J Periodont Res 2015; 50: 864–869 © 2015 John Wiley & Sons A/S.

All rights reserved Published by John Wiley & Sons Ltd

JOURNAL OF PERIODONTAL RESEARCH

doi:10.1111/jre.12280

D. F. Kinane1, P. Zhang1,
Experimental gingivitis, M. Benakanakere1, J. Singleton2,
A. Biesbrock3, C. Nonnenmacher4,

bacteremia and systemic T. He3

1
Department of Periodontics, School of Dental
Medicine, University of Pennsylvania,

biomarkers: a randomized Philadelphia, PA, USA, 2Department of

Periodontics, Endodontics and Dental Hygiene,
Center for Oral Health and Systemic Disease,

clinical trial
University of Louisville School of Dentistry,
Louisville, KY, USA, 3Health Care Research
Center, Procter & Gamble Company, Mason,
OH, USA and 4Institute of Medical
Microbiology and Hygiene, Philipps University
Marburg, Marburg, Germany
Kinane DF, Zhang P, Benakanakere M, Singleton J, Biesbrock A, Nonnenmacher
C, He T. Experimental gingivitis, bacteremia and systemic biomarkers: a
randomized clinical trial. J Periodont Res 2015; 50: 864–869.
© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

Background and Objective: Bacteremia and systemic inflammatory markers are

associated with periodontal and systemic diseases and may be linking mecha-
nisms between these conditions. We hypothesized that in the development of
gingival inflammation, systemic markers of inflammation and bacteremia would
increase.
Material and Methods: To study the effect of bacteremia on systemic inflamma-
tory markers, we recruited 80 subjects to participate in an experimental gingivi-
tis study. Subjects were stratified based on gender, smoking and the number of
bleeding sites and then randomized to one of two groups: control group
(n = 40) or experimental gingivitis group (n = 40). Subjects in the control group
conducted an oral hygiene regimen: brushing twice daily with a regular sodium
fluoride cavity protection dentifrice and a standard manual toothbrush, flossing
twice daily, and mouth rinsing with an anti-cavity fluoride rinse once daily. The
experimental group stopped brushing and flossing, and used only the fluoride
anti-cavity mouth rinse for 21 d.

Results: Seventy-nine of 80 subjects were evaluable. One subject in the control

group was excluded from the results due to antibiotic use during the study. Our
data showed the experimental gingivitis group exhibited a significant (p < 0.05)
increase in dental plaque level and gingival inflammatory indices relative to base-
line and the control group but a decrease in bacteremia and soluble intercellular
Denis F. Kinane, BDS, PhD, Department of
adhesion molecule-1 levels vs. baseline. Bacteremia was negatively correlated Periodontics, School of Dental Medicine,
with gingival inflammatory indices and soluble intercellular adhesion molecule-1 University of Pennsylvania, 240 South 40th
levels in the experimental gingivitis group, thus negating our hypothesis. Street, Philadelphia, PA 19004, USA
Tel: 215 898 1038
Conclusion: We conclude that there are marked differences in systemic cytokine Fax: 215 573 4075
e-mail: dfkinane@dental.upenn.edu
levels over the course of short-term experimentally induced gingivitis and further
Key words: bacteremia; experimental gingivitis;
conclude that a long-term periodontitis study must be considered to address
periodontal disease; systemic inflammation
mechanisms whereby oral diseases may affect systemic diseases.
Accepted for publication March 14, 2015

typically induced by dental plaque, a including diabetes, atheroma and car-

Introduction
complex microbial biofilm (1). Several diovascular diseases (1–6). The causal
Gingivitis and periodontitis are inflam- other conditions and complications are mechanism linking periodontal disease
matory disorders of the periodontium associated with periodontal disease, and systemic diseases is yet unknown

but proposed to share several risk fac-
tors, including infiltration of hyperin-
flammatory immune cells from various
sources (7). Bacteremia can occur
through simple tooth brushing as well
as scaling and root planing (8). Daily
episodes of bacteremia originating
from periodontal lesions can cause
changes in systemic markers in peri-
odontitis (9). Moreover, the associa-
tion between periodontitis and
coronary heart disease is particularly
affected by the periodontal microbial
pathogen burden (10). A number of
studies have shown that chronic peri-
odontitis is associated with increased
serum levels of inflammatory media-
tors, including C-reactive protein
(CRP), soluble intercellular adhesion
molecule-1 (sICAM-1) and interleukin-
6 (IL-6) (11–14). However, there are
also contradictory reports indicating
no significant differences in serum
CRP levels following periodontal treat-
ment (15,16).
The fact that gingivitis is reversible
and can be experimentally induced
makes it an ideal condition for studies
addressing susceptibility to bacteria-
induced inflammation (17). In view of
this, we designed an experimental gin-
givitis study to: (i) evaluate the effect
of oral hygiene, including flossing, on
the prevention of gingival inflamma-
tion; (ii) explore the relationship
between gingival inflammation and
bacteremia, as well as levels of circulat-
ing inflammatory biomarkers (includ-
ing CRP, sICAM, tumor necrosis
factor-alpha [TNF-a] and IL-8) that
are associated with both the develop-
ment of gingival inflammation and
systemic inflammatory biomarkers.
Material and methods
Subjects
The present study comprised 80
subjects (both male and female, ages
18–65 years) recruited by the investi-
gators from the periodontology clinic
of the University of Louisville, School
of Dentistry (Louisville, KY, USA) in
2005. Sample size was determined
based on using Fisher’s exact test,
whereby a sample size of 80 subjects
with 40 in each of two treatment
Early gingivitis & systemic inflammation
groups would have 80% power, with
a two-tailed alpha of 0.05, to detect
differences in group 1 ranging from
2.5% to 10% and in group 2 from
25% to 38%. All subjects were in
good general health, had a minimum
of 16 natural teeth with scorable
facial and lingual surfaces and had a
diagnosis of chronic gingivitis with
> 20% bleeding sites or equivalent
and no obvious chronic periodontitis.
Subjects had to also agree to return
for the scheduled visits and to follow
study procedures, to refrain from
using any non-study oral care prod-
ucts or receiving elective dental treat-
ment (including dental prophylaxis),
and to avoid participating in any
other clinical study while the current
study was in progress. Exclusion crite-
ria included the presence of four or
more teeth in any two quadrants with
pockets > 5 mm; any systemic disease
or significant medical conditions such
as a history of diabetes, or use of
medication(s) known to affect peri-
odontal status; any requirement for
antibiotic premedication before dental
procedures; being pregnant, lactating
or not using birth control; or, having
used systemic antibiotics within 1 mo
before enrollment. The study was
approved by the University of Louis-
ville Institutional Review Board. Par-
ticipants in this study had signed and
received a copy of the Informed Con-
sent Form, including HIPAA authori-
zation.
Dental treatment
Two weeks pre-baseline, scaling was
performed on all subjects and they
were given oral hygiene instructions.
At baseline, subjects were assessed for
gingivitis, gingival bleeding and pla-
que by an experienced calibrated
examiner using the modified gingival
index (MGI), gingival bleeding index
(GBI) and Turesky modification of
the Quigley–Hein plaque index
(TMQHPI), respectively (18–20). Sub-
jects then received a complete dental
prophylaxis. Subjects were stratified
based on gender, smoking and the
number of bleeding sites and then
randomized within these strata to one
of two groups (control group or
865
experimental gingivitis group) in
blocks of four, using an encoded pro-
gram on site that was supplied by the
study sponsor. The assignment pro-
cess was performed in a protected
area to ensure blinding of the examin-
ers. Subjects in the control group
received intensive oral hygiene
instruction and were instructed to
brush twice daily for 2 min with an
ADA-reference manual toothbrush
and regular fluoride dentifrice (Crest
Cavity Protection; Procter & Gamble,
Mason, OH, USA), and to floss twice
daily after brushing (Oral-B Glide
Comfort Plus, Procter & Gamble) for
21 d. They were also instructed to
rinse with 10 mL of 0.05% neutral
sodium fluoride mouthrinse (ACT
Anti-Cavity mouthrinse; Chattem
Inc., Chatanooga, TN, USA) for 30 s,
once daily, for the 21 d. Subjects in
the experimental gingivitis group
stopped brushing and flossing and
only rinsed with 10 mL of the 0.05%
neutral sodium fluoride mouth rinse
for 30 s, once daily, for 21 d. The
examiners were blinded to product
assignment and distribution, which
was conducted by site staff. Product
was supplied in kit boxes that were
labeled with the subject number and
initials on the outside of the boxes.
Subjects were recalled at day 21, at
which time MGI, GBI and TMQHPI
scores were again recorded, and a
scaling and prophylaxis was provided.
At each visit, an oral soft tissue exam-
ination was performed and any
adverse events found, or reported,
were noted (Fig. 1).
Blood collection and genomic DNA
extraction
At baseline and day 21, peripheral
blood was drawn from enrolled sub-
jects 10 min after starting the scaling
procedure to maximize the level of
induced bacteremia (8). All blood
samples were drawn in BD Vacutain-
erÒ tubes (Becton, Dickinson and
Company, Franklin Lakes, NJ, USA)
containing 15% K3EDTA. Sample
tubes were put on ice immediately
and sent to the lab for plasma collec-
tion by standard procedure and geno-
mic DNA extraction using a QIAamp
866 Kinane et al.
Fig. 1. Flow of participants through the study. GBI, gingival bleeding index; MGI, modi-
fied gingival index; OST, Oral soft tissue; TMQHPI, Turesky modification of the Quigley–
Hein plaque index.
blood mini kit (QIAGEN Inc, Valen-
cia, CA, USA).
Real-time polymerase chain reaction
for total bacteria number in
peripheral blood
Primers and probes used for universal
bacteria detection were selected using
PRIMER EXPRESS software V 1.0
(Applied Biosystems International,
Foster City, CA, USA) and were
based on species-specific highly con-
served regions from the 16S rRNA
gene. The sequences of primers and
probes used in this study for the 16s
rRNA gene and the methods for total
bacteria detection have been previ-
ously described (21).
Luminex multiplex cytokine assay
The level of biomarkers, including
IL-1b, IL-6, IL-8, TNF-a and sI-
CAM-1 in plasma samples, were
determined by the Luminex 100 IS
Instrument System and LUMINEX100 IS
software (versions 2.3) (Luminex
Corp., Austin, TX, USA) using Bead-
lyteÒ human multi-cytokine detection
system containing multiple bead-
matesTM (Millipore Upstate, Char-
lottesville, VA, USA). Samples were
performed in triplicate and processed
using the Multiple Cytokine Detection
Protocol B in accordance with the
manufacturer’s instructions. The
results were analyzed and generated
by Upstate Beadview Multiplex data
analysis software (version 1.0) (Milli-
pore Upstate).
Enzyme-linked immunosorbent
assay
The levels of CRP in plasma samples
were assessed using an enzyme-linked
immunosorbent assay kit (Alpha
Diagnostic International, San Anto-
nio, TX, USA). The assay procedure
was carried out according to the man-
ufacturer’s instructions. Detection was
performed in triplicate.
Statistics
MGI, GBI and TMQHPI data were
summarized into a single whole
mouth average score for each subject
at each visit and presented as
mean  SD. The bacteria counts
were summarized as the median and
interquartile range. The plasma levels
of CRP (ng/mL), sICAM-1 (pg/mL),
TNF-a (pg/mL) and IL-8 (pg/mL)
were presented as mean  SEM or
median and interquartile range based
on the analyses of sample distribu-
tion by software GRAPHPAD INSTAT
(GraphPad Software Inc., San
Diego, CA, USA). IL-1b and IL-6
were not included in the analysis
because their levels in the majority
of samples were below the detection
limit. All statistical tests were two-
sided and comparison results
between groups were assessed by
ANCOVA using the SAS software
package (SAS Software, Cary, NC,
USA) and were reported as statisti-
cally significant if the test p-value
was ≤ 0.05.
Results
Of 80 subjects enrolled, one was
excluded due to antibiotic therapy
during the study. In total, 40 experi-
mental gingivitis and 39 control sub-
jects between 18 and 65 years of age
were included in the study results
(Table 1). All analyses performed are
reported below.
MGI and GBI scores significantly
increased after 21 d in the experimen-
tal gingivitis group relative to baseline
and compared to the control group
(Table 2, p < 0.01). TMQHPI scores
also significantly increased in the
experimental gingivitis group relative
to baseline and compared to the con-
trol group (Table 2, p < 0.01). Base-
line and day 21 MGI and GBI scores
were comparable (p > 0.33) for sub-
jects in the control group, indicating
that the oral hygiene regimen was
effective in the control of gingival
inflammation. There were four
adverse events (aphthous ulcer) in the
study and they were all resolved.
Quantitative polymerase chain reac-
tion (PCR) of total bacteria revealed
a significant decrease in the experi-
mental gingivitis group at 21 d
(Table 2, p = 0.04). We noted a sig-
nificant decrease in sICAM-1 in the
experimental gingivitis group
(p < 0.01) and IL-8 in the control
group (p < 0.01) at day 21 compared
to baseline. No significant change was
observed in CRP or TNF-a over the
21 d period.
 Early gingivitis & systemic inflammation 867

Table 1. Study population with oral status

Age No. of teeth

n Mean SD Median Min, max Female (%) Smoker (%) Mean SD Median Min, max

Control 39 39.6 13.42 41 19, 67 59 23 25.4 3.40 27 16, 28

Experimental gingivitis 40 39.6 11.81 39.5 18, 70 60 25 26.9 1.52 28 23, 28

Table 2. Average MGI, GBI, TMQHPI (mean  SD) and total bacterial count (median and interquartile range), and plasma levels of
CRP, sICAM-1 and IL-8 (median and interquartile range) in EG and control group for baseline and day 21

Clinical indices Grouping Baseline Day 21 Difference p value

MGI Control 0.15  0.09 0.19  0.11 0.04  0.04 0.33

EG 0.15  0.13 0.56  0.39 0.41  0.04 < 0.01
p value 0.92 < 0.01
GBI Control 21.64  15.12 27.33  24.80 5.69  6.85 0.44
EG 20.60  12.70 103.18  64.70 82.58  6.77 < 0.01
p value 0.74 < 0.01
TMQHPI Control 1.18  0.71 1.40  0.64 0.20  0.07 0.03
EG 1.30  0.64 2.46  0.63 1.18  0.07 < 0.01
p value 0.42 < 0.01
Total bacterial count Control 183.37 (245.86) 139.79 (79.64) 20.38 (190.99) 0.20
EG 238.86 (237.57) 220.16 (79.35) 35.72 (220.27) 0.04
p value 0.93 0.99
CRP (ng/mL) Control 4.98 (9.26) 5.06 (7.40) 0.08 (2.02) 0.90
EG 3.13 (3.55) 2.51 (3.35) 0.63 (1.88) 0.10
p value 0.44 0.22
sICAM-1 (pg/mL) Control 1860.00 (4089.00) 1160.00 (3449.00) 90.00 (925.00) 0.08
EG 1175.00 (1756.00) 884.00 (1050.00) 165.50 (1108.00) < 0.01
p value 0.41 0.42
TNF-a (pg/mL) Control 1.28 (18.10) 2.15 (22.70) 0.00 (1.84) 0.42
EG 3.25 (18.70) 4.50 (14.10) 0.00 (5.39) 0.93
p value 0.36 0.49
IL-8 (pg/mL) Control 6.23 (6.74) 5.69 (3.7) 1.32 (5.49) < 0.01
EG 5.33 (5.11) 4.61 (3.55) 0.37 (4.47) 0.34
p value 0.21 0.14

Control, control group; CRP, C-reactive protein; EG, experimental gingivitis group; GBI, gingival bleeding index; IL, interleukin; MGI,
modified gingival index; sICAM-1, soluble intercellular adhesion molecule-1; TMQHPI, Turesky modification of the Quigley–Hein plaque
index; TNF, tumor necrosis factor.

PCR technique, all samples in this

Correlation between total bacteria
with clinical indices and systemic
biomarkers
Interestingly, total bacteremia was
inversely correlated with MGI and
GBI at day 21 in the experimental
gingivitis group (Table 3, p < 0.05).
The total bacteremia were inversely
correlated with MGI at baseline for
all 79 subjects (p = 0.04) and posi-
tively with TMQHPI at both baseline
and day 21 in the control and experi-
mental gingivitis groups (p < 0.01).
Total bacteremias correlated posi-
tively with TNF-a at baseline in both
the control and experimental gingivitis
groups (p ≤ 0.01) and inversely with
IL-8 (p < 0.01) and sICAM-1
(p = 0.02) in the experimental gingivi-
tis group (Table 3).
Discussion
Bacteremia frequently occurs after
dental treatment and is detectable by
conventional microbiological culture
techniques as well as by specific PCR
and/or sequencing of the 16s rRNA
gene, including deep sequencing (22).
DNA sequencing (16s rRNA) follow-
ing BACTEC culturing provides more
accurate identification of a diverse
population of bacteria from bactere-
mia following dental extractions (23).
It is evident that PCR assessments
cannot discriminate live from dead
bacteria but remain a very sensitive
approach, which correlates with cul-
ture techniques and because of its sen-
sitivity, PCR quantification is the
preferred method for the current
study (8). Using the highly sensitive
study were positive for some bactere-
mia although individual levels differed
considerably.
Forner et al. reported an associa-
tion between bacteremia and gingival
index, plaque index and number of
sites with bleeding on probing, but
not with probing pocket depth mea-
surements (24). In the current experi-
mental gingivitis study, all clinical
indices, including MGI, GBI and
TMQHPI, increased significantly
upon termination of oral hygiene for
21 d. In contrast, the total bacteremia
and circulating levels of CRP,
sICAM-1 and IL-8 decreased,
although these declines were modest.
Plasma acute-phase proteins are usu-
ally present in low levels and upon
bacterial infections the levels drasti-
868 Kinane et al.

Table 3. Spearman rank correlation for total bacteremia with MGI, GBI, TMQHPI and reported no difference in serum CRP
with TNF-alpha, IL-8 and sICAM-1 in peripheral blood levels before and after treatment (16).
Baseline Day 21
Our study is consistent with these
findings, reporting no significant dif-
Clinical indices Grouping Spearman r p value Spearman r p value ference in plasma CRP after 21 d of
developing experimental gingivitis.
MGI Control 0.11 0.50 0.34 0.04
Prolonged vascular activation due
EG 0.28 0.09 0.33 0.04
Total 0.24 0.04 0.18 0.12 to inflammation increased serum lev-
els of sICAM (15), and circulating
GBI Control 0.16 0.33 0.22 0.19 levels of sICAM-l are elevated in
EG 0.08 0.63 0.39 0.01
smokers (32–34). The present study
Total 0.15 0.20 0.40 0.01
showed a significant decrease in
TMQHPI Control 0.44 < 0.01 0.44 < 0.01 plasma sICAM-1 after 21 d of devel-
EG 0.70 < 0.01 0.45 < 0.01 oping experimental gingivitis. A
Total 0.27 < 0.01 0.40 < 0.01
recent study shows that gingivitis and
TNF-alpha (pg/mL) Control 0.50 < 0.01 0.333 0.04 periodontitis operate as antagonistic
EG 0.40 0.01 0.071 0.66 modulators of gingival perfusion,
Total 0.46 < 0.01 0.204 0.07 which is positively related to the
IL-8 (pg/mL) Control 0.23 0.16 0.08 0.65 extent of gingivitis and negatively
EG 0.25 0.11 0.43 < 0.01 related to the extent of periodontitis
Total 0.21 0.02 0.24 0.03 (35). The perfusion difference may
CRP (ng/mL) Control 0.12 0.48 0.03 0.86 explain the decrease in these systemic
EG 0.16 0.32 0.20 0.22 inflammatory molecules during gingi-
Total 0.12 0.31 0.08 0.46 vitis contrasting with their increase in
sICAM-1 (pg/mL) Control 0.01 0.95 0.03 0.85 chronic periodontitis (35).
EG 0.31 0.05 0.36 0.02 We anticipated a positive associa-
Total 0.13 0.27 0.15 0.17 tion between local inflammation,
systemic markers and bacteremia.
Control, control group; CRP, C-reactive protein; EG, experimental gingivitis group; GBI,
However, the present study negates
gingival bleeding index; IL, interleukin; MGI, modified gingival index; sICAM-1, soluble
intercellular adhesion molecule-1; TMQHPI, Turesky modification of the Quigley–Hein this hypothesis, at least for short-term
plaque index; TNF, tumor necrosis factor. experimental gingivitis with a statisti-
cally sufficient number of subjects in a
cally increase (25–27). It is feasible 60 and 120 min (28). However, our randomized clinical trial. We showed
that bacteremia levels were so low time point was determined as optimal, a statistically significant decrease in
and of such short duration that they based on our previous study (8), for levels of sICAM-1 (p < 0.01) and bac-
did not influence systemic inflamma- assessing bacteremia changes and teremia (p = 0.04) and a directional
tory biomarkers. However, there were although it may not be optimal for but not significant decrease in levels
significant differences in acute-phase detecting changes in systemic biomar- of CRP (p = 0.10) and IL-8 (p = 0.34)
markers such as sICAM and IL-8, kers we did, however, see significant at 21 d in the experimental gingivitis
which negatively correlated with gin- changes in our experimental gingivitis group as the oral indices worsened. A
gival inflammation in the experimen- study. Findings from this study chal- limitation of the current study is that
tal gingivitis group. These findings lenge our current paradigm that the more time points and a longer time
contradict our original hypothesis and more inflammation one exhibits, the frame might have allowed us better to
the literature, which suggests that the greater the bacteremia. Beck et al., detect acute-phase proteins (28).
extent of bacteremia directly relates to however, stated that it is not the clini- In conclusion, bacteremia was neg-
the severity of gingival inflammation. cal indices per se that correlate with atively correlated with gingival inflam-
A positive correlation between the risk of systemic disease but the matory indices, bleeding indices and
TMQHPI, TNF-a and bacteremia ability of the host to mount an peripheral blood IL-8 levels thus
was found at baseline for both immune response against periodontal negating our working hypothesis and
groups. This is consistent with the lit- bacteria (29). the current literature that supports an
erature where TNF-a has been Previous studies showed that exten- association between chronic periodon-
reported as a proinflammatory media- sive periodontitis (periodontal disease titis and circulating systemic markers
tor during the short-term effect of > 4 mm in > 30% of the sites mea- of inflammation. We conclude that
intervention (28); Ide et al. performed sured) was associated with elevated there are marked differences in the
a short-term time course study of sys- CRP levels (30,31). Although a large systemic effects of short-term
temic marker changes following scal- body of evidence indicates that experimentally induced gingivitis and
ing in patients with periodontitis, systemic inflammation occurs in long-term chronic periodontitis, which
sampling venous blood at 0, 15, 30, periodontal disease, a meta-analysis must be considered in planning stud-

ies to address mechanisms whereby
oral diseases may affect systemic dis-
eases.
Acknowledgements
This investigation was supported by
DE017384 from the National Institute
of Dental and Craniofacial Research,
NIH, and Procter and Gamble
(Mason, OH, USA).
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