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: [email protected]
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
 Early gingivitis & systemic inflammation 865

but proposed to share several risk fac- groups would have 80% power, with experimental gingivitis group) in
tors, including infiltration of hyperin- a two-tailed alpha of 0.05, to detect blocks of four, using an encoded pro-
flammatory immune cells from various differences in group 1 ranging from gram on site that was supplied by the
sources (7). Bacteremia can occur 2.5% to 10% and in group 2 from study sponsor. The assignment pro-
through simple tooth brushing as well 25% to 38%. All subjects were in cess was performed in a protected
as scaling and root planing (8). Daily good general health, had a minimum area to ensure blinding of the examin-
episodes of bacteremia originating of 16 natural teeth with scorable ers. Subjects in the control group
from periodontal lesions can cause facial and lingual surfaces and had a received intensive oral hygiene
changes in systemic markers in peri- diagnosis of chronic gingivitis with instruction and were instructed to
odontitis (9). Moreover, the associa- > 20% bleeding sites or equivalent brush twice daily for 2 min with an
tion between periodontitis and and no obvious chronic periodontitis. ADA-reference manual toothbrush
coronary heart disease is particularly Subjects had to also agree to return and regular fluoride dentifrice (Crest
affected by the periodontal microbial for the scheduled visits and to follow Cavity Protection; Procter & Gamble,
pathogen burden (10). A number of study procedures, to refrain from Mason, OH, USA), and to floss twice
studies have shown that chronic peri- using any non-study oral care prod- daily after brushing (Oral-B Glide
odontitis is associated with increased ucts or receiving elective dental treat- Comfort Plus, Procter & Gamble) for
serum levels of inflammatory media- ment (including dental prophylaxis), 21 d. They were also instructed to
tors, including C-reactive protein and to avoid participating in any rinse with 10 mL of 0.05% neutral
(CRP), soluble intercellular adhesion other clinical study while the current sodium fluoride mouthrinse (ACT
molecule-1 (sICAM-1) and interleukin- study was in progress. Exclusion crite- Anti-Cavity mouthrinse; Chattem
6 (IL-6) (11–14). However, there are ria included the presence of four or Inc., Chatanooga, TN, USA) for 30 s,
also contradictory reports indicating more teeth in any two quadrants with once daily, for the 21 d. Subjects in
no significant differences in serum pockets > 5 mm; any systemic disease the experimental gingivitis group
CRP levels following periodontal treat- or significant medical conditions such stopped brushing and flossing and
ment (15,16). as a history of diabetes, or use of only rinsed with 10 mL of the 0.05%
The fact that gingivitis is reversible medication(s) known to affect peri- neutral sodium fluoride mouth rinse
and can be experimentally induced odontal status; any requirement for for 30 s, once daily, for 21 d. The
makes it an ideal condition for studies antibiotic premedication before dental examiners were blinded to product
addressing susceptibility to bacteria- procedures; being pregnant, lactating assignment and distribution, which
induced inflammation (17). In view of or not using birth control; or, having was conducted by site staff. Product
this, we designed an experimental gin- used systemic antibiotics within 1 mo was supplied in kit boxes that were
givitis study to: (i) evaluate the effect before enrollment. The study was labeled with the subject number and
of oral hygiene, including flossing, on approved by the University of Louis- initials on the outside of the boxes.
the prevention of gingival inflamma- ville Institutional Review Board. Par- Subjects were recalled at day 21, at
tion; (ii) explore the relationship ticipants in this study had signed and which time MGI, GBI and TMQHPI
between gingival inflammation and received a copy of the Informed Con- scores were again recorded, and a
bacteremia, as well as levels of circulat- sent Form, including HIPAA authori- scaling and prophylaxis was provided.
ing inflammatory biomarkers (includ- zation. At each visit, an oral soft tissue exam-
ing CRP, sICAM, tumor necrosis ination was performed and any
factor-alpha [TNF-a] and IL-8) that adverse events found, or reported,
Dental treatment
are associated with both the develop- were noted (Fig. 1).
ment of gingival inflammation and Two weeks pre-baseline, scaling was
systemic inflammatory biomarkers. performed on all subjects and they
Blood collection and genomic DNA
were given oral hygiene instructions.
extraction
At baseline, subjects were assessed for
Material and methods
gingivitis, gingival bleeding and pla- At baseline and day 21, peripheral
que by an experienced calibrated blood was drawn from enrolled sub-
Subjects
examiner using the modified gingival jects 10 min after starting the scaling
The present study comprised 80 index (MGI), gingival bleeding index procedure to maximize the level of
subjects (both male and female, ages (GBI) and Turesky modification of induced bacteremia (8). All blood
18–65 years) recruited by the investi- the Quigley–Hein plaque index samples were drawn in BD Vacutain-
gators from the periodontology clinic (TMQHPI), respectively (18–20). Sub- erÒ tubes (Becton, Dickinson and
of the University of Louisville, School jects then received a complete dental Company, Franklin Lakes, NJ, USA)
of Dentistry (Louisville, KY, USA) in prophylaxis. Subjects were stratified containing 15% K3EDTA. Sample
2005. Sample size was determined based on gender, smoking and the tubes were put on ice immediately
based on using Fisher’s exact test, number of bleeding sites and then and sent to the lab for plasma collec-
whereby a sample size of 80 subjects randomized within these strata to one tion by standard procedure and geno-
with 40 in each of two treatment of two groups (control group or mic DNA extraction using a QIAamp
866 Kinane et al.

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

Fig. 1. Flow of participants through the study. GBI, gingival bleeding index; MGI, modi- Of 80 subjects enrolled, one was
fied gingival index; OST, Oral soft tissue; TMQHPI, Turesky modification of the Quigley– excluded due to antibiotic therapy
Hein plaque index. during the study. In total, 40 experi-
mental gingivitis and 39 control sub-
jects between 18 and 65 years of age
blood mini kit (QIAGEN Inc, Valen- lottesville, VA, USA). Samples were were included in the study results
cia, CA, USA). performed in triplicate and processed (Table 1). All analyses performed are
using the Multiple Cytokine Detection reported below.
Protocol B in accordance with the MGI and GBI scores significantly
Real-time polymerase chain reaction
manufacturer’s instructions. The increased after 21 d in the experimen-
for total bacteria number in
results were analyzed and generated tal gingivitis group relative to baseline
peripheral blood
by Upstate Beadview Multiplex data and compared to the control group
Primers and probes used for universal analysis software (version 1.0) (Milli- (Table 2, p < 0.01). TMQHPI scores
bacteria detection were selected using pore Upstate). also significantly increased in the
PRIMER EXPRESS software V 1.0 experimental gingivitis group relative
(Applied Biosystems International, to baseline and compared to the con-
Enzyme-linked immunosorbent
Foster City, CA, USA) and were trol group (Table 2, p < 0.01). Base-
assay
based on species-specific highly con- line and day 21 MGI and GBI scores
served regions from the 16S rRNA The levels of CRP in plasma samples were comparable (p > 0.33) for sub-
gene. The sequences of primers and were assessed using an enzyme-linked jects in the control group, indicating
probes used in this study for the 16s immunosorbent assay kit (Alpha that the oral hygiene regimen was
rRNA gene and the methods for total Diagnostic International, San Anto- effective in the control of gingival
bacteria detection have been previ- nio, TX, USA). The assay procedure inflammation. There were four
ously described (21). was carried out according to the man- adverse events (aphthous ulcer) in the
ufacturer’s instructions. Detection was study and they were all resolved.
performed in triplicate. Quantitative polymerase chain reac-
Luminex multiplex cytokine assay
tion (PCR) of total bacteria revealed
The level of biomarkers, including a significant decrease in the experi-
Statistics
IL-1b, IL-6, IL-8, TNF-a and sI- mental gingivitis group at 21 d
CAM-1 in plasma samples, were MGI, GBI and TMQHPI data were (Table 2, p = 0.04). We noted a sig-
determined by the Luminex 100 IS summarized into a single whole nificant decrease in sICAM-1 in the
Instrument System and LUMINEX100 IS mouth average score for each subject experimental gingivitis group
software (versions 2.3) (Luminex at each visit and presented as (p < 0.01) and IL-8 in the control
Corp., Austin, TX, USA) using Bead- mean  SD. The bacteria counts group (p < 0.01) at day 21 compared
lyteÒ human multi-cytokine detection were summarized as the median and to baseline. No significant change was
system containing multiple bead- interquartile range. The plasma levels observed in CRP or TNF-a over the
matesTM (Millipore Upstate, Char- of CRP (ng/mL), sICAM-1 (pg/mL), 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 Discussion
study were positive for some bactere-
with clinical indices and systemic
Bacteremia frequently occurs after mia although individual levels differed
biomarkers
dental treatment and is detectable by considerably.
Interestingly, total bacteremia was conventional microbiological culture Forner et al. reported an associa-
inversely correlated with MGI and techniques as well as by specific PCR tion between bacteremia and gingival
GBI at day 21 in the experimental and/or sequencing of the 16s rRNA index, plaque index and number of
gingivitis group (Table 3, p < 0.05). gene, including deep sequencing (22). sites with bleeding on probing, but
The total bacteremia were inversely DNA sequencing (16s rRNA) follow- not with probing pocket depth mea-
correlated with MGI at baseline for ing BACTEC culturing provides more surements (24). In the current experi-
all 79 subjects (p = 0.04) and posi- accurate identification of a diverse mental gingivitis study, all clinical
tively with TMQHPI at both baseline population of bacteria from bactere- indices, including MGI, GBI and
and day 21 in the control and experi- mia following dental extractions (23). TMQHPI, increased significantly
mental gingivitis groups (p < 0.01). It is evident that PCR assessments upon termination of oral hygiene for
Total bacteremias correlated posi- cannot discriminate live from dead 21 d. In contrast, the total bacteremia
tively with TNF-a at baseline in both bacteria but remain a very sensitive and circulating levels of CRP,
the control and experimental gingivitis approach, which correlates with cul- sICAM-1 and IL-8 decreased,
groups (p ≤ 0.01) and inversely with ture techniques and because of its sen- although these declines were modest.
IL-8 (p < 0.01) and sICAM-1 sitivity, PCR quantification is the Plasma acute-phase proteins are usu-
(p = 0.02) in the experimental gingivi- preferred method for the current ally present in low levels and upon
tis group (Table 3). study (8). Using the highly sensitive 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-
 Early gingivitis & systemic inflammation 869

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