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BACTERIAL CONTAMINATION OF TOOTHBRUSHES
HYGIENE AND PREVENTION

 
Andrea Felloni, Eugenio Brambilla, Maria Grazia Cagetti, Laura Strohmenger
University of Milan, Department of Medicine,
Surgery and Odontology, License in Odontology and Dental Prosthetics, Chair of Pedodontics
Full Professor L. Strohmenger
SUMMARY:The toothbrush is the most important and common means of prevention
against pathologies caused by bacterial plaque but, by coming into contact with
saprophytic and pathogenic microorganisms found in the oral cavity,
it undergoes consistent contamination, which represents an infectious load for uses
successive. With a view to horizontal prevention of dental caries, the authors
examined residual microbial contamination of Streptococcus mutans after use
daily, comparing a traditional brush with a silver head brush through
an experimental clinical study. Although the antimicrobial activity of the silver coating
of the removable head of this brush has already been confirmed by in vitro studies, it was necessary
to collect further data regarding wild strains of Streptococcus mutans.
A case-control clinical study was carried out on 20 randomized patients (mean age 24+_0.3)
in an experimental group (n=10) and a control group (n=10). After a period of use at
home for 3 days, the toothbrushes were returned and the heads were kept in
atmosphere saturated with water vapor to prevent changes to the bacterial flora.
We therefore carried out two samplings, at the end of the clinical phase and after a period of 6 hours,
on different tufts for each head, seeded on MSB agar plate and incubated.
At the end of the incubation, the developed colonies were counted. The data obtained puts
highlight the colonization capacity of the toothbrush surfaces as well as the activity
antibacterial silver ions deposited on the surface of the head after normal use of the brush at home.
KEY WORDS: silver, prevention, toothbrush, Streptococcus mutans

L

Caries prevention is based on the control and eliminationation of bacterial plaquene from the surfaces of the tooth.
The toothbrush represents the most important and common means of prevention against pathologies
caused by dental plaque.
The ita marketlink offers a wide variety of models, different in shape, material and
technical characteristics; significant differences can be noted in the elimination efficiency
of plaque and in the abrasive effect on hard and soft oral tissues.
A problem whose importance has increased in recent years is contamination
residual from the brush after use.
Each brush, according to the specifications of the British Standard1, is made up of different parts,
each with a well-defined name:
HYGIENE AND PREVENTION
The brush (the part of the toothbrush that includes the tufts of bristles).
Tufts of hair (the aggregate of filaments fixed in a hole on the base).
The base (the extension of the handle which supports the tufts of hair).
The head (the whole formed by the base and the tufts of hair).
The handle (the part of the toothbrush except the head).
The head in particular comes into contact, in the oral cavity, with a multiplicity of saprophytic microorganisms and,
in certain specific pathogenic cases, undergoing more or less consistent contamination.
In addition to the infectious load with which it comes into contact, residual contamination of the head arises from certain
variables linked to its structure, and which substantially express the capacity to retain water between the hairs, and therefore
to keep contaminating microorganisms vital, after use1-5.
Experimental studies have demonstrated how the quantity of microorganisms retained and kept vital
also depends on the treatment to which the toothbrush is subjected after use, i.e. rinsing and drying6.
Most of the microorganisms with which toothbrushes become contaminated during normal use are shown
by saprophytic species in the oral cavity; these can, however, become the vector of microorganisms
pathogens when the brushes are used by subjects affected by infectious systemic pathologies such as
viral hepatitis or AIDS 1,3,5,6.
Regarding dental caries, it should be emphasized how Streptococcus mutans, the cariogenic microorganism
most importantly, can be easily transferred from the oral cavity of one subject to that of another subject through
using the same toothbrush. The microorganism demonstrates, in fact, that it can survive for periods
relatively long on the toothbrush, maintaining sufficient vitality and infectious load to be
re-implanted in the oral cavity of the person who subsequently uses the instrument2; this phenomenon is of importance
particular in the longitudinal transmission (mother - son) of S infection. mutans
On the national and international market there are toothbrushes that have antibacterial activity and
specific instruments to reduce residual contamination, indicator of the sensitivity of certain companies to
health needs expressed by society.
The present study aims to examine the residual microbial contamination of S. mutans after daily use,
comparing a traditional toothbrush and a brush with a silver head.
MATERIALS AND METHODS
We selected 20 patients (average age 24+_0.3), with a minimum of 24 dental elements, relating to the Clinic
Odontostomatology from the Department of Medicine, Surgery and Odontology.
In order to be included in the experimental protocol, subjects must not have been subjected to antibiotic therapy
for at least 30 days, nor having taken other medications which could have directly or indirectly influenced the balance
oral flora; they should not be subjected to topical treatment with clorexidine or fluorides;
they should not wear removableviable prostheses, implants or orthodontic appliances.
For the purpose of the study, two toothbrush models on sale on the Italian market were taken into consideration:
one with silver head (Silvercare, Spazzolificio Piave, Onara di Tombolo) and a traditional type control brush
with the number of tufts and the dimensions of the head similar, for each model 10 samples were purchased.
On the day the study began, selected subjects were asked to complete and sign the informed consensus for
participation in research.
The subjects were therefore randomly assigned to the Silvercare group (n = 10) and the control group (n = 10)
and everyone was given a toothbrush corresponding to their group. Participants were asked
use your own toothbrush twice a day (morning and evening) using the modified Bass brushing technique,
for the overall duration of 3 minutes, refraining from the use of toothpaste.
After a period of use equivalent to 3 days, the toothbrushes were returned to the laboratory,
after the last brushing carried out just before the start of microbiological procedures.
Under the laminar flow hood and under sterile conditions, the heads of each brush were separated from the handle thereof,
then they were placed separately in sterile test tubes containing 0.5 ml of sterile PBS (Sigma Chemicals, St. Louis, MO, USA)
in order to create an atmosphere saturated with water vapor to prevent the evaporation of liquids retained by the sample.
BACTERIAL CONTAMINATION OF TOOTHBRUSHES
HYGIENE AND PREVENTION

 
Andrea Felloni, Eugenio Brambilla, Maria Grazia Cagetti, Laura Strohmenger
University of Milan, Department of Medicine,
Surgery and Odontology, Degree in Odontology and Dental Prosthetics, Chair of Pedodontics
Full Professor L. Strohmenger
SUMMARY:The toothbrush is the most important and common means of prevention
against pathologies caused by bacterial plaque but, by coming into contact with
saprophytic and pathogenic microorganisms found in the oral cavity,
it undergoes consistent contamination, which represents an infectious load for uses
successive. With a view to horizontal prevention of dental caries, the authors
examined residual microbial contamination of Streptococcus mutans after use
daily, comparing a traditional brush with a silver head brush through
an experimental clinical study. Although the antimicrobial activity of the silver coating
of the removable head of this brush has already been confirmed by in vitro studies, it was necessary
to collect further data regarding wild strains of Streptococcus mutans.
A case-control clinical study was carried out on 20 randomized patients (mean age 24+_0.3)
in an experimental group (n=10) and a control group (n=10). After a period of use at
home for 3 days, the toothbrushes were returned and the heads were kept in
atmosphere saturated with water vapor to prevent changes to the bacterial flora.
We therefore carried out two samplings, at the end of the clinical phase and after a period of 6 hours,
on different tufts for each head, seeded on MSB agar plate and incubated.
At the end of the incubation, the developed colonies were counted. The data obtained puts
highlight the colonization capacity of the toothbrush surfaces as well as the activity
antibacterial silver ions deposited on the surface of the head after normal use of the brush at home.
KEY WORDS: silver, prevention, toothbrush, Streptococcus mutans

L

Prevention of caries is based on the control and eliminationation of bacterial plaquene from the surfaces of the tooth.
The toothbrush represents the most important and common means of prevention against pathologies
caused by dental plaque.
The ita marketlink offers a wide variety of models, different in shape, material and
technical characteristics; significant differences can be noted in the elimination efficiency
from plaque and in the abrasive effect on hard and soft oral tissues.
A problem whose importance has increased over timeurs of recent years is contamination
residual of the brush after use.
Each brush, according to the specifications of the British Standard1, is made up of different parts,
each with a well-defined name:
HYGIENE AND PREVENTION
The brush (the part of the toothbrush that includes the tufts of bristles).
Tufts of hair (the aggregate of filaments fixed in a hole on the base).
The base (the extension of the handle which supports the tufts of hair).
The head (the whole formed by the base and the tufts of hair).
The handle (the part of the toothbrush except the head).
The head in particular comes into contact, in the oral cavity, with a multiplicity of saprophytic microorganisms and,
in certain specific pathogenic cases, undergoing more or less consistent contamination.
In addition to the infectious load with which it comes into contact, residual contamination of the head arises from certain
variables linked to its structure, and which substantially express the capacity to retain water between the hairs, and therefore
to keep contaminating microorganisms vital, after use1-5.
Experimental studies have demonstrated how the quantity of microorganisms retained and kept vital
also depends on the treatment to which the toothbrush is subjected after use, i.e. rinsing and drying6.
Most of the microorganisms with which toothbrushes become contaminated during normal use are shown
by saprophytic species in the oral cavity; these can, however, become the vector of microorganisms
pathogens when the brushes are used by subjects affected by infectious systemic pathologies such as
viral hepatitis or AIDS 1,3,5,6.
Regarding dental caries, it should be emphasized how Streptococcus mutans, the cariogenic microorganism
most importantly, can be easily transferred from the oral cavity of one subject to that of another subject through
using the same toothbrush. The microorganism demonstrates, in fact, that it can survive for periods
relatively long on the toothbrush, maintaining sufficient vitality and infectious load to be
re-implanted in the oral cavity of the person who subsequently uses the instrument2; this phenomenon is of importance
particular in the longitudinal transmission (mother - son) of S infection. mutans.
On the national and international market there are toothbrushes that have antibacterial activity and
specific instruments to reduce residual contamination, indicator of the sensitivity of certain companies to
health needs expressed by society.
The present study aims to examine the residual microbial contamination of S. mutans after daily use,
comparing a traditional toothbrush and a brush with a silver head.
MATERIALS AND METHODS
We selected 20 patients (mean age 24+_0.3), with a minimum of 24 dental elements, relating to the Clinic
Odontostomatology from the Department of Medicine, Surgery and Odontology.
In order to be included in the experimental protocol, subjects must not have been subjected to antibiotic therapy
for at least 30 days, nor having taken other medications which could have directly or indirectly influenced the balance
oral flora; they should not be subjected to topical treatment with clorexidine or fluorides;
they should not wear removableviable prostheses, implants or orthodontic appliances.
For the purpose of the study, two toothbrush models on sale on the Italian market were taken into consideration:
one with silver head (Silvercare, Spazzolificio Piave, Onara di Tombolo) and a traditional type control brush
with the number of tufts and the dimensions of the head similar, for each model 10 samples were purchased.
On the day the study began, selected subjects were asked to complete and sign the informed consensus for
participation in research.
The subjects were therefore randomly assigned to the Silvercare group (n = 10) and the control group (n = 10)
and everyone was given a toothbrush corresponding to their group. Participants were asked
use your own toothbrush twice a day (morning and evening) using the modified Bass brushing technique,
for the overall duration of 3 minutes, refraining from the use of toothpaste.
After a period of use equivalent to 3 days, the toothbrushes were returned to the laboratory,
after the last brushing carried out just before the start of microbiological procedures.
Under the laminar flow hood and under sterile conditions, the heads of each brush were separated from the handle thereof,
then they were placed separately in sterile test tubes containing 0.5 ml of sterile PBS (Sigma Chemicals, St. Louis, MO, USA)
in order to create an atmosphere saturated with water vapor to prevent the evaporation of liquids retained by the sample.
We then carried out two samplings, respectively at the end of the clinical phase (time 0)
and after a period of 6 hours (time 1), introducing in the center of a tuft of each
HYGIENE AND PREVENTION
T-TEST RESULTS TABLE 1
VARIANCE ANALYSIS

GL

Sum of squares

Mean of squares

F

p

λ

Power

Toothbrushes

1

0.096

0.096

5,779

0.0272

5,779

0.621

Subject (Group)

18

0.300

0.017

Category for Time

1

1.215

1.215

69,679

<0.0001

69,679

1,000

Category for Time* Toothbrush

1

0.084

0.084

4,799

0.0419

4,799

0.537

Category for Time* Subject Group

18

0.314

0.017

Account

Average

DS

ES

Control

20

1,570

0.193

0.043

Silvercare

20

1,472

0.251

0.056

TABLE 2
sample 10 μl of sterile physiological solution and recover them using a micropipette. The recovered liquid
was diluted in 1 ml of physiological solution and 20 μl of the final suspension was inoculated on platelets of
MSB agar, incubated for 48 hours at 37°C.
At the end of the incubation, the developed colonies were counted.
STATISTICAL ANALYSIS
For statistical analyses, the Statview 5.0 program for Windows was used (SAS Institute Ine, Cary, North Carolina, USA).
To assess the comparability of the two groups at time 0, a t-test for unpaired data was carried out.
To evaluate the reduction in concentration over time, the ANOVA test for repeated measures was used.
The level of significance was set for a probability p < 0.05.
RESULTS
In order to assess the comparability of the data, we carried out the static analysis at time 0 (table 1) using the t-test
for unpaired data; from this datas we can observe er that the averages of the relative concentrations
to the two toothbrushes at the end of the clinical period are superimposable (p = 0.919). From ANOVA
data
(table 2) we note that, although the reduction in concentrations present on the tufts of hair depends
mainly of the time variable (p < 0.0001), the two types of brushes express different behavior
statistically significant (p = 0.027) (figure 1).
DISCUSSION AND CONCLUSIONS
Preventive dentistry, in concomitance with the decline of caries pathology in industrialized countries
as a result of the efforts made over the previous twenty years and thanks to recent scientific research,
has identified in recent years a new objective on which to focus available resources: identification
and the treatment of “subjects at risk”, that is to say the particular group of individuals who present a development
pathology significantly higher than the norm.
Biological research has identified S. mutans as the main etiological agent of the disease because of its capacity
to produce these metabolite acids responsible for the dissolution of dental enamel at the beginning of development
of the carious cavity, even if in a broader concept of multifactorial pathology it has been demonstrated that other
species of bacteria have cariogenic power7.
The different response of the organismme in relation to the altered balance of the oral ecosystem in subjects identified as
at risk makes it essential, in addition to the identification of said subjects, the implementation of preventive measures capable of reducing
“infection” from pathogenic bacteria.
The concept of transmissibility of caries pathology8 has regained force in recent years in association with
instruments intended for personal oral hygiene, in the absence of structures and controls capable of preventing cross-contamination.
While the implementation of preventive measures during pregnancy9, in order to reduce the vertical transmission of
infection of S. mutans, is easy to achieve, on the other hand the means capable of reducing horizontal transmission are
few in number. To reduce residual bacterial contamination on toothbrushes to prevent contamination
crossed between different subjects in a community context, such as the family community or like communities
school, the industry has tried to satisfy this health need by researching different techniques for hygienizing toothbrushes.
The use of metals with microbicidal activity dates back to the end of the last century when the first experimental observations were made
subsequently confirmed at the beginning of the century through a more precise oligodynamic classification of metal ions10.
The mechanism of action of metals in an aqueous solution, colloidal metals, on microbial particles is multiple,
and more recent studies11 attribute the microbicidal activity to the penetration of the mcitoplasmatic embrane and inhibition
of the enzymatic activity of some protein classes. The antimicrobial activity of the silver coating of the removable head
of the Silvercare brush compared to the numerous pathogenic and saprophytic bacterial species present in the cavity
buccal has already been experimentally confirmed in vitro10, but in relation to S. mutans further data was required
compared to in vivo contamination with wild strains of said bacterial species. The data obtained by the protocol
experimental relating to the clinical colonization of S. mutans show that said microbial species is able to
colonize the surfaces of instruments used during daily oral hygiene operations at home.
It can further be deduced that the activity of the silver ions deposited on the surface of the Silvercare head is able to reduce
in a statistically significant manner, after a period of time equivalent to the normal time interval which elapses
between oral hygiene maneuvers at home, the concentration of S. mutans present on the head of the toothbrush itself.
Although our data only relates to the colonization of tufts of hair, they can be compared with those
in vitro studies. The observed variability must be sought in the possible interaction of the agonist active ingredients by
relation to colonization itself. Studies available in the literature provide little indication about the effect of
toothpastes2 on the bacterial colonization pattern; in fact, the adhesive capacity of different bacterial species
may undergo variations due to the use of toothpastes with antibactericidal, surfactant components, etc. in quantity
such that they modify the decontamination pattern that we have noted.
Correspondence to: dott. Andrea Felloni Department of Medicine,
Surgery and Odontology,
Chair of Pedodontics
Via Beldiletto 1/3, 20142 Milano e-mail: Andrea.Felloni@unimi.it
BIBLIOGRAPHY
  1. Dayoub MB, Rusilko D, Gross A. Mi-crobial contamination of toothbrushes. J Dent Res 1977;56(6):706-7.
  2. Svanberg M. Contamination of too-thpaste and toothbrush by Streptocoaus Scand J Dent Res 1978;86(5):412-4.
  3. Glass RT, Lare MM. Toothbrush contamination: a potential health risk? Quintessence Int 1986;17(l):39-42.
  4. Apiou J, Gueguen MM, Doleux S, Bon-naure-Mallet M. Evaluation of a new toothbrush concept with regard to
  5. bacterial elimination. Imprint study using scanning electron microscopy. J Clin Pe-riodontol 1994; 21(5):347-50.
  6. Muller HP, Lange DE, Muller RE Ac-tinobacillus actinomycetemcomitam contamination of toothbrushes funi patients
  7. harbouring the organism.J Clin Periodon-tol 1989;16(6):388-90.
  8. Kozai K, IwaiT, Miura K. Residual contamination of toothbrushes by mi-croorganisms. ASDC J Dent Children 1989;56(3):201-4.
  9. Hardie JM.Oral microbiology: current concepts in the microbiology of dental caries and periodontal disease.
  10. Br Dent J 1992;172 (7):271-8.
8.Tanzer JM. Dental caries is a transmissible infectious disease: the Keyes and Fitzgerald revolution.J Dent Res 1995;74(9):1536-42.
  1. Brambilla E, Felloni A, Gagliani M, Malerba A, Garcia-Godoy F, Strohmen-ger L. Caries prevention during pregnancy:
  2. results of a 30-month study [see comments], JADA 1998; 129(7): 871-7.
  3. Simonetti D'Arca A, Capozzi L. Sul potere di autodepurazione dello spaz-zolino denti con particolare riguardo al tipo
  4. a testina argentata. Riv It Stomat 1983;52(4):285-92.
  5. Goodman G, Gilman G. The pharma-cological basis of therapeutics. New York: Mc-Millan, 1980.
In writing since September 1999 Dm
IL DENTISTA MODERNO
The recovered liquid
was diluted in 1 ml of physiological solution and 20 μl of the final suspension was inoculated on platelets of
MSB agar, incubated for 48 hours at 37°C.
At the end of the incubation, the developed colonies were counted.
STATISTICAL ANALYSIS
For statistical analyses, we used the Statview 5 program.0 for Windows (SAS Institute Ine, Cary, North Carolina, USA).
To assess the comparability of the two groups at time 0, a t-test for unpaired data was carried out.
To evaluate the reduction in concentration over time, the ANOVA test for repeated measures was used.
The level of significance was set for a probability p < 0.05.
RESULTS
In order to assess the comparability of the data, we carried out the static analysis at time 0 (table 1) using the t-test
for unpaired data; from these data we can observe er that the averages of the relative concentrations
to the two toothbrushes at the end of the clinical period are superimposable (p = 0.919). From ANOVA
data
(table 2) we note that, although the reduction in concentrations present on the tufts of hair depends
mainly of the time variable (p < 0.0001), the two types of brushes express different behavior
statistically significant (p = 0.027) (figure 1).
DISCUSSION AND CONCLUSIONS
Preventive dentistry, in concomitance with the decline of caries pathology in industrialized countries
as a result of the efforts made over the previous twenty years and thanks to recent scientific research,
has identified in recent years a new objective on which to focus available resources: identification
and the treatment of “subjects at risk”, that is to say the particular group of individuals who present a development
pathology significantly higher than the norm.
Biological research has identified S. mutans as the main etiological agent of the disease because of its capacity
to produce these metabolite acids responsible for the dissolution of dental enamel at the beginning of development
of the carious cavity, even if in a broader concept of multifactorial pathology it has been demonstrated that other
species of bacteria have cariogenic power7.
The different response of the organismme in relation to the altered balance of the oral ecosystem in subjects identified as
at risk makes it essential, in addition to the identification of said subjects, the implementation of preventive measures capable of reducing
“infection” from pathogenic bacteria.
The concept of transmissibility of caries pathology8 has regained force in recent years in association with
instruments intended for personal oral hygiene, in the absence of structures and controls capable of preventing cross-contamination.
While the implementation of preventive measures during pregnancy9, in order to reduce the vertical transmission of
infection of S. mutans, is easy to achieve, on the other hand the means capable of reducing horizontal transmission are
few in number. To reduce residual bacterial contamination on toothbrushes to prevent contamination
crossed between different subjects in a community context, such as the family community or like communities
school, the industry has tried to satisfy this health need by researching different techniques for hygienizing toothbrushes.
The use of metals with microbicidal activity dates back to the end of the last century when the first experimental observations were made
subsequently confirmed at the beginning of the century through a more precise oligodynamic classification of metal ions10.
The mechanism of action of metals in an aqueous solution, colloidal metals, on microbial particles is multiple,
and more recent studies11 attribute microbicidal activity to penetration of the citoplasmatic membrane and inhibition
of the enzymatic activity of some protein classes. The antimicrobial activity of the silver coating of the removable head
of the Silvercare brush compared to the numerous pathogenic and saprophytic bacterial species present in the cavity
buccal has already been experimentally confirmed in vitro10, but compared to S. mutans we required further data
compared to in vivo contamination with wild strains of said bacterial species. The data obtained by the protocol
experimental relating to the clinical colonization of S. mutans show that said microbial species is able to
colonize the surfaces of instruments used during daily oral hygiene operations at home.
It can further be deduced that the activity of the silver ions deposited on the surface of the Silvercare head is able to reduce
in a statistically significant manner, after a period of time equivalent to the normal time interval which elapses
between oral hygiene maneuvers at home, the concentration of S. mutans present on the head of the toothbrush itself.
Although our data only relates to the colonization of tufts of hair, they can be compared with those
in vitro studies. The observed variability must be sought in the possible interaction of the agonist active ingredients by
relation to colonization itself. Studies available in the literature provide little indication about the effect of
toothpastes2 on the bacterial colonization pattern; in fact, the adhesive capacity of different bacterial species
may undergo variations due to the use of toothpastes with antibactericidal, surfactant components, etc. in quantity
such that they modify the decontamination pattern that we have noted.
Correspondence to: dott. Andrea Felloni Department of Medicine,
Surgery and Odontology,
Chair of Pedodontics
Via Beldiletto 1/3, 20142 Milano e-mail: Andrea.Felloni@unimi.it
BIBLIOGRAPHY
  1. Dayoub MB, Rusilko D, Gross A. Microbial contamination of toothbrushes. J Dent Res 1977;56(6):706-7.
  2. Svanberg M. Contamination of too-thpaste and toothbrush by Streptocoaus Scand J Dent Res 1978;86(5):412-4.
  3. Glass RT, Lare MM. Toothbrush contamination: a potential health risk? Quintessence Int 1986;17(l):39-42.
  4. Apiou J, Gueguen MM, Doleux S, Bon-naure-Mallet M. Evaluation of a new toothbrush concept with regard to
  5. bacterial elimination. Imprint study using scanning electron microscopy. J Clin Pe-riodontol 1994; 21(5):347-50.
  6. Muller HP, Lange DE, Muller RE Ac-tinobacillus actinomycetemcomitam contamination of toothbrushes funi patients
  7. harbouring the organism.J Clin Periodontol 1989;16(6):388-90.
  8. Kozai K, IwaiT, Miura K. Residual contamination of toothbrushes by microorganisms. ASDC J Dent Children 1989;56(3):201-4.
  9. Hardie JM. Oral microbiology: current concepts in the microbiology of dental caries and periodontal disease.
  10. Br Dent J 1992;172 (7):271-8.
8.Tanzer JM. Dental caries is a communicable infectious disease: the Keyes and Fitzgerald revolution.J Dent Res 1995;74(9):1536-42.
  1. Brambilla E, Felloni A, Gagliani M, Malerba A, Garcia-Godoy F, Strohmen-ger L. Caries prevention during pregnan-cy:
  2. results of a 30-month study [see comments], JADA 1998; 129(7): 871-7.
  3. Simonetti D’Arca A, Capozzi L. It is possible to self-depurate tooth particles with special cleaning agents
  4. a testina argentata. Riv It Stomat 1983;52(4):285-92.
  5. Goodman G, Gilman G. The pharmacological basis of therapeutics. New York: McMillan, 1980.
In writing since September 1999 Dm
IL DENTISTA MODERNO
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