ORIGINAL ARTICLE

Influence of thermoplastic retainers on

Streptococcus mutans and Lactobacillus
adhesion

rı Tu
Çag € z,a Nehir Canigu
€ rko € r Bavbek,b Selin Kale Varlik,c and Gu
€ lçin Akçad
Ankara, Turkey

Introduction: This study was designed to test the hypothesis that thermoplastic retainers influence oral micro-
bial flora during the retention period because they prevent the flushing effect of saliva on dental and mucous
tissues. Methods: Twenty-four orthodontic patients finished the study. After debonding, the patients were given
thermoplastic retainers (Essix ACE 0.040-in plastic, Dentsply International, York, Pa) for both jaws and instructed
to wear them all day. Plaque samples from tooth surfaces and saliva samples were collected from each patient
just after debonding (T0), and on day 15 (T1), day 30 (T2), and day 60 (T3) of retention. The jaws were divided into
6 regions, and the data for each region were evaluated separately. Total viable Lactobacillus and Streptococcus
mutans colonies were counted, and the numbers of the viable microorganisms were calculated. Results: The
numbers of Lactobacillus colonies at T3 were higher than at T0, T1, and T2, and the difference between T0
and T3 was statistically significant (P \0.05). The numbers of S mutans colonies at T3 were higher than at
T0, T1, and T2, and the differences between T0 and T1, and T1and T2 were statistically significant (P \0.05).
Conclusions: Retention with thermoplastic retainers might create oral conditions conducive to S mutans and
Lactobacillus colonization on dental surfaces. (Am J Orthod Dentofacial Orthop 2012;141:598-603)

R
etention is an important phase of orthodontic In studies concerning oral hygiene during or after
treatment during which tooth movements after orthodontic treatment, special attention is attributed to
active treatment are stabilized. There are numer- cariogenic populations such as Streptococcus mutans
ous types of retention methods for clinical use, and ther- and Lactobacillus.
moplastic retainers are widely preferred for their Many studies in the literature have evaluated the
advantages: removable, more esthetic, cost effective, changes in S mutans and Lactobacillus levels during
and less time-consuming in the laboratory. orthodontic therapy with either acrylic removable
Identification of the changes in the oral cavity of pa- appliances or orthodontic bands and brackets.5-7
tients who have orthodontic therapy, including the re- Besides, most of these studies concentrated on the
tention period, is important. Previous data about oral active orthodontic treatment phase with fixed or
hygiene of orthodontic patients held removable and removable appliances. However, microbiologic data
fixed orthodontic appliances responsible for worsening during retention period are limited.8,9 Yet, as far as we
the oral hygiene1,2 and promoting the differentiation know, there is no information about the effects of
of microbial flora of the oral cavity3,4 that might result thermoplastic retainers on oral microbiota, although
in caries, white spot lesions, and gingival inflammation. they are frequently preferred. In addition, there are no
data evaluating the oral cavity by separating it into
specific zones during retention with thermoplastic
From Gazi University, Ankara, Turkey.
a retainers. Therefore, the purpose of this study was to
Assistant professor, Department of Orthodontics.
b
Research assistant, Department of Orthodontics. test the hypothesis whether thermoplastic retainers
c
Associate professor, Department of Orthodontics. influence oral microbial flora during the retention
d
Assistant professor, Department of Microbiology.
period because they prevent the flushing effect of
The authors report no commercial, proprietary, or financial interest in the prod-
ucts or companies described in this article. saliva on dental and mucous tissues.
Reprint requests to: Ça

grı T€
urk€ €
oz, Gazi Universitesi Diş Hekimli

gi Fak€
ultesi Orto-
donti, AD 06510 Emek, Ankara, Turkey; e-mail, cturkoz@hotmail.com.
Submitted, October 2011; revised and accepted, November 2011. MATERIAL AND METHODS
0889-5406/$36.00
Copyright Ó 2012 by the American Association of Orthodontists. The initial study population comprised 40 subjects
doi:10.1016/j.ajodo.2011.11.021 with an age range of 14 to 20 years, who had fixed
598
T€
urk€oz et al 599

orthodontic treatment at the Department of Orthodon-

tics of Gazi University in Ankara, Turkey, and had started
to wear thermoplastic retainers in the retention period.
Exclusion criteria included caries-active patients, the
use of oral antimicrobial agents or antibiotics within
the past 3 months, smoking habit, periodontal or sys-
temic disease, and prosthodontic appliances. Unfortu-
nately, 16 subjects did not attend their periodic visits,
and the study population was narrowed to 24 subjects.
All patients included in the study signed informed con-
sent forms. Thermoplastic retainers (Essix ACE 0.040-in
plastic; Dentsply International, York, Pa) covering all
tooth surfaces up to 2 mm above the gingival margin
and the occlusal surfaces were prepared for both jaws.
The patients were instructed to wear their retainers all
day and brush them after toothbrushing. The subjects
had refrained from eating or drinking beverages for at
least 2 hours before sample collection and had been
told not to brush their teeth before the examinations.
Plaque samples from tooth surfaces and saliva samples
were collected from each patient just after debonding
(T0), and at day 15 (T1), day 30 (T2), and day 60 (T3)
of retention with the thermoplastic retainers. First, un-
stimulated saliva samples were collected from the pa-
tients. They rinsed their mouth out well with drinking
water and were advised to sit relaxed for 5 minutes. After
this procedure, they were asked to spit about 5 mL of sa-
liva into 50-mL sterile tubes. The jaws were divided into Fig. Region identification in oral cavity.
6 regions and named as shown in the Figure. Plaque
samples were collected with sterile swabs from the gin-
gival margin and enamel surface of each tooth at the Lactobacillus colonies were counted, and the numbers
vestibule and the palatal-lingual sides, and the data of the viable microorganisms were calculated by means
for each region were evaluated separately. The entire of colony-forming units per milliliter of volume (cfu/
surfaces of all teeth and 1 to 2 mm of gingiva in each re- ml). The data and the statistics were done according to
gion were swabbed. After scraping the dental plaque, the 1 mL amount of volume as colony-forming units
the samples were put into presterilized test tubes con- per milliliter. Homogeneities of the group variances
taining 1 ml of trypticase soy broth (Merck, Darmstadt, were analyzed with the Levene test; because they were
Germany) separately and vortexed for 60 seconds. normally distributed, significance was determined with
Then, the suspensions were diluted to 10 1, 10 2, and 1-way analysis of variance (ANOVA).
10 3 serially, and 100-mL amounts of each primer
suspension and the diluted suspensions were put onto
de Man, Rogosa and Sharpe (MRS) agar for Lactobacil- RESULTS
lus selective agar (Merck) and tryptone-yeast extract- Means of the sums of all regions were evaluated. The
cysteine-sucrose-bacitracin agar for the isolation of S mean amount of Lactobacillus at T3 (14.49 cfu/mL) was
mutans.10 The plates were incubated in an incubator higher than at T0, T1, and T2, and the difference be-
at 37 C for 3 to 4 days in microaerophilic conditions tween T0 and T3 was statistically significant (P \0.05)
(air 1 5% carbon dioxide). After collecting the unstimu- (Table I). The mean amount of S mutans at T1 (43.72
lated saliva, 1 mL of saliva was put in another sterilized cfu/mL) was higher than at T0, T2, and T3, and the dif-
tube and diluted from 10 1 to 10 3 serially with steril- ferences between T0 and T1, and T1 and T2 were statis-
ized distilled water. After vortexing the tubes for 15 sec- tically significant (P \0.05) (Table I).
onds, 100 mL of each diluted saliva was put onto the S mutans and Lactobacillus counts in the saliva
agar media separately and incubated as mentioned be- showed no significant differences among T0, T1, T2,
fore. After these processes, total viable S mutans and and T3 (Table II).

American Journal of Orthodontics and Dentofacial Orthopedics May 2012  Vol 141  Issue 5
600 T€
urk€oz et al

Table I. Total regional changes in Lactobacillus and S mutans levels from T0 to T3

T0 T1 T2 T3

Mean SD Mean SD Mean SD Mean SD

Lactobacillus 5.82 25.52 8.45 39.51 10.59 23.74 14.49 33.43
S mutans 28.21 66.53 43.72 91.91 32.2 44.8 39.46 78.31
Means are in units of cfu/mL.

DISCUSSION
Table II. Saliva changes in Lactobacillus and S mu-
tans levels from T0 to T3 The use of removable appliances for small move-
ments, retention, and myofunctional therapies results
Mean (cfu/mL) SD P
in greater biofilm accumulation on dental surfaces.7 Hi-
Lactobacillus
T0 10.42 24.68 0.371
bino et al11 believed that removable orthodontic appli-
T1 37.54 131.83 ances could hinder patients from maintaining good oral
T2 4.57 8.7 hygiene and prevent plaque removal either naturally or
T3 14.25 22.88 mechanically. Removable retainers can be fabricated
S mutans from acrylic resins, stainless steel wires, and thermoplas-
T0 46.67 51.2 0.811
T1 70.21 135.41 tic. Recently, the use of thermoplastic retainers has in-
T2 53.21 74.14 creased because of their easy fabrication and good
T3 51.04 73.71 esthetic appearance. Thermoplastic retainers cover all
palatal, lingual, labial, and buccal surfaces of the teeth
and gingiva; thus, they might influence oral microbial
Overall, the mean amount of Lactobacillus in the flora during the retention period because they prevent
mandibular right vestibular region was the highest at the flushing effect of saliva on dental and mucous tissues.
all times, whereas it was the lowest in the maxillary ante- Microfloral changes are directly related to the design
rior region and the mandibular anterior region (Table III). of the removable appliance. According to Battoni et al,7
On the other hand, the highest mean amount of S the creation of extra retention sites with removable ap-
mutans was found in the maxillary left and mandibular pliances can favor the adhesion and colonization of S
palatal-lingual regions, but it was the lowest at the max- mutans. Similarly, Addy et al12 showed that palatal pla-
illary anterior labial region and the mandibular anterior que scores were significantly higher in removable appli-
region at all times (Table IV). Nevertheless, the differ- ance wearers compared with a control group without
ences in the numbers of both microbial agents were orthodontic treatment. They also reported that the buc-
not statistically different among the regions where the cal scores were significantly lower compared with the
samples were taken at T0, T1, T2, and T3 (P .0.05) control group. It might be due to the appliance design
(Tables III and IV). of most of the acrylic removable appliances, since they

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T€
urk€oz et al 601

Table III. Comparison of the mean differences in Lactobacillus levels for each region from T0 to T3
1A 1B 2A 2B 3A 3B

Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD

T0 4.96 6.94 4.17 7.86 2.71 5.55 2.00 5.02 15.29 60.90 2.75 4.87
T1 3.58 6.39 8.83 21.89 1.21 3.18 1.54 2.73 8.42 16.70 7.54 12.03
T2 12.17 27.87 11.17 24.79 7.58 18.47 5.04 11.59 12.25 20.66 10.33 19.5
T3 20.63 47.12 19.48 46.8 7.75 15.91 8.33 18.42 13.29 27.77 12.21 29.31
4A 4B 5A 5B 6A 6B

Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD

T0 7.83 13.63 2.71 4.96 1.21 4.35 0.63 2.08 17.29 60.61 3.75 5.68
T1 10.17 23.97 9.63 24.83 3.08 7.55 2.96 7.05 7.33 11.84 8.00 15.80
T2 19.96 36.05 15.75 28.59 6.83 17.07 3.88 9.04 16.71 39.72 11.17 21.93
T3 17.38 35.89 18.75 43.80 6.63 20.97 7.92 23.00 22.92 43.80 19.00 38.96
Means are in units of cfu/mL. T0, P 5 0.378; T1, P 5 0.304; T2, P 5 0.520; T3, P 5 0.748.
1A, Right maxillary posterior buccal; 1B, right maxillary posterior palatinal; 2A, maxillary anterior labial; 2B, maxillary anterior palatinal; 3A, left
maxillary posterior buccal; 3B, left maxillary posterior palatinal; 4A, left mandibular posterior buccal; 4B, left mandibular posterior lingual; 5A,
mandibular anterior labial; 5B, mandibular anterior lingual; 6A, right mandibular posterior buccal; 6B, right mandibular posterior lingual.

Table IV. Comparison of the mean differences in S mutans levels for each region from T0 to T3
1A 1B 2A 2B 3A 3B

Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD

T0 57.21 202.27 28.67 35.65 19.54 27.25 25.21 26.16 25.04 36.40 33.25 37.89
T1 34.79 64.63 42.83 82.10 28.96 67.34 60.17 155.99 45.92 83.31 53.67 96.42
T2 29.92 44.06 32.58 42.05 26.04 35.54 28.29 35.37 29.63 35.19 41.17 43.76
T3 43.04 68.85 51.08 88.26 26.00 39.84 41.96 91.31 43.71 57.51 55.33 93.18
4A 4B 5A 5B 6A 6B

Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD

T0 23.21 32.08 39.00 65.93 12.25 13.95 10.67 14.35 21.83 34.53 24.21 41.58
T1 44.96 85.17 47.75 95.73 20.50 32.16 21.04 36.77 44.88 92.26 52.67 99.58
T2 31.92 40.73 40.83 51.43 19.13 25.47 19.04 37.54 30.88 46.79 35.75 51.21
T3 20.88 29.96 44.75 105.94 16.42 29.24 37.71 112.63 33.38 58.73 47.67 110.07
Means are in units of cfu/mL. T0, P 5 0.635; T1, P 5 0.905; T2, P 5 0.750; T3, P 5 0.864.
1A, Right maxillary posterior buccal; 1B, right maxillary posterior palatinal; 2A, maxillary anterior labial; 2B, maxillary anterior palatinal; 3A, left
maxillary posterior buccal; 3B, left maxillary posterior palatinal; 4A, left mandibular posterior buccal; 4B, left mandibular posterior lingual; 5A,
mandibular anterior labial; 5B, mandibular anterior lingual; 6A, right mandibular posterior buccal; 6B, right mandibular posterior lingual.

cover the palatal-lingual sides only, in contrast to ther-
moplastic retainers. Microorganism colonies on remov-
able orthodontic appliances attack the dental surfaces
as well; thereby, the appliances can alter the normal
oral microbiota.13 Also, insufficient saliva secretion re-
duces the self-cleaning mechanisms of the oral cavity
and limits the antimicrobial effects of the residual sa-
liva.14 In this study, like most routine clinical applica-
tions, thermoplastic retainers covered all tooth surfaces
including 1 to 2 mm of the gingiva. The patients were
instructed to wear their retainers for 24 hours a day,
and this protocol might make mechanical cleaning
more difficult because it prevents the washing effect of
saliva, especially in the gingival margin where the sam-
ples were taken. Supporting this, statistically significant
changes in S mutans and Lactobacillus levels in plaque
samples were recorded at the different times (Table I),
but no significant changes were recorded in S mutans
and Lactobacillus levels in saliva (Table II). These find-
ings agree with the results of Lara-Carrillo et al.5 This
might be due to an insufficient flushing effect of saliva
on dental and gingival tissues, since there was no signif-
icant change in S mutans and Lactobacillus levels in the
saliva.
S mutans and Lactobacillus levels in the different re-
gions of the oral cavity were also evaluated in this study.

American Journal of Orthodontics and Dentofacial Orthopedics May 2012  Vol 141  Issue 5
602
No statistical difference was observed between the re-
gions of the mandible and the maxilla for S mutans and
Lactobacillus levels during retention with thermoplastic
retainers (Tables III and IV). These results address the
alteration of oral microbiota equally in the oral cavity
after thermoplastic retainers were started to be worn.
However, this result does not specifically mean that
plaque accumulation would be equal as well, because it
is known that the buccal surfaces of the maxillary
molars and the lingual surfaces of the mandibular
molars accumulate more plaque than do other sites.15
In this study, S mutans and Lactobacillus levels were
distributed in a wide range with high standard deviation
values. Although it might not seem to be reliable at first
sight, the formation and the microbial composition of
plaque is also associated with individual variations to
some extent.16 Zee et al17 previously reported that the
proportions of bacterial species were significantly differ-
ent between rapid and slow plaque formers. Likewise,
Haffajee et al18 found remarkable variability in the total
numbers of organisms in their subjects. They also
pointed out that, while examining changes in the com-
position of oral biofilms, individual influences such as
diet, oral hygiene habits, and genetic background are of-
ten neglected.
The composition of microbial flora is a determinative
factor for cariogenic activity. Dental plaque on tooth
surfaces acts as an infective agent for dental caries and
periodontal inflammation.19 S mutans had been chosen
as a representative oral bacterium because it is also found
in early plaque and is a known important cariogenic
agent.20 For this reason, the evaluation of S mutans
levels during any treatment is mandatory for understand-
ing the caries risk factor. Different levels of S mutans
adhesion to orthodontic materials have been previously
reported.16,21 Our results showed a significant increase
in cumulative Lactobacillus levels from T0 to T3,
whereas S mutans levels showed a significant increase
from T0 to T1 and a significant decrease from T1 to T2
(Table I). Despite this, saliva levels showed no significant
changes (Table II). Gram-positive cocci such as S mutans
are known to be the early colonizers of plaque because
they can withstand high oxygen concentrations. As the
numbers of plaque layers increase, or the plaque becomes
more mature, the oxygen levels decrease, and the anaer-
obic flora become dominant.22 Syed and Loeshe23 also
showed that S mutans predominates in early (0-1
week) plaque, but its concentration dramatically dropped
in the 2-to-3 week period. The authors also reported that,
as plaque matured, Actinomyces species started to dom-
inate its composition and S mutans adhesion was sup-
pressed. This may be the reason of the decrease in S
mutans levels at T3 in this study. Although the patients
May 2012  Vol 141  Issue 5 American Journal of Orthodontics and Dentofacial Orthopedics
T€
urk€oz et al
in this study were instructed to brush their teeth and re-
tainers every day, irregular surfaces provide niches and
reservoirs for bacterial species, thus promoting regrowth
of existing microbiota.24 Low et al16 found bacterial col-
onies inside the thermoplastic retainers with scanning
electron microscope images even when they were
brushed every day. We also found maturation of plaque
inside the appliance, in agreement with the study of
Syed and Loeshe.23 It is known that mechanical cleaning
of dental biofilm is provided by both toothbrushing and
flushing of saliva. Even if all patients in this study
brushed their teeth regularly, the S mutans and Lactoba-
cillus counts on the biofilm showed mature plaque pro-
files. This might be because the thermoplastic retainers
hinder the buffering effect of saliva.
Not only the treatment modality but also the surface
characteristics of the used material is definitive for bac-
terial adhesion. According to Lee et al,21 2 surface char-
acteristics affect the amount of bacterial adhesion to
orthodontic materials: surface roughness and surface
free energy. A rough surface provides suitable niches
for bacterial colonization, and a material with high
free surface energy attracts more bacteria. Lessa et al13
reported that S mutans colonies were observed on all
noninfected acrylic base plates; therefore, the acrylic
surface of removable orthodontic appliances acts as
a sponge for microbial colonization. Low et al16 recently
reported that thermoplastic appliances were not com-
pletely smooth but had microabrasions and irregularities
that might contribute to bacterial adhesion. In this
study, colonization on thermoplastic retainers was not
evaluated because our main purpose was to investigate
oral bacterial changes. Further studies that examine bac-
terial colonization on thermoplastic materials and their
interactions with oral microbiota are needed for a better
understanding of microbial changes during the reten-
tion period.
CONCLUSIONS
These results demonstrate that retention with ther-
moplastic retainers might have a positive effect on S
mutans and Lactobacillus colonization on dental sur-
faces. In the evaluation of the data, the possible effects
of several factors—eg, frequency of toothbrushing and
a cariogenic diet during this period—should not be
neglected. It might be beneficial to advise patients to
take extra oral hygiene and dental care during retention
with thermoplastic retainers.
REFERENCES
1. Tufekci E, Dixon JS, Gunsolley JC, Lindauer SJ. Prevalence of white
spot lesions during orthodontic treatment with fixed appliances.
Angle Orthod 2011;81:206-10.
T€
urk€oz et al
2. Klukowska M, Bader A, Erbe C, Bellamy P, White DJ, Anastasia MK,
et al. Plaque levels of patients with fixed orthodontic appliances
measured by digital plaque image analysis. Am J Orthod Dentofa-
cial Orthop 2011;139:e463-70.
3. Ristic M, Vlahovic Svabic M, Sasic M, Zelic O. Effects of fixed or-
thodontic appliances on subgingival microflora. Int J Dent Hyg
2008;6:129-36.
4. Turkkahraman H, Sayin MO, Bozkurt FY, Yetkin Z, Kaya S, Onal S.
Archwire ligation techniques, microbial colonization, and peri-
odontal status in orthodontically treated patients. Angle Orthod
2005;75:231-6.
5. Lara-Carrillo E, Montiel-Bastida NM, Sanchez-Perez L,
Alanis-Tavira J. Effect of orthodontic treatment on saliva, plaque
and the levels of Streptococcus mutans and Lactobacillus. Med
Oral Patol Oral Cir Bucal 2010;15:e924-9.
6. Papaioannou W, Gizani S, Nassika M, Kontou E, Nakou M. Adhe-
sion of Streptococcus mutans to different types of brackets. Angle
Orthod 2007;77:1090-5.
7. Batoni G, Pardini M, Giannotti A, Ota F, Giuca MR, Gabriele M,
et al. Effect of removable orthodontic appliances on oral colonisa-
tion by mutans streptococci in children. Eur J Oral Sci 2001;109:
388-92.
8. Rosenbloom RG, Tinanoff N. Salivary Streptococcus mutans levels
in patients before, during, and after orthodontic treatment. Am J
Orthod Dentofacial Orthop 1991;100:35-7.
9. Al Groosh D, Roudsari GB, Moles DR, Ready D, Noar JH, Pratten J.
The prevalence of opportunistic pathogens associated with intrao-
ral implants. Lett Appl Microbiol 2011;52:501-5.
10. Kidd EA, Ricketts DN, Beighton D. Criteria for caries removal at the
enamel-dentine junction: a clinical and microbiological study. Br
Dent J 1996;180:287-91.
11. Hibino K, Wong RW, H€agg U, Samaranayake LP. The effects of or-
thodontic appliances on Candida in the human mouth. Int J
Paediatr Dent 2009;19:301-8.
12. Addy M, Shaw WC, Hansford P, Hopkins M. The effect of ortho-
dontic appliances on the distribution of Candida and plaque in ad-
olescents. Br J Orthod 1982;9:158-63.
American Journal of Orthodontics and Dentofacial Orthopedics
603
13. Lessa FC, Enoki C, Ito IY, Faria G, Matsumoto MA,
Nelson-Filho P. In-vivo evaluation of the bacterial contami-
nation and disinfection of acrylic baseplates of removable ortho-
dontic appliances. Am J Orthod Dentofacial Orthop 2007;131:
705.e11-7.
14. Hahnel S, Rosentritt M, Handel G, Burgers R. Influence of saliva
substitute films on initial Streptococcus mutans adhesion to
enamel and dental substrata. J Dent 2008;36:977-83.
15. Furuichi Y, Lindhe J, Ramberg P, Volpe AR. Patterns of de novo
plaque formation in the human dentition. J Clin Periodontol
1992;19:423-33.
16. Low B, Lee W, Seneviratne CJ, Samaranayake LP, H€agg U. Ultra-
structure and morphology of biofilms on thermoplastic orthodon-
tic appliances in ‘fast’ and ‘slow’ plaque formers. Eur J Orthod
2011;33:577-83.
17. Zee KY, Samaranayake LP, Attstrom R. Predominant cultivable
supragingival plaque in Chinese “rapid” and “slow” plaque formers.
J Clin Periodontol 1996;23:1025-31.
18. Haffajee AD, Teles RP, Patel MR, Song X, Veiga N, Socransky SS.
Factors affecting human supragingival biofilm composition. I.
Plaque mass. J Periodontal Res 2009;44:511-9.
19. Orstavik D, Kraus FW, Henshaw LC. In vitro attachment of strepto-
cocci to the tooth surface. Infect Immun 1974;9:794-800.
20. Nyvad B, Kilian M. Comparison of the initial streptococcal micro-
flora on dental enamel in caries-active and in caries-inactive indi-
viduals. Caries Res 1990;24:267-72.
21. Lee SP, Lee SJ, Lim BS, Ahn SJ. Surface characteristics of ortho-
dontic materials and their effects on adhesion of mutans strepto-
cocci. Angle Orthod 2009;79:353-60.
22. Sbordone L, Bortolaia C. Oral microbial biofilms and plaque-related
diseases: microbial communities and their role in the shift from oral
health to disease. Clin Oral Investig 2003;7:181-8.
23. Syed SA, Loesche WJ. Bacteriology of human experimental gingi-
vitis: effect of plaque age. Infect Immunol 1978;21:821-9.
24. Teughels W, Van Assche N, Sliepen I, Quirynen M. Effect of mate-
rial characteristics and/or surface topography on biofilm develop-
ment. Clin Oral Implants Res 2006;17(Suppl 2):68-81.
May 2012  Vol 141  Issue 5