernetics, Silver Spring, Maryland) to record mean lesion depth.
Figure 14. Measuring technique used for eroded lesions.
30
Control (n = 19)
Average Depth = 128 µm
Std Dev = 27
Crest (n = 14)
Average Depth = 61.9 µm
SD = 16.3
Renew (n = 15)
Average Depth = 66.1µm
SD = 13.7
PreviDent 5000 Plus (n = 13)
Average Depth = 38.9 µm
SD = 10.4
Oravive (n = 15)
Average Depth = 127.1 µm
SD = 32.2
Figure 15. Composite of polarized light microscopy images of representative lesions.
31
Table 1. Descriptive statistics of mean lesion depth by treatment group.
Results of one-way ANOVA revealed a significant effect for the type of
preventive treatments on the white spot lesion depths (p<0.0001). The post-hoc
Bonferroni multiple comparison test indicated that the mean lesion depths observed in
Control and Oravive groups were significantly greater than the other three treatment
groups, while the mean lesion depth observed in Prevident was significantly lower than
those in Renew and Crest. Moreover, no significant difference was found between
Control and Oravive or between Renew and Crest. Table 2 presents the results of the
post-hoc Bonferroni multiple comparison test.
32
*** means with the same letter are not significantly different using post-hoc Bonferroni
multiple comparison test (P>.05)
Table 2. Mean lesion depth by treatment group.
33
Figure 16. Mean lesion depth comparison between groups
groups.
The Oravive group showed only a 0.8% reduction in mean lesion depth compared
with the Control group, while the Renew and Crest groups had 48.4% and 51.7%
reduction respectively. The Prevident 5000 Plus group demonstrated a 69.7% reduction
in mean lesion depth compared with the Control group and a 39
39%
% reduction, compared
with the Renew/Crest group (Figure 17).
34
Figure 17. Percent (%) reduction of mean lesion depth compared to the Control group.
35
DISCUSSION
The purpose of this study was to compare in vitro the effectiveness of Novamin
powered technology Oravive and TopexRenew with Crest and Colgate’s PreviDent 5000
Plus in decreasing demineralization and preventing white spot lesion formation on
extracted human teeth. It was hypothesized that Oravive and TopexRenew would,
respectively, be more effective than Crest and Prevident 5000 Plus in the prevention of
enamel demineralization.
Eighty-two extracted non-carious human third molar teeth without observable
white-spot lesions, decalcification, or dental fluorosis were selected for this in vitro
study. Throughout the study 6 teeth were lost during the sectioning process. The final
n=76 proved to be adequate, since enough statistical power was achieved to show a
statistically significant difference between the samples. It is not clear whether Novamin’s
mechanism of action is more effective in preventing demineralization or in enhancing
remineralization. Thus, it was rationalized that by conducting the treatment applications
before and after periods of demineralization, one would be allowing both scenarios to
occur.
The amount of time spent in both solutions throughout 10 days was 240 hours,
divided as follows: 80 hours in the demineralization solution, 150 hours in the
remineralization solution, and 10 hours in the treatment application protocol. After ten
days the cycling was terminated, teeth were sectioned and the slices were examined under
the microscope and measured.
Statistical analysis indicated that the mean lesion depths observed in the Control
and Oravive groups were significantly greater than in the Crest, Renew and Prevident
5000 Plus groups, while the mean lesion depth observed in Prevident 5000 Plus was
significantly lower than those in Renew and Crest. In addition, no significant difference
was found between Controls and Oravive or between Renew and Crest.
36
Some of the results encountered were expected, such as the Prevident 5000 Plus
(69.7%) showing a higher reduction in mean lesion depth when compared to the Crest
toothpaste (51.7%). Since the amount of fluoride present in the Crest toothpaste is
significantly less than the amount present in the Prevident 5000 Plus, which makes F
much less available to prevent demineralization and promote remineralization of white
spot lesions in the Crest group. Through comparison of the mean lesion depth reduction
in the Control (0 ppm F), Crest(1100 ppm F) and Prevident 5000 Plus (5000 ppm
F)groups, it can be concluded that the fluoride dose response was effective under the
model utilized in this study. It was also expected that Renew (48.4%) showed superior
performance than Oravive (0.8%). Although both products contain 5% Novamin, Renew
also contained 5000 ppm F.
Some of the results, however, were quite different from those expected. It was
hypothesized that Oravive (5% Novamin) would be more effective than Crest (1100 ppm
F) in preventing demineralization and/or enhancing remineralization, and the results
showed that the Crest group had a significantly higher reduction in mean lesion depth
when compared to the Oravive group. In fact, perhaps the most unexpected finding was
the fact that the Oravive group performed almost exactly the same as the Control group,
with no statistically significant difference between both groups.
Due to the combination of Novamin and fluoride, and according to the literature
and manufacturer advertisements, it was expected that the Renew group (5% Novamin;
5000 ppm Fl) would be more effective and have stronger results than the Prevident 5000
Plus group (5000 ppm Fl), hence our hypothesis. However, based on the results when
comparing the Control group against both groups containing Prevident 5000 Plus or
Renew, Prevident 5000 Plus had a significantly more effective performance in reducing
mean lesion depth, compared to Renew (69.7% reduction versus 48.4% for the Renew
group). The Renew had also been expected to be more effective than the Crest group,
37
since it has a higher F concentration in addition to 5% Novamin, but no statistically
significant difference was found between these two groups.
A common pattern observed showed that the Novamin-containing products used
in this study (Oravive and Renew) did not perform as expected, indicating that, under the
model chosen in this study, Novamin was not an effective agent in preventing
demineralization and/or enhancing remineralization.
This could also be explained due to the fact that when Calcium and Phosphate
ions (existing in Novamin) are applied in conjunction with fluoride, their low solubility
could lead to precipitation of these ions, which in turn could make it more difficult for
these ions to reach the tooth surface (Wefel, 2009).
In disagreement with these findings, Alaudin and Fontana (2007) compared
dentifrice containing 5% Novamin and fluoride (MFP) to a commercially available
dentifrice with MFP only, and concluded that the Novamin-containing dentifrice was
statistically more significant in decreasing the lesion area than the commercial dentifrice
containing MFP alone. One of the differences between our study and Alaudin’s is that
we used NaF instead of MFP fluoride.
When considering the clinical application of these particular products, some
important factors must also be taken into account. While Crest, Renew, and Prevident
5000 Plus have all been shown in this study to decrease mean lesion depth, Oravive did
not present any statistically significant difference, compared to the Control group; thus,
under the model utilized in this study, it would be fair to say that we do not recommend
its use in the prevention of demineralization and/or enhancement of remineralization.
While both Crest and Renew proved to be effective agents in the prevention of
demineralization and/or enhancement of remineralization, no significant difference was
found in lesion depth reduction between them (51.7% versus 48.4%, respectively).
Therefore, it can be concluded that it is more cost effective and convenient to use the
38
non-prescription Crest toothpaste rather than the more expensive, prescription-only
Renew toothpaste.
When comparing both prescription-only toothpastes Prevident 5000 Plus and
Renew, our study showed a statistically significant difference in the reduction of lesion
depth (69.7% versus 48.4%, respectively). As the cost of both products is comparable,
one might lean towards the use of Prevident 5000 Plus.
However it should be taken into consideration that despite Renew being less
effective than Prevident 5000 Plus in the prevention of white spot lesions, and equally as
effective as the non-prescription Crest toothpaste, some Novamin-containing products
have also been shown to be effective desensitization agents, according to Burwell (2006).
An orthodontic patient with sensitivity, in particular an adult, could profit from this added
benefit and may be more compliant using a single preventive product rather than multiple
products.
There were also several inherent limitations to this study. The fact that it was
conducted in vitro is one of them. It is quite difficult to recreate the oral environment in a
laboratory study outside of the mouth. Saliva has several components that are not part of
the remineralization solution used in this study, such as proteins and bacteria. It is also
impossible to precisely account for the salivary flow that continuously remineralizes the
oral environment. Naturally the cycling simulated in our study happens in a much more
varied manner in the oral cavity, being affected not only by schedule but also by diet.
Certain foods such as sugary snacks and drinks can potentially increase demineralization,
while other calcium-rich products such as milk can inhibit demineralization.
Also, the absence of plaque on the teeth used in the study makes it difficult to
fully evaluate the effectiveness of Novamin. Tai BJ et al (2006) conducted a randomized,
double-blinded pilot clinical trial to evaluate the anti-gingivitis and anti-plaque effects of
a Novamin containing dentifrice. 100 patients had a baseline measurement of their Plaque
Index(PLI) and Gingival Bleeding Index (GBI) and 6 wks later following use of the
39
Novamin containing dentifrice or a placebo control toothpaste. Patients were told not to
perform any oral hygiene for 8h prior to the baseline and 6 wks examination. Following
the clinical assessments, all subjects received a supragingival prophylaxis and were given
either the test-dentifrice or the placebo formulation. The results showed a statistically
significant difference in the reduction of gingival bleeding (58%) and reduction in plaque
growth (16%) in the Novamin group.
Another limitation relates to the short term period in which it was conducted (10
days). For that reason, the experimental conditions were intentionally more extreme in
order to create lesions within this short period of time. Ogaard et all (1988) has shown
that white spot lesions can normally develop anywhere from 4 wks to several months.
Both this study and Ogaard’s study are examples of harsher acidogenic challenges in
significantly shorter periods of time, when compared to the naturally slower and milder
demin/remin cycle over the course of 2 years in the orthodontic patient. Novamin’s
mechanism of action might require a longer treatment time period and/or a less
acidogenic challenge to be effective. The accelerated and stronger conditions in our study
might have led to a limitation of Novamin’s ability to prevent demineralization and/or
enhance remineralization.
Certainly additional research is warranted to help understand the exact optimal
conditions under which Novamin-containing products can be more effective in the
prevention of demineralization and enhancement of remineralization. Ideal formulation
and delivery methods need to be further developed and investigated and then tested in
randomized clinical trials, in order to fully explore the potential of this promising agent.
40
SUMMARY AND CONCLUSIONS
The primary goal of orthodontic treatment is to improve the patient’s smile and
facial esthetics, while providing a great functional result for the patient. Several factors
are involved in achieving that goal, such as adequate diagnosis, treatment planning,
execution of treatment in a timely fashion, and patient cooperation. Patient cooperation
is one of the biggest challenges faced by the orthodontist. If proper care doesn’t occur in
relation to diet and oral hygiene, the development of white spot lesions is very likely to
happen.
The purpose of this study was to compare in vitro the effectiveness of Novamin
powered technology Oravive and TopexRenew with Crest and Colgate’s PreviDent 5000
Plus in decreasing demineralization and preventing white spot lesion formation on
extracted human teeth. After completion of the cycling protocol, teeth were analyzed
under a polarized light microscope and sectioned.
The following conclusions can be made from this study:
1.
The fluoride dose response was effective under this model,
demonstrating progressively smaller lesion depths with greater
fluoride concentration.
2.
We expected a synergistic effect between Novamin and
fluoride, however this was not evident, as our data showed that
Renew (5% novamin; 5000 ppm F) performed at the same level as Crest (1100
ppm F) and inferior than Prevident 5000 Plus (5000 ppm F).
41
3.
Novamin by itself (Oravive, 5%Novamin) performed at the
same level as the non-fluoridated Control group.
4.
Prevident 5000 Plus (5000 ppm F) was the most effective
product in this study for the prevention of enamel
demineralization and white spot lesion formation.
5.
It is difficult to determine Novamin’s full potential in an
vitro study not including bacteria, dental plaque and natural
saliva.
6.
Further research is needed to further evaluate Novamin’s
capability in prevention of demineralization and/or
enhancement of remineralization.
42
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