Mode of action studies of a new desensitizing mouthwashcontaining 0.8% arginine, PVM/MA copolymer,pyrophosphates, and 0.05% sodium fluoride

Sarita V. Mello a,*, Evangelia Arvanitidou a, Michael A. Stranick a, Ramon Santana a,Yasemin Kutes b, Bryan Huey b

aColgate-Palmolive Technology Center, 909 River Road, Piscataway, NJ, USAbUniversity of Connecticut, Chemical, Materials & Biomolecular Engineering Department, 97 North Eagleville Road,

Unit 3136, Storrs, CT 06269-3136, USA

j o u r n a l o f d e n t i s t r y 4 1 s ( 2 0 1 3 ) s 1 2 – s 1 9

a r t i c l e i n f o

Article history:

Received 18 July 2012

Received in revised form

30 October 2012

Accepted 1 November 2012

Keywords:

Mouthwash

Arginine

Tooth sensitivity

Dentine occlusion

a b s t r a c t

Objective: The mode of action of an arginine mouthwash using the Pro-ArginTM Mouthwash

Technology, containing 0.8% arginine, PVM/MA copolymer, pyrophosphates and 0.05%

sodium fluoride, has been proposed and confirmed as occlusion using a variety of

in vitro techniques.

Methods: Quantitative and qualitative laboratory techniques were employed to investigate the

mode of action of the new arginine mouthwash. Confocal laser scanning microscopy (CSLM)

and atomic force microscopy (AFM) investigated a hydrated layer on dentine surface. Electron

spectroscopy for chemical analysis (ESCA), secondary ion mass spectroscopy (SIMS) and near-

infrared spectroscopy (NIR) provided information about its chemical nature.

Results: CLSM was used to observe the formation of a hydrated layer on exposed dentine

tubules upon application of the arginine mouthwash. Fluorescence studies confirmed pene-

tration of the hydrated layer in the inner walls of the dentinal tubules. The AFM investigation

confirmed the affinity of the arginine mouthwash for the dentine surface, supporting its

adhesive nature. NIR showed the deposition of arginine after several mouthwash applications,

and ESCA/SIMS detected the presence of phosphate groups and organic acid groups, indicating

the deposition of copolymer and pyrophosphates along with arginine.

Conclusion: The studies presented in this paper support occlusion of the dentine surface

upon the deposition of an arginine-rich layer together with copolymer and phosphate ions

from an alcohol-free mouthwash containing 0.8% arginine, PVM/MA copolymer, pyropho-

sphates and 0.05% sodium fluoride.

# 2012 Elsevier Ltd.

Available online at www.sciencedirect.com

journal homepage: www.intl.elsevierhealth.com/journals/jden

Open access under CC BY-NC-ND license.

1. Introduction

Tooth sensitivity has been recognized as one of the earliest

maladies of the mouth reported in history. Tooth sensitivity is

* Corresponding author at: Colgate-Palmolive Technology Center, 909

fax: +1 7328787365.E-mail address: sarita_mello@colpal.com (S.V. Mello).

0300-5712 # 2012 Elsevier Ltd.

http://dx.doi.org/10.1016/j.jdent.2012.11.001

Open access under CC BY-NC-ND license.

a common name for dentine hypersensitivity or root sensitiv-

ity. It is estimated that up to 57% of the population in

the United States suffers from dentine hypersensitivity.1

Dentine hypersensitivity is characterized by short, sharp pain

arising from exposed dentine in response to stimuli, typically

River Road, Piscataway, NJ 08855-1343, USA. Tel.: +1 7328787883;

j o u r n a l o f d e n t i s t r y 4 1 s ( 2 0 1 3 ) s 1 2 – s 1 9 S13

thermal, evaporative, tactile, osmotic or chemical and which

cannot be ascribed to any other dental defect or pathology.2

Diverse in-office treatments have been reported to address

dentine hypersensitivity, such as desensitizing pastes, fluo-

ride varnishes, and restorative resins.3–13 Over-the-counter

products that treat dentine hypersensitivity are normally

toothpastes formulated with potassium ions or dentine

occlusion agents.14–21 The mode of action of desensitizing

products can be divided in two main groups: (1) nerve

desensitization by potassium ions and (2) decrease in dentine

permeability by occlusion agents. Historically, marketed anti-

hypersensitivity toothpastes were recommended for a mini-

mum of two weeks use, in order to allow the accumulation of

an effective concentration of potassium ions to desensitize the

nerve endings. The over-the-counter occlusion treatment

approach has been traditionally reported as the deposition of

solid particles or mineralized crystals, such as stannous,

strontium and oxalates.4,5 More recently, a breakthrough

occlusion technology containing arginine and calcium car-

bonate has been formulated into toothpaste and in-office

products.7,20–24

In vitro investigations of dentine occlusion systems are

commonly carried out via Pashley’s hydraulic conductance

method25–29 and surface analysis techniques.3,23 Also, Kolker

et al.30 studied the in vitro performance of different synthetic

resins, showing effective reduction of hydraulic conductance

in the range of 40–60%, for different coatings with diverse

chemical compositions. In contrast to mineral solids occlu-

sion, the mechanism of action of adhesive coatings depends

on the physical adsorption of organic materials, their diffusion

through porous dentine, the chemical bonding or curing and

electrostatic interactions. The viscosity and contact time of

the polymeric layer are also important to allow the coating

effect (film-forming) and ensure enough cohesive forces to

increase the life-time of the coating. Most recently, hydraulic

conductance studies have demonstrated a significant reduc-

tion in fluid flow for a toothpaste23 and for a mouthwash.31

Although a variety of dental products have been employed

successfully to address dentine hypersensitivity, there is still

an absence of effective mouthwash products and only a few

scientific papers have been published.32–35 Herein we present

the in vitro evaluation of a new mouthwash designed to

effectively reduce dentine hypersensitivity through the

occlusion of the dentine surface via a clinically proven

formulation of arginine, PVM/MA copolymer and pyropho-

sphates.36,37 Polymethylvinyl ether/maleic acid (PVM/MA)

copolymer and pyrophosphates have been associated with

dentine occlusion in toothpaste formulations by Miller et al.38

and Mason et al.39 based on in vitro measurements of

hydraulic conductance and surface analysis techniques. The

mechanism of occlusion presented here differs from Miller

et al.38 due to several factors, especially the presence of

arginine, other formula ingredients, pH, and delivery in the

absence of brushing (toothpaste vs mouthwash). The mode of

action of the new mouthwash containing the Pro-ArginTM

Mouthwash Technology is based on occlusion of the dentine

tubules from the deposition of arginine, PVM/MA copolymer

and polyphosphates onto the dentine, which after repeated

application, decreases dentine permeability and reduces pain-

causing stimuli. The mechanism of action was investigated via

several laboratory techniques, namely confocal laser scanning

microscopy (CSLM), atomic force microscopy (AFM), electron

spectroscopy for chemical analysis (ESCA), secondary ion

mass spectroscopy (SIMS) and near-infrared spectroscopy

(NIR).

2. Materials and methods

2.1. Test products

The test mouthwash (‘‘arginine’’) consisted of 0.8% arginine,

polyvinylmethyl ether/maleic acid (PVM/MA) copolymer,

pyrophosphates and 0.05% sodium fluoride (Colgate-Palmol-

ive Company, New York, NY) in an alcohol-free base. The

control mouthwash (negative control) (Colgate-Palmolive

Company, New York, NY) did not contain any of the

ingredients mentioned above. In addition, a commercial

fluoride toothpaste (Colgate-Palmolive Company, New York,

NY) was employed during one of the evaluations.

2.2. Dentine disc preparation

Dentine discs used in all tests were prepared from extracted

human molars. An average of 3 discs per molar were cut and

prepared accordingly to each to be used in different experi-

ments. At least 6 discs were randomly assigned per sample in

all tests. Specimens, 0.8 mm thick, were obtained by using a

slow-speed saw turning a diamond wafering blade (Isomet,

Lake Bluf, USA). Afterwards, discs were sanded 600 grit paper

discs (Carbimet, Buehler, Lake Bluf, USA) and polished with

soft plane cloth (P4000, Silicon Carbide using a Variable Speed

Grinder-Polisher EcoMet1 3000, Buehler, Lake Bluf, USA).

Each dentine disc was sonicated in deionized water and

dipped into 6% (w/v) citric acid for 1 min to remove the smear

layer.28

2.3. Surface and chemical characterization

2.3.1. Confocal laser scanning microscopy (CLSM)A Leica TCS SP confocal laser scanning microscope equipped

with a spectral detection system was used to visualize the

dentine surfaces. The 488 nm line from an argon laser along

with a PLO APO 50� objective was used in all experiments.

Dentine discs were prepared according to the procedure

described above. Images were taken with a 50� objective and

optical zoom of 2�. Distinct treatment procedures were

developed for each detection mode in order to assess the

relevant aspect of the coating mechanism, i.e. a thicker

coating to allow the tubule blocking visualization (10 applica-

tions) and a thinner coating (2 applications) for the fluor-

ophore detection inside the tubules.

Reflectance mode: on dentine discs pre-conditioned in

phosphate buffer saline (PBS), 15 mL of mouthwash was

applied 10 times for 1 min, with intervals of 10 min

between each application.

Fluorescent mode: fluorescein–cadaverine (molecular probes)

was added to both mouthwash samples prior to treatment,

to a final concentration of 0.4 mM. A mouthwash aliquot of

j o u r n a l o f d e n t i s t r y 4 1 s ( 2 0 1 3 ) s 1 2 – s 1 9S14

15 mL was added to the dentine surface and left to dry for

10 min. The same procedure was repeated one more time

followed by a gentle rinse with PBS.

2.3.2. Atomic force microscopy (AFM)Atomic force microscopy (AFM) is a high-resolution, non-

destructive laboratory technique that allows imaging of nano

structures by mapping topography and/or other properties

using a tip with a nanoscale apex.40 When working with soft

samples such as polymer, biological and dental materials,41–

43 the AFM is typically operated in ‘AC’ mode, where the probe

is vibrated near a specimen such that they measurably

interact �10,000–300,000 times per second. A constant

interaction amplitude (and thus force gradient) is then

maintained by raising or lowering the tip as needed, which

when scanning the sample thus provides images of the

surface height with sub-nanometer resolution in the x, y, and

z directions.44

For the specimens considered here, the arginine mouth-

wash was coated on tooth substrates with a spin coating

technique to create uniform layers.45 Each layer was spun with

3000 rpm for 30 s. The samples were then mounted on a glass

slide and kept at 37 8C until the images were taken.

2.3.3. Near infrared (NIR)Baseline spectra were taken prior to arginine mouthwash.

Samples were evaluated in triplicate. Treatment was per-

formed as follows:

1. 10 mL of mouthwash was applied to disc surface and left for

10 min on a hot plate at 37 8C.

2. Disc was rinsed with PBS buffer (3� dipping for 1 s).

3. Buffer excess was removed with air stream parallel to the

disc surface and taken immediately to the NIR instru-

ment.

4. Following spectra acquisition, a new mouthwash treatment

was applied to the disc.

2.3.4. Electron spectroscopy for chemical analysis (ESCA) andsecondary ion mass spectroscopy (SIMS)All dentine discs were analysed prior to treatment to obtain

baseline compositions. For ESCA, three separate areas per disc

were analysed, while for SIMS one area encompassing most of

the disc was analysed. The same discs were analysed by both

ESCA and SIMS. For the untreated dentine, the ESCA results

represent the average compositions for the four discs studied,

while the SIMS values are the averages for two of the discs

studied. The ESCA and SIMS results shown for the treated

discs represent the average values for two discs of each

treatment regimen.

The Test treatment consisted of the commercial fluoride

toothpaste and the arginine mouthwash, and the control

treatment consisted of the commercial fluoride toothpaste

and the negative control mouthwash.

Treatments were performed as follows:

1. Dentine surface was wetted with PBS.

2. Surface was gently brushed with toothpaste for 30 s.

3. Disc was air dried for 2 min, and then moved to a beaker

containing PBS with mild agitation for 1 min.

4. Disc was removed from PBS, tapped dry on the back

(untreated surface) to remove excess fluid and then placed

in beaker containing mouthwash for 2 min under mild

agitation.

5. Disc was removed from the mouthwash and air dried for

2 min. Excess mouthwash was gently removed with a small

brush.

6. Two drops of PBS was placed on the surface and the excess

of liquid was gently removed from the surface with a small

soft brush. Film was air dried for 30–40 min. Steps 4–6 were

repeated once.

3. Results

3.1. CLSM

Images shown in Figs. 1 and 2 represent dentine discs after ten

1-min treatments using the arginine mouthwash and the

negative control mouthwash, respectively. There was a 10-

min interval between each treatment. Reflectance mode

(Fig. 1a and b) was used to assess the surface effect while

fluorescent mode (Fig. 2a and b) investigated the penetration

of the Arginine mouthwash into open tubules. Fig. 1a shows a

continuous image after treatment with the arginine mouth-

wash, indicating complete coverage of the open dentine

tubules. The solid line of the xz view indicates the surface

effect, i.e. the light source cannot penetrate the tubules. Fig. 1b

shows the open tubules before and after treatment with

negative control mouthwash. A non-continuous line on the xz

view confirms the openness of the tubules.

Fig. 2a shows the fluorescence images taken after treat-

ment with the arginine mouthwash. The bright points indicate

the presence of the arginine mouthwash throughout the

surface. The side view shows the penetration of the arginine

mouthwash inside the tubule walls, indicating the affinity of

the mouthwash to the dentine surface. The same experiment

was performed with the negative control mouthwash shown

in Fig. 2b. In contrast to the arginine mouthwash, very little

evidence of mouthwash on the surface (xy image) in the

tubules (xz image) could be visualized for the negative control

mouthwash. These images prove that the arginine mouth-

wash penetrates inside the tubules as well as deposits on the

surface.

3.2. NIR

Arginine has a unique fingerprint in the NIR region

(�1530 nm), allowing its identification on the dentine surface.

Fig. 3 shows the spectra obtained on the dentine surface before

treatment (baseline) and after 3, 7 and 13 treatments with the

arginine mouthwash. The presence of arginine molecules was

identified by the 3rd treatment, and their concentration

increased upon deposition with subsequent treatments.

3.3. AFM

The ability of the Arginine mouthwash to deposit onto

dentine surfaces has also been explored by AFM. The high

resolution of AFM even allowed for imaging the fine structure

in the vicinity of a single dentine tubule (Fig. 4). The

Fig. 1 – (a) CLSM images (reflectance mode) of dentine surface treated with arginine mouthwash. Image scale

100 mm T 100 mm. (b) CLSM images (reflectance) of dentine surface treated with negative control mouthwash. Image scale

100 mm T 100 mm.

j o u r n a l o f d e n t i s t r y 4 1 s ( 2 0 1 3 ) s 1 2 – s 1 9 S15

5 mm � 5 mm images of single tubules, before and after

coating, indicate that the arginine mouthwash is deposited

onto the dentine surface. The occlusion occurs by a buildup

mechanism of the arginine mouthwash in the tubules after

several applications.

3.4. ESCA/SIMS

ESCA/SIMS assessed the chemical composition on dentine

after treatment with a mouthwash in conjunction with a

commercial fluoride toothpaste, in order to mimic human use.

As shown in Table 1, the compositions of the discs before

treatment were consistent with those for demineralized,

Table 1 – ESCA results.

Sample Atomic

C O N Ca

ESCA – surface composition

Untreated dentine

2, 3, 10, 11 Avg. 61.30 21.16 16.30 0.74

Fluoride toothpaste/Negative Control MW – 20 treatments

2, 10 Avg. 36.88 39.85 5.77 9.43

Fluoride toothpaste/Arginine MW – 20 treatments

3, 11 Avg. 58.03 30.04 2.28 2.24

untreated dentine: high surface protein with low calcium (Ca)

and phosphorus (P) levels. After treatment, the sodium (Na)

levels for the Test discs were greater than those for Negative

Control discs. Potassium (K) was detected for Test discs only.

The P/Ca ratio was also greater for Test discs compared to

negative control discs. The Na, K and P/Ca results indicate the

deposition onto the dentine surface of the tetrapotassium

pyrophosphate and tetrasodium pyrophosphate (pyropho-

sphates) present only in the arginine mouthwash. Elevated

carbon (C) levels with reduced N, Ca, and P were observed for

the Test discs compared to the negative control discs. This is

indicative of the presence of an organic material on the Test

discs. Also, the nitrogen (N) spectra for the Test discs differed

percent Ratio

P Na F Si K P/Ca N+/N

0.50 – – 0.00 – 0.67 0.030

6.92 0.25 0.44 0.93 – 0.71 0.019

2.97 1.16 0.11 1.86 1.30 1.49 0.095

Fig. 2 – (a) CLSM images (fluorescence mode) of dentine surface treated with arginine mouthwash. Image scale

100 mm T 100 mm. (b) CLSM images (fluorescence mode) of dentine surface treated with the negative control mouthwash.

Image scale 100 mm T 100 mm.

j o u r n a l o f d e n t i s t r y 4 1 s ( 2 0 1 3 ) s 1 2 – s 1 9S16

in line shape from those for the negative control discs, because

of the greater amount of charged nitrogen, N+. This difference

is reflected in the N+/N ratios shown in Table 1. Higher N+ is

consistent with that observed in data for arginine reference

Fig. 3 – NIR spectra of dentine surface t

films, and indicates the presence of arginine on the surfaces of

the Test discs. SIMS analysis (Table 2) of the treated dentine

revealed the presence of a mass peak at 175 amu after

treatment with the Test treatment. This peak was not

reated with Arginine mouthwash.

Fig. 4 – AFM topography images of a dentinal tubule before and after treatment with the arginine mouthwash.

j o u r n a l o f d e n t i s t r y 4 1 s ( 2 0 1 3 ) s 1 2 – s 1 9 S17

observed on untreated dentine or on dentine treated with the

negative control mouthwash. The mass peak at 175 amu is

consistent with that for the arginine ion and confirms

deposition of arginine on the surfaces of the Test treated

discs. SIMS analysis of PVM/MA copolymer reference films

revealed mass peaks from fragments of the copolymer at 45

and 59 amu. SIMS analysis of the Test treated discs revealed an

increase in the 45/43 and 59/43 peak intensity ratios, compared

to the negative control treated discs. The increases in these

ratios above the background values for the baseline suggests

PVM/MA copolymer deposition.

4. Discussion

A new alcohol-free mouthwash, with 0.8% arginine, PVM/MA

copolymer, pyrophosphates, and 0.05% sodium fluoride has

been designed to alleviate dentine hypersensitivity by occlu-

sion. To the best of our knowledge, this is the first time

a mouthwash has been effective on addressing dentine

hypersensitivity via occlusion mechanism with a unique

combination of a complex formed by copolymer/aminoacid/

pyrophosphate salts. Laboratory studies indicate the forma-

tion a micro-adhesive layer on the dentine surface comprised

arginine, PVM/MA copolymer and pyrophosphates. Arginine

and the PVM/MA copolymer form an adhesive complex, which

Table 2 – SIMS results.

Sample Gantrez pe

45/43

Untreated dentine

10, 11 Avg. 0.098

Fluoride toothpaste/Negative Control MW – 20 treatments

2, 10 Avg. 0.069

Fluoride toothpaste/Arginine MW – 20 treatments

3,11 avg. 1.823

PVM/MA copolymer – fragment positive ions at 45 and 59 amu; hydroca

175 amu.

deposits onto the dentine surface. The pyrophosphates

promote the complex’s stability in solution and are deposited

together onto dentine surface, leading to a coherent layer after

multiple mouthwash applications to dentine specimens. The

overall result is an arginine/copolymer/phosphate deposit on

the demineralized dentine surface. Experimental challenges

of characterizing a hydrated polymeric layer have been

addressed by using a range of qualitative and quantitative

techniques already established to investigate dentine occlu-

sion as shown in the work of Petrou et al.24.

In addition, hydraulic conductance studies have shown

that this new mouthwash provides a significant reduction in

fluid flow, consistent with an occlusion mechanism.31 The

occlusion of dentine tubules with this arginine mouthwash is

the basis of the dentine hypersensitivity relief demonstrated

in two clinical studies.36,37 In this paper, CLSM confirmed the

ability of the Arginine mouthwash to form a layer on the

dentine surface (reflectance) as well as within the internal

walls of the dentine tubules (fluorescence). Based on the

fluorophore chemistry, which is designed to interact with

carboxylic groups, the fluorescence detected is due to arginine

and copolymer deposition inside the dentine tubules as well as

on the dentine surface. One important aspect of the CLSM

study was the ability to study the deposition of the arginine

mouthwash onto dentine in open air and after a relatively

short time after treatment (30 min). Arginine is drawn to the

ak intensity ratios Arginine peak (amu)

59/43 175

0.164 �

0.041 �

0.514 +

rbon reference peak at 43 amu. Arginine – molecular positive ion at

j o u r n a l o f d e n t i s t r y 4 1 s ( 2 0 1 3 ) s 1 2 – s 1 9S18

dentine surface, serving as the anchor for subsequent

copolymer deposition. Studies using NIR spectroscopy confirm

arginine deposition on the dentine surface upon application of

the arginine mouthwash. As seen using AFM, the images of a

single tubule indicate that the arginine mouthwash is present

on the dentine surface, and occlusion happens by a buildup

mechanism of the arginine mouthwash in the tubules after

several applications. This may explain the lack of a hyper-

sensitivity effect within the first 30 min of using the arginine

mouthwash for the first time.37

Finally, the ESCA and SIMS results indicate the presence of

an organic material on the discs with the Test treatment. Also,

the nitrogen spectra for these discs differed in line shape from

those for the negative control treated discs. The discs treated

with the Test treatment containing the arginine mouthwash

exhibited a greater amount of charged nitrogen than the

Negative Control treated discs.

5. Conclusion

A new hypersensitivity mouthwash based on the Pro-ArginTM

Mouthwash Technology containing 0.8% arginine, PVM/MA

copolymer, pyrophosphates and 0.05% sodium fluoride has

been developed. In vitro studies confirm the mechanism of

action as occlusion. CLSM captured images of the hydrated

layer on the dentine surface. Fluorescence studies confirmed

penetration of the hydrated layer in the inner walls of the

dentine tubules. ESCA confirmed deposition of arginine and

phosphates onto the dentine surface. AFM confirmed this

observation, even identifying tubules that are spanned by the

mouthwash. SIMS analysis confirmed the presence of arginine

and organic groups which are indicative of copolymer

deposition. NIR confirmed the presence of arginine on dentine

surface upon treatment with the arginine mouthwash. The

occlusion from the arginine mouthwash has been achieved

due to a formula design that allows for the deposition of

arginine and phosphates embedded in a thin polymeric

matrix. This unique formulation is an effective system to

address dentine hypersensitivity.

Conflicts of interest

None declared.

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