Title
Author(s)
Citation
Issued Date
URL
Rights
Remineralizing action of CPP-ACP reagents on artificial carious
lesions
Buckshey, Sakshi.
Buckshey, S.. (2011). Remineralizing action of CPP-ACP
reagents on artificial carious lesions. (Thesis). University of
Hong Kong, Pokfulam, Hong Kong SAR. Retrieved from
http://dx.doi.org/10.5353/th_b4684891.
2011
http://hdl.handle.net/10722/144146
The author retains all proprietary rights, (such as patent rights)
and the right to use in future works.
Remineralizing action of CPP-ACP reagents
on artificial carious lesions
a thesis submitted as partial fulfilment for the degree
Master of Dental Surgery
by
Dr. Sakshi Buckshey
The University of Hong Kong
Faculty of Dentistry
Paediatric Dentistry and Orthodontics
2011
i
Abstract
Remineralizing action of CPP-ACP reagents on artificial carious
lesions
Objectives: the objectives of this in vitro study were to evaluate and compare the efficacy of
Abstract
two CPP-ACP containing pastes; Clinpro® (500ppm tri-calcium phosphate with 950ppm
sodium fluoride) and Tooth Mousse Plus® (10% CPP-ACP with 900ppm fluoride) for
remineralizing artificial enamel lesions, using a 10-day pH cycling model involving topical
applications for three minutes. Methods: Fifty extracted human third molars were cleaned
of soft tissue and debris, inspected for any cracks, caries or hypoplasia. The teeth were
painted with an acid resistant nail varnish leaving a 1mm window on the buccal and lingual
surfaces and then immersed in a demineralizing solution, for 96 hours, to produce artificial
carious lesions 90-180μm deep. Subsequently, the teeth were sectioned longitudinally
through the lesion to produce sections that were approximately 100-150μm thick. These
sections were then painted with an acid resistant nail varnish leaving only the outer surface
of the lesion exposed. The specimens were then randomly divided into six groups (n=25) to
receive treatment thrice daily. Specimens were treated with either a non-fluoridated paste,
Vicco®; or a fluoridated paste, Colgate Total® (1000ppm), or one of the two CPP-ACP
containing pastes, namely Clinpro® containing 500ppm tri-calcium phosphate with 950ppm
sodium fluoride and Tooth Mousse Plus® containing 10% CPP-ACP with 900ppm fluoride.
Clinpro® and Tooth Mousse Plus® were also used in combination with the fluoridated
dentifrice. All the specimens were subjected to a 10-day pH cycling model. The lesion depth
(LD) and mineral content (Vmax) for each specimen was evaluated using polarized light
microscopy (PLM) and microradiography (MRG); both before and after the pH cycle. Paired
t-tests and ANOVA tests were employed to make comparisons within and among the
different groups.
ii
Results: Specimens treated with Colgate Total® showed the maximum increase in Vmax
followed by the specimens treated by the combination of Clinpro® plus Colgate Total®. On
comparing the reduction in the lesion depth between the different treatment groups, there
was a trend for the specimens treated with the combination of Clinpro® plus Colgate Total®
to show the greatest reduction in lesion depth followed by Clinpro® and then the
combination of Tooth Mousse Plus® plus Colgate Total®, even though the differences were
not statistically significant.
Conclusions: Statistically significant differences (p< 0.01) were evident when comparisons
were made between the pre-treatment and post-treatment LD for all the therapies tested.
The combination of Clinpro® plus Colgate Total® proved most effective in decreasing lesion
depth, followed by Clinpro® and then the Tooth Mousse Plus® plus Colgate Total® group,
even though the differences were not statistically significant. There was a trend, though not
statistically significant, for the three minute application of Clinpro® to exhibit a higher
efficacy in remineralizing artificial enamel carious lesions than Tooth Mousse Plus® .
Word count: 449
iii
Acknowledgements
I would like to express my sincere heartfelt gratitude to my teacher and mentor, Professor
Nigel M King, Professor in Paediatric Dentistry at the Faculty of Dentistry, The University of
Hong Kong, for accepting me into this prestigious program and giving me the opportunity to
learn and broaden my horizons. His constant encouragement, guidance and support have
Abstract
helped and enabled me to successfully complete
this research project. It has been a
privilege to complete my training under his supervision and guidance and I consider myself
extremely lucky to have been given this opportunity.
The past two years in Paediatric Dentistry and Orthodontics have been the best years of my
life and I have enjoyed every single day, be it the clinical sessions, the GA sessions, or the
journal club discussions. None of this would have been possible without the wonderful
supervisors and the ever smiling dental surgery assistants and nurses.
I would like to express my appreciation to my senior colleague, Dr. Robert Anthonappa, for
his patience and guidance that was instrumental in the successful completion of my
research project.
I would also like to extend my sincere gratitude to Mr. Yip Chui and Mr. Shadow Yeung for
their technical expertise and for answering my innumerable queries patiently.
To my colleagues and friends: especially Charanya, Sadia and Hajar for helping me cope with
this challenge and making the experience enjoyable. A special thanks to all the secretarial
iv
staff in Paediatric Dentistry and Orthodontics, especially Ms. Frances Chow for her efficient
secretarial work.
Most importantly this would not have been complete without the love, encouragement,
prayers and constant support of my mom and dad to whom I will forever be grateful and
also my brother, Arjun for his constant encouragement and love.
v
Table of Contents
Abstract
ii
Acknowledgements
iv
Table of Contents
Abstract
vi
List of Tables
viii
List of Figures
ix
Abbreviations
xii
Chapter 1.0
Statement of the problem
1
1.1 Objectives
8
1.2 Null hypotheses
9
Chapter 2.0 Literature review
10
Chapter 3.0 Materials and methods
31
3.1 Formation for demineralizing/remineralizing solutions
32
3.2 Artificial carious lesion formation
32
3.3 Grouping
33
3.4 Agent preparation
34
3.5 The pH cycling protocol
34
3.6 Evaluation techniques
35
3.6.1 Qualitative evaluation
35
3.6.2 Quantitative evaluation
36
3.7 Statistical analysis
Chapter 4.0 Results
37
48
4.1 Quantitative evaluations
49
4.1.1 Lesion depth (LD) changes in the artificial enamel
vi
carious lesions after pH cycling
49
4.1.2 Mineral content changes in the surface zone (Vmax)
of the artificial enamel carious lesions after pH cycling.
50
Chapter 5.0 Discussion
60
Chapter 6.0 Conclusions
65
Chapter 7.0 References
70
Chapter 8.0 Appendices
Appendix I Lesion depth and mineral content changes before
85
and after pH cycling with different agents.
Appendix II Time schedule and pH cycling protocol.
101
Appendix III Statistical analysis and raw data.
108
Appendix IV Abstract of paper presentation at the 23 rd Congress
of the International Association of Paediatric Dentistry
Appendix V
in Athens, Greece.
117
List of published studies on CPP-ACP.
120
vii
List of Tables
Table 3.1:
The treatment protocols for the six experimental groups.
Table 4.1:
Mean values (+SD) of the lesion depth (LD) and the maximum
33
59
mineral content (Vmax) of the samples in the six treatment groups
both before and after the 10-day pH cycle.
Abstract
Table V.1:
Evidence from in vitro studies.
121
Table V.2:
Evidence from in vivo studies.
124
Table V.3:
Evidence from clinical trials that used CPP-ACP and have been
131
published in the English literature.
Table V.4:
Evidence from literature reviews on the use of CPP-ACP that have
133
been published in the English literature.
Table V.5:
Evidence from published studies investigating CPP-ACP for more than 136
anticariogenic reasons that have been published in the English
literature.
viii
List of Figures
Figure 3.1: The reagents used to prepare the demineralizing solution.
37
Figure 3.2: Dentifrices used in the study.
38
Figure 3.3: Treatment agents.
38
Figure 3.4: The sequence of steps involved
in the formation of artificial carious lesions, 39
Abstract
(a) painted specimen (b) teeth immersed in the demineralizing solution for
96 hours, and (c) a 1mm wide window left on the buccal and lingual surfaces
of the tooth.
Figure 3.5: The various steps involved in preparing the tooth sections for the pH cycle, 40
(a) the microtome (Leica® 1600 saw microtome, Germany), (b) the tooth
section and (c) the micrometer.
Figure 3.6: Quantitative evaluation of the artificial carious lesion using
41
microradiography, (a) the sectioned tooth specimen, (b) X-ray machine
(Softex® ISR-20, JIRA, Japan) and (c) microradiograph of a tooth specimen.
Figure 3.7: Qualitative evaluation using polarized light microscopy technique showing
42
(a) the sectioned tooth specimen, (b) polarizing light microscope (Nikon
Eclipse LV100POL, Nikon®, Japan) and (c) a polarized light photomicrograph
of a specimen.
Figure 3.8: The pH calibration system.
43
Figure 3.9: The centrifuge machine (Beckman®, Avanti J-251, USA).
44
Figure 3.10: Schematic representation of the six different groups and their respective
45
treatment protocols.
Figure 3.11: The reagents used to prepare the remineralizing solution.
46
Figure 3.12: Diagram illustrating the sequence of steps in the pH cycling model.
47
Figure 4.1: Polarized light photomicrographs of a specimen in Group A treated
51
with the non-fluoridated dentifrice Vicco®, (a) before and (b) after
the 10-day pH cycling period. An increase in lesion depth is evident.
Figure 4.2: Polarized light photomicrographs of a specimen in Group B treated with
51
ix
the fluoridated dentifrice Colgate Total®, (a) before and (b) after the 10-day
pH cycling period. An increase in lesion depth is evident.
Figure 4.3: Polarized light photomicrographs of a specimen in Group C treated with
51
Clinpro®, (a) before and (b) after the 10-day pH cycling period. A decrease
in lesion depth is evident.
Figure 4.4: Polarized light photomicrographs of a specimen in Group D treated with
52
Tooth Mousse Plus®, (a) before and (b) after the 10-day pH cycling period. A
slight increase in lesion depth is evident.
Figure 4.5: Polarized light photomicrographs of a specimen in Group E treated with
52
Clinpro® plus Colgate Total®, (a) before and (b) after the 10-day pH cycling
period. A decrease in lesion depth is evident.
Figure 4.6: Polarized light photomicrographs of a specimen in Group F treated with
52
Tooth Mousse Plus® plus Colgate Total®, (a) before and (b) after the 10-day
pH cycling period. A slight decrease in lesion depth is evident.
Figure 4.7: Graph showing the relationship between the lesion depth (LD) on x-axis
53
in µm and % maximum mineral content (Vmax) on the y-axis, both
before and after the 10-day pH cycle, for a specimen in Group A
(non-fluoridated dentifrice, Vicco®).
Figure 4.8: Graph showing the relationship between the lesion depth (LD) on x-axis
54
in µm and % maximum mineral content (Vmax) on the y-axis, both
before and after the 10-day pH cycle, for a specimen in Group B
(fluoridated dentifrice, Colgate Total®).
Figure 4.9: Graph showing the relationship between the lesion depth (LD) on x-axis
55
in µm and % maximum mineral content (Vmax) on the y-axis, both
before and after the 10-day pH cycle, for a specimen in Group C (Clinpro®).
Figure 4.10: Graph showing the relationship between the lesion depth (LD) on x-axis
56
in µm and % maximum mineral content (Vmax) on the y-axis, both
before and after the 10-day pH cycle, for a specimen in Group D
x
(Tooth Mousse Plus®).
Figure 4.11: Graph showing the relationship between the lesion depth (LD) on x-axis
57
in µm and % maximum mineral content (Vmax) on the y-axis, both before
and after the 10-day pH cycle, for a specimen in Group E
(Clinpro® + Colgate Total®).
Figure 4.12: Graph showing the relationship between the lesion depth (LD) on x-axis
58
in µm and % maximum mineral content (Vmax) on the y-axis, both before
and after the 10-day pH cycle, for a specimen in Group F
(Tooth Mousse Plus® + Colgate Total®).
xi
Abbreviations
CPP-ACP
casein phosphopeptide amorphous calcium phosphate
ACP
amorphous calcium phosphate
TCP
tri-calcium phosphate
TM
Tooth Mousse®
TMP
Tooth Mousse Plus®
PLM
polarized light microscopy
Ca
calcium
PO4
phosphate
SEM
scanning electron microscopy
ANOVA
analysis of variance
CaCl2
calcium chloride
CaF2
calcium fluoride
F
fluoride
HKU
The University of Hong Kong
KCL
potassium chloride
KH2PO4
potassium dihydrogen phosphate
KOH
potassium hydroxide
kV
kilovolt
L
litre
LD
lesion depth
min
minutes
s
seconds
ml
mililitre
mM
milimolar
Abstract
xii
MRG
microradiography
mV
milivolt
PLM
polarized light microscopy
ppm
parts per million
SD
standard deviation
Vmax
maximum mineral content
ΔZ
delta z
µm
micrometer
%
percent
C
centigrade
w/v
weight per volume
w/w
weight per weight
rpm
revolutions per minute
WSL
white spot lesion
◦
xiii
Chapter 1
Statement of the problem
1
Dental caries is one of the most prevalent infectious diseases to afflict mankind (Hicks et al,
1993). It has been defined as the localized destruction of susceptible dental hard tissues by
acidic by-products produced from the bacterial fermentation of dietary carbohydrates
(Marsh et al, 1999). Endogenous bacteria in the biofilm (dental plaque), mainly Streptococci
(S. mutans and S. sobrinus) and Lactobacillus species, produce weak organic acids as
metabolic by-products of the fermentation of carbohydrates. These acids cause the local pH
to fall below a critical value, diffuse through the plaque into the porous enamel or dentine
and dissociate to produce hydrogen ions. These ions further dissolve the hydroxyapatite,
freeing calcium and phosphate ions (Featherstone, 1983); thus causing demineralization.
The resting pH of the saliva determines the caries experience and the salivary buffering
capacity of an individual. Individuals who have a resting salivary pH of approximately 7, tend
to have either low caries activity or no activity, while those with a pH of 5.5 tend to have a
high caries experience.
The process of caries development takes place over a considerable period of time, maybe as
long as several months or even years (Pearce, 1998; Marsh, 1999; Zero, 1999; Walsh, 2000).
With repeated and prolonged exposure to a low pH, the plaque buffering capacity and
supersaturation with respect to the calcium and phosphate ions will be compromised,
leading to eventual demineralization of the tooth structure. In the early stages, this process
can be reversed if the pH of the biofilm is restored by the saliva. The calcium, phosphate
and fluoride ions that are present in the biofilm are able to restore the tooth structure
by remineralization (Selwitz et al, 2007). Thus, remineralization can be defined as the
natural repair process of the body for subsurface non-cavitated carious lesions (ten Cate et
al, 1991). It can be explained as being the process whereby calcium and phosphate ions are
2
supplied from an internal or external source, in order to promote ion deposition into the
crystal voids present in the demineralized enamel so as to cause a net gain in the mineral
content. Human saliva has the ability to remineralize these enamel crystals due to its ability
to supply calcium and phosphate ions to the tooth. This process was first studied by Head, in
1912, who reported that lesions would re-harden in the presence of saliva. It has
subsequently been confirmed repeatedly in various studies that at a physiological pH, saliva
is supersaturated in terms of the presence of calcium ions (Larsen and Pearce, 2003).
However, this remineralization maybe small and a very slow process with the maximum
level of mineral deposition occurring on the surface of the lesion due to the low ion
concentration gradient from saliva into the lesion (Silverstone, 1972). This process of
remineralization can be explained as a process that takes place by two main routes:
precipitation of calcium phosphates from salivary sources, and the use of salivary calcium
and phosphate for the growth of the remaining enamel and dentine crystallites.
Conversely, demineralization, can be observed in various forms such as, surface loss or
erosion, surface-softening, or white-spot lesion (WSL) formation. The lesion looses as much
as half of its original mineral content and appears to be covered by “an intact surface layer”
(Silverstone, 1973). If the diffusion of calcium, phosphate and carbonate ions is
allowed to continue, without proper remineralization, cavitation eventually occurs
(Seow et al, 1998).
Fluoride and its various agents have been regarded for many years, as the mainstay of the
non-invasive management of non-cavitated carious lesions. The discovery of the
reduced solubility of enamel and dentine, after treatment with fluoride, lead to further
studies related to the anti-cariogenic properties of fluoride (Volker, 1939). Various in vitro
3
and in vivo studies have highlighted the anticariogenic action of fluoride in drinking water
(Newbrun, 1989) and also as a topically applied remineralizing agent. Its anticariogenic
action in dentifrices, mouthrinses, chewing gums and professionally applied products has
been extensively documented (Marinho et al, 2003). Fluoride acts by inhibiting
demineralization, enhancing remineralization (ten Cate, 1999) and inhibiting the growth of
plaque micro-organisms (Whitford, 1977). These agents supply fluoride ions, which combine
with the calcium and phosphate ions produced during the process of demineralization and
lead to the formation of fluorapatite (ten Cate, 1999). However, for every two fluoride ions,
ten calcium and six phosphate ions are required to form one unit of fluorapatite
[Ca10(Po4)6F2]. For this reason, topical fluoride containing agents may have insufficient
calcium and phosphate ions present to produce net remineralization.
In spite of the widespread success of fluorides, several new agents have been proposed and
recommended for personal and professional applications (Pulido et al, 2008; Reynolds,
2008). Casein phosphopeptides (CPP) with the sequence -Ser(P)-Ser(P)-Ser(P)-Glu-Gluthat stabilizes amorphous calcium phosphate in solution (Cross et al, 2005) are such
agents . They form nanocomplexes at the tooth surface, thus providing a reservoir of nonstructurally bound calcium and phosphate ions which favour remineralization during a
cariogenic attack (Reynolds, 1998). Calcium and phosphate, either from the saliva, or from
other topical sources diffuse into the tooth and build on existing crystal remnants rather
than forming new crystals (Featherstone, 2000).
Currently, there are three main calcium phosphate based remineralization systems
in use, for which the manufacturers claim that the specific form of calcium phosphate
present helps to overcome the limited bioavailability of calcium and phosphate ions for the
4
remineralization process. The first technology involves the use of casein phosphopeptide
stabilized amorphous calcium phosphate (CPP-ACP, Recaldent) where the casein
phosphopeptides stabilize high concentrations of calcium and phosphate ions, together with
fluoride ions at the tooth surface by binding to the salivary pellicle and plaque. Although the
calcium, phosphate and fluoride ions are stabilised by the CPP, they are freely bioavailable
to diffuse down the concentration gradient into the subsurface lesions in enamel thereby
promoting remineralization in vivo. The second technology which uses unstabilized
amorphous calcium phosphate (ACP), and is marketed as Enamelon, supplies calcium ions
(calcium phosphate) and phosphate ions (ammonium phosphate) separately so that
amorphous calcium phosphate, or amorphous fluoride calcium phosphate forms intraorally, that helps to rebuild the enamel through remineralization. This technology is used in
bleach-based whitening products. Alternatively, the third technology is a bioactive glass
containing calcium sodium phosphosilicate (NovaMin) where the glass particles release
calcium and phosphate ions intra-orally to promote remineralization.
For the purpose of remineralization CPP-ACP has been used in various forms and
incorporated into products such as; xylitol or sorbitol based sugar-free chewing gums (Iijima
et al, 2004), milk (Walker et al, 2006), lozenges (Cai et al, 2003) and mouthrinses (Reynolds
et al, 2008) and dental creams (Andersson et al, 2007).
From the contemporary literature it appears that CPP-ACP may exhibit synergistic effects
with fluoride, which reduces the caries activity due to the formation of CPP-stabilized
amorphous calcium fluoride phosphate (Cross et al, 2004). This results in increased
incorporation of fluoride ions into plaque with increased amounts of calcium and phosphate
ions. In an in vitro study by Reynolds and co-workers (2008), the combination of 2% CPP-ACP
5
and 1100ppm F was found to produce the highest level of remineralization. CPP-ACP at 0.5%
w/v was found to have a caries preventive effect similar to that produced by 500ppm
fluoride and when used together they produced a higher reduction in the caries rate than
either of them alone at the same concentrations (Reynolds et al, 1995). CPP-ACP was found
to significantly increase the level of calcium and phosphate ions in plaque and to promote
remineralization of subsurface lesions in enamel, when delivered in the form of a
mouthrinse (Reynolds et al, 2003). It was also found to be superior to other forms of calcium
phosphate including unstabilized ACP when used in products such as Tooth Mousse® (Kariya
et al, 2004).
Various CPP-ACP containing products are available currently such as; Tooth Mousse
Plus® (MI Paste Plus®) (10% CPP-ACP with 900ppm fluoride), Clinpro® (950ppm sodium
fluoride with 500ppm tri-calcium phosphate), Tooth Mousse® (MI Paste®) (CPP-ACP),
Enamelon (ACP) and chewing gums such as Trident Xtracare® (CPP-ACP), Recaldent® (xylitol
and CPP-ACP) and Orbit professional® (calcium carbonate).
Fluoride containing products exert their remineralizing effect primarily on the surface of
a lesion (Reynolds et al, 2008; Cochrane et al, 2010). Products such as Tooth Mousse Plus®
contain high levels of calcium and phosphate ions in combination with fluoride. These ions
are released into saliva to substantially increase the salivary concentrations of calcium,
inorganic phosphate and fluoride. This high concentration of ions is further stabilized by the
presence of CPP which prevents spontaneous precipitation, phase transformation and
allows penetration of the ions deep into the subsurface lesion causing an increase in
remineralization throughout the body of the lesion.
Conversely, products such as Clinpro® which contains TCP (tricalcium phosphate) have a low
6
level of available (acid soluble) calcium phosphate. Several studies have demonstrated that
fluoride ions are unstable in dentifrice formulations, especially in those containing poorly
soluble calcium-based abrasives (Noren and Harse, 1974; Sullivan et al, 2001). The
preparation leads to the development of sodium monofluorophosphate (MFP). Clinpro®
contains sodium fluoride, which, in the absence of a stabilizer can be susceptible to a
decrease in its bioavailability especially in the presence of added calcium phosphate. The
calcium phosphate that has been incorporated into Clinpro® is in the form of beta-tricalcium
phosphate that has been ball milled with sodium lauryl sulphate to produce particles within
the size range of 1–15 µm (Karlinsey et al, 2010).The poor solubility, the large particle size
and low amount of calcium phosphate explains the reason for the poor release from
Clinpro®, and its inability to significantly increase salivary calcium and inorganic phosphate
levels and also the failure to show an enhanced rate of remineralization (Shen et al, 2011).
Therefore, in view of the wide range of products available for use, clinicians and patients
face a dilemma as to which product should be used, or which product can be expected to
produce the highest remineralization rate. After a comprehensive search of the literature,
it was found that even though there are many papers in the literature on the efficacy of
CPP-ACP as a remineralizing agent and its use in various forms; there is not much evidence
based on direct comparisons of the efficacy of one product against another. Thus, at present
there is insufficient scientific evidence, based on comparisons and studies, that can be used
by clinicians’ to make recommendations to patients and parents.
7
1.1 Objectives
The objectives of this in vitro study were to evaluate the efficacy of CPP-ACP
containing pastes; Tooth Mousse Plus® (10% CPP-ACP with 900ppm fluoride) and
Clinpro® (950ppm sodium fluoride with 500ppm tri-calcium phosphate) for the
remineralization of artificial enamel carious lesions using a 10-day pH cycling model
when applied:
i.
as a topical cream for three minutes,
ii.
as a topical cream for three minutes following a one minute treatment
with a 1000ppm F containing dentifrice
and to compare all of these findings to a standard 1000ppm F containing dentifrice.
8
1.2 Null hypotheses:
I.
When applied topically for three minutes, Clinpro® will not exhibit a level
of remineralization comparable to a one minute treatment with a
1000ppm F dentifrice.
II.
When applied topically for three minutes, Tooth Mousse Plus® will not
exhibit a level of remineralization comparable to a one minute treatment
with the 1000ppm F dentifrice.
III.
When applied topically for three minutes, following a one minute
treatment with a 1000ppm F dentifrice, Tooth Mousse Plus® will not
exhibit a level of remineralization greater than that of a one minute treatment
with the 1000ppm F dentifrice.
IV.
When applied topically for three minutes, following a one minute
treatment with a 1000ppm F dentifrice, Clinpro® will not exhibit a level
of remineralization greater than that of a one minute treatment with the
1000ppm F dentifrice.
9
Chapter 2
Literature Review
10
2.1 Dental caries: initiation and prevention
On the basis of its location, dental caries can be classified into coronal and root surface
caries. The major difference between these two is the chemical environment of the
tissues leading to the generation of the carious lesion. Coronal caries occurs in enamel,
which has a higher mineral content and higher crystallinity as compared to dentine, where
root caries occurs (Hoppenbrouwers et al, 1987). Due to this reason, under an acid attack
dentine would be expected to be more soluble than enamel.
Until recently, the conventional treatment of a carious tooth involved the removal of the
carious tooth structure and its replacement with the appropriate restorative material.
Nowadays, a contemporary non-invasive approach has been adopted for the management
of such affected teeth. Lesions can be arrested if the cariogenic challenges are controlled
and certain therapeutic agents can be applied to encourage substantial tissue healing to
take place (Burke, 2003). Fluoride delivery methods such as varnishes, gels and creams have
been in use for several years to remineralize high risk tooth areas. However, certain agents
based on milk products have now been developed to enhance the remineralization of
enamel and dentine under cariogenic conditions. These new agents may contain calcium
phosphate compounds such as amorphous calcium phosphate (ACP), casein
phosphopeptides (CPP) or bioactive glasses based on calcium sodium phosphosilicate
compounds known as Novamin (CSP) (Reynolds, 1997; Featherstone et al, 2007).
2.2 CPP-ACP: introduction
In spite of the widespread and long term usage of fluorides, several new agents have been
proposed and recommended for personal and professional use (Pulido et al, 2008; Reynolds
et al, 2008). In the early 1960’s, dietary supplements of calcium and phosphate were
proposed for the prevention of dental caries. Koulourides and his co-workers (1968) in an in
11
vitro study demonstrated that acid softened enamel surfaces would reharden after the
application of calcium phosphate solutions. Furthermore, Silverstone (1975) in an in vitro
study showed that carious lesions or caries-like lesions showed a significant degree of
remineralization after exposure to calcifying solutions. After histological examination, the
lesions exhibited features that showed that they were at a much earlier stage in
development than they were before exposure to the calcifying solution. In animal studies, it
was found that dicalcium phosphates reduced the caries rate by 90% in hamsters (Stralfors,
1961) and calcium lactate significantly reduced the caries rate in rats (McClure, 1960).
Reynolds and Black (1987) attempted to incorporate casein into confectionaries in order to
decrease their cariogenic effect. Unfortunately, the addition of casein into the confectionary
products made them unpalatable. However, when the level of casein was reduced to 2%
w/w to make the confectionary palatable, the caseinate was found to be ineffective as an
anticariogenic agent (Reynolds, 1989).
For several years, dairy products have been linked to good oral health due to their proven
anticariogenic properties (Reynolds and Johnson, 1981; Rosen et al, 1984; Harper et al,
1986). Krobicka and co-workers demonstrated anticariogenic effects of cheddar cheese in
rats who had their parotid ducts tied and the submandibular and sublingual salivary glands
surgically removed. Similarly, Harper and co-workers studied the anticariogenicity of four
different cheeses and concluded that their caries preventive properties could be attributed
to their phosphoprotein casein and the calcium phosphate content (Harper et al, 1986).
Most cheeses contain around 20% w/w casein, that when rapidly degraded by the bacterial
proteolytic activity releases phosphopeptides that are relatively stable to further
degradation. Therefore, it is likely that the calcium phosphate complexes present in these
peptides maybe partially responsible for the cariostatic activity of the cheese and other such
dairy products (Reynolds, 1987; Reynolds and Riley, 1989). Casein phosphopeptides were
12
also found to increase the calcification of cultured embryonic rat bone in vitro by increasing
the concentration of soluble calcium phosphate (Gerber and Jost, 1986). Furthermore,
a decrease in enamel softening was demonstrated with the use of a water extract of
cheddar cheese in a human caries model by Silva and co-workers (1987). The cheese extract
increased the calcium level in the experimental plaque, thus causing a decrease in
demineralization and/or an increase in remineralization. Reynolds (1987) used an in vitro
model to show that solutions containing tryptic casein peptides were able to reduce the rate
of enamel demineralization significantly. Subsequently, it was concluded that the tryptic
peptides responsible for the remineralizing action of the caseinates were the calcium
phosphate stabilizing casein phosphopeptides (Reynolds et al, 1995). The anticariogenic properties of milk which have been demonstrated in various in vitro and animal
studies (Reynolds, 1998) are attributed to the presence of calcium, casein and phosphate
(McDougall, 1977; Morr and Roda, 1983; Reynolds, 2003). Casein is the major
protein present in milk and accounts for 80% of the total protein content. This casein forms
a protective coating on the enamel surface; thus, preventing demineralization while also
providing calcium and phosphate ions that help in remineralization. It also resists the
proteolytic action of the enzymes and stabilizes calcium phosphate complexes. However,
the majority of these ions are present in the form of micelles and their size and low
availability of ions limits their remineralization potential. More recent studies have shown
that the addition of 0.2% (w/v) and 0.3% (w/v) CPP-ACP to normal milk results in an extra
6.5mM and 9.75mM of calcium respectively; thus, increasing the remineralization potential
of the milk (Walker et al, 2009).
2.2.1 CPP-ACP: structure
Casein phosphopeptides (CPP) which contains a cluster of phophoseryl residues, has the
sequence -Ser(P)-Ser(P)-Ser(P)-Glu-Glu- , increases the solubility of calcium phosphate by
13
stabilizing amorphous calcium phosphate in solution (Reeves and Latour, 1958; Reynolds,
1995 and 1998). The ability of casein to release calcium and phosphate ions resides in these
sequences that can be released as small peptides, called casein phosphopeptides, when
they undergo enzymatic digestion. The multiple Ser(P) remain bound to form nanoclusters
of ACP in supersaturated solutions, thus preventing the growth of these clusters to the
critical size for phase transformation. The mineral deposited in the tooth structure after
exposure to CPP-ACP containing agents has been found to have a higher resistance to acid
attacks as compared to normal tooth structure (Iijima, 2004). This is due to the fact that
the CPP-ACP localizes at the tooth surface, where it buffers the free calcium and phosphate
ion activities, and maintains a state of supersaturation in the enamel. As a result, it prevents
demineralization and enhances remineralization (Reynolds, 1999).
This finding led to the development of remineralization technology based on caseinphosphopeptide stabilized amorphous calcium phosphate complexes (Reynolds et al, 1995;
Cross et al, 2005) and casein phosphopeptide amorphous calcium fluoride phosphate
complexes (Cross et al, 2004; Cochrane et al, 2008; Reynolds et al, 2008). This complex is a
nanocluster of ACP with four multi-phosphorylated peptides that prevent its growth for
further phase transformation (Reynolds, 1998; Huq et al, 2004).
2.2.2 Mechanism of action
The proposed anticariogenic mechanism of CPP-ACP is by the incorporation of
nanocomplexes into the plaque and onto the tooth surface. The CPP-ACP nanocomplexes
then buffer the free calcium and phosphate ion activities, maintaining a state of
supersaturation with respect to the tooth enamel; this results in the promotion of
remineralization whilst preventing demineralization .
14
The activity of the neutral ion species, CaHPO4 is significantly related to the degree of
remineralization (Reynolds, 1997). CPP-ACP has been found to be a reservoir of calcium
phosphate ions including the neutral ion species CaHPO4, formed in the presence of acid. On
the production of acid by the plaque bacteria, the CPP-ACP buffers the plaque pH and
dissociates to produce calcium phosphate ions. This increase in the level of calcium
phosphate ions counteracts any fall in the pH, thus preventing demineralization.
Their mechanism of remineralization is based on the localization and supply of
calcium, phosphate and fluoride ions in the correct ratio at the tooth surface by the CPP
(Reynolds et al, 2003; Cross et al, 2004) in order to drive the diffusion of the ions into the
subsurface enamel. Thus, CPP supplies the ions and also prevents their spontaneous
transformation at the enamel surface. This increases the concentration of the ions and
results in the formation of hydroxyapatite or fluorapatite at the crystal growth level
(Reynolds, 1997).
CPP-ACP nanocomplexes have the ability to bind to the tooth surface and the subgingival
plaque and significantly increase the levels of calcium and phosphate ions (Reynolds et al,
2003). An in vivo study was conducted to measure the incorporation of calcium and
phosphate into the plaque after 5 days of rinsing with either water, unstabilized calcium
and phosphate or solutions containing either 2% or 6% CPP-ACP. The results demonstrated
similar levels of calcium and phosphate ions after rinsing with water or the unstabilized
calcium and phosphate. However, both the CPP-ACP containing solutions caused a higher
uptake of calcium and phosphate ions in the plaque (Reynolds, 2003). These findings
confirmed the results presented by Rose (2000) who showed that the CPP-ACP
nanocomplexes attached to the streptococcus mutans and the plaque to produce a reservoir
of calcium ions. This had earlier been demonstrated by Schupbach and co-workers (1996)
15
using an in vitro model and in animal studies. He reached the conclusion that CPP-ACP
inhibited the binding of the streptococcus mutans to enamel, leading to its incorporation
into the pellicle and plaque. This causes an ecological transition of the bacterial population,
which along with the remineralizing action of CPP-ACP changes the cariogenic potential of
plaque.
The mechanism of the binding of CPP-ACP to plaque has been hypothesised to be either due
to calcium cross-linking (Rose, 2000; Reynolds, 2003) or hydrophobic and hydrogen bond
mediated interactions (Reynolds et al, 2003). Reynolds and co-workers (2008) in an in vivo
study comparing the use of a CPP-ACP and a fluoride mouthrinse versus a fluoride
mouthrinse alone showed a greater increase in the supragingival plaque fluoride ion content
by the use of the CPP-ACP and fluoride containing mouthrinse than with a mouthrinse
containing only fluoride. However, this process could have been driven by the low pH. With
the increase in bacterial acid production, the plaque pH decreases, facilitating the release of
calcium, phosphate and fluoride ions from the CPP-ACP nanocomplexes. Furthermore, CPP
incorporation into the salivary pellicle not only increases its remineralizing potential
but also inhibits the incorporation of the cariogenic streptococci by affecting their
adherence (Schupbach et al, 1996). This finding confirmed the results of earlier studies by
Guggenheim and co-workers (1994) and Reynolds and Wong (1983).
Several authors have reported that an inverse relationship may exist between the calcium
and phosphate levels in plaque and the caries experience of an individual, with higher
calcium and phosphate levels indicating a low caries experience and vice versa (Dawes and
Jenkins, 1962; Ashley, 1975; Schamschula et al, 1977; Shaw et al, 1983). Higher levels of
calcium and phosphate ions may result in a higher degree of saturation in the enamel
leading to an increase in the level of remineralization with a simultaneous decrease in the
16
level of demineralization (Moreno and Margolis, 1988).
A significant concern about solutions that affect the mineralization of plaque is the
formation of calculus; however, CPP-ACP solutions do not promote calculus formation
because they prevent the transformation of amorphous calcium phosphate into the
crystalline phase. Instead, they provide a reservoir of soluble calcium phosphate ions that
are capable of diffusing into the subsurface enamel and promoting remineralization (Holt
and van Kemenade, 1989).
2.2.3 Anticariogenic activity of CPP-ACP
The anticariogenicity of the CPP-ACP nanocomplexes was first demonstrated in a rat model
by Reynolds and co-workers (1995). Solutions containing different concentrations of CPPACP were applied to the animal’s molar teeth twice daily while the other animals received
the same amount of either a 500ppm fluoride containing solution or distilled water. All of
the animals were given a high cariogenic diet which did not contain any dairy products. The
results showed a significant reduction in the caries activity caused by the CPP-ACP in a dose
related fashion. The 0.1% CPP-ACP containing solution caused a 14% reduction in the caries
activity compared to a 55% reduction by the 1% CPP-ACP containing solution. The solution
containing both 0.5% CPP-ACP and 500ppm fluoride produced a significantly greater
reduction in caries activity than either of the solutions alone at the same
concentrations.
In a human in vivo demineralization study, a 1% w/v CPP-ACP solution used twice daily
after frequent sugar solution exposures, produced a 51 +19% reduction in the enamel
mineral loss (Reynolds, 1998). It also resulted in a 144% increase in the calcium level and a
17
160% increase in the level of inorganic phosphate. Therefore, these results indicated the
anticariogenic mechanism of the CPP-ACP, where the CPP stabilized and localized the ACP at
the tooth surface, buffered the plaque pH, thus slowing demineralization of the enamel
and increasing remineralization. In plaque, the CPP-ACP probably acts as a reservoir of the
calcium and phosphate ions, thus maintaining a state of supersaturation. The anti-cariogenic
activity of CPP-ACP is greatest when the peptides are delivered at the same time as the
cariogenic challenge. CPP, which is a natural derivative of milk, can be added to sugar
containing foods, a property that fluorides do not possess.
2.2.4 CPP-ACP as a remineralizing agent
The remineralizing effect of casein phosphopeptides has been illustrated in various in vitro
(Reynolds, 1997; Cochrane et al, 2008), human (Reynolds, 1987; Reynolds, 1991; Cochrane
et al, 1998; Shen et al, 2001; Cai et al, 2007; Pai et al, 2008; Reynolds, 2008; Wu et al, 2010)
and animal studies (Reynolds et al, 1995; Reynolds, 1997; Reynolds et al, 2003). Throughout
the published literature, CPP-ACP has been used in in vitro studies in varying forms and for
different time periods; as a solution for 10 days (Reynolds, 1997), as a topically applied
cream for time periods of 1.5min (Rehder-Neto, 2009), 2min (Pulido, 2008), 3min (Pai et al,
2008) and 5min (Rahiotis et al, 2007) and even as a 10 times diluted solution (Yamaguchi,
2006).
The in vitro pH cycling model (ten Cate and Duijusters, 1982) used in all the above
mentioned studies replicates the in vivo conditions and hence exposes the specimens to a
variety of remineralizing and demineralizing conditions. This system has been used in a
variety of studies in order to compare the mineral content and lesion depth of artificial
enamel lesions (ten Cate and Duijusters, 1982; Itthagarun et al, 2000; Kumar et al, 2008).
Cochrane and co-workers (2008) studied the remineralizing ability of CPP-ACP at different
18
pH values of 7.0, 6.5, 6.0, 5.5, 5.0 and 4.5. On testing tooth specimens, it was found that at
pH <5.5 greater levels of remineralization were seen. An in vitro study by Rehder-Nato and
co-workers showed that specimens treated with CPP-ACP containing pastes showed a
greater degree of remineralization than the controls tested over a 5 day period using a
topical application for 90 seconds (Rehder-Nato et al, 2009). A protective effect against
demineralization after a 10min application of a CPP-ACP containing paste has also been
shown (Yamaguchi et al, 2007). According to Wu and co-workers who treated specimens for
3, 6, 9 and 12 weeks with a 5min application of Tooth Mousse® and compared the results to
a fluoridated paste, a significant reduction in the grey demineralized areas was seen for
both the groups under polarizing light microscopy (Wu et al, 2010). However, this effect was
more significant with the CPP-ACP containing paste and when used in combination with a
fluoridated paste, the effect was strengthened. This remineralizing ability was further
illustrated in an in vitro study by Zhao and Cai (2001), who studied the degree of
remineralization and its increase over a period of 10 days. The CPP-ACP containing
remineralizing solution replaced 9.19%, 14.27%, 29.07%, 38.45% of the lost mineral after 1,
3, 5 and 10 days respectively. Therefore, an increase in the level of remineralization was
seen as the days progressed. This was again reaffirmed in an in vitro study by Pai and coworkers (2009) who evaluated the remineralizing ability of a CPP-ACP containing paste using
laser fluorescence and scanning electron microscopy over a period of 14 days. Their results
confirmed the higher levels of remineralization seen in samples treated with CPP-ACP. A
comprehensive synopsis of similar in vitro studies that illustrated the remineralizing
potential of CPP-ACP are shown in Appendix V (Table V.1).
This remineralizing ability of CPP-ACP has also demonstrated in various in vivo studies
(Shen et al, 2001; Cai et al, 2003; Reynolds et al, 2003; Iijima et al, 2004; Reynolds et al,
2007; Manton et al, 2008; Bailey et al, 2009). The CPP-ACP nanocomplexes are far superior
19
to other forms of calcium phosphate for remineralizing enamel subsurface lesions in situ
(Reynolds et al, 2003). This effect is attributed to the their ability to stabilize calcium
phosphate in solution, by binding the amorphous calcium phosphate (ACP) to their
phosphoserine residues and also by localizing the calcium and phosphate ions at the tooth
surface so producing an effective concentration gradient into the subsurface enamel. This
leads to the formation of small CPP-ACP clusters which suppress demineralization, enhance
remineralization or even exert both these effects (Reynolds, 1997). After a 24 month clinical
trial to assess the remineralizing ability of a CPP-ACP containing sugar-free chewing gum in
comparison to a control sugar-free gum, Morgan and co-workers (2008) concluded that the
CPP-ACP containing gum caused an 18% reduction in the caries progression with a 53%
greater remineralization of the baseline lesions as compared to the control gum.
As far as the use of CPP-ACP in children is concerned, it is apparent that the most commonly
used forms are the topically applied remineralizing pastes with the application time ranging
from the manufacturer’s recommended time of three minutes and even upto thirty minutes
in some studies. Caruana and co-workers (2008) showed that the addition of CPP-ACP in
Tooth Mousse® caused a greater reduction in the plaque pH following a carbohydrate
challenge. Furthermore, a clinical trial comparing the efficacy of three dentifrices; a placebo,
a 1190ppm fluoride preparation and a 2% CPP-ACP with calcium carbonate containing paste
showed that approximately 70% of the children using the CPP-ACP containing dentifrice
remained caries-free as compared to 53.2% of those who used the fluoride paste and 31.1%
of those who used the placebo (Rao et al, 2009). In another clinical trial, a dentifrice
containing 2% CPP-ACP was found to have a greater remineralizing potential than a
1100ppm fluoridated dentifrice and the remineralizing potential was similar to that of a
2800ppm fluoridated dentifrice. However, the combination of 2% CPP-ACP with 1100ppm
fluoride proved to have superior remineralizing potential (Reynolds, 2008). Similar in vivo
20
studies and clinical trials are mentioned in Appendix V (Tables V.2 and V.3).
2.2.5 Synergistic effect of fluoride and CPP-ACP
The synergistic remineralizing potential of CPP-ACP and fluoride has been proven in
several animal, in vitro and in vivo studies (Reynolds et al, 1995; Suge et al, 1995; Hodnett,
2007; Cochrane et al, 2008; Reynolds et al, 2008). In the rat model by Reynolds and his coworkers (1995) rats treated with the combination of CPP-ACP and fluoride exhibited a
greater reduction in caries activity than rats treated with either CPP-ACP or fluoride alone.
This effect maybe due to the formation of amorphous calcium fluoride phosphate at
the enamel surface, which results in an increased incorporation of fluoride ions into plaque
and an increased concentration of bioavailable calcium and phosphate ions. Therefore, the
presence of plaque is essential for the synergistic effect of CPP-ACP and fluoride (Reynolds
et al, 2008). Also, fluoride was found to enhance the conversion of dicalcium phosphate
dihydrate (DCPD), a normal component of enamel, dentine and bone to hydroxyapatite. Not
only did CPP increase fluoride incorporation into plaque, it also increased the incorporation
of fluoride into the subsurface enamel and enhanced the remineralization of subsurface
enamel lesions as compared to the use of fluoride alone. The remineralizing ability of these
CPP-ACP containing agents is also related to the concentration of the calcium ions even
though the remineralizing fluids have an identical calcium to phosphate ratio (1:1.67). The
addition of fluoride to the calcifying fluids markedly affects the degree and extent of
remineralization (Silverstone 1981, 1983 and 1984). On using 1mM and 3mM calcifying
solutions, the addition of a minimal quantity of 1ppm fluoride resulted in a three-fold
reduction in the lesion area. This reduction was more marked with the 1mM solution
(3.3 fold) as compared to the 3mM solution (3 fold). Without fluoride, the lesion area
reduction was 22%, but after the addition of 1ppm fluoride to the 1mM calcium solution,
the reduction increased to almost 72%. A similar effect was also seen with the addition of
21
10ppm fluoride.
Studies have also shown that the concentration of fluoride in the remineralizing and
demineralizing solutions is of more importance than its concentration within enamel (Wefel,
1990). Several studies have shown that not only did CPP increase fluoride incorporation into
plaque but also increased the incorporation of fluoride into subsurface enamel and as a
result increased the remineralization significantly as compared to the use of fluoride alone
(Reynolds et al, 2008). This belief was further strengthened by the work of Schemehorn and
co-workers who tested the bio-availability of fluoride from Enamelon toothpaste
(Schemehorn et al, 1999). The results indicated that the calcium and phosphate salts
delivered by the remineralizing Enamelon dentifrice increased the bioavailability of fluoride
to a level that substantially exceeded that of the clinically proven standard dentifrice.
Therefore, he stated that the calcium and phosphate ions present in the dentifrice increased
the availability of the fluoride ions on the lesion surface; thus, effectively increasing
remineralization. The addition of 2% CPP-ACP to a fluoride dentifrice increased the
remineralizing potential by 156% when compared to the same fluoride dentifrice alone. This
may be due to the fact that the remineralization caused by the fluoride dentifrice may be
calcium and phosphate limited and for this reason, not all of the fluoride ions that diffuse
into the subsurface lesion get incorporated into the mineral phase and the excess fluoride
stays as fluoride ions that eventually got adsorbed onto crystallites (Arends and
Christoffersen, 1990). These findings were further confirmed in a study by Lennon and coworkers (2006) who demonstrated a decrease in the level of enamel loss after treatment
with a paste containing casein calcium phosphate followed by the use of a 250ppm
fluoridated toothpaste as compared to treatment with either of them alone.
This may be attributed to the formation of fluorapatite and a subsequent increase in the
22
degree of remineralization.
Therefore, the ability to deliver calcium, phosphate and fluoride ions in the correct
ratio to a subsurface enamel lesion maybe due to the ability of CPP-ACP to stabilize the ions
on the tooth surface in the correct ratio of Ca:PO4:F of 5:3:1 (Cross et al, 2004). The
increased remineralization exhibited by dentifrices containing higher levels of fluoride was
further illustrated by Biesbrock and co-workers (2001) who showed that a 2800ppm F
dentifrice reduced the caries experience by 20.4% which was a 85% greater reduction than
that obtained by a 1700ppm F dentifrice. It has also been reported that the CPP-ACP
nanocomplexes interact with fluoride ions to produce a novel ACFP phase (Cross et al, 2004;
Reynolds et al, 2008). The anticariogenic effect of CPP-ACP nanocomplexes and F may be
due to the localization of ACFP at the tooth surface by CPP which also localizes calcium,
phosphate and fluoride ions in the correct molar ratio to form fluorapatite. Kumar and his
co-workers (2008) in their in vitro study demonstrated a 13% reduction in lesion depth in
lesions that received both CPP-ACP and fluoride while the group treated with CPP-ACP
showed only a 10% reduction and a 7% reduction was seen in the fluoride group.
2.2.6 Other uses of CPP-ACP
Apart from the remineralizing potential, several other applications of CPP-ACP have
been studied. These include reduction in erosion, hardening of enamel that has been
softened by acidic drinks, reduction in white spot lesions and increase in surface
microhardness after micro-abrasion.
The demineralization of enamel adjacent to orthodontic brackets is a significant clinical
problem with a prevalence between 2% and 96% (Mizrahi, 1982; Mitchell, 1992). Even in a
23
population with a low prevalence of dental caries and with a preventive program in place,
almost 61% may develop white spot lesions during orthodontic treatment (Ogaard et al,
2001). This may be due to the irregular surface of the brackets, bands or wires and other
such attachments that create stagnation areas for plaque and consequently make tooth
cleaning more difficult. They also limit the naturally occurring self-cleansing mechanism
such as the movement of the oral musculature and saliva (Rosenbloom and Tinanoff, 1991).
This leads to a lower plaque pH, accelerates the rate of plaque accumulation and maturation
while also favouring colonization of bacteria such as streptococcus mutans and lactobacilli.
The demineralization of white spot lesions may accelerate after the initiation of orthodontic
treatment due to alterations in the environment. Some of these lesions may remineralize
and return to either normal or, at least to a visually acceptable appearance. Some lesions
may persist and even require restorative treatment. Therefore, the optimum management
of these lesions involves the prevention of demineralization and/or increasing the rate of
remineralization. Topical fluorides are a valuable aid for reducing enamel demineralization
around orthodontic brackets (Benson et al, 2005). In this regard, it has been proven that
fluorapatite formation from either a monthly high dose of fluoride (use of orthodontic
elastomeric ligature ties which contain fluoride) or a continuous low dose of fluorides (from
fluoride containing adhesive cements) can be advantageous for reducing enamel
decalcification during fixed orthodontic appliance therapy (Wiltshire, 1999). Casein
phosphopeptide amorphous calcium phosphate is ideal for the prevention of enamel
demineralization as there appears to be an inverse relationship between plaque calcium and
phosphate and measured caries experience (Reynolds et al, 2003). These nanocomplexes
buffer free calcium and phosphate ions in the plaque, so as to maintain a state of
supersaturation of ACP (Reynolds, 1997).
24
Remineralization of these white spot lesions with fluorides, particularly on the labial aspect
of maxillary anterior teeth is often calcium limited and ineffective. It has been shown, in
numerous studies that the use of products such as Tooth Mousse® (TM) can be beneficial to
patients with enamel demineralization because not only does it remineralize existing
enamel lesions, but it also prevents the development of further white spot lesions. The
combination of topical fluoride gel with TM has provided the greatest preventive effect
against white spot lesion development due to the synergistic action of both the fluoride and
the TM (Sudjalim et al, 2007). This may minimize aesthetic damage and prevent the need for
further restorative treatment. Studies have showed that the fluoride content of the intact
surface layer over a white spot lesion in enamel is greater than the adjacent sound enamel,
which indicates that the presence of fluoride in solution during demineralization is a
significant factor (Dawes and Jenkins, 1957; Little and Steadman, 1966). A clinical trial
carried out by Bailey and co-workers (2009) studied the effect of a CPP-ACP containing paste
on the regression of white spot lesions in orthodontic patients when compared to a
fluoridated dentifrice over a 12-week period. The results showed that the combination of a
fluoridated dentifrice and a paste containing CPP-ACP produced a beneficial effect in the
reduction of white spot lesions (WSL). A similar effect was reported by Zhou and co-workers
(2009) who studied the reduction of WSL’s in the post-orthodontic phase after a twice daily
application of a CPP-ACP containing paste after 1 and 2 months. An in vitro study by
Andersson and co-workers (2007) compared the remineralizing effect produced by the use
of a CPP-ACP dentifrice for three months as compared to a 0.05% sodium fluoride
mouthwash followed by a three month period of daily tooth brushing with a fluoridated
dentifrice. Clinical scoring and laser fluorescence assessment after 1, 3, 6 and 12 months
suggested, that both regimens could promote the regression of WSL after debonding of
fixed orthodontic appliances. Therefore, after debonding, saliva-mediated remineralization
25
takes place and this can be further accentuated by periodic topical fluoride applications
(Kleber et al, 1999; Alexander and Ripa, 2000; Bergstrand and Twetman, 2003; Bensen et al,
2005). Regular exposure to various forms of fluoride has a more profound effect on the
prevention of demineralization than it does on remineralization. This is due to the ability of
the fluoride to decrease the solubility of minerals in the enamel crystal lattice with it helping
in mineral formation (Jeansonne et al, 1979). This process substitutes hydroxide ions with
fluoride ions in the hydroxyapatite and thus decreases the critical pH for the dissolution of
enamel containing fluorapatite (Hicks et al, 2004).
The visual evaluation of white spot lesions suggests an aesthetically more favourable
outcome with the amorphous calcium phosphate treatment. However, some investigators
have proved the opposite. Brochner and co-workers (2010) studied the effect of a local
application of a CPP-ACP containing paste on WSL’s created during orthodontic treatment;
with the results being assessed using laser fluorescence. The mean area of the lesions
decreased by 58% in the CPP-ACP group, compared to only 26% in the fluoridated dentifrice
group after a four week treatment period. However, the improvement was inferior to the
regression noted after the daily use of a fluoridated dentifrice.
Another extremely effective method for preventing white spot lesion formation is the use
of glass ionomer cements (GIC) in place of composites for bracket bonding (Norevall et al,
1996). However, there are certain drawbacks such as the lower shear and tensile bond
strengths of GIC. Studies have shown that the incorporation of 1.56% w/w CPP-ACP into GIC
leads to an increase in the compressive and microtensile bond strengths and also enhances
the release of calcium, phosphate and fluoride ions thus, leading to an increase in the level
of remineralization (Mazzaoui et al, 2003). Therefore, the clinician should take into
26
consideration the benefits of fluoride release from the cement whilst keep in the mind the
possibility of increased bond failure of the brackets.
In addition to the remineralizing effect, recent literature has shown that casein
phosphopeptide amorphous calcium phosphate (CPP-ACP) containing pastes have a
protective effect against erosion. The in vitro study by Ranjitkar and co-workers (2009)
tested enamel specimens using a tooth wear machine where the machine was stopped
every 120s and a CPP-ACP containing paste was applied for 5min in one group, and was
compared to a non CPP-ACP paste of the same formulation in the second group. The results
showed that the mean wear rate in the CPP-ACP treated specimens was lower, and the
wear facets were smoother and more highly polished. Therefore, they concluded that the
remineralizing and lubricating properties of the CPP-ACP paste contribute to the wear
reduction in enamel. Similar results were reported by Tantbirojn and co-workers (2008), and
Piekarz and co-workers (2008) who showed an increase in the hardness of enamel exhibiting
erosion after treatment with CPP-ACP for 48 hours. They concluded that products such as
Tooth Mousse®, in addition to preventing erosive demineralization also remineralize
eroded enamel and dentine crystals.
The ability of CPP-ACP containing dental creams to reduce surface roughness of enamel
has been stated in various studies. Mathias and co-workers (2009) compared the surface
roughness of enamel after micro-abrasion with and without the application of CPP-ACP
containing pastes. They found that a combination of the micro-abrasion procedure and
CPP-ACP application reduced the enamel surface roughness to a greater extent than microabrasion alone. In addition, an in vitro study conducted by Kowalczyk and co-workers (2006)
showed that Tooth Mousse® had sufficient effectiveness and short-term therapeutic benefit
27
for treating hypersensitivity of dentine. This reduction in pain is probably an additional
remineralizing effect of CPP-ACP. This property was utilized by Ng and Manton (2007) who
showed that treatment with CPP-ACP decreased tooth sensitivity after bleaching and
whitening procedures.
The increase in the hardness of enamel after CPP-ACP application was considered to be
a benefit after bleaching. This benefit was illustrated in an in vitro study by Bayrak and his
co-workers (2009) who found an increase in the microhardness of bleached enamel
specimens after local application of CPP-ACP and CPP-ACFP containing pastes.
In order to overcome the problems associated with xerostomia, affected patients have
adopted different strategies which include sipping water frequently, chewing gum,
sucking sweets and using artificial saliva. These therapies may be used either in isolation or
in combination with each other (Morton et al, 1997). Whilst, water keeps the oral cavity
wet, most patients claim that it is a poor lubricant and has a drying sensation when used as
a moistener. It has been reported that casein derivatives with calcium phosphate are
equivalent to sodium fluoride in their caries preventive efficacy when used in patients with
xerostomia. Since CPP-ACP preparations are non-toxic, they have a clear advantage over
fluoride based preparations as they can be swallowed and there are no contra-indications
to their use more than three times per day (Hay and Thomson, 2002). According to the
study performed by Hay and Morton in 2003, Dentacal (3.6% w/w CPP-ACP complex) when
diluted, and used as an atomised spray for mouth moistening provided good lubrication and
lasted longer than conventional moisteners. In comparison to sugar free chewing gums and
the parasympathetic sialogogue pilocarpine, products containing CPP-ACP offered greater
benefits because of their significant anticariogenic effects and mouth moistening properties
when used regularly in dentate patients with xerostomia (Hay and Thomson, 2002; Hay and
28
Morton, 2003).
2.2.7 Toxicity of fluoride and CPP-ACP
It is well documented that prolonged use of fluoride at recommended levels does not
produce any harmful physiological effects. However, there are safe limits for fluoride
beyond which harmful effects can occur. The permissible daily dose of fluoride has been
suggested to be between 0.05-0.07 mg F/kg body weight (Villa et al, 1999). If the fluoride
level in the body reaches 5mg/kg body weight or more, immediate intervention is needed.
This has been determined as the toxic dose of fluoride (Whitford, 1996). These harmful
effects can be classified into acute and chronic toxicity. The management of an overdose
differs on the basis of the level of fluoride and the systemic symptoms.
In cases of acute toxicity, symptoms occur rapidly. There is diffuse abdominal pain, diarrhea,
vomiting, excess salivation, and thirst. If the level of fluoride is around 5mg/kg body weight,
administration of milk and 5% calcium lactate or gluconate to the child to induce vomiting is
the treatment of choice. In cases with fluoride levels between 5-15mg/kg body weight,
intravenous administration of calcium gluconate and lactate along with milk is suggested
and in cases with levels exceeding this range, immediate hospital admission and constant
monitoring in addition to the above mentioned measures is required.
When ingested in elevated doses over a period of time, fluoride gets deposited in the bones
and teeth, causing changes known as fluorosis. It can be described as a diffuse symmetric
hypomineralization disorder of the ameloblasts. Fluorosis is irreversible and only occurs
with exposure to fluoride when enamel is developing. It is thus, a toxic manifestation of
chronic (low-dose, long-term) fluoride intake. Therefore, to prevent fluorosis from occurring
in the most prominent and/or most susceptible teeth, the most critical time to avoid
29
fluoride exposure is the first three to six years of a child's life. Hence, it has been
recommended that only a pea-sized amount of fluoridated dentifrice should be used by
children when brushing; also that they should be under the supervision of a mature adult
(Walsh et al, 2010) so as to prevent ingestion of the dentifrice (Marinho et al, 2003).
In this regard, one major advantage of CPP-ACP containing products over fluoride containing
products, which pose a risk if ingested in a significant quantity (Hawkins et al, 2003) is that
they are ingestible. However, the potential side effects from the consumption of casein
derived proteins in people with immunoglobulin E allergies to milk proteins should be taken
into consideration. However, CPP-ACP is digestible even by individuals with lactose
intolerance.
In summary, the anticariogenic potential of CPP-ACP has been demonstrated in animal
models, in various in vivo and in vitro studies and in human trials. CPP-ACP stabilizes and
localizes ACP and ACFP at the tooth surface, buffers the plaque pH and as a result decreases
the rate of enamel demineralization and enhances remineralization. When used in
combination with fluoride, a synergistic anticariogenic effect is evident as the CPP localizes
the calcium, phosphate and fluoride ions on the tooth surface, thus producing a greater
anticariogenic effect.
30
Chapter 3
Materials & methods
31
3.1 Formation of demineralizing / remineralizing solutions
The demineralizing solution was prepared from 2.2mM calcium chloride (CaCl2), 2.2mM
monopotassium phosphate (KH2PO4), 0.05 acetic acid and deionized water (Figure 3.1). The
pH of this solution was further adjusted using 1M potassium hydroxide (KOH) to 4.4.
The remineralizing solution contained 1.5mM calcium chloride (CaCl2), 0.9mM monosodium
phosphate (NaH2PO4), 0.15M potassium chloride (KCl) and the pH was adjusted using 5M
potassium hydroxide (KOH) to 7 (Figure 3.11). These solutions were similar to the one used
by ten Cate and Duijsters (1982).
3.2 Artificial carious lesion formation
Fifty extracted human third molars with intact buccal and lingual surfaces were cleansed of
any soft tissue and debris and further inspected under a stereomicroscope for any evidence
of hypoplasia, cracks or caries. Sticky wax (Model Cement®, Dentsply) was then used to
cover the roots of the teeth upto the amelo-dentinal junction, in order to seal the apical
foramen and the furcation area. After this, the teeth were painted with an acid resistant nail
varnish (Revlon®, USA) leaving a 1mm window on both the buccal and lingual surfaces and
left to dry overnight . On the following day, a second coat of varnish was applied, so as
to ensure complete coverage. The teeth were then immersed in the demineralizing solution
(10ml/tooth) for 96 hours to produce artificial carious lesions varying from 90-180 µm in
depth (Figure 3.4). The teeth were then sectioned longitudinally through the carious lesions,
using a hard tissue saw microtome (Leica®, 1600 saw microtome, Germany, see Figure 3.5),
to produce sections that were approximately 100-150 µm thick. The thickness of each
section was then confirmed using a micrometer (Figure 3.5). A piece of dental floss was
attached to each section using nail varnish (Revlon®, USA) in such a way that it was placed
away from the lesion in order to make manipulation easier. The specimens were then
32
painted under the stereomicroscope using an acid resistant nail varnish (Revlon®, USA). It
was essential to coat all the surfaces of the lesion with the varnish, leaving only the superior
margin exposed. Following this, the sections were stored in 100% humidity until the start of
the pH cycle.
3.3 Grouping
One hundred and fifty specimens were randomly assigned to six treatment groups and
subjected to the treatment protocols shown in Table 3.1.
Table 3.1: The treatment protocols for the six experimental groups.
Test groups
Group A
(negative
control)
Agent tested
Non-fluoridated
dentifrice*
Group B
(positive
control)
Fluoridated
dentifrice
(1000ppm F) **
Group C
Mode of application
Supernatant
Time of application
60s
Supernatant
60s
Clinpro®***
(CPP-ACP with
950ppm fluoride)
Topical coating
180s
Group D
Tooth Mousse
Plus®****
(CPP-ACP with
900ppm fluoride)
Topical coating
180s
Group E
Fluoridated
dentifrice
+
Clinpro®
Fluoridated
dentifrice
+
Tooth Mousse
Plus®
Supernatant
60s
+
Topical coating
Supernatant
+
180s
60s
+
Topical coating
+
180s
Group F
33
* Vicco® Laboratories, Goa, India.
** Colgate Total®, Bangkok, Thailand.
*** Clinpro®, 3M ESPE, USA.
**** Tooth Mousse Plus® GC Corp, Tokyo, Japan.
3.4 Agent preparation
Dentifrice supernatants were prepared by adding 15g of each of the pastes to 45ml of
deionized water to achieve a 1:3 ratio (dentifrice: deionized water). These suspensions were
then thoroughly stirred for one minute by mechanical agitation using a vortex mixer (Super
Mixer®, Lab Line Instruments Inc, Illinois, USA) and centrifuged at room temperature for 20
minutes at 4000rpm (Beckman®, Avanti J-251, California, USA), see figure 3.9. Following
this, the higher sediment layer was discarded and only the supernatant solutions were used
for treating the specimens. Clinpro® and Tooth Mousse Plus® were directly dispensed (as a
paste) onto the surface of the lesions in Groups C, D, E and F respectively.
3.5 The pH cycling model
All tooth specimens were subjected to a 10-day pH cycling model. All solutions were
freshly prepared before each phase of the pH cycle and the pH values for the demineralizing
and remineralizing solutions were checked prior to inserting the specimens during each
phase. During pH cycling, all specimens were placed on an orbital shaker (Labnet®,
Woodbridge, USA) to ensure that they were fully immersed in the treatment solutions, so as
to simulate the oral environment. The daily cycle involved three hours of demineralization
twice a day, with two hours of remineralization in between. After the daily cycle, the tooth
specimens were placed in a remineralizing solution overnight. The specimens received
treatment thrice a day; before the first demineralization and before and after the second
demineralization, respectively. The treatment protocol for Groups A and B involved
34
immersion of the specimens in the respective dentifrice supernatant solutions for one
minute with an average of 5ml of solution being available per specimen. The sections in
Groups C and D were placed directly in contact with the Clinpro® and Tooth Mousse Plus®
for three minutes. Whereas, the specimens in Groups E and F were treated with the
dentifrice supernatant solution for one minute followed by a topical application of the
respective CPP-ACP pastes for three minutes again. After the 10-day pH cycle, the nail
varnish was removed carefully from all the specimens using Acetone (Advanced Technology
& Instruments Co. Ltd, Hong Kong) so that all of the lesions could be evaluated.
3.6 Evaluation techniques
The specimens were subjected to evaluation both before and after the 10-day pH cycling
model. Digital polarizing light microscopy (PLM) was used to perform qualitative evaluation
while microradiography (MRG) was used to perform quantitative evaluation of the
specimens.
3.6.1 Qualitative evaluation: polarizing light microscopy (PLM)
A Nikon Eclipse LV100POL microscope (Nikon®, Japan, LV-UEPI) with a rotating stage,
polarizer and analyzer was used to qualitatively evaluate the body of the lesion in each of
the specimens (Figure 3.7). They were first imbibed in deionized water and then subjected
to digital PLM by using this microscope at a magnification of 5X. All specimens were
expected to show a clear demarcation between sound enamel and the initial carious lesion
(Wefel and Jensen, 1992). The PLM images were taken at a fixed standard magnification for
all of the specimens and captured in the computer (software NIS-Elements AR 3.0) before
and after the pH cycle to allow visual comparisons between the pre-treatment and posttreatment effects and then analysed for any changes in lesion depth (Figure 3.7).
35
3.6.2 Quantitative evaluation: microradiography (MRG)
This MRG technique was used to quantitatively evaluate all of the tooth specimens.
Specimens were mounted on high resolution electron microscopy film (Kodak electron
microscopy film 4489, Kodak®, USA) and exposed to Cu (Kα) X-rays (Softex® IRS-20, JIRA,
Japan) at 10kV voltage and a current of 3mA for 15 seconds (figure 3.6). Subsequently, each
film was immersed in the developing solution for 60 seconds, rinsed for 60 seconds in
running water, after which they were placed in the fixing solution for 60 seconds. Each
microradiograph was washed, air dried and then mounted onto a glass slide. The
specimens were subjected to the same procedure both before and after the 10-day pH
cycle. The images of the microradiographs were scanned at 12800 dpi resolution using a
flatbed colour image scanner (Epson® Perfection 3200 Photo, Japan). Subsequently, the
scanned images were used with an image analysis system, Image J software, version 1.37
(Bethesda, Maryland, USA) to plot the mineralization profile and quantify the changes in the
LD (lesion depth) and Vmax (mineral content) and mineral distribution of all the specimens
before and after the 10-day pH cycling procedure. Changes in both these parameters were
expressed as a percentage change in mineral content (Vmax) or lesion depth (LD). The
software calculated the relative mineral density on the basis of data from sound enamel
(Chow et al, 1991; Jordan et al, 1991). This analysis was carried out for all the specimens
within the same group and between different groups.
3.7 Statistical analysis
In order to calculate the before and after changes in the LD (lesion depth) and Vmax
(mineral content) within the different groups, the paired t-test was used. For between
group changes in the above mentioned parameters, One-way ANOVA was employed. All of
the statistical analyses were performed using the Graph Pad InstatR software (Version 3.00,
Windows 95, Graph Pad Software, San Diego, California, USA).
36
Figure 3.1: The reagents used to prepare the demineralizing solution, calcium chloride
(CaCl2), monopotassium phosphate (KH2PO4), acetic acid, potassium hydroxide (KOH), and
deionized water.
37
(a)
(b)
Figure 3.2: Dentrifices used in the study: (a) negative control, non-fluoridated dentifrice
(Vicco®) and (b) positive control, fluoridated dentifrice (Colgate Total®).
(a)
(b)
Figure 3.3: Treatment agents, (a) Clinpro® (950ppm sodium fluoride with tri-calcium
phosphate) and (b)Tooth Mousse Plus® (900ppm fluoride with 10% CPP-ACP).
38
(a)
(b)
(c)
Figure 3.4: The sequence of steps involved in the formation of artificial carious lesions, (a)
painted specimen (b) teeth immersed in the demineralizing solution for 96 hours, and (c)
1mm wide window left on the buccal and lingual surfaces of the tooth.
39
(a)
(b)
(c)
Figure 3.5: The various steps involved in preparing the tooth sections for the pH cycle, (a)
the microtome (Leica® 1600 saw microtome, Germany), (b) the sectioned tooth specimen
and (c) the micrometer.
40
(a)
(b)
(c)
Figure 3.6: Quantitative evaluation of the artificial carious lesion using microradiography,
(a) the sectioned tooth specimen, (b) X-ray machine (Softex® ISR-20, JIRA, Japan) and (c)
microradiograph of a tooth specimen.
41
(a)
(b)
(c)
Figure 3.7: Qualitative evaluation using polarized light microscopy technique showing (a)
the sectioned tooth specimen, (b) polarized light microscope (Nikon Eclipse LV100POL,
Nikon®, Japan) and (c) a polarized light photomicrograph of a specimen.
42
Figure 3.8: The pH calibration system.
43
Figure 3.9: The centrifuge machine (Beckman®, Avanti J-251, USA).
44
60s supernatant
60s supernatant
180s topical application
180s topical application
Specimen
n=150
60s supernatant and 180s topical application
60s supernatant and 180s topical application
Figure 3.10: Schematic representation of the six different groups and their respective
treatment protocols.
45
Figure 3.11: The reagents used to prepare the remineralizing solution, calcium chloride
(CaCl2), monosodium phosphate (NaH2PO4), potassium chloride (KCl), potassium hydroxide
(KOH) and deionized water.
46
Treatment I
Demineralization: 3 hours
Remineralization: 2 hours
Treatment II
Demineralization: 3 hours
Treatment III
Remineralization: overnight
Back to treatment I
Figure 3.12: Diagram illustrating the sequence of steps in the pH cycling model.
47
Chapter 4
Results
48
4.1 Quantitative evaluations
4.1.1 Lesion depth (LD) changes in the artificial enamel carious lesions after pH cycling
No statistically significant difference was detected between the lesion depths (LD) within
and between each of the six treatment groups before the 10-day pH cycle (Group A,
p= 0.78; Group B, p= 0.36; Group C, p= 0.43; Group D, p= 0.24; Group E, p= 0.12; Group F,
p= 0.24; ANOVA).
On comparing the pre-treatment and post-treatment lesion depths between the different
test groups, specimens treated with Clinpro® (Group C), with Tooth Mousse Plus® plus
Colgate Total® (Group F) and with Clinpro® plus Colgate Total® (Group E) exhibited
statistically significant differences (p<0.01, paired t-test). Conversely, specimens treated
with Vicco® (Group A), Colgate Total® (Group B) and Tooth Mousse Plus® (Group D) showed
no statistically significant difference between the pre-treatment and post-treatment lesion
depths. Though statistically insignificant, these three agents caused an increase in lesion
depth, which was greatest for Vicco® followed by Colgate Total® and Tooth Mousse Plus®.
On comparing the changes in the lesion depth between the six treatment groups, no
statistically significant differences were evident. However, it was seen that three of the
groups showed a decrease in the lesion depth while the remaining three showed an
increase. The maximum decrease in lesion depth was seen in the specimens treated with
Clinpro® plus Colgate Total®, followed by Clinpro® which was followed by Tooth Mousse
Plus® plus Colgate Total® group. The remaining three treatment groups, which were Vicco®,
Colgate Total® and Tooth Mousse Plus® caused an increase in the lesion depth with Vicco®
causing the greatest followed by the other two test agents in decreasing order.
49
4.1.2 Mineral content changes in the surface zone (Vmax) of the artificial enamel carious
lesions after pH cycling.
There was no statistically significant difference between the pre-treatment mineral content
values within each treatment group and between the six different treatments prior to the
10-day pH cycle (Group A, p= 0.56; Group B, p= 0.21; Group C, p= 0.10, Group D, p= 0.45,
Group E, p= 0.58, Group F, p= 0.67; ANOVA).
On comparing the changes in the Vmax before and after the 10 day pH cycle, the specimens
treated with Colgate Total® showed the greatest increase in Vmax followed by the
combination of Clinpro® plus Colgate Total®, Clinpro®, Tooth Mousse Plus®, Tooth Mousse
Plus® plus Colgate Total® and then Vicco®. However, none of the changes in the Vmax were
statistically significant.
Polarized light photomicrographs, illustrating the changes in Vmax and LD, of the specimens
in the different groups before and after pH cycling are depicted in Figures 4.1 to 4.6.
Graphical representation of specimens representing the different treatment groups before
and after the 10 day pH cycling period are illustrated in Figures 4.7 to 4.12.
50
(a) pre-treatment
(b) post-treatment
Figure 4.1: Polarized light photomicrographs of a specimen in Group A treated with the nonfluoridated dentifrice Vicco®, (a) before and (b) after the 10-day pH cycling period. An
increase in the lesion depth is evident.
(a) pre-treatment
(b) post-treatment
Figure 4.2: Polarized light photomicrographs of a specimen in Group B treated with the
fluoridated dentifrice Colgate Total®, (a) before and (b) after the 10-day pH cycling period.
An increase in lesion depth is evident.
(a) pre-treatment
(b) post-treatment
Figure 4.3: Polarized light photomicrographs of a specimen in Group C treated with Clinpro®,
(a) before and (b) after the 10-day pH cycling period. A decrease in lesion depth is evident.
51
(a) pre-treatment
(b) post-treatment
Figure 4.4: Polarized light photomicrographs of a specimen in Group D treated with Tooth
Mousse Plus®, (a) before and (b) after the 10-day pH cycling period. A slight increase in
lesion depth is evident.
(a) pre-treatment
(b) post-treatment
Figure 4.5: Polarized light photomicrographs of a specimen in Group E treated with Clinpro ®
plus Colgate Total®, (a) before and (b) after the 10-day pH cycling period. A decrease in
lesion depth is evident.
(a) pre-treatment
(b) post-treatment
Figure 4.6: Polarized light photomicrographs of a specimen in Group F treated with Tooth
Mousse Plus® plus Colgate Total®, (a) before and (b) after the 10-day pH cycling period. A
slight decrease in lesion depth is evident.
52
100
90
80
% mineral 70
content
60
50
40
30
20
10
0
0
0.1
0.2
0.3
Lesion depth
0.4
0.5
Pre-treatment
Post-treatment
Figure 4.7: Graph showing the relationship between the lesion depth (LD) onPost
x-axis in µm
and % maximum mineral content (Vmax) on the y-axis, both before and afterpo
the 10-day pH
cycle, for a specimen in Group A (non-fluoridated dentifrice; Vicco®).
53
100
90
80
% mineral
content
70
60
50
40
30
20
10
0
0
0.1
0.2
0.3
0.4
0.5
Lesion depth
Figure 4.8: Graph showing the relationship between the lesion depth (LD) on x-axis in µm and
% maximum mineral content (Vmax) on the y-axis, both before and after the 10-day pH cycle, for
a specimen in Group B (fluoridated dentifrice; Colgate Total®).
54
100
90
80
%
mineral
content
70
60
50
40
30
20
10
0
0
0.1
0.2
0.3
0.4
0.5
Lesion depth
Figure 4.9: Graph showing the relationship between the lesion depth (LD) on x-axis in µm
and % maximum mineral content (Vmax) on the y-axis, both before and after the 10-day pH
cycle, for a specimen in Group C (Clinpro®).
55
100
90
80
% mineral
content
70
60
50
40
30
20
10
0
0
0.1
0.2
0.3
0.4
0.5
Lesion depth
Figure 4.10: Graph showing the relationship between the lesion depth (LD) on x-axis in µm
and % maximum mineral content (Vmax) on the y-axis, both before and after the 10-day pH
cycle, for a specimen in Group D (Tooth Mousse Plus®).
56
100
90
80
% mineral
content
70
60
50
40
30
20
10
0
0
0.1
0.2
0.3
0.4
0.5
Lesion depth
Figure 4.11: Graph showing the relationship between the lesion depth (LD) on x-axis in µm
and % maximum mineral content (Vmax) on the y-axis, both before and after the 10-day pH
cycle, for a specimen in Group E (Clinpro® + Colgate Total®).
57
100
90
80
% mineral
content
70
60
50
40
30
20
10
0
0.0
0.1
0.2
0.3
0.4
0.5
Lesion depth
Figure 4.12 : Graph showing the relationship between the lesion depth (LD) on x-axis in µm
and % maximum mineral content (Vmax) on the y-axis, both before and after the 10-day pH
cycle, for a specimen in Group F (Tooth Mousse Plus® + Colgate Total®).
58
Table 4.1: Mean values (+SD) of the lesion depth (LD) and the maximum mineral content
(Vmax) of the samples in the six treatment groups both before and after the 10-day pH
cycle.
Groups
n
Lesion depth (LD)
Pre-op
Group 1:
25
Non-fluoridated
dentifrice (Vicco® )
Group 2:
25
25
25
Tooth Mousse Plus®
+ fluoridated
dentifrice
35.37+12.14
209.68 +49.95
248.44 +48.21
268.56 +45.80
227.60 +45.47
236.24 +43.19
238.68 +41.70
% change: 1.81 +13.10
25
Clinpro® + fluoridated
dentifrice
Group 6:
288.84 +41.98
% change: -13.62 +19.38
Tooth Mousse Plus®
Group 5:
Pre-op
% change: 21.52 +19.89
Clinpro®
Group 4:
Post-op
% change: 38.80 +37.18
Fluoridated dentifrice
(Colgate Total® )
Group 3:
221.64 +60.18
273.92 +59.03
230.36 +48.14
% change: -13.84 +17.16
25
258.56 +49.01
Maximum mineral content
(Vmax)
240.08 +45.64
% change: - 6.63 +9.62
Post-op
33.54 +12.80
% change: -6.19 +11.61
17.89 +10.29
30.23 +9.75
% change: 99.62 +72.14
16.54 +6.78
26.50 +8.11
% change: 74.84 + 62.47
29.39 +11.71
30.39 +9.12
% change: 10.74 +28.18
17.77 +9.88
29.35 +10.82
% change: 95.58 +90.41
28.85 +9.76
30.45 +10.35
% change: 7.08 +21.01
59
Chapter 5
Discussion
60
Several studies have demonstrated the reliability of the pH cycling model for the evaluation
of lesion progression and mineral changes of artificial enamel carious lesions (ten Cate and
Duijsters, 1982; Damato et al, 1990; Itthagarun et al, 2000). This model is considered to be
appropriate for evaluating the extent of remineralization, because it simulates to a major
extent the in vivo conditions leading to the process of carious lesion development
(Featherstone et al, 1986; ten Cate, 1990; Argenta et al, 2003). Nevertheless, it does not
completely simulate the in vivo processes of demineralization and remineralization that
occurs in separate phases. Hence, the results from this in vitro study cannot be directly
compared to the clinical scenarios (Wefel and Harless, 1984). This maybe due to the fact
that under in vivo conditions changes in the pH occur throughout the course of 24 hours,
which in turn determines the caries experience of an individual. However, in a pH cycling
model, the periodic changes from demineralization to remineralization significantly
influence the end product and are therefore likely to exaggerate the final results (Duff,
1976). Furthermore, the deposition of mineral during a shorter period of remineralization
would be significantly different from remineralization that occurs for a longer duration.
Conversely, in vivo studies have the advantage of an oral environment with the
presence of saliva and plaque. However, a potential drawback is the lack of exposure to
acid and other demineralizing environments because the participants were instructed
to remove the appliances while eating and drinking (Shen et al, 2001; Reynolds et al,
2003; Manton et al, 2008).
The pH cycling model which was utilized in this study has been used in several in vitro
studies is considered by many authorities to be appropriate for evaluating the
remineralizing ability of various caries preventive agents, it also offers a degree of
standardization that in vivo studies lack. The process of enamel demineralization is directly
61
related to the pH and the ionic content of calcium, phosphate and fluoride in the oral cavity
which in turn determines the degree of tooth mineral saturation (Fejerskov and Clarkson,
1996). Therefore, a subsaturated environment would lead to the dissolution of
hydroxyapatite and diffusion of calcium and phosphate ions towards the enamel surface
whereas a supersaturated environment would facilitate remineralization, thus stimulating
the formation of an intact surface layer (Arnold et al, 2006).
In the present study, artificial enamel carious lesions were produced by using solutions
similar to those proposed by ten Cate and Duijsters (1982). Similar demineralizing solutions
used in the present study have been used in several other in vitro studies (Itthagarun and
Wei, 1996; Itthagarun et al, 1999, 2000; Thaveesangpanich et al, 2005; Rana et al, 2007;
Kumar et al, 2008; Rana et al, 2008). Artificial carious lesions are considered to be more
reproducible than natural carious lesions and thus make the experimental model more
reliable (Silverstone, 1983). They facilitate the testing of multiple areas in any lesion at
different time intervals, in order to assess the remineralizing phenomena (Arends
and Christoffersen, 1986). Prior to the start of the pH cycle the two parameters, lesion
depth (LD) and the maximum mineral content (Vmax) were studied and found to be
statistically insignificant. This precluded the possibility of a sampling bias at baseline.
In the interest of standardization, all of the specimens in the present study were subjected
to 10 days of pH cycling which involved three hours of demineralization twice a day, with
two hours of remineralization in between. During the pH cycle, the specimens were treated
thrice daily; before the first demineralization, and before and after the second
demineralization. This also to a greater extent mimicked the daily eating patterns that occur
in vivo.
62
The specimens were divided into six different groups with varying treatment protocols,
which involved an one minute application of a non-fluoridated or fluoridated dentifrice
(supernatant), a three minute topical application of either of the two CPP-ACP
containing pastes, (Clinpro® and Tooth Mousse Plus®) or a combination of an one minute
application of a fluoridated dentifrice supernatant followed by a three minute topical
application of either of the two CPP-ACP containing pastes. The manufacturers’ of the test
agents recommended application times for Clinpro® and Tooth Mousse Plus® of three
minutes, and these recommendations were followed in this in vitro study.
Tooth Mousse Plus®, contains high levels of calcium and phosphate ions that together with
the fluoride ion are released into saliva to substantially increase the salivary concentrations
of calcium, inorganic phosphate and fluoride. These ions are stabilized by the presence of
the CPP, which prevents spontaneous precipitation and phase transformation thus
permitting a deeper penetration of the ions into the subsurface of carious lesions. This
results in increased remineralization throughout the body of the lesion (Reynolds et al,
2008; Cochrane et al, 2010). Conversely, Clinpro® contains TCP (tricalcium phosphate) which
has a low level of available (acid soluble) calcium phosphate. Several studies have
demonstrated that fluoride ions are unstable in dentifrice formulations, especially in those
containing poorly soluble calcium-based abrasives (Noren and Harse, 1974; Sullivan et al,
2001). Subsequently, this instability of fluoride ions leads to the development of sodium
monofluorophosphate (MFP). Clinpro® contains sodium fluoride, which in the absence of a
stabilizer would be susceptible to a decrease in its bioavailability especially in the presence
of added calcium phosphate. The calcium phosphate that has been incorporated into
Clinpro® is in the form of beta-tricalcium phosphate that has been ball milled with sodium
lauryl sulphate to produce particles within the size range of 1–15 µm (Karlinsey et al,
2010). The poor solubility, the large particle size and the small amount of calcium phosphate
63
explains the reason for its inability to significantly increase salivary calcium and inorganic
phosphate levels and its failure to show an enhanced rate of remineralization (Shen et al,
2011). In the present study, in an attempt to overcome the poor solubility of Clinpro® it was
used as a topically applied paste rather than as a slurry or supernatant. However, this was
not in accordance with the manufacturers recommended method of usage.
The qualitative evaluation which involved imbibing the specimens in water and examining
them under the PLM provided qualitative information on mineral loss and gain; however,
they are difficult to assess quantitatively (Arends and Bosch, 1992) and this was found to
be the case. Therefore, quantitative evaluation was carried out using MRG’s.
Statistically significant differences were evident when comparisons were made between the
pre-treatment and post-treatment lesion depths within the Clinpro®, Tooth Mousse Plus®
plus Colgate Total® and Clinpro® plus Colgate Total® groups. Though not statistically
significant, the combination of Clinpro® plus Colgate Total® proved to be the most effective
preparation for reducing the lesion depth, this was followed in effectiveness by Clinpro®,
and then the combination of Tooth Mousse Plus® plus Colgate Total®. Therefore, it appears
that the combination of fluoride with CPP-ACP produces a beneficial effect. There was a
trend, though not statistically significant, for the three minute application of Clinpro® to
exhibit a higher efficacy for remineralizing artificial enamel carious lesions than Tooth
Mousse Plus®. This finding could imply that Clinpro®, when applied topically, could be more
effective as a remineralizing agent than when applied in a diluted form that occurs when it is
used as a dentifrice.
Inevitably, certain limitations are associated with in vitro studies such as the present study.
These include the lack of saliva, plaque and the salivary pellicle which would be present in
64
the oral cavity. These variations in the characteristics and quantities of these factors, which
vary between individuals need equalisation in in vivo studies. Nevertheless, the lack of
these factors could have influenced the results of the present study, because the main
mechanism of action of CPP-ACP is for it to bind to the dental plaque and provide a reservoir
of calcium and phosphate ions. Thus, inhibiting demineralization and enhancing the
remineralization process (Rose, 2000; Reynolds, 2003).
Another limitation maybe that although the samples were randomly divided into the six
treatment groups, to prevent a sampling bias, we cannot exclude the possibility that one
group was dominated by multiple sections from the same tooth. Furthermore, some teeth
can be expected to have greater susceptibility than others to demineralization due to the
age of the donor and exposure to environmental factors such as fluoride. Also, the
specimens in the pH cycle were subjected to repeated cycles of remineralization and
demineralization (ten Cate and Duijsters, 1982) which is more aggressive than the acid
attacks that a tooth is exposed to, on a daily basis, in the oral cavity. Although there are
limitations in the methodology in respect of this pH cycling model, and the use of the test
reagents which were not used as recommended by the respective manufacturers’, the
conditions were at least standardised and repeatable.
Conclusions: based on the findings of this study, it can be concluded that CPP-ACP agents
are effective in remineralizing artificial enamel carious lesions in vitro.
Clinical relevance: CPP-ACP pastes such as Clinpro® and Tooth Mousse Plus®, could be
useful additions to the clinicians’ armamentarium for the prevention and management of
carious lesions, especially for those individuals with a high caries risk.
65
Chapter 6
Conclusions
66
Conclusions:
Within the limitations, and based on the findings of this in vitro study, the following
conclusions can be drawn:
1. Statistically significant differences were evident when comparisons were made
between the pre-treatment and post-treatment lesion depths (LD) within the
Clinpro®, Tooth Mousse Plus® plus Colgate Total® and Clinpro® plus Colgate Total®
groups.
2. Though not statistically significant, the combination of Clinpro® plus Colgate Total®
proved most effective in decreasing lesion depth, followed by Clinpro® and then the
combination of Tooth Mousse Plus® plus Colgate Total®.
3. There was a trend, though not statistically significant, for the three minute
application of Clinpro® to exhibit a higher efficacy in remineralizing artificial enamel
carious lesions than Tooth Mousse Plus®.
67
Null hypotheses:
It is proposed to consider the following null hypotheses based on the findings of this in vitro
study:
I.
The null hypotheses that stated “When applied topically for three minutes,
Clinpro® will not exhibit a level of remineralization comparable to a one
minute treatment with the 1000ppm F dentifrice” can be rejected because
there was a greater reduction in lesion depth with the use of Clinpro® even
though it was not statistically significant.
II.
The null hypotheses that stated “When applied topically for three minutes,
Tooth Mousse Plus® will not exhibit a level of remineralization comparable to
a one minute treatment with the 1000ppm F dentifrice” can be rejected
because when applied topically for three minutes, Tooth Mousse Plus®
produced a greater decrease in the lesion depth.
III.
The null hypotheses that stated “When applied topically for three minutes
following a one minute treatment with a 1000ppm F dentifrice, Tooth
Mousse Plus® will not exhibit a level of remineralization greater than that of
a one minute treatment with the 1000ppm F dentifrice” can be rejected
because even though statistically insignificant, there was a greater reduction
in lesion depth with the use of Tooth Mousse Plus® in combination with
a 1000ppm F dentifrice.
68
IV.
The null hypotheses that stated “When applied topically for three minutes
following a one minute treatment with a 1000ppm F dentifrice, Clinpro® will
not exhibit a level of remineralization greater than that of a one minute
treatment with the 1000ppm F dentifrice” can be rejected as even though
not statistically significant the combination of Clinpro® with Colgate Total®
reduced the lesion depth to a greater extent as compared to the fluoridated
dentifrice alone.
69
Chapter 7
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87
Appendix I
Lesion depth and mineral content changes
before and after pH cycling with different
agents
88
Changes in the lesion depth (LD) of the artificial enamel carious lesions both
before and after the 10-day pH cycle.
Group 1: Non-fluoridated dentifrice (Vicco®).
Specimen
Pre-cycle LD
Post-cycle LD
% change
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
257
235
106
124
234
165
206
261
287
108
271
264
254
175
212
276
254
176
308
311
245
267
165
231
149
379
224
251
262
342
287
287
271
309
216
305
284
272
212
287
289
286
267
334
329
321
334
285
345
243
47.47
-4.68
136.79
111.129
46.15
73.94
39.32
3.83
7.67
100.00
12.55
7.58
7.09
21.14
35.38
4.71
12.60
51.70
8.44
5.79
31.02
25.09
72.73
49.35
63.09
n
mean
SD
Max
Min
25
221.64
60.18
311.00
106.00
25
288.84
41.98
379.00
212.00
25
38.80
37.18
136.79
- 4.68
89
Group 2: Fluoridated dentifrice (Colgate Total®).
Specimen
Pre-cycle LD
Post-cycle LD
% change
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
194
212
184
242
196
154
251
176
298
203
276
208
114
204
227
176
196
314
235
214
254
237
165
98
214
255
267
242
278
269
198
297
217
253
244
342
244
198
278
291
251
218
362
249
163
234
265
212
143
241
31.44
25.94
31.52
14.88
37.24
28.57
18.33
23.30
-15.10
20.20
23.91
17.31
73.68
36.27
28.19
42.61
11.22
15.29
5.96
-23.83
-7.87
11.81
28.48
45.92
12.62
n
Mean
SD
Max
Min
25
209.68
49.95
314.00
98.00
25
248.44
48.21
362.00
143.00
25
21.52
19.89
73.68
-23.83
90
Group 3: Clinpro®.
Specimen
Pre-cycle LD
Post-cycle LD
% change
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
289
198
237
298
276
298
312
345
261
289
254
308
265
268
243
308
279
254
302
311
249
314
176
231
149
278
212
219
184
235
261
258
286
142
180
265
227
236
224
275
248
265
276
178
172
254
276
237
178
124
-3.81
7.07
-7.59
-38.26
-14.86
-12.42
-17.31
-17.10
-45.59
-37.72
4.33
-26.30
-10.94
-16.42
13.17
-19.48
-5.02
8.66
-41.06
-44.69
2.01
-12.10
34.66
-22.94
-16.78
n
Mean
SD
Max
Min
25
268.56
45.80
345.00
149.00
25
227.60
45.47
286.00
124.00
25
-13.62
19.38
34.66
-45.59
91
Group 4: Tooth Mousse Plus®.
Specimen
Pre-cycle LD
Post-cycle LD
% change
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
212
232
234
194
321
232
223
226
247
256
186
197
287
226
295
293
194
281
309
198
235
199
276
164
189
214
284
196
203
307
224
213
229
251
249
192
202
274
215
284
289
192
264
299
202
224
208
291
166
295
0.94
22.41
-16.24
4.64
-4.36
-3.45
-4.48
1.33
1.62
-2.73
3.23
2.54
-4.53
-4.87
-3.73
-1.37
-1.03
-6.05
-3.24
2.02
-4.68
4.52
5.43
1.22
56.08
n
Mean
SD
Max
Min
25
236.24
43.19
321.00
164.00
25
238.68
41.70
307.00
166.00
25
1.81
13.10
56.08
-16.24
92
Group 5: Clinpro® + Colgate Total®.
Specimen
Pre-cycle LD
Post-cycle LD
% change
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
290
312
237
295
336
294
223
285
234
216
194
368
291
283
394
361
274
312
283
198
297
195
291
231
154
248
224
232
254
225
221
228
175
210
194
165
185
248
278
321
277
298
342
275
185
186
221
175
227
165
-14.48
-28.21
-2.11
-13.90
-33.04
-24.83
2.24
-38.60
-10.26
-10.19
-14.95
-49.73
-14.78
-1.77
-18.53
-23.27
8.76
9.62
-2.83
-6.57
-37.37
13.33
-39.86
-1.73
7.14
n
Mean
SD
Max
Min
25
273.92
59.03
394.00
154.00
25
230.36
48.14
342.00
165.00
25
-13.84
17.16
13.33
-49.73
93
Group 6: Tooth Mousse Plus® + Colgate Total®.
Specimen
Pre-cycle LD
Post-cycle LD
% change
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
275
312
204
326
242
287
245
184
232
298
225
235
285
274
312
253
306
252
316
356
248
218
234
181
164
264
291
194
224
238
290
198
178
254
265
234
198
220
227
265
272
289
248
324
328
254
194
224
176
153
-4.00
-6.73
-4.90
-31.29
-1.65
1.05
-19.18
-3.26
9.48
-11.07
4.00
-15.74
-22.81
-17.15
-15.06
7.51
-5.56
-1.59
2.53
-7.87
2.42
-11.01
-4.27
-2.76
-6.71
n
Mean
SD
Max
Min
25
258.56
49.01
356.00
164.00
25
240.08
45.64
328.00
153.00
25
-6.63
9.62
9.48
-31.29
94
Changes in the maximum mineral content (Vmax) before and after the 10day pH cycle with different agents.
Group 1: Non-fluoridated dentifrice (Vicco®).
Specimen
Pre-cycle Vmax
Post-cycle Vmax
% change
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
43.29
39.60
42.35
44.56
46.75
21.98
62.54
52.34
51.45
33.98
21.34
27.65
44.78
34.56
24.58
21.23
27.68
36.98
32.20
42.31
22.35
17.64
20.70
24.57
46.78
48.91
42.60
39.67
39.72
43.13
23.85
64.83
47.15
47.84
32.90
13.57
24.12
42.74
32.13
22.17
23.12
24.34
32.87
31.23
40.43
21.25
11.24
21.76
22.32
44.54
12.98
7.58
-6.33
-10.86
-7.74
8.51
3.66
-9.92
-7.02
-3.18
-36.41
-12.77
-4.56
-7.03
-9.80
8.90
-12.07
-11.11
-3.01
-4.44
-4.92
-36.28
5.12
-9.16
-4.79
n
Mean
SD
Max
Min
25
35.37
12.14
62.54
17.64
25
33.54
12.80
64.83
11.24
25
-6.19
11.61
12.98
-36.41
95
Group 2: Fluoridated dentifrice (Colgate Total®).
Specimen
Pre-cycle Vmax
Post-cycle Vmax
% change
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
43.29
11.23
9.65
11.24
16.57
6.54
14.35
21.45
11.49
7.86
7.34
27.65
11.24
21.20
31.78
21.34
27.69
31.23
19.78
13.27
7.98
8.54
5.68
35.46
23.45
34.73
23.46
24.70
23.88
46.64
20.64
28.65
29.76
41.34
25.64
12.95
34.63
22.86
41.98
42.23
26.47
34.56
42.78
39.34
33.24
17.23
21.27
12.34
41.23
33.25
-19.77
108.90
155.96
112.46
181.47
215.60
99.65
38.74
259.79
226.21
76.43
25.24
103.38
98.02
32.88
24.04
24.81
36.98
98.89
150.49
115.91
149.06
117.25
16.27
41.79
n
Mean
SD
Max
Min
25
17.89
10.29
43.29
5.68
25
30.23
9.75
46.64
12.34
25
99.62
72.14
259.79
-19.77
96
Group 3: Clinpro®.
Specimen
Pre-cycle Vmax
Post-cycle Vmax
% change
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
36.81
27.84
12.34
22.46
12.34
21.98
18.53
13.34
8.76
17.35
8.56
9.84
13.56
16.87
22.46
21.24
6.78
13.65
18.68
12.65
15.47
8.76
17.68
14.30
21.34
45.24
22.23
23.88
33.85
21.20
36.54
22.34
37.84
27.65
33.24
28.24
21.23
22.40
26.18
32.56
34.78
12.24
28.75
14.55
19.82
28.65
12.56
32.23
24.27
19.84
22.90
-20.15
93.52
50.71
71.80
66.24
20.56
183.66
215.64
91.59
229.91
115.75
65.19
55.19
44.97
63.75
80.53
110.62
-21.57
56.68
85.20
43.38
82.30
69.72
-7.03
n
Mean
SD
Max
Min
25
16.54
6.78
36.81
6.78
25
26.50
8.11
45.24
12.24
25
74.84
62.47
229.91
-21.57
97
Group 4: Tooth Mousse Plus®.
Specimen
Pre-cycle Vmax
Post-cycle Vmax
% change
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
48.19
18.13
32.30
14.21
22.29
33.91
13.93
37.42
12.86
43.01
30.87
45.03
42.07
36.94
39.67
45.48
17.02
38.25
37.50
16.83
23.47
19.40
30.40
22.79
12.80
31.34
22.23
31.23
18.56
24.80
32.12
28.76
32.29
16.90
41.32
32.24
48.60
44.64
39.65
31.23
43.25
24.37
42.24
32.45
26.75
24.30
18.90
32.50
24.50
14.50
-34.97
22.61
-3.31
30.61
11.26
-5.28
106.46
-13.71
31.42
-3.93
4.44
7.93
6.11
7.34
-21.28
-4.90
43.18
10.43
-13.47
58.94
3.54
-2.58
6.91
7.50
13.28
n
Mean
SD
Max
Min
25
16.54
6.78
36.81
6.78
25
30.39
9.12
48.60
14.50
25
10.74
28.18
106.46
-34.97
98
Group 5: Clinpro® + Colgate total®.
Specimen
Pre-cycle Vmax
Post-cycle Vmax
% change
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
9.54
18.15
7.65
9.70
11.42
7.39
13.65
7.85
34.79
18.65
15.76
17.94
21.48
33.65
36.74
29.60
16.82
39.14
17.53
6.88
13.72
9.46
17.45
21.83
7.54
21.34
25.64
28.64
7.23
24.35
22.65
39.12
22.34
32.34
29.84
22.54
32.14
40.23
29.12
51.23
42.21
42.45
34.56
47.65
22.34
33.76
32.31
22.56
18.56
8.65
123.69
41.19
274.38
-25.46
113.22
206.50
186.59
184.59
-7.04
60.00
43.02
79.15
87.29
-13.46
39.44
42.60
152.38
-11.70
171.82
224.71
146.06
241.54
29.28
-14.98
14.72
n
Mean
SD
Max
Min
25
17.77
9.88
39.14
6.88
25
29.35
10.82
51.23
7.23
25
95.58
90.41
274.38
-25.46
99
Group 6: Tooth Mousse Plus® + Colgate Total®.
Specimen
Pre-cycle Vmax
Post-cycle Vmax
% change
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
28.65
19.57
41.62
17.90
23.47
16.53
24.50
23.56
25.96
17.49
43.40
18.96
27.65
51.32
43.71
28.82
39.97
38.55
36.88
32.65
29.70
19.76
21.64
19.83
29.21
32.24
18.35
52.24
18.69
24.34
31.23
18.45
22.45
32.25
18.35
51.37
21.45
28.86
46.89
38.14
32.56
41.67
39.70
38.76
31.60
24.65
22.45
19.87
22.40
32.24
12.53
-6.23
25.52
4.41
3.71
88.93
-24.69
-4.71
24.23
4.92
18.36
13.13
4.38
-8.63
-12.74
12.98
4.25
2.98
5.10
-3.22
-17.00
13.61
-8.18
12.96
10.37
n
Mean
SD
Max
Min
25
28.85
9.76
51.32
16.53
25
30.45
10.35
52.24
18.35
25
7.08
21.01
88.93
-24.69
100
Appendix II
Time schedule and pH cycling protocol
101
Time
9 am : Treat
Group A
Treat:
Vicco® (60s)
Group B
Treat:
Colgate
Total®
(60s)
Group C
Treat:
Clinpro®
(180s)
Wash
Wash
Wash
Wash
Demin
Place in
DEMIN
Place in
DEMIN
Group E
Treat:
Colgate
Total® (60s)
+ Clinpro®
(180s)
Group F
Treat:
Colgate
Total® (60s)
+
TM Plus®
(180s)
Wash
Wash
Wash
Place in
DEMIN
Place in
DEMIN
Place in
DEMIN
Place in
DEMIN
Remove
DEMIN
Remove
DEMIN
Remove
DEMIN
Remove
DEMIN
Remove
DEMIN
Remove
DEMIN
Wash
Wash
Wash
Wash
Wash
Wash
Wash
Remin
Place in
REMIN
Remove
REMIN
Place in
REMIN
Remove
REMIN
Place in
REMIN
Remove
REMIN
Place in
REMIN
Remove
REMIN
Place in
REMIN
Remove
REMIN
Place in
REMIN
Remove
REMIN
Treat
Vicco®(60s)
Treat:
Colgate
Total®(60s)
Treat:
Clinpro®
(180s)
Treat:
TM Plus®
(180s)
Treat:
Colgate
Total®(60s)
+ Clinpro®
(180s)
Treat:
Colgate
Total® (60s)
+ TM Plus®
(180s)
Wash
Wash
Wash
Wash
Wash
Wash
Demin
Place in
DEMIN
Place in
DEMIN
Place in
DEMIN
Place in
DEMIN
Place in
DEMIN
Place in
DEMIN
Demin
Remove
DEMIN
Remove
DEMIN
Remove
DEMIN
Remove
DEMIN
Remove
DEMIN
Remove
DEMIN
Treat
Treat
Vicco®(60s)
Treat:
Colgate
Total®(60s)
Treat:
Clinpro®
(180s)
Treat:
TM Plus®
(180s)
Treat:
Colgate
Total® (60s)
+ Clinpro®
(180s)
Treat:
Colgate
Total® (60s)
+ TM plus®
(180s)
Wash
Wash
Wash
Wash
Wash
Wash
Wash
Place in
REMIN
Place in
REMIN
Place in
REMIN
Place in
REMIN
Place in
REMIN
Place in
REMIN
12 pm: Demin
2pm
Remin
Treat
Wash
5pm
Remin
Group D
Treat:
Tooth
Mousse
Plus®
(180s)
102
Time
9
am
Group
1a & 1b
2a & b
3a & b
4a & b
5a & b
6a & b
Time
12pm
Group
1a&b
2a&b
3a&b
4a&b
5a&b
Treatment
 Vicco® supernatant:60s
 Wash
 Put in Demin
 Colgate Total®
supernatant:60s
 Wash
 Put in Demin
 Clinpro® paste: 180s
 Wash
 Put in Demin
 TMP® paste: 180s
 Wash
 Put in Demin
 Colgate Total®
supernatant:60s
 Clinpro® paste: 180s
 Wash
 Put in Demin
 Colgate Total®
supernatant:60s
 TMP®: 180s
 Wash
 Put in Demin
Treatment
 Remove from
Demin
 Wash
 Place in Remin
 Remove from
Demin
 Wash
 Place in Remin
 Remove from
Demin
 Wash
 Place in Remin
 Remove from
Demin
 Wash
 Place in Remin
 Remove from
Demin
 Wash
 Place in Remin
103
6a&b



Time
2pm
Group
1a&b
2a&b
3a&b
4a&b
5a &b
6a &b
Remove from
Demin
Wash
Place in Remin
Treatment
 Remove from remin
 Vicco® supernatant:60s
 Wash
 Put in Demin
 Remove from remin
 Colgate Total®
supernatant:60s
 Wash
 Put in Demin
 Remove from Remin
 Clinpro® paste:180s
 Wash
 Put in Demin
 Remove from remin
 TMP®: 180s
 Wash
 Put in demin
 Remove from remin
 Colgate Total®
supernatant:60s
 Clinpro® paste: 180s
 Wash
 Put in demin
 Remove from remin
 Colgate Total®
supernatant: 60s
 TMP®: 180s
 Wash
 Put in demin
104
Time
5 pm
Group
1a &b
2a& b
3a&b
4a&b
5a&b
6a&b
Treatment
 Remove from demin
 Vicco® supernatant:60s
 Wash
 Put in remin
 Remove from demin
 Colgate Total®
supernatant:60s
 Wash
 Put in remin
 Remove from demin
 Clinpro® paste:180s
 Wash
 Put in remin
 Remove from demin
 TMP®: 180s
 Wash
 Put in remin
 Remove from demin
 Colgate Total®
supernatant:60s
 Clinpro® paste: 180s
 Wash
 Put in remin
 Remove from demin
 Colgate Total®
supernatant: 60s
 TMP®: 180s
 Wash
 Put in remin
105
Treatment
Prep
CENTRIFUGE
SUPERNATANT
Working solution
Preparation
COLGATE TOTAL®
 150g paste + 450
ml DI (1:3 ratio)
 Mix in beaker
 Separate into two
containers (by
weight)
VICCO®
 60g paste + 180ml
DI
 Mix in beaker
 Separate into two
containers by
weight
4000 rpm; 20 mins; temp
25®C
COLGATE TOTAL®: 5ml x
75=375 ml
VICCO®= 5 x 25=125 ml
3.4 litres/day
9am:
Prepare DEMIN
 300ml Demin stock
 Add water till 3
litres (10 times
dilution)
 Calibrate pH meter
with 4 and 7
 Measure pH
 Add KOH till 4.4 pH
 Add DI till 3.4 litres
12pm:
Prepare REMIN
 300ml Remin stock
 Add water till 3
litres (10 times
dilution)
 Calibrate pH meter
with 4 and 7
 Measure pH
 Add KOH till 7 pH
 Add DI till 3.4 litres
2pm:
Prepare DEMIN
 300ml Demin stock
 Add water till 3
litres (10 times
dilution)
106

REMIN/DEMIN
SOLUTION
Calibrate pH meter
with 4 and 7
Measure pH
Add KOH till 4.4 pH
Add DI till 3.4 litres



5pm:
Prepare REMIN
 300ml Remin stock
 Add water till 3
litres (10 times
dilution)
 Calibrate pH meter
with 4 and 7
 Measure pH
 Add KOH till 7 pH
 Add DI till 3.4 litres
GRP 1:250 ML
GRP2: 250 ML
GRP 3:250 ML
GRP 3:250 ML
GRP 4:250 ML
GRP 5:250 ML
GRP 6:250 ML
107
Appendix III
Statistical analysis and raw data
108
109
110
111
112
113
114
115
116
Appendix IV
Abstract of the paper presentation at the
23rd Congress of the International Association of
Paediatric Dentistry in Athens, Greece.
(oral presentation)
117
REMINERALIZING ACTION OF CPP-ACP REAGENTS ON ARTIFICIAL CARIOUS
LESIONS
Sakshi Buckshey1, Robert Anthonappa1, Nigel King1, Anut Itthagarun2
1
Paediatric Dentistry, Faculty of Dentistry, The University of Hong Kong, Hong Kong SAR.
2
Paediatric Dentistry, School of Dentistry and Oral Health, Griffith University, Queensland,
Australia.
Aim: To evaluate the efficacy of CPP-ACP containing pastes; Clinpro® (500ppm tri-calcium
phosphate +950ppm sodium fluoride) and Tooth Mousse Plus® (10% CPP-ACP+900ppm
sodium fluoride) in remineralizing artificial enamel carious lesions.
Design: Fifty extracted human third molars were cleaned of soft tissue debris and inspected
for any cracks, caries or hypoplasia. The teeth were painted with an acid resistant nail
varnish leaving a 1mm window on the buccal and lingual surfaces and then immersed in a
demineralizing solution for 96 hours, to produce artificial carious lesions 90-180μm deep.
Subsequently, the teeth were sectioned longitudinally through the lesion to produce
sections that were approximately 100-150μm thick. The tooth specimens were randomly
divided into four groups (n=29) and treated with a non-fluoridated or fluoridated paste (for
1 min), Clinpro® or TM Plus® pastes (for 3 min) in a 10 day pH cycling model. Lesion depth
(LD) and mineral content (Vmax) for each specimen were evaluated using polarized light
microscopy (PLM) and microradiography (MRG) before and after the pH cycle. Paired t-test,
ANOVA and Student-Newman-Keuls tests were employed to make comparisons within, and
between the different treatment groups.
Results: Significant differences were evident when comparisons were made between the
pre- and post-treatment specimens within each group. When multiple comparisons were
made, specimens treated with Clinpro® exhibited the greatest reduction in LD and increase
118
in Vmax as compared to the other treatment groups.
Conclusion: A three minute application of Clinpro® exhibited a higher efficacy in
remineralizing artificial enamel carious lesions than TM Plus®.
Word count: 250
119
Appendix V
List of published studies on CPP-ACP
120
Table V.1: Evidence from in vitro studies
Author/year
Aim
Sample
Intervention
Reynolds,
1997
To evaluate
Extracted
human
third
molars
Remineralizing
solutions for 10
days
-the ability of CPPACP solution to
remineralize
-the effect of
concentration
-the effect of pH
Yamaguchi
et al, 2006
To evaluate
mechanical
properties of
enamel
remineralized by
CPP-ACP using
ultrasonic device
Extracted
bovine
incisors
Outcome
measures
Microradiography
SEM
Conclusion
CPP-ACP is
effective in
remineralization
1% CPP- pH 7
0.1% CPP- pH 7
0.5% CPP- pH 7
0.5% CPP- pH 7.5
0.5% CPP- pH 8
0.5% CPP- pH 8.5
0.5% CPP- pH 9
Microphotometry
Dose dependent
efficacy and
highest at pH 7
All specimens
stored in
artificial saliva (4
weeks) and
subject to 10 min
in demineralizing
agent twice a
day
SEM
CPP-ACP was
effective
Sonic velocity of
ultrasonic waves
-preventing
demin
-enhancing remin
2 groups:
Treatment grp:
10 mins in 10
times diluted
CPP-ACP before
demin twice a
day
Yamaguchi
et al, 2007
To evaluate
mechanical
properties of
dentine
remineralized by
CPP-ACP using
ultrasonic device
Extracted
bovine
incisors
Placebo grp: 10
min in placebo
paste before
demin twice a
day
All specimens
stored in
artificial saliva
and subjected to
Treatment
group: 10 min in
10 times diluted
CPP-ACP before
demin twice a
day
SEM
Sonic velocity of
ultrasonic waves
CPP-ACP was
effective in
-preventing
demin
-enhancing remin
of dentine
substrate
121
Oshiro et al.,
2007
Rahiotis et
al., 2007
To evaluate the
Extracted
effect of CPP-ACP
bovine
paste on
incisors
demineralization of
bovine enamel and
dentine
To evaluate the
effect of CPP-ACP
paste on
Extracted
human
third
molars
-Demineralization
of sound human
dentin
-remineralization
of artificial caries
lesions on dentin
surfaces
Pai et al 2008
Evaluate remin of
incipient enamel
lesions by topical
application of CPPACP
Extracted
human
premolar
s and
third
molars
Placebo group:
10 min in
placebo paste
before demin
twice a day
All specimens
stored in
artificial saliva
and subjected to
FE-SEM
CPP-ACP was
effective in
preventing
demineralization
of enamel and
dentine
substrates
compared to
placebo
Surface analysis
CPP-ACP
treatment
Treatment
group: 10 min in
10 times diluted
CPP-ACP before
demin twice a
day
Placebo group:
10 min in
placebo paste
before demin
twice a day
Group A and B
Treatment prior
to demin
Grp A: TM (5
min)
Grp B:no agent
Treatment after
demin
Grp C: TM
applied (5 min)
Grp D:no agent
Test group: once
daily -3 min
application of
tooth mousse for
14 days
-decreased
dentine
demineralization
-increased
dentine
remineralization
SEM
Laser
fluorescence
Topical
application of
CPP-ACP cream
results in
significant
remineralization
of enamel lesions
122
Rees et al
2007
To examine
whether a single
topical application
of proenamel or
tooth mousse
would prevent
enamel erosion
Extracted
unerupte
d third
molars
Three groups
with 10 samples
each:
Grp A:Tooth
mousse-15 min
Surface
profilometry
Tooth mousse
and proenamel
may offer a
degree of
protection from
erosion of
permanent
enamel
Grp B:
Proenamel15min
Grp C: water
(control group)
Specimens were
then exposed to
an erosive
challenge of
250 ml of a 0.2%
citric acid
solution for 1 h.
(recommends
longer
application time)
123
Table V.2: Evidence from in vivo studies
Author / year
Study design
Shen et al,
2001
Aim
Sample
population
Intervention
Control
Outcome
Conclusion
Determine the
ability of CPPACP in sugar
free gum to
remineralize
enamel
lesions
30 subjects
3 types of
gum:
Crossover
trial
MRG
-Sorbitol
based pellet
14 day test
period with
one week
washout
between
interventio
ns
Addition of CPPACP to chewing
gums produces
dose related
increase in
remineralization
Extracted
human
third
molars
-Sorbitol
based slab
removal
palatal
appliance
-Xylitol based
pellet
Independent of
the gum type
and the weight
Different conc
of CPP-ACP:
0.19mg,
10mg,18.8mg,
56.4mg
20 min
chewing +20
min exposure
Cai et al, 2003
Effect of CPPACP present in
a sugar free
lozenge on
enamel
remineralizati
on
10 subjects
Extracted
human
third
molars
4 times a
day/14 days
Lozenge
containing
-56.4 mg CPPACP
-18.8mg CPPACP
Removable
palatal
appliance
-no CPP-ACP
Crossover
trial:
14 day test
period with
a one week
washout in
between
interventio
ns
MRG
Dose related
increase in
remineralization
Lozenges are a
suitable system
for the delivery
of CPP-ACP
124
Reynolds et al
2003
Retention in
plaque and
remineralizati
on of enamel
lesions by
calcium in a
mouthrinse or
a sugar free
gum
30 subjects
Extracted
human
third
molars
Control: no
lozenge, nil
treatment
Mouthrinse
trial
-2% CPP-ACP
-6% CPP-ACP
-Ca+Po4 slurry
Removable
palatal
appliance
-de-ionised
water
Crossover
trial
14 day test
period with
one week
washout
between
interventio
ns
Mouthrinse
trial
-plaque
calcium
and
inorganic
PO4
analysis
-plaque
CPP
analysis
Chewing gum
trial
-pellet or slab
containing
Chewing gum
studies:
-CPP detected in
plaque 3 hrs
after chewing
Chewing
gum study
-CPP-ACP
superior to
other forms of
Ca in
remineralizing
-CPP level
in plaque
CaHPO4/
CaCo3
Only CPP-ACP
mouthrinse
increased
plaque calcium
and Po4 levels
-EM
CaCo3
CPP-ACP
Mouthrinse
study:
-enamel
remineraliz
ation
Enamel lesions.
MRG
CPP-ACP gum
produced twice
Pellet: 20 min
chewing 4x
day/14 days
Slab: 5 min.
Chewing 7x
day/7 days
Iijima et al
2004
Acid
resistance of
10 subjects
Chewed 2
pellets
containing 9.5
mg of CPP-ACP
per piece for
20 min 3 x
day/ 4 days
Treatment:
Crossover
125
enamel
lesions
remineralized
by a sugar
free chewing
gum
containing
CPP-ACP
Extracted
human
third
molars
Removable
palatal
appliance
Sugar free
gum
containing
18.8 mg of
CPP-ACP
Control: gum
with no CPPACP
trial
the
remineralization
14 day test
period with
one week
washout
between
interventio
ns
Mineral
remineralized
with CPP is
more resistant
to subsequent
acid challenge
20 min
chewing 4
times a day/14
days
Itthagarun et
al 2005
Effects of gum
containing
urea, urea
with dicalcium
phosphate or
urea with
CPP-ACP
complex for
20 min after
food on the
remineralizati
on of artificial
caries lesions
12 subjects
Extracted
human
third
molars
After each
treatment,
half of the
lesions were
acid
challenged for
8 or 16 hrs
Three gums:
All sugar free
with 30 mg
urea per piece
-no calcium
phosphate
Removable
lower
appliance
21 day test
period
-25mg
dicalcium
phosphate
dehydrate
-47mg CPPACP
5 day
washout
between
interventio
ns
MRG
Gum containing
no calcium
showed no
significant
difference in
lesion depth
Calcium
containing gum
decreases lesion
depth but to a
lesser extent as
compared to
CPP-ACP
containing gum
2 pieces of
gum for 20
min (5 times a
day)
126
Cai et al 2007
Effect of CPPACP in a sugar
free chewing
gum on
enamel
remineralizati
on
10 subjects
Extracted
human
third
molars
Removable
palatal
appliance
Three
treatments
Crossover
trial
Sugar free
gums (2
pellets)
containing
14 days
test period
MRG
Microdensitomet
ry
-20mg citric
acid and 18.8
mg CPP-ACP
All gums
resulted in
remineralization
Addition of CPPACP resulted in
highest
remineralization
Level of mineral
after acid
challenge was
significantly
greater for gum
containing CPPACP
-only 20mg
citric acid
-neither CPPACP nor citric
acid
Chewed 20
min 4 times/
day x 14 days
Schirrmeister
et al 2007
determine the
effects of 4
chewing gums
on artificial
subsurface
lesions
15 subjects
for study
Extracted
bovine
incisors
Removable
lower
appliance
with
buccal
wing
Appliance
retained for 40
min after
chewing. After
each
treatment
period, half of
each
remineralized
block was acid
challenged for
16 h at 37◦C
4 chewing
Crossover
gums:
trial
Two chewing
gums
(plus/minus
zinc
citrate)contain
ed:
-dicalcium
phosphate
(3.9%)
-calcium
gluconate
14 day test
period
One week
washout
between
interventio
ns
MRG
No significant
difference
between
calcium
containing and
calcium free
chewing gums
with regard to
mineral change
and lesion
depth
No significant
difference
between
treatment and
127
(1.8%)calcium
lactate (0.45%)
control group
CPP-ACP
(0.7%)
No calcium
Control: No
chewing gum
Chewed 20
min 4
times/day x 14
days
Manton et al
2008
Walker et al
2009
Compare
efficacy of
three
commercially
available
gums in
remineralizing
lesions in situ
Compare
remineralizati
on of
subsurface
enamel
lesions in situ
after drinking
100 ml of milk
containing
0.02% or 0.3%
CPP-ACP for
30 sec once
daily for 15
days
10 subjects
Extracted
human
third
molars
Removable
palatal
appliance
10 subjects
Extracted
human
molars
Appliance
worn 20 min
after chewing
Treatment
Trident white
(CPP-ACP 10
mg per serve)
Crossover
trial
14 day test
period
MRG
Microdensi
-tometry
Orbit: xylitol
gum
Orbit
professional:
xylitol
Chewed 20
min 4
times/day for
14 days
Bovine milk
containing
Treatment
Crossover
trial
15 day test
period
MRG
Microdensi
-tometry
Chewing of
Trident white
gum (10 mg
CPP-ACP)
produced
greater
remineralization
of subsurface
lesions than
Orbit and Orbit
professional
gum
Dose related
increase in
remineralization
compared to
control
0.2% CPP-ACP
Removable
palatal
appliance
0.3% CPP-ACP
Control: No
added CPPACP
One week
washout
between
interventio
ns
Addition of CPPACP significantly
increases the
remineralization
potential of milk
100 ml milk
128
sipped (5-8
sips) in 30 secs
once a day for
15 days
Cai et al 2009
Compare the
remineralizati
on efficacy of
four
Sugar-free
gums, two
containing
calcium in a in
situ model
10 subjects
Extracted
human
molars
Removable
palatal
appliance
Appliance
retained in
mouth for 40
min following
milk
Four sugar
free gums:
Trident Xtra
Care: contains
CPP-ACP
Orbit
professional:
added calcium
carbonate
with citric acid
Crossover
trial
14 day test
period
MRG
Microdensi
tometry
One week
washout
between
interventio
ns
No correlation
between
stimulated
saliva flow rates
or saliva calcium
levels and
measurements
of enamel
remineralization
Orbit: sugar
free gum
Extra: sugar
free gum
Walker et al
2010
Investigate
the potential
of CPP-ACP
when added
to candy to
slow the
progression of
enamel
subsurface
lesions in an
in situ model
Study 1: 10
subjects
Chewed 20
min 4 times/
day for 14
days
Candy
containing
Crossover
trial
Study 2: 14
subjects
-sugar
(control) 65%
sugar + 33%
glucose
10 day test
period
Extracted
human
molars
Removable
palatal
appliance
-sugar free
(control)
-sugar + 0.5%
CPP-ACP
-sugar + 1.0%
CPP-ACP
One week
washout
between
interventio
ns
MRG
Consumption of
sugar candy
containing CPPACP caused
significant
remineralization
of lesions
Dose related
response with
CPP-ACP
Sugar + 1.0%
CPP-ACP
remineralization
> sugar free
candy
-sugar free +
129
0.5% CPP-ACP
Consume 1
candy 6
times/day x 10
days (average
dissolution
time : 10min)
Incorporation of
CPP-ACP into
sugar candy can
reduce their
cariogenicity
130
Table V.3: Evidence from clinical trials that used CPP-ACP and have been
published in the English literature.
Author/year
Aim
Subject
Intervention
Control
Outcome
Conclusion
Morgan et al
2008
Progression
and regression
of approximal
caries in
subjects
chewing gum
with and
without CPPACP
2720 healthy
children
Treatment
grp:
Sorbitol
based
gum
chewed 3
times
daily for
10 min
per
session
Caries
progression
or
regression
at 24
months
Treatment
group: odds of
the surface
experiencing
caries 18% <
control gum
Approximal
caries
diagnosed
with bitewing
radiograph
CPP-ACP sugar
free gum
slowed
progression
and enhanced
regression of
approximal
caries
Following
bracket
removal
Following
bracket
removal
Laser
fluorescenc
e
Treatment:
twice daily
application
of CPP-ACP
as
toothpaste
(3 months)
followed by
normal use
of F
dentrifice
(1000 ppm)
Conventi
onal 0.5%
NaF rinse
once
daily
along
with
standard
F
dentrifice
for 6
months
No significant
difference in
clinical WSL
scores but
more no. of
sites
disappeared
after 12
months with
CPP-ACP
Andersson
et al 2007
Randomised
Test : 1369
Control: 1351
Compare
26 healthy
effect of CPPadolescents
ACP cream
and Fluoride
mouthwash on
regression of
white spot
lesions
Gum
containing
54 mg CPPACP chewed
3 times daily
for 10 min
per session
CPP-ACP has a
more
aesthetically
favourable
outcome
(3 months)
Bailey et al
2009
To test lesion
regression in a
post
45 healthy
adolescents
After
bracket
removal
After
bracket
removal
Clinical
visual
assessment
Lesions with
severity codes
2 and 3: more
131
orthodontic
population
using F
toothpastes
and using F
mouthrinses,
with a CPPACP cream as
compared to a
placebo
Twice daily
application
of 1g 10%
CPP-ACP
(TM) for 12
weeks (after
brushing
with a 1000
ppm NaF
dentrifice)
Twice
daily
similar
applicatio
n of
placebo
cream
s according
to ICDAS II
criteria
Transitions
between
examinatio
n coded
between
progressing
, regressing
and stable
regressed with
CPP-ACP
cream at 12
weeks
No significant
difference in
transition
scores
between grps
Greater
remineralizatio
n in CPP-ACP
grp.
132
Table V.4: Evidence from literature reviews on the use of CPP-ACP that have
been published in the English literature.
Author/year
Aim
Reynolds 2008
To determine scientific
evidence for 3
remineralization
systems in the non
invasive treatment of
early carious lesions
Methods
Scientific
evidence based
on published
original studies
(papers and
abstracts)
reporting the
-CPP-ACP (Recaldent)
testing of the
-unstabilized ACP (ACP three systems
or enamelon)
and their use in
-bioactive glass
randomised
containing calcium
control caries
sodium phosphosilicate trials
(Novamin)
Presentation
Conclusion
Tabulated
evidence, quotes,
papers and result
(yes/no) under:
Enamelon:
anticariogenic efficacy
for root caries
Animal studies
In-vitro:
-promoting
remineralization
-preventing
demineralization
In-vivo:
promoting
remineralization
-preventing
demineralization
Recaldent: slowing
progression and
promoting regression
of coronal caries
Calcium phosphate
based
remineralization
technologies as an
adjunctive to F
therapy
Randomised
control clinical
trial
Azarpazooh
and Limeback
2008
Systematically review
the clinical trials of
casein derivatives used
in dentistry
Includes
randomised
and quasirandomised
controlled trials
testing efficacy
of casein
derivatives in
dental
applications
12 original studies
9: caries
prevention
1: WSL regression
1:survey on relief
of dry mouth
symptoms
1:effect on
dentine
hypersensitivity
Measured
strength and
quality of
evidence
Quantity and quality
of clinical trial
evidence are
insufficient to make
conclusions regarding
long term
effectiveness of casein
derivatives,
specifically CPP-ACP in
preventing caries in
vivo and treating
dentine
hypersensitivity and
dry mouth
133
DT Zero 2009
Reynolds 2009
Yengopal and
Mickenautsch
2009
To independently
confirm findings of the
in-situ models using
other methods that
closely model clinically
relevant conditions
Included in
vivo/in situ
studies and
clinical trials
To determine the
scientific evidence to
support a role for this
CPP-ACP
remineralization
technology as an
adjunct to F treatment
Includes
original studies
in various
caries model
systems and
clinical trials
Analyse if CPP-ACP
when introduced into
oral environment
provide caries benefit
superior to any other
intervention or placebo
using meta-analysis
English
language
clinical trials
(randomised or
quasirandomised; in
situ or in vivo)
or systematic
reviews (with
or without
meta analysis)
of published
trials reporting
on efficacy of
CPP-ACP using
any mode of
delivery
Studies were
reviewed and
Three in situ
demin models
Eight in situ remin
models
Tabulated along
with the results
discusses
remineralization
increment and
critically views
studies in relation
to methodology,
type of appliance,
simulation of
actual oral
situation
Identified and
divided studies
based on model
and factor
studied.
Includes result as
yes/no
Five in-situ RCT
pooled for meta
analysis
Pooled results
show weighted
mean difference
of percentage
remineralization
The clinical effects of
CPP-ACP in paste form
with or without F have
not yet been
substantiated by
scientific evidence.
Topically applied F
remains the standard
of care for anti-caries
effectiveness
Evidence exists to
support the clinical
use of the CPP-ACP
technology as an
adjunct to F treatment
in the non invasive
management of early
carious lesions
Within the limitations
of this systematic
review with metaanalysis, the results of
the clinical in situ
trials indicate a short
term remineralization
effect of CPP-ACP
CPP-ACP (18.8
mg)> CPP-ACP (10
mg)> plain gum
>placebo
In vivo RCT results
suggest a caries
preventing effect for
long term clinical CPPACP use
One long term
RCT: odds of
caries is 18% less
in subjects
chewing 54mg
Further RCT are
needed in order to
confirm these initial
results in vivo
134
quality
assessed
independently
CPP-ACP than
controls
135
Table V.5: Evidence from published studies investigating CPP-ACP for more
than anti-cariogenic reasons that have been published in the English
literature.
Author/year
Aim
Sample population
Intervention
Outcome
measure
Conclusions
Effect of
incorporating CPPACP into GIC
Extracted human
molars
Box shaped
cavity filled with
GIC acid demin
test
MALDI-MS
(laser
desorption/
mass
spectroscopy)detect CPP in
soln
Incorporation of
CPP-ACP increased
compressive and
microtensile bond
strength
Study
design
Mazzaoui et
al 2003
In vitro
study
1.56% CPP-ACP
incorporated into GIC
Cylinders- to test
comprehensive
strength
Disc-test setting time
Lesion formation:
4 days
ten Cate
&Duijusters 1982
Bar- (half dentine and
half GIC): microtensile
bond strength
Ramalingam Determine
concentration of
et al 2005
CPP-ACP added to
In-vitro
a sports drink that
study
would eliminate invitro erosion
Extracted human third
molars
SEMmicrostructure
of cement
Compressive
strength; microtensile bond
strength
Specimens
exposed to
solution for 30
min
Powerade sports
drink (PA)
PA+CPP-ACP at 4
conc
0.063%,0.09%,
0.125% and
0.25%
SEM
Enhanced release of
Ca, P and F ions and
enhanced protection
of adjacent dentine
from
demineralization
pH increased and
titrable acidity
decreased with
increasing conc of
CPP-ACP
except 0.063% CPPACP, all other
concentrations
effectively eliminate
erosive step lesions
136
Lennon et al Effect of tooth
casein/calcium
2006
phosphate (CaSCP)
In vitro
against erosion
study
Extracted lower
bovine incisors
All specimens
rinsed with 1%
citric acid (pH
2.3) for 30 min
six times a day
(14 days)
Stylus
profilometry
Highly concentrated
AmF gel can protect
enamel against
erosion while 5%
CaSCP and 250 ppm
F provide little
protection when
used individually or
in combination
Stereomicrosco
py to study
enamel
TM reduced erosion
depth in enamel and
dentin/cementum
by 30%
Rinsed
continuously in
artificial saliva
between erosions
Treatment (120
sec twice a day)
Grp 1 :control
Grp 2:CasCP
(120secs)
Grp 3: 250 ppm F
Grp 4: CasCP
+250 ppm F
Grp 5:amine F
(12500 ppm)
Piekarz et al
2008
In-vitro
study
Tantbirojn
et al 2008
In vitro
study
Effectiveness of TM
in reducing erosion
of coronal enamel
and radicular
dentine/cementum
in vitro simulating a
wine judging
session
Extracted human
maxillary premolars
Evaluate if CPP-ACP Extracted bovine
can reharden
incisors
enamel softened by
a cola
Effect of different
Dipped in wine
and artificial
saliva for 1 min
each alternately
using dipping
machine 1500
times
TM effective in
reducing wine
erosion in both
enamel and
dentin/cementum
Test grp:TM
applied every 20
cycles for 4 min,
wash for 2 min
Phase 1:
All specimens
immersed in cola
for 8 min
Knoop hard ness
number
Softened enamel
hardened after 4
applications of CPPACP paste along with
continous artificial
137
saliva substitutes
on enamel
hardness
Treatment:
saliva for 48 hours
CPP-ACP applied:
3 min
Presence of 1 ppm F
did not enhance
hardness
At 0,8,24,36,hrs
Biotene mouthwash
softened enamel
further
Control: no
treatment
Phase II: placed
in 0.4 ml/min
drip with various
saliva
substitutes( 48
hrs)
-saliva with soln
(SLS)
-SLS with 1 ppm F
-Biotene
mouthwash
Mathias et
al 2009
In vitro
study
Evaluate surface
hardness of enamel
after micro
abrasion with or
without using
remineralization
agent, CPP-ACP
Extracted human
anterior teeth
Three groups:
All stored in
artificial saliva for
30 days
Grp
A:Microabrasion
(11% HCl acid
and fine
powdered
pumice slurry)
Surface
profilometric
analysis
A combination of the
micro abrasion
procedure and CPPACP application
reduced the enamel
surface roughness
significantly as
compared to
microabrasion done
alone
Grp B:
microabrasion
followed by CPPACP once a day
for 3 min x 30
days
Grp C: No
preparation
138
Srinivasan
et al 2010
In situ study
Tang and
Miller 2010
Clinical trial
Compare the
remineralization
potential of pastes
containing CPP-ACP
and CPP-ACP with
900 ppm fluoride
on human enamel
softened by a cola
drink
Test if a chewing
gum containing
(CPP-ACP) was
effective in
reducing tooth
sensitivity
associated with inoffice whitening
procedures
Five subjects
Three groups:
Removable midpalatal appliance
CPP-ACP(TM)
Extracted human third
molars
Erosion:
Immersed in 5ml of
fresh cola drink (Coca
Cola, India) (pH 2.3)
for 8 min at room
temperature
88 healthy patients
Surface
microhardness
CPP-ACP with
900 ppm F (TM
plus)
Percentage surface
hardness increases
from post-erosion
stage:
CPP-ACP +900 ppm F
>CPP-ACP >Control
Control group:
saliva
Confirmed the
synergistic effect of
F with CPP-ACP on
remineralization of
eroded enamel
Single application
per day for 3 min
Appliance wore
10 hours/day
removed during
meals
Single visit, in
office whitening
treatment with
15% hydrogen
peroxide
augmented by
light for one hour
Three study
groups:
Grp A: sugar free
gum with CPPACP
Grp B: no agent
Grp C: sugar free
gum without
CPP-ACP
24 hour post
whitening
questionnaire
Sensitivity following
whitening
procedure:
Presence,
duration,
intensity of
sensitivity and
effect of agent
Group A and
C<group B (no
significant difference
between Grp A and
C)
Using sugar free gum
(with and without
CPP-ACP) could
reduce intensity of
tooth sensitivity
associated with in
office whitening
procedures
Chewing one
piece of gum for
10 minutes every
hour – 12 pieces
139
Adebayo et
al 2009
Evaluate
Four groups
20 days
(a) the surface
morphology of acid
etched/conditioned
enamel following
carbamide
peroxide bleaching
with/without
CPP-ACP containing
Tooth Mousse
Grp 1:no treatment
Applied for 60
min daily for 7
days
b) the nature of the
bonded resin–
enamel interfaces
formed with a selfetching primer
adhesive.
four subgroups for
etching/conditioning:
Grp 2:16% carbamide
peroxide bleaching;
Grp 3:CPP-ACP paste;
Grp 4: bleaching and
CPP-ACP paste
Grp A:CSE Primer
only;
The use of a CPPACP paste with or
without prior
bleaching did not
inhibit enamel
etching.
Enamel
etching/conditioning
may help improve
bonding efficiency of
the self-etching
primer adhesive
after CPP-ACP
treatment.
Grp B: 30–40%
phosphoric acid and
CSE primer;
Grp C:15% EDTA and
CSE primer;
Grp D:20% polyacrylic
acid and CSE primer
140