For this substudy, 17,356 patients with the diagnosis heart failure according to ICD-9 (4280–4289) and ICD-10 (I50.0 – I50.9) classification were selected and assessed for self-reported oral health.
Assessment of oral health, cardiovascular disease and lifestyle factorsParticipants completed a touchscreen questionnaire at the baseline visit, providing information on their self reported oral health indicators including mouth ulcers, painful gums, bleeding gums, loose teeth, denture treatment, and toothache.
Patients were classified as having a compromised oral health status if any of these conditions were present (i.e.
Composite of oral health indicators).
In a basic model (model 1), we adjusted for age and sex.
UK biobank analysis
The UK Biobank is a prospective cohort of approximately 500,000 participants enrolled between 2006 and 2010 aged between 40–69 years who lived ~25 miles from one of the 22 assessment centers located across the UK15. With their consent, they provided detailed information about their lifestyle, physical measures and had blood, urine and saliva samples collected and stored for future analysis. The UK Biobank cohort study obtained full ethical approval from the North West Multicentre Research Ethics Committee (10 May 2016). The study is conducted according to the Declaration of Helsinki. For this substudy, 17,356 patients with the diagnosis heart failure according to ICD-9 (4280–4289) and ICD-10 (I50.0 – I50.9) classification were selected and assessed for self-reported oral health. Use of data for this study was approved by the UK Biobank (Application number 88924).
Assessment of oral health, cardiovascular disease and lifestyle factors
Participants completed a touchscreen questionnaire at the baseline visit, providing information on their self reported oral health indicators including mouth ulcers, painful gums, bleeding gums, loose teeth, denture treatment, and toothache. Patients were classified as having a compromised oral health status if any of these conditions were present (i.e. Composite of oral health indicators). In addition, socioeconomic status (assessed using the Townsend deprivation index), and education level (categorized as holding a university degree or not) as well as lifestyle factors, including smoking status, alcohol consumption, and food intake were collected. Furthermore, medical conditions were assessed according to ICD-9 and ICD-10 classification (i.e. majority of all patients was diagnosed according to ICD-10 classification).
Mouse model
All animal experiments were approved by the LANUV-NRW Germany (protocol number AZ 81-02.04.2022.A162). All experiments were performed in accordance with the German legislation governing animal studies following the Guide for the Care and Use of Laboratory Animals (NIH publication, 8th edition, 2011) and the 2010/63/EU Directive on the protection of animals used for scientific purposes (Official Journal of the European Union, 2010). Male C57BL/6J mice were obtained from Janvier Labs and bred in the animal facility of the University Hospital, RWTH Aachen University, and placed with one to three animals per cage in a 12-hour day-night cycle at libitum access to food and water. At the age of 20 weeks, mice underwent transverse aortic constriction (TAC) surgery as previously described16 to induce pressure overload-induced cardiac hypertrophy or sham procedure as control. Mice with a body weight of 32.2 to 36.9 g were anesthetized and analgesia was applied. Mice were anesthetized with isoflurane (2%) and received preoperative analgesia with buprenorphine (0.1 mg/kg body weight) 30 minutes prior surgery. After intubation, the chest was opened by a small incision in the second intercostal space. Transverse aortic constriction was performed by tying a ligature around a 27G needle. In control mice, a sham procedure was conducted, where the thread was positioned but not tightened. All mice were sacrificed 10 weeks after surgery by cervical dislocation under isoflurane anesthesia. In total, 6 mice underwent transverse aortic constriction (TAC), while 3 mice underwent a sham procedure.
Echocardiography measurement
Transthoracic echocardiography was performed using a Visualsonics Vevo 3100 system with MX550D transducer (Fujifilm VisualSonics) 8 weeks after surgery. Mice were placed on a heated table (37°C) and heart rate, respiration rate, and body temperature were continuously measured. Anesthesia with 1-1.5% Isoflurane was adjusted to sustain a target heart rate between 415–450 bpm. Left ventricular ejection fraction was obtained from parasternal long axis B-Mode and analyzed using VevoLab (v5.8, Fujifilm VisualSonics). All parameters were measured at least three times and means were calculated.
PCR
PCR was conducted as previously described17. In brief, total ribonucleic acid (RNA) from gingiva was isolated with the RNeasy Mini Kit (#74106) (Qiagen, Hilden, Germany) and RNA preparation was followed by DNase digestion (#18068015) and reverse transcription into complementary DNA (cDNA) (#18080051) (both Thermo Fisher Scientific, Massachusetts, USA). Gene expression was quantified by use of SYBR Green reagent (#11760500) (Thermo Fisher Scientific, Massachusetts, USA) with a ViiATM 7 Real-Time PCR System (Applied Biosystems, Massachusetts, USA). Measurements were conducted in duplicates under standard reaction conditions and normalized to b-actin (Actb). Genes were selected based on their pathophysiological/physiological function (fibrosis: Tgfb and inflammation: Tnf, Il1b, Adgre1).
Micro-computertomographie (micro-CT) assessment
Maxillae from both SHAM and TAC groups were scanned using a high-resolution micro-CT system (Skyscan 1272, Bruker Micro-CT, Belgium) at 70 kV and 130 µA with a 0.5 mm aluminum filter, an exposure time of 1870 ms and a nominal resolution of 53 µm3. Post-reconstruction in NRecon (Bruker Micro-CT, Belgium), datasets were registered to a reference dataset in DataViewer (Bruker Micro-CT, Belgium) according to previously described methods18 for consistent positioning of the studied volume of interest across cohorts, focusing on the region from the first to the third molar as a reference zone. Subsequent analysis was conducted in CTAn (Bruker Micro-CT, Belgium).
Two volumes of interest (VOIs) were defined around the first molar tooth (M1): one encompassing the periodontal ligament (PDL) space and another within the alveolar bone socket of M1. After binarization based on global thresholding, PDL boundaries were delineated using seed-based segmentation for the tooth surface and a closing algorithm for the alveolar bone at the PDL-bone interface. The PDL space was defined as the region with x-ray opacity below that of alveolar bone or dentin, located between tooth surface and alveolar bone periphery, with PDL thickness determined via algorithms for trabecular thickness. For alveolar bone analysis, teeth were segmented out from VOIs, followed by virtual closure of bone peripheries to assess morphology through measurements of trabecular thickness (Tr. Thickness) and trabecular number per section (Tr. Number). Data normality was assessed using GraphPad Prism (version 10.3.1), and correlations with left ventricular ejection fraction were evaluated.
Statistical analysis
The baseline characteristics are reported as means with standard deviations in case of continuous variables, and frequencies with percentages in case of binary variables. For comparison between two groups, categorical variables were analyzed using the chi-square test, while continuous variables were compared using Student’s t-test or the Mann–Whitney U test, as appropriate. For logistic regression models, continuous variables were tested for normal distribution using the Kolmogorov–Smirnov test, visually assessed by histograms and log transformed before analysis when required. Odds ratios (HRs) and 95% confidence intervals (95% CIs) were calculated and adjusted for potential confounders as indicated. In a basic model (model 1), we adjusted for age and sex. In a second model (model 2), we additionally adjusted coronary artery disease (CAD), presence of diabetes (according to ICD), and presence of hypertension (according to ICD). Next, we additionally adjusted for hsCRP. In a final model we adjusted for tobacco use, food intake, academic qualifications, income, and alcohol use (model 5). All statistical tests were 2-sided, and a p-value <0.05 was considered significant. The analyses were performed using R version v. 4.2.2 and Graphpad Prism v10.
