Methods

Study design and participants

The Scottish Health Survey is a cross sectional survey (typically every three to five
years) that draws a nationally representative sample of the general population living in
Scottish households. For the present analysis we combined data from the 1995, 1998,
and 2003 surveys in adults aged 35 and older as previously described elsewhere.

Assessment of oral health behaviours and covariates

Survey interviewers visited eligible households and collected data on demographics
and health behaviours (such as smoking, physical activity, and oral health behaviour)
and measured height and weight. Physical activity interviews inquired about activity
in the four weeks before the interview (1998 and 2003) or during a typical week
(1995). Frequency of participation (for at least 20 minutes per occasion) was assessed
across three domains of activity: leisure time sports (such as cycling, swimming,
running, aerobics, dancing, and ball sports such as football and tennis); walking for
any purpose; and domestic physical work (such as heavy housework, home
improvement activities, manual and gardening work). The total physical activity
frequency score was converted into sex specific thirds of weekly activity episodes.
Oral health behaviour was assessed in all survey years from self reported frequency of
visits to a dentist (at least once every six months, every one to two years, or
rarely/never), and toothbrushing (brushing teeth twice a day, once a day, less than one
a day). On a separate visit nurses collected information on medical history and family
history of cardiovascular disease, blood pressure, and blood samples from consenting
adults. The definition of hypertension was based on a self reported diagnosis from a
doctor or a clinic blood pressure reading of >140/90 mm Hg. Diabetes was based on a
doctor’s diagnosis. The covariables included in the present study were selected in a
pre-specified protocol because they are all well established risk factors for
cardiovascular disease. Detailed information on the survey methods can be found
elsewhere.

Follow-up for clinical events

The surveys were linked to a database of hospital admissions and deaths in patients
with follow-up until December 2007 (Information Services Divisions (ISD),
Edinburgh). The database has been shown to be 94% accurate and 94% complete
when samples of computerized records for cardiovascular disease from the database
were compared with the original case notes from patients. Information on deaths was
ascertained from the general registrar office for Scotland. Classification of the
underlying cause of death is based on information collected on cause of death from
the medical certificate together with any additional information provided
subsequently by the certifying doctor.
Our primary and point was composite of fatal and non-fatal cardiovascular disease
events. Mortality from cardiovascular causes was coded according to ICD-9
(International Classification of Diseases, ninth revision) codes 390-459 and ICD-10
(10th revision) codes I01-I99, and non-fatal events included hospital admissions
related to cardiovascular disease, incorporating acute myocardial infarction, coronary
artery bypass surgery, percutaneous coronary angioplasty, stroke, and heart failure.
Blood analyses
In a subsample of 4830 participants we collected peripheral blood samples in citrate
and serum tube for the assessment of C reactive protein and fibrinogen. This was
optional, and several participants did not consent to having blood taken. Participants
who did not consent to blood had poorer health risk profiles with higher body mass
index (BMI) and a higher prevalence of hypertension, cardiovascular disease, and
smoking. All blood samples were frozen at -70oC until assay. The analysis of C
reactive protein concentrations from serum was performed with the N Latex high
sensitivity C reactive protein mono-immunoassay on a Behring Nephelometer II
analyser. The limit of detection was 0.17 mg/l, and the coefficient of variation was
less than 6% for this assay. Fibrinogen concentrations were determined with the
Organon Teknika MDA 180 analyser, with a modification of the Clauss thrombin
clotting method, with a coefficient of variation of less than 10%. All analyses were
carried out in the same laboratory according to standard operating procedures by state
registered medical laboratory scientific officers.

Statistical analysis
Cox proportional hazards models were used, with months as the time scale, to
estimate the risk of cardiovascular disease events or death according to oral hygiene.
The data were censored to December 2007 in event-free survivors. Initially,
toothbrushing habit was included in the Cox model as a covariate, with adjustment for
age and sex. In further multivariate models was adjusted for socioeconomic group
using the registrar general classification (professional/intermediate, skilled non-
manual, skilled manual, part skilled/unskilled), smoking (never, ex-smoker, current
smoker), sex specific thirds of physical activity, frequency of dentist visits (at least
once every six months, once every one or two years, rarely or never), BMI
(underweight <18.5, normal weight 18.5-25.0, overweight 25.01-30.0, obese 30.01-
40.0, morbidly obese >40.0), and family history of cardiovascular disease,
hypertension, and diabetes. The proportional hazards assumption was examined by
comparing the cumulative hazard plots grouped on exposure, although no violations
were noted.
We examined the association between frequency of toothbrushing and inflammatory
markers (C reactive protein and fibrinogen) using general linear models with
adjustments for age, sex, socioeconomic group, smoking, visits to dentist, BMI,
family history of cardiovascular disease, hypertension, and diabetes, and acute
infections (including influenza, pneumonia, bronchitis, and log transformed to
normalize the data. All analyses were performed with SPSS (version 14), and all tests
of significance were based on two sided probability.

Results
We removed from the analyses 3685 participants who were edentulous (no natural
teeth) and 386 with existing cardiovascular disease. Participants with missing
demographic data (n=204) were also excluded, leaving a final sample size of 11☐869
(46.1% men, mean age 50.0 (SD 11.0)). The excluded edentate participants were
older and more likely to be women and smokers.
Oral health behaviour was generally good, with about 62% (14☐718) of participants
reporting regular (at least every six months) visits to a dentist and 71% (8481)
reporting good oral hygiene (brushing teeth twice a day). Participants who brushed
their teeth less often than twice a day were slightly older, more likely to be men, and
of lower social status and had a high prevalence of risk factors including smoking,
physical inactivity, obesity, hypertension, and diabetes (table 1). Participants from the
different survey years were comparable in terms of demographics and risk factors.
View this table:

Table 1

Characteristics of study population in relation to oral hygiene. Figures are numbers

(percentage) unless stated otherwise.

There were 555 cardiovascular disease events over an average of 8.1 (SD 3.4) years to
follow-up, of which 170 were fatal. In about 74% (411) of cardiovascular disease
events the principal diagnosis was coronary heart disease. The mean age of event-free
survivors compared with participants with a recorded cardiovascular disease event
was 49.6 (SD 10.9) and 57.0 (SD 10.3), respectively (P<0.001), at baseline. In age
and sex adjusted analyses participants reporting poor dental hygiene had an increased
risk of cardiovascular disease events and cardiovascular disease death (tables 2 and
3). In further multivariate models the associations were attenuated, although they
remained significant in the case of cardiovascular disease events. Participants who
reported less frequent toothbrushing had a 70% increased risk of cardiovascular
disease event in fully adjusted models compared with participants who brushed their
teeth twice a day. The other independent predictors of cardiovascular disease events
included smoking (hazard ratio 2.4, 95% confidence interval 1.9 to 2.9), hypertension
(1.7,1.4 to 2.0), and diabetes (1.9, 1.4 to 2.7).

Table 2

Cox regression models for toothbrushing and cardiovascular disease (CVD) events
(fatal and non-fatal combined)

Table 3
Cox regression models for toothbrushing and death from cardiovascular disease
(CVD)

As some previous studies have suggested effect modifications by age, sex, and
smoking status in relation to oral health and cardiovascular disease, we performed
various sensitivity analyses. There were no clear sex differences in our results; the age
adjusted hazard ratio for cardiovascular disease events in relation to toothbrushing
less than one a day was 2.2 (1.6 to 3.1) in men and 3.6 (1.7 to 7.7) in women. There
was also no difference by age; the age/sex adjusted hazard ratio was 2.7 (1.4 to 5.4) in
participants aged 65 or above compared with 2.2 (1.6 to 3.0) in those aged less than
65. There were no differences between smokers (2.0,1.3 to 3.0) and those who had
never smoked (2.0,1.1 to 3.6).
There were significant associations between frequency of toothbrushing and markers
of low grade systemic inflammation (table 4). Participants who brushed their teeth
less often had increased concentrations of both C reactive protein and fibrinogen.
These associations remained significant after multiple adjustments. In the subsample
of participants with available biological data we re-ran the survival analyses to
examine if the association between toothbrushing and cardiovascular disease was
weakened by the inclusion of inflammatory markers (see appendix on bmj.com). In
these analyses there were 161 cardiovascular disease events and the age and sex
adjusted associations were similar compared with the main sample. The introduction
of C reactive protein and fibrinogen did partly attenuate the point estimates, thus
suggesting a possible mediating role.

Table 4

Adjusted regression coefficients (95% CI) of toothbrushing on inflammatory markers

(n=4830)
The Association Between Toothbrushing Habit with
Cardiovascular Disease and Inflammation

 MATERIALS AND METHODS

Materials:
1. Data from the 1995,1998,and 2003 surveys in adults aged
35 and older
2. Participants 11.269 men and woman aged 50 in average
3. General population living in scottish household

Methods:
1. Assessments of oral health behaviours and covariates
a. Survey interviewers visited ellgible household
b. Collected data on demographics and health
behaviours
c. Measure height and weight
d. Physical activity interview
e. Assessed frequency of visits to a dentist and
toothbrushing
f. Collected information of medical history
2. Follow up for clinical events
3. Blood analysis
4. Statistical analysis

 MAIN RESEARCH QUESTION

Is toothbrushing habit associated with cardiovascular disease
and inflammation?

 RESEARCH QUESTION
1. How many events of cardiovascular disease are there?

2. Have cardiovascular disease events also increased

concentrations of both c reactive protein and fibrinogen?

3. What is the principal diagnosis of cardiovascular disease

events?
 RESEARCH FINDING & RESULT
1. There are 555 cardiovascular disease events which 170
were fatal

2. Cardiovascular disease events also have increased

concentrations of bot c reactive protein and fibrinogen

3. The principal diagnosis of cardiovascular disease events is

coronary heart disease