Development of an in vitro biofilm model of the human supra-gingival microbiome for Oral microbiome transplantation.

The high prevalence of dental caries and periodontal disease place a significant burden on society, both socially and economically. Recent advances in genomic technologies have linked both diseases to shifts in the oral microbiota - a community of >700 bacterial species that live within the mouth. The development of oral microbiome transplantation draws on the success of fecal microbiome transplantation for the treatment of gut pathologies associated with disease. Many current in vitro oral biofilm models have been developed but do not fully capture the complexity of the oral microbiome which is required for successful OMT. To address this, we developed an in vitro biofilm system that maintained an oral microbiome with 252 species on average over 14 days. Six human plaque samples were grown in 3D printed flow cells on hydroxyapatite discs using artificial saliva medium (ASM). Biofilm composition and growth were monitored by high throughput sequencing and confocal microscopy/SEM, respectively. While a significant drop in bacterial diversity occurred, up to 291 species were maintained in some flow cells over 14 days with 70% viability grown with ASM. This novel in vitro biofilm model represents a marked improvement on existing oral biofilm systems and provides new opportunities to develop oral microbiome transplant therapies.

A snapshot of intraoperative conditions to predict prolonged postoperative ileus after colorectal surgery.

BACKGROUND: The cause of prolonged postoperative ileus (PPOI) is multifactorial. The influence of preoperative factors on PPOI has been well documented, but little is known about the impact of intraoperative conditions. The aim of this study was to investigate the influence of intraoperative factors on PPOI in patients undergoing colorectal surgery. METHODS: The LekCheck study database of the Colorectal Unit at the Royal Adelaide Hospital was analysed. Per patient, over 60 data points were prospectively collected between March 2018 and July 2020. Intraoperative data were collected in theatre during a one-off snapshot measure. Univariate and multivariable logistic regression analyses were performed. RESULTS: Data of 336 patients were included. The median age was 66 years and 58.3% were male. Ninety-three patients (27.7%) developed PPOI. Univariate analysis identified the following intraoperative variables as risk-factors of PPOI: greater volumes of intraoperative IV fluid administration (464 versus 415 mL/h for those without PPOI; p = 0.04), side-to-side anastomosis orientation (53.8 versus 41.2%; p = 0.04) and increased perioperative opioid use (6.73 versus 4.11 mg/kg morphine equivalents for patients with and without PPOI, respectively; p = 0.02). Upon multivariable analysis, increased perioperative opioid use remained significant (p = 0.05), as well as the preoperative factors anticoagulation use (p = 0.04) and higher levels of serum total protein (p = 0.02). CONCLUSION: This study suggests that intraoperative factors may also contribute to the development of PPOI, but this could not be confirmed in the multivariate analysis. Further studies including larger patient numbers will be required to determine the impact of intraoperative conditions on the development of PPOI.

Probiotic Lactobacillus rhamnosus GG prevents alveolar bone loss in a mouse model of experimental periodontitis.

AIM: This study investigated the role of Lactobacillus rhamnosus GG (LGG) on bone loss and local and systemic inflammation in an in vivo mouse model of experimental periodontitis (PD). MATERIALS AND METHODS: Experimental PD was induced in mice by oral inoculation with Porphyromonas gingivalis and Fusobacterium nucleatum over a period of 44 days. The probiotic LGG was administered via oral inoculation or oral gavage prior to, and during disease induction. The antimicrobial activity of LGG on the inoculum was also tested. Alveolar bone levels and gingival tissue changes were assessed using in vivo microcomputed tomography and histological analysis. Serum levels of mouse homologues for IL-8 were measured using multiplex assays. RESULTS: Pre-treatment with probiotics either via oral gavage or via oral inoculation significantly reduced bone loss (p < .0001) and gingival inflammation (p < .0001) when compared with PD group. Oral gavage treatment group had significantly less tartrate-resistant acid phosphatase positive cells (p < .02) then PD group. LGG showed no antimicrobial activity against P. gingivalis and F. nucleatum. CONCLUSIONS: Lactobacillus rhamnosus GG effectively suppresses bone loss in a mouse model of induced PD irrespective of the mode of administration.

Compromised inflammatory cytokine response to P. gingivalis LPS by fibroblasts from inflamed human gingiva.

OBJECTIVES: The aims of this study were to compare the in vitro cytokine response of gingival fibroblasts (GF's) from healthy and inflamed human gingival tissues and to assess whether GF's from inflamed gingivae are capable of mounting a secondary inflammatory response after exposure to P. gingivalis LPS. MATERIALS AND METHODS: GF's were obtained from healthy donors and periodontitis patients and cultured in vitro. Cells were exposed to P. gingivalis LPS for 24h before measurement of MCP-1, GRO, IL-6, IL-8 and VEGF using a bead-based multiplex assay. Statistical comparisons were made between LPS-exposed GF's and unstimulated cells as well as the two patient groups by two-way ANOVA. RESULTS: GF's exposed to P. gingivalis LPS significantly increased their production of MCP-1, GRO, IL-6, IL-8 and VEGF compared to unstimulated cells. GF's isolated from inflamed tissue from periodontitis patients demonstrated consistently less cytokine production after exposure to P. gingivalis LPS, most notably for GRO and IL-6. CONCLUSIONS: The current study demonstrates that GF's play an active role in the inflammatory response in periodontal disease by producing a number of chemokines and cytokines. Furthermore, inflamed GF's may be compromised in their ability to mount an adequate secondary immune response in relation to chemokine/cytokine production. CLINICAL RELEVANCE: The compromised inflammatory cytokine response of inflamed human gingival fibroblasts to P. gingivalis LPS may impact on their ability to recruit and activate inflammatory cells while maintaining persistent inflammation, a key feature of periodontal disease.

Abnormal pregnancy outcomes in mice using an induced periodontitis model and the haematogenous migration of Fusobacterium nucleatum sub-species to the murine placenta.

OBJECTIVES: To investigate if there is subspecies specific migration to the placenta by Fusobacterium nucleatum (Fn) and to determine whether experimentally induced periodontitis results in adverse pregnancy outcomes (APO) in mice. METHODS: Periodontitis was induced in pregnant mice using an inoculum of Fn and Porphyromonas gingivalis. In parallel, four sub-species of Fn were individually injected into the circulatory system. At day 18 of gestation, the placenta, liver, spleen and blood were harvested and litter size, number of viable fetuses and resorptions, maternal, fetal and placenta weights were recorded. For the direct inoculation group, some mice were allowed to deliver for assessment of length of gestation, litter size, maternal, placental and pup weight. The presence of Fn was assessed by PCR and inflammatory mediators were measured by ELISA or multiplex analysis. RESULTS: Mice with alveolar bone loss, a marker of periodontitis, demonstrated significantly higher fetal weights (p = 0.015) and fetal/placental weight ratios (p = 0.030). PCR analysis of maternal organs did not identify Fn in any extracted tissues. In mice that received direct injection of Fn subspecies, varying degrees of APO were observed including preterm birth, intrauterine growth restriction, and fetal loss. Haematogenous spread of only Fn subsp. nucleatum to the placenta was confirmed. Litter size was significantly smaller (p = 0.023) and the number of resorptions was higher in inoculated versus control groups. Mice injected with subsp. nucleatum had significantly increased circulating CRP levels (p = 0.020) compared to controls while the mice with induced periodontitis had increased levels of IL-6 (p = 0.047) and IL-8 (p = 0.105). CONCLUSIONS: Periodontitis in mice elevated fetal weight and the fetal weight/placental weight ratio. This study found that subsp. nucleatum migrated haematogenously to the placenta, leading to APO in mice. The study supports the potential role of Fn in the association between periodontitis and APO.

Local and systemic biomarkers in gingival crevicular fluid increase odds of periodontitis.

AIM: To determine the independent and combined associations of interleukin-1beta (IL-1beta) and C-reactive protein (CRP) in gingival crevicular fluid (GCF) on periodontitis case status in the Australian population. MATERIALS AND METHODS: GCF was collected from 939 subjects selected from the 2004-2006 Australian National Survey of Adult Oral Health: 430 cases had examiner-diagnosed periodontitis, and 509 controls did not. IL-1beta and CRP in GCF were detected by enzyme-linked immunosorbent assays. Odds ratios (OR) and 95% confidence intervals (CIs) were calculated in bivariate and stratified analysis and fully adjusted ORs were estimated using multivariate logistic regression. RESULTS: Greater odds of having periodontitis was associated with higher amounts of IL-1beta (OR=2.4, 95% CI=1.7-3.4 for highest tertile of IL-1beta relative to lowest tertile) and CRP (OR=1.9, 95% CI=1.5-2.5 for detectable CRP relative to undetectable CRP). In stratified analysis, there was no significant interaction between biomarkers (p=0.68). In the multivariate analyses that controlled for conventional periodontal risk factors, these relationships remained (IL-1beta OR=1.8, 95% CI=1.1-2.6; CRP OR=1.7, 95% CI=1.3-2.3). CONCLUSIONS: Elevated odds of clinical periodontitis was associated independently with each biomarker. This suggests that people with elevated biomarkers indicative of either local (IL-1beta) or systemic (CRP) inflammation are more likely to suffer from periodontal disease.

Effect of smoking on concentrations of receptor activator of nuclear factor kappa B ligand and osteoprotegerin in human gingival crevicular fluid.

AIM: To compare the levels of the soluble receptor activator of nuclear factor kappa B ligand (sRANKL), osteoprotegerin (OPG) and their relative ratio in gingival crevicular fluid (GCF) among periodontitis patients with varying smoking histories. MATERIAL AND METHODS: GCF samples were collected from 149 periodontitis patients who were never smokers (n=58), former smokers (n=39) and current smokers (n=52). sRANKL and OPG concentrations in GCF were measured by enzyme-linked immunosorbent assays. RESULTS: sRANKL, OPG and their relative ratio were not statistically significant among the never smokers, former smokers and current smokers. However, OPG was significantly reduced and subsequently the sRANKL:OPG ratio was significantly increased in the high pack-years group as compared with never smokers. The positive correlation between pack-years and the sRANKL:OPG ratio remained statistically significant after adjusting for age and current smoking status. CONCLUSION: Increased lifetime exposure to cigarette smoking above a minimum threshold suppresses OPG production and leads to increased sRANKL:OPG. This may partially explain increased bone loss in smoking-related periodontitis.

Physical activity, inflammatory biomarkers in gingival crevicular fluid and periodontitis.

AIMS: To examine the associations of physical activity with interleukin 1-beta (IL-1beta), C-reactive protein (CRP) and periodontitis and to investigate whether any relationship between physical activity and inflammatory mediators differs between periodontitis cases and non-cases. MATERIAL AND METHODS: In this population-based case control study of Australians aged 18+ years, dentists conducted oral epidemiologic examinations identifying cases with moderate or severe periodontitis and periodontally healthy controls. Gingival crevicular fluid samples collected during examinations were analysed for inflammatory biomarkers. Subject-completed questionnaires assessed leisure-time physical activity. Exposure odds ratios (ORs) were estimated in multivariable logistic regression models adjusting for periodontitis risk indicators. RESULTS: Of 751 subjects (359 cases, 392 controls), those meeting a prescribed threshold for leisure-time physical activity had lower adjusted odds of elevated IL-1beta: OR=0.69, (95% CI=0.50-0.94) and detectable CRP: OR=0.70 (0.50-0.98) than less active adults. Physical activity was not associated with periodontitis: OR=1.14 (0.80-1.62). Periodontitis modified the association between levels of physical activity and detectable CRP. Increasing quartiles of physically activity were associated with decreasing probability of detectable CRP, but the effect was limited to periodontitis cases and was not apparent among non-cases. CONCLUSION: Leisure-time physical activity may protect against an excessive inflammatory response in periodontitis.