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TheImpactoftheStoneAgeDiet
on Gingival Conditions in the Absence
of Oral Hygiene
Stefan Baumgartner,* Thomas Imfeld,
†
Olivier Schicht,
†
Christian Rath,
†
Rigmor E. Persson,*
‡§
and G. Rutger Persson*
‡§i
Background: The objective of this study was to assess the oral
microbiota and clinical data in subjects without access to traditional
oral hygiene methods and who ate a diet available in the Stone Age.
Methods: Ten subjects living in an environment replicating the
Stone Age for 4 weeks were enrolled in this study. Bleeding on probing
(BOP), gingival and plaque indices, and probing depth (PD) were
assessed at baseline and at 4 weeks. Microbiologic samples were col-
lected at the mesio-buccal subgingival aspects of all teeth and from
the dorsum of the tongue and were processed by checkerboard
DNA-DNA hybridization methods.
Results: No subject had periodontitis. Mean BOP decreased from
34.8% to 12.6% (P<0.001). Mean gingival index scores changed
from 0.38 to 0.43 (not statistically significant) and mean plaque
scores increased from 0.68 to 1.47 (P<0.001). PD at sites of subgin-
gival sampling decreased (mean difference: 0.2 mm; P<0.001). At
week 4, the total bacterial count was higher (P<0.001) for 24 of 74
species, including Bacteroides ureolyticus,Eikenella corrodens,Lac-
tobacillus acidophilus,Capnocytophaga ochracea,Escherichia coli,
Fusobacterium nucleatum naviforme,Haemophilus influenzae,Helico-
bacter pylori,Porphyromonas endodontalis,Staphylococcus aureus
(two strains), Streptococcus agalactiae,Streptococcus anginosis,and
Streptococcus mitis. Bacterial counts from tongue samples were higher
at baseline (P<0.001) for 20 species, including Tannerella forsythia
(previously T. forsythensis), Aggregatibacter actinomycetemcomitans
(previously Actinobacillus actinomycetemcomitans; serotype a), and
Streptococcus spp.
Conclusions: The experimental gingivitis protocol is not applicable if
the diet (e.g., Stone Age) does not include refined sugars. Although pla-
que levels increased, BOP and PD decreased. Subgingival bacterial
counts increased for several species not linked to periodontitis, whereas
tongue bacterial samples decreased during the study period. JPeri-
odontol 2009;80:759-768.
KEY WORDS
Diet; gingivitis; microbiology; periodontitis.
Dental plaque is a com-
plex biofilm accumulat-
ing on teeth and oral
tissues. Environmental and ge-
netic factors are influential in the
development of dental plaque bio-
films.
1,2
Although dental plaque
is made up of a large variety of
bacterial species, the coloniza-
tion follows a regimented pat-
tern: adhesion of initial colonizers
to the acquired pellicle, followed
by secondary colonization through
interbacterial adhesion.
3
It is
well established that many of
the early colonizing bacteria
in the development of dental
biofilms include Actinomyces,
Lactobacillus,andStreptococcus
species.
4
However, microbial
communities in the oral cavity
are polymicrobial.
5,6
Many of
the individual microorganisms
in such communities cannot sur-
vive outside that community.
The role of dental plaque in
gingivitis is well established.
7-11
Thus, within 8 days of the begin-
ning of plaque accumulation, an
early lesion exhibiting many fea-
tures characteristic of delayed
hypersensitivity develops.
12
In-
creased gingival crevicular fluid
during gingivitis affects pellicle
formation, and increased plasma
proteins in the pellicle may modify
* Laboratory of Oral Microbiology, Department of Clinical Research, School of Dental Medicine,
University of Bern, Bern, Switzerland.
† Department of Preventive Dentistry, Periodontology, and Cariology, Center for Dental Medicine,
University of Zu
¨rich, Zu
¨rich, Switzerland.
‡ Regional Clinical Dental Research Center, University of Washington, Seattle, WA.
§ Department of Oral Medicine, University of Washington.
iDepartment of Periodontics, University of Washington.
doi: 10.1902/jop.2009.080376
J Periodontol • May 2009
759
bacterial attachment and early dental plaque com-
position.
13
The complement system may also be ac-
tivated in gingival crevice material from inflamed
gingiva.
14
Animal studies
15,16
showed that diet may play an
important role in the development of gingivitis. Stud-
ies
17,18
in humans demonstrated that subjects on a
high-carbohydrate diet developed gingivitis com-
pared to subjects on a low-carbohydrate diet. An in-
crease in dietary sucrose has been associated with
more accumulation of plaque and evidence of gingi-
vitis in humans.
19
Thus, an assessment of sucrose in-
take followed by appropriate changes in diet seems
appropriate in clinical efforts to reduce the extent of
gingivitis. Studies
20,21
also demonstrated that exper-
imental gingivitis and chronic gingivitis may not be
comparable. After 4 weeks of experimental gingivitis,
more plaque accumulation and higher interleukin
(IL)-1blevels, but lower IL-8 levels, were demon-
strated in the gingival crevicular fluid.
21
Data also
demonstrated that levels of aspartate aminotrans-
ferase in gingival crevicular fluid were much higher
in experimental gingivitis than in chronic gingivitis.
22
A decrease in the prevalence of gingivitis has been
recognized through the analysis of trends over 30
years.
23
Nevertheless, high prevalence scores for
supragingival plaque and gingivitis have been re-
ported in adolescent subjects.
24,25
The prevalence
of periodontitis seems to have remained virtually
constant during the past 3,000 years in Great Britain,
despite considerable changes in the oral environ-
ment.
26,27
In an isolated community (Isla Grande, Co-
lombia) with no dental services and a low educational
level, a Community Periodontal Index of Treatment
Needs (CPITN) score of 1 (presence of bleeding on
probing [BOP]) was found in only 18% of subjects,
and 11% presented with probing depths (PDs) ‡5mm
(CPITN =4).
28
To the best of our knowledge, there
are no studies on the periodontal and oral microbio-
logic conditions in subjects without access to oral hy-
giene measures or a modern diet.
The purpose of the present longitudinal case series
was to assess the oral microbiota and clinical data in a
cohort of 10 subjects with no access to traditional oral
hygiene methods and who ate a diet available to Stone
Age humans over a 4-week period.
MATERIALS AND METHODS
In 2007, Swiss National Television approached fac-
ulty members at the University of Bern and at the Uni-
versity of Zu
¨rich to monitor a group of subjects who
had committed themselves to living in a confirmed en-
vironment replicating that of the Stone Age. Archeo-
logic experts were consulted about the environment,
which included replication of housing, clothing, uten-
sils, and food (fishing and hunting) known from ar-
cheologic sites in Switzerland. The project received
ethics approval (University of Zu
¨rich), and all subjects
signed approved informed consent/assent forms.
These subjects were confined to living conditions that
archeologic experts had identified as typical for Stone
Age humans along the Rhine River. Nutrition and
health experts monitored the daily activities and eat-
ing habits of the participants.
Ten subjects were enrolled, including two families
(husband, wife, and two children each) and two single
young adult males. These volunteers lived under
Stone Age conditions for 4 weeks. Daily television
reports were broadcast about their experiences. A
thorough medical (data not shown) and dental exam-
ination was performed before the study and 4 weeks
later.
All subjects were examined before and after the
4-week study at the University of Zu
¨rich, School of
Dentistry; assessments included extra- and intraoral
examinations, periodontal screening index, and
partial-mouth recording of plaque index (PI)
29
and
gingival index (GI).
30
Thus, PI was recorded in the
first (upper right) and third quadrants, whereas GI
was recorded in the second and fourth quadrants
(lower left) at the University of Zu
¨rich (by OS and
CR). Bitewing radiographs were taken (only at base-
line) of areas for which no recent radiographic docu-
mentation was available, and dental photographs
were taken. At baseline and at week 4, faculty mem-
bers at the University of Bern (GRP and REP) collected
subgingival microbiologic samples at the mesio-
buccal aspects of all teeth present in each subject.
The same examiner took the bacterial samples and
recorded the measurements of the subject at both
visits. The examiners had no access to baseline data
or the results from the other center. It was not possible
to mask them in terms of the order of examination.
None of the subjects had access to toothbrushes,
toothpaste, dental floss, toothpicks, or other oral hy-
giene products during the study period. No advice was
given about how to clean teeth without access to such
oral hygiene aids; the use of twigs and any other nat-
ural material was allowed. Swiss television crews and
security guards ensured that all subjects maintained
the appropriate lifestyle for Stone Age humans.
Stone Age Diet and Living Conditions
The study subjects signed a contract with the Swiss
television system. The environment was developed
by anthropologists to be as similar as possible to what
had been identified in archeologic findings from an
area close to the Rhine River in Switzerland and dated
early Stone Age or between ;4000 and 3500 BC. Liv-
ing quarters, clothing, tools, and types of food stock
were provided as known from archeologic findings
in the region. Therefore, the diet was restricted to
Natural Experimental Gingivitis Volume 80 • Number 5
760
include a basic supply of whole grains of barley,
wheat, spelt (‘‘einkorn,’’ ‘‘emmer’’ =local ancient ag-
ricultural wheat), some salt, herbs, honey, milk, and
meat from domestic animals (goats and hens). A
hunter would shoot one of the goats at the partici-
pants’ request. This food supply would not provide
the participants with a full diet over 4 weeks. Hence,
they were forced to seek supplemental food from na-
ture, including berries, edible plants, and fish without
nets. A sports medicine physician monitored the sub-
jects. The location was within a nature reservation,
and subjects were restricted in how they could move
and use natural resources. The subjects had to make
fire by themselves. They had no access to refined sug-
ars or modern kitchen utensils. Huts were available for
them as living quarters.
Microbiologic Sampling
All sampled areas were isolated from saliva contam-
ination. Supragingival plaque was gently removed
with a curet.
¶
Subgingival plaque samples were col-
lected with sterile endodontic paper points (size 55)
inserted into the pocket for 20 seconds. Samples were
placed individually in 1.5-ml natural flat-cap micro-
centrifuge tubes free of DNase and DNA and sterile.
#
Bacterial samples were also collected from the back
of the tongue using sterile swabs,** which were
placed in labeled sterile containers. All samples were
placed in boxes at 4C, transported within 4 hours to
the laboratory, and immediately frozen at -20C.
Following bacterial sampling, clinical PD measure-
ments were made, using Michigan periodontal probes,
††
at the mesio-buccal aspect of all teeth that had
been sampled. In addition, the extent of bleeding on
probing (BOP) was assessed 10 seconds after prob-
ing. All clinical examinations were performed with
no access to previous data and with no information
about the results of the examinations at the University
of Zu
¨rich.
Microbiologic Processing
We analyzed the samples with the checkerboard
DNA-DNA hybridization technique. Seventy-four
bacterial species were included in the checkerboard
panel (Table 1). The checkerboard DNA-DNA hy-
bridization method was performed as described by
Socransky et al.
31,32
For processing, 0.15 ml Tris
EDTA buffer (10 mM Tris-HCl, 1.0 mM EDTA, pH
7.6) was added to each vial with a subgingival bacte-
rial sample; 0.10 ml 0.5 M NaOH was added to each
Eppendorf tube with an oral bacterial sample; and 300
ml Tris EDTA buffer (10 mM Tris-HCl, 1.0 mM EDTA
and pH 7.6) was added to each Eppendorf tube with a
tongue bacterial sample. After 10 minutes, these
samples were sonicated for 10 seconds. Subse-
quently, 200 ml freshly made 0.5 M NaOH was added
to each vial, and the swab was removed.
Bacterial DNA was extracted, concentrated on ny-
lon membranes,
‡‡
and fixed by cross-linking using ul-
traviolet light.
§§
The membranes with fixed DNA were
placed in a multichannel incubation chamber.
ii
A gel
and blot imaging system
¶¶
for chemifluorescence-
based methods was used for quantification using a
setting of 200 mm and 600 V. The digitized information
was analyzed by a software program
##
allowing com-
parison of the density of 19 sample lanes against the
two standard lanes (10
5
or 10
6
cells). Signals were
converted to absolute counts by comparisons with
these standards.
30
The bacteria assayed are identified
as panel 1 and panel 2 (Table 1).
Statistics
The paired ttest was used to assess changes in the
clinical indices (PD, BOP, GI, and PI) over time. Sub-
ject-based mean values were calculated for each bac-
terial species. The paired-samples ttest was used to
compare bacterial counts between baseline and
week 4. Tongue samples were analyzed with the
Wilcoxon signed-sum rank test. Significance was de-
clared at the P<0.001 level. A statistical software
package was used for the analysis.***
RESULTS
Subjects
Subject characteristics and clinical data are presented
in Table 2. No subject presented with clinical evidence
of periodontitis. Between baseline and week 4, the
mean percentages of sites with BOP decreased from
34.8% to 12.6% (P<0.001) (Fig. 1). Decreases in
PD were also found at sites from which bacterial sam-
ples were taken (mean difference, 0.2 mm; 95% con-
fidence interval: 0.1 to 0.3 mm; P<0.001). The mean
PI at baseline and 4 weeks (partial-mouth recordings)
was 0.68 and 1.47, respectively (P<0.001). The mean
GI (partial-mouth recordings) at baseline and 4 weeks
was 0.38 and 0.43, respectively (not statistically sig-
nificant). Anterior clinical images are presented for
one of the subjects at baseline (Fig. 2) and at 4 weeks
(Fig. 3).
Microbiology
Tongue samples. The baseline and 4-week distri-
butions of Tannerella forsythia (previously T. forsyth-
ensis), Aggregatibacter actinomycetemcomitans (a)
(previously Actinobacillus actinomycetemcomitans),
and Streptococcus gordonii for the 10 subjects are
¶ Gracey curet 4R/4L, Deppeler, Rolle, Switzerland.
# Starlab, Ahrensburg, Germany.
** Catch-All Sample Collection Swabs, Epicentre, Madison, WI.
†† PCP 11, Hu-Friedy, Chicago, IL.
‡‡ Roche Diagnostics, Mannheim, Germany.
§§ Stratalinker 1800, Stratagene, La Jolla, CA.
ii Miniblotter 45, Immunetics, Cambridge, MA.
¶¶ Storm 840 Fluor-Imager, Amersham Biosciences, Piscataway, NJ.
## ImageQuant, Amersham Biosciences.
*** SPSS 16.0, SPSS, Chicago, IL.
J Periodontol • May 2009 Baumgartner, Imfeld, Schicht, Rath, Persson, Persson
761
Table 1.
Bacterial Species and Subspecies Included in the DNA-DNA Checkerboard Kit
Species Panel 1 Collection Species Panel 2 Collection
1a. A. actinomycetemcomitans (a) ATCC 29523 1. A. neuii GUH 550898
1b. A. actinomycetemcomitans (Y4) ATCC 43718 2. Aerococcus christensenii GUH 070938
2. Actinomyces israelii ATCC 12102 3. A. vaginalis GUH 290486
3. A. naeslundii (type I +II) ATCC 43146 4. Atopobium parvulum GUH 160323
4. A. odontolyticus ATCC 17929 5. A. vaginae GUH 010535
5. C. gracilis ATCC 33236 6. B. ureolyticus GUH 080189
6. C. rectus ATCC 33238 7. B. biavatii GUH 071026
7. C. showae ATCC 51146 8. Bifidobacterium bifidum GUH 070962
8. Capnocytophaga gingivalis ATCC 33612 9. Bifidobacterium breve GUH 080484
9. C. ochracea ATCC 33596 10. B. longum GUH 180689
10. C. sputigena ATCC 33612 11. Corynebacterium aurimucosum
pseudogenitalium
GUH 071035
11. E. corrodens ATCC 23834 12. Corynebacterium nigricans GUH 450453
12. E. saburreum ATCC 33271 13. Dialister sp. GUH 071045
13a. Fusobacterium nucleatum nucleatum ATCC 25586 14a. E. faecalis GUH 170812
13b. Fusobacterium nucleatum
polymorphum
ATCC 10953 14b. E. faecalis ATCC 29212
13c. F. nucleatum naviforme ATCC 49256 15. E. coli GUH 070903
14. Fusobacterium periodonticum ATCC 33693 16. G. vaginalis GUH 080585
15. L. acidophilus ATCC 11975 17. H. influenzae ATCC 49247
16. L. buccalis ATCC 14201 18. H. pylori ATCC 43504
17. N. mucosa ATCC 33270 19. L. crispatus GUH 160342
18. P. intermedia ATCC 25611 20. L. gasseri GUH 170856
19. P. micra ATCC 19696 21. Lactobacillus iners GUH 160334
20. P. melaninogenica ATCC 25845 22. L. jensenii GUH 160339
21. P. nigrescens ATCC 33563 23. Lactobacillus vaginalis GUH 0780928
22. P. acnes (type I +II) ATCC 11827/28 24. M. curtisii GUH 070927
23. P. gingivalis ATCC 33277 25. Mobiluncus mulieris GUH 070926
24. S. noxia ATCC 43541 26. Peptoniphilus sp. GUH 550970
25. S. aureus ATCC 25923 27. P. anaerobius GUH 160362
26. S. anginosus ATCC 33397 28. P. endodontalis ATCC 35406
27. S. constellatus ATCC 27823 (M32b) 29. Prevotella bivia GUH 450429
28. S. gordonii ATCC 10558 30. P. disiens GUH 190184
29. S. intermedius ATCC 27335 31. P. mirabilis GUH 070918
Natural Experimental Gingivitis Volume 80 • Number 5
762
presented in Figures 4 through 6, respectively. Anal-
ysis by Wilcoxon signed-sum rank test demonstrated
that higher bacterial counts at baseline (P<0.001)
were found for 20 species, including Actinomyces
neuii,Atopobium vaginae,A. actinomycetemcomi-
tans (serotype a), Actinomyces naeslundii,Campylo-
bacter rectus,Eubacterium saburreum,Leptotrichia
buccalis,Parvimonas micra (previously Peptostrepto-
coccus micros or Micromonas micros), Peptoniphilus
sp., Pseudomonas aeruginosa,Selenomonas noxia,
Staphylococcus aureus,S. gordonii,Streptococcus
intermedius,Streptococcus mitis,Streptococcus
Table 1. (continued)
Bacterial Species and Subspecies Included in the DNA-DNA Checkerboard Kit
Species Panel 1 Collection Species Panel 2 Collection
30. S. mitis ATCC 49456 32. P. aeruginosa ATCC 33467
31. S. oralis ATCC 35037 33a. S. aureus (yellow) GUH 070921
32. S. sanguinis ATCC 10556 33b. S. aureus (white) GUH 070922
33. S. mutans ATCC 25175 34. Staphylococcus epidermidis GUH 130381
34. T. forsythia ATCC 43037 (338) 35. Staphylococcus haemolyticus GUH 071047
35. T. denticola ATCC 35405 36. S. agalactiae GUH 230282
36. T. socranskii D40DR2 37. Varibaculum cambriense GUH 070917
37. Veillonella parvula ATCC 10790
ATCC =AmericanType Culture Collection;D =sample fromForsyth Institute, Boston,Massachusetts; GUH=Ghent University HospitalCollection, Ghent, Belgium.
Ta b l e 2 .
Subject Characteristics at Baseline (BL) and Week 4 (W4)
Subject Age (years) Teeth (N) Time PI
BOP
(% sites)
PD
(mm; mean)
PD
(mm; SD)
Father family 1 46 28 BL 0.2 35.7 2.5 0.7
W4 0.8 35.7 2.1 0.7
Mother family 1 45 26 BL 0 34.6 1.7 0.6
W4 0.9 0.0 2.2 0.6
Older child (A) family 1 (female) 18 28 BL 0 17.9 2.1 0.5
W4 0.2 3.6 1.9 0.5
Younger child (B) family 1 (male) 8 23 BL 0.8 47.8 2.1 0.4
W4 0.2 17.4 1.7 0.8
Father family 2 44 28 BL 0.1 10.7 2.6 0.6
W4 0.1 10.7 2.1 0.7
Mother family 2 44 26 BL 0.8 75.0 2.4 0.6
W4 0.5 14.3 2.5 0.6
Older child (C) family 2 (female) 12 26 BL 0.1 0.0 2.4 0.8
W4 0.4 11.5 2.2 0.6
Younger child (D) family 2 (female) 11 23 BL 0 27.3 1.8 0.6
W4 0.2 4.4 1.8 0.7
Young male 1 25 28 BL 0.8 27.3 2.0 0.6
W4 1.5 17.9 2.0 0.6
Young male 2 21 28 BL 0.31 71.4 2.7 0.6
W4 0.53 10.7 2.4 0.5
J Periodontol • May 2009 Baumgartner, Imfeld, Schicht, Rath, Persson, Persson
763
mutans,Streptococcus oralis,T. forsythia,Treponema
denticola,Treponema socranskii, and for the total
bacterial count. Trends (P<0.01) for higher bacterial
counts at baseline were found for Lactobacillus acid-
ophilus,S. oralis,Campylobacter gracilis,Campylo-
bacter showae,Streptococcus constellatus,S.
mutans,Propionibacterium acnes, Prevotella melani-
nogenica,Streptococcus anginosus,Anaerococcus
vaginalis,Bacteroides ureolyticus,Bifidobacterium
biavatii,Lactobacillus gasseri, and Mobiluncus
curtisii. Higher bacterial counts at week 4 (P<0.001)
were found for Helicobacter pylori and Lactobacillus
crispatus. Trends (P<0.01) for higher bacterial counts
at week 4 were found for Porphyromonas gingivalis
and Enterococcus faecalis.
Subgingival samples. The total bacterial count of
all 74 species was significantly higher at 4 weeks
(P<0.001). The bacterial counts were specifically
higher (P<0.001) for 24 of 74 species, including Ac-
tinomyces odontolyticus,A. vaginae,B. ureolyticus,
Eikenella corrodens,L. acidophilus,Capnocytophaga
ochracea,Dialister sp., Escherichia coli,Fusobacte-
rium nucleatum naviforme,Gardnerella vaginalis,
Haemophilus influenzae,H. pylori,L. crispatus,
Lactobacillus jensenii,N. mucosa,Peptoniphilus
sp., Porphyromonas endodontalis,Prevotella disiens,
Figure 1.
BOP at baseline (BL) and at week 4 for each subject. Data for the
children are given after their parents.
Figure 2.
Anterior clinical image for one of the subjects at baseline.
Figure 3.
Anterior clinical image for the same subject in Figure 2 at 4 weeks.
Figure 4.
Counts of T. forsythia in tongue samples from baseline and week 4.
Only one sample was taken at each time point.
Figure 5.
Counts of A. actinomycetemcomitans (a) in tongue samples from
baseline and week 4. Only one sample was taken at each time point.
Natural Experimental Gingivitis Volume 80 • Number 5
764
Prevotella mirabilis,Staphylococcus aureus (two
strains), Streptococcus agalactiae,S. anginosus,and
S. mitis and illustrated for three selected species with
significant differences between baseline and week 4
and based on bacterial distributions among the 10
subjects (Figs. 7 through 9). Trends of higher counts
(P<0.01) at week 4 were also noted for the following
species: Capnocytophaga sputigena,Peptostrepto-
coccus anaerobius,Prevotella intermedia,Prevotella
nigrescens,S. oralis,andStreptococcus sanguinis.
At week 4, lower counts (P<0.001) were found for A.
naeslundii,Bifidobacterium longum,andL. buccalis.
DISCUSSION
Through daily television broadcastings it became ob-
vious that the participating subjects were mainly oc-
cupied by tasks such as searching for and preparing
food, as well as conserving energy. The lack of access
to detergents and water was obvious. They had been
provided a stock supply of cereals, wild fruits, nuts,
herbs, wild mushrooms, honey, some salt, and dried
meat. They had no access to refined sugars or modern
kitchen utensils and had to prepare food over an open
Figure 6.
Counts of S. gordonii in tongue samples from baseline and week 4.
Only one sample was taken at each time point.
Figure 7.
Box plot diagram illustrating the distribution of subgingival samples of
P. endodontalis at baseline (BL) and P. endodontalis at week 4 (W4)
as calculated from all sites examined in each subject (=outlier value;
*=extreme outlier value). Unit of measure for y axis =·10
5
bacterial
cells.
Figure 8.
Box plot diagram illustrating the distribution of subgingival samples of
F. nu c l e a t um nav i f o r m e at baseline (BL) and at week 4 (W4) as
calculated from all sites examined in each subject (=outlier value; * =
extreme outlier value). Unit of measure for y axis =·10
5
bacterial cells.
Figure 9.
Box plot diagram illustrating the distribution of subgingival samples of
H. pylori at baseline (BL) and at week 4 (W4) as calculated from all
sites examined in each subject (=outlier value; * =extreme outlier
value). Unit of measure for y axis =·10
5
bacterial cells.
J Periodontol • May 2009 Baumgartner, Imfeld, Schicht, Rath, Persson, Persson
765
fire. Some of the subjects used twigs to clean their
teeth. The data demonstrated that the use of such oral
hygiene tools was highly insufficient to prevent further
accumulation of supragingival dental plaque, as il-
lustrated by an increase in PI. Although some of
the clinical measurements were taken only from the
mesio-buccal aspects, the use of twigs was also insuf-
ficient at these sites, which might have been the easi-
est ones to clean.
The consequence of having no access to modern
oral hygiene methods is reflected by the increase in
supragingival plaque scores. However, this increase
was not accompanied by an anticipated increase in
the severity of gingival inflammation. The insignifi-
cant increases in the subjects’ mean GI from 0.38 to
0.43, with a decrease in BOP scores as well as a slight
decrease in PDs, was not expected.
Different patterns of bacterial changes were ob-
served between the tongue and subgingival samples.
For tongue samples, higher bacterial counts of S.
gordonii and S. mitis were found at baseline, whereas
higher counts at week 4 compared to baseline in sub-
gingival samples were found only for S. mitis. This dif-
ference may have to do with differences in nutrition
and the requirement for sugars between these two
bacteria. In contrast, P. endodontalis was found at higher
levels in tongue and subgingival samples at week 4
compared to baseline. S. oralis was found at higher
counts at baseline samples from the tongue, but at
higher counts at week 4 in subgingival samples. This
may also have to do with differences in access to
nutrients as an effect of diet changes among the sub-
jects. Counts of A. actinomycetemcomitans (Y4) and
T. denticola were higher at baseline than at week 4 in
samples from the tongue; they did not undergo
changes in the subgingival samples. S. mutans and
S. gordonii were found at higher counts in samples
from the tongue at baseline, but no changes were
noted in subgingival samples. The reductions of T.
forsythia (Fig. 4) and S. gordonii (Fig. 6) in the tongue
samples were consistent among all subjects. The
changes in the levels of A. actinomycetemcomitans
(a) were not as obvious. The adolescent and children
in the study (subjects 3, 4, 7, and 8) did not seem to
differ from the adults in terms of bacterial changes.
The fact that a more pathogenic microbiota, com-
monly found in tooth decay and gingivitis/periodonti-
tis, was not present may have several explanations.
The lack of access to refined sugars could have had
an impact on sugar-fermenting bacteria and biofilm
development associated with disease. Honey was
the primary source of sugar. The findings reported
here are consistent with earlier studies
15-17,19
on car-
bohydrate restriction. Dietary sugar restriction seems
to be an appropriate measure to reduce the extent of
gingivitis, whereas a carbohydrate-rich diet increases
the severity of gingivitis.
18
A diet rich in sucrose
seems to enhance plaque accumulation, whereas a
glucose-rich diet seems to have marginal effects.
15
The assessment of dietary habits was difficult be-
cause an analysis of the percentage or weight of car-
bohydrates, fat, proteins, and calories for the specific
stock supply of dried fruits, nuts, grains, mushrooms,
dried meat, and so forth was not possible. The sub-
jects lost between 1 and 5 kg of body weight and a re-
duction in blood pressure was noted (data not shown)
during the 1-month period.
These subjects had been eating a normal Swiss diet
prior to enrollment in the project. The Swiss diet is
commonly rich in carbohydrates and fat. We hypoth-
esized that the changes in the clinical and microbio-
logic parameters were the effects of diet and life
conditions in general.
Although honey contains glucose and sucrose, it
seems to have antibacterial properties against viri-
dans streptococci.
33
Phenolic compounds in honey
may exert antibacterial activity.
34
Through its capac-
ity to reduce levels of reactive oxygen species, honey
seems to have antibacterial properties against P.
aeruginosa.
35
However, in the present study, counts
of P. aeruginosa increased in subgingival samples.
P. aeruginosa has been associated with periodonti-
tis.
36
Data have also shown that honey reduces blood
lipids, homocysteine, and C-reactive protein in nor-
mal and hyperlipidemic subjects.
37
Reductions in sys-
tolic and diastolic blood pressures were found among
the participating subjects (data not shown).
Dog rose, sour cherries, blackberries, strawberries,
raspberries, and blueberries are high in antioxi-
dants.
38
Flavonoids are commonly found in fruit,
vegetables, nuts, seeds, stems, flowers, tea, wine,
and honey. For centuries, preparations containing
these compounds have been used to treat human dis-
eases. Various structures of flavonoids may possess
antifungal, antiviral, and antibacterial activity.
39,40
Cereals and berries were primary food sources for
the subjects. Cereals and berries are rich in polyphe-
nols with anti-inflammatory properties.
41
Berries
found in nature and cereals were sought by the sub-
jects. They also consumed mushrooms. Data suggest
that mushrooms can modulate immune responses,
resulting in more enhanced innate and acquired dis-
ease resistance.
42
It was not assessed whether psychologic stress was
a major component during the 4 weeks. The 4 weeks
away from daily life might have been a positive factor
for the participants. The current level of stress and
psychosocial variables indicative of stress suscepti-
bility do not seem to account for variability in plaque
accumulation and gingival inflammation during ex-
perimental gingivitis in young adults.
43
The impact
of stress on experimental or persistent gingivitis might
Natural Experimental Gingivitis Volume 80 • Number 5
766
also be very different.
44,45
Furthermore, the impact on
inflammation in experimental gingivitis may be differ-
ent from that of persistent gingivitis.
21
The design of experimental gingivitis usually in-
cludes an initial phase in which subjects are provided
instruction about oral hygiene to reduce gingival in-
flammation, followed by a period of no hygiene.
7
Sub-
jects entered into the present study without an initial
phase of attempting oral hygiene improvements to re-
duce gingival inflammation. This was reflected by an
average BOP score of 34.8%. Therefore, in our first
hypothesis, prior to the initiation of the study, we an-
ticipated that abstinence or further insufficient oral hy-
giene measures would result in a significant increase
in PD, BOP, GI, and PI. The data demonstrated that this
was only true for PI. Therefore, we believe that other fac-
tors compensated for the lack of access to toothbrushes,
toothpaste, dental floss, toothpicks, and mouthrinses.
Among the volunteers who participated in the
4-week experiment that qualifies as a modified exper-
imental gingivitis study, there was no clinical evi-
dence of increased gingival inflammation, despite
increasedsupragingival plaquelevels. Despite increas-
ing plaque scores, PD and BOP values decreased over
time.
Four of the participants were children or adoles-
cents. Thus, it is possible that the impact of transition
from deciduous teeth to permanent teeth or hormonal
effects in young individuals had an impact on the
measurements. However, we found no trends of differ-
ences that specifically suggested that changes in con-
ditions among these four subjects were different from
the older subjects.
The subgingival microbiota did not change with an
enhanced bacterial colonization; increases in the
counts of bacteria associated with periodontitis or
tooth decay were not found. The primary explanation
for why these subjects did not develop evidence of in-
creased gingivitis must be the sugar intake restriction
and the intake of food items rich in antibacterial and
anti-inflammatory components. Thus, the traditional
experimental model published in hundreds of studies
may only be applicable if the subjects maintain a
Western diet rich in sugar and low in anti-inflamma-
tory foods. These data are based on a small sample,
however. Nevertheless, the findings are worthy of fur-
ther research to assess how diet restriction in experi-
mental gingivitis studies influences the results. The
presented hypothesis that sugar restriction and the
supplemental intake of antioxidants and flavonoids
are important dietary guidelines for subjects with gin-
givitis and periodontitis may be extremely important.
Studies of indigenous populations with regard to gin-
gival inflammation and oral bacterial colonization
may be crucial to understand the development of gin-
givitis and its progression to periodontitis.
CONCLUSIONS
The present observations support the concept that di-
etary factors are important in the control or develop-
ment of gingivitis, in the absence of oral hygiene
measures, over 4 weeks. Therefore, the experimental
gingivitis model commonly used without dietary con-
trol may have significant limitations. Although plaque
levels increased, decreases in BOP and PDs were
found. Diet restriction, coupled with abstinence from
oral hygiene, did not result in increased gingival in-
flammation; however, it did result in increases in bac-
terial counts in subgingival samples and decreases in
counts in samples from the tongue. The increase in
subgingival samples did not include species commonly
associated with periodontitis. Specifically, counts of
Streptococcus species decreased in tongue samples
over time.
ACKNOWLEDGMENTS
The Clinical Research Foundation, University of Bern,
supported this study. The authors express their ap-
preciation to Ms. Marianne Weibel and Ms. Regula
Hirschi-Imfeld, Laboratory of Oral Microbiology,
University of Bern, for their dedicated laboratory
processing of the study material. The authors report
no conflicts of interest related to this study.
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Correspondence: Dr. Thomas Imfeld, Department of Pre-
ventive Dentistry and Oral Epidemiology, Center for Dental
Medicine, University of Zu
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Zu
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Submitted July 10, 2008; accepted for publication De-
cember 19, 2008.
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