Manal Mustafa

Ratio of Pro-Resolving and Pro-Inflammatory Lipid Mediator Precursors as Potential Markers for Aggressive Periodontitis

Aggressive periodontitis (AgP) is a rapidly progressing type of periodontal disease in otherwise healthy individuals which causes destruction of the supporting tissues of the teeth. The disease is initiated by pathogenic bacteria in the dental biofilm, and the severity of inflammation and attachment loss varies with the host response. Recently, there has been an increased interest in determining the role of lipid mediators in inflammatory events and the concept of pro-inflammatory and pro-resolution lipid mediators has been brought into focus also in periodontal disease. The present study aimed to determine the profile of omega-3 or n3- as well as omega-6 or n6- polyunsaturated fatty acids (PUFAs) and PUFA-metabolites of linoleic acid, arachidonic acid (AA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in gingival crevicular fluid (GCF), saliva and serum in AgP patients and healthy controls. In total, 60 selected n3- and n6-PUFAs and various PUFA metabolites were measured using high performance liquid chromatography-tandem electrospray ionisation mass spectrometry (HPLC-ESI-MS-MS). Of these, 51 could be quantified in this study. The concentrations of the majority were low in saliva samples compared with serum and GCF, but were mainly higher in AgP patients compared with healthy controls in all three kinds of sample. Ratios of n3- to n6-PUFAs (DHA + EPA)/AA were significantly lower in the GCF of AgP patients than in the healthy controls. Furthermore, various ratios of the direct precursors of the pro-resolution lipid mediators (precursors of resolvins and protectins) were calculated against the precursors of mainly pro-inflammatory lipid mediators. These ratios were mainly lower in GCF and saliva of AgP patients, compared with healthy controls, but only reached significance in GCF (P<0.05). To conclude, the ratios of precursors of pro-resolution/pro-inflammatory lipid mediators seem to be more relevant for describing the disease status of AgP than the concentration of specific lipid mediators.

Effect of compressive force on human osteoblast-like cells and bone remodelling: an in vitro study.

OBJECTIVE The aim of this study was to determine the effect of continuous compressive force (CF) on expression by human alveolar bone-derived osteoblasts (HOBs) of some specific molecules involved in bone remodelling. DESIGN HOBs were cultured with or without CF (control, 2.0, 4.0gcm(-2)) for 1, 3 and 7 days. Expression of alkaline phosphatase (ALP), type I collagen (Col I), osteopontin (OPN), osteocalcin (OCN), transcription factor Runx2, receptor activator of nuclear factor κB ligand (RANKL), osteoprotegerin (OPG) and prostaglandin E2 (PGE2) was analysed by real-time-polymerase chain reaction (RT-PCR), enzyme-linked immunosorbent assay (ELISA) and/or immunostaining. RESULTS The results revealed that CF upregulated ALP and Col I expression at both messenger RNA (mRNA) and protein levels but did not affect expression of OPN and OCN mRNA. Runx2 mRNA was inhibited by CF, which also altered the expression of molecules involved in osteoclastogenesis, by enhancing RANKL expression and suppressing OPG expression. At 4.0gcm(-2) of CF, the expression of RANKL and PGE2 was significantly upregulated. CONCLUSION The results suggest that initial application of CF on HOBs can simultaneously affect expression of markers related to both osteogenesis and osteoclastogenesis.

Impact of Chronic Periodontitis on Levels of Glucoregulatory Biomarkers in Gingival Crevicular Fluid of Adults with and without Type 2 Diabetes

The relationship between diabetes and periodontal disease is bidirectional, but information about the effect of chronic periodontitis on the levels of the glucoregulatory biomarkers locally in gingival crevicular fluid (GCF) is limited. The aim of this study was to compare the levels of 10 glucoregulatory biomarkers in GCF, firstly in subjects with type 2 diabetes (T2DM) presenting with and without chronic periodontitis and secondly, in subjects without diabetes, with and without chronic periodontitis. The material comprised a total of 152 subjects, stratified as: 54 with T2DM and chronic periodontitis (G1), 24 with T2DM (G2), 30 with chronic periodontitis (G3) and 44 without T2DM or periodontitis (G4). The levels of the biomarkers were measured using multiplex biometric immunoassays. Periodontal pocket depths were recorded in mm. Subsets G1 and G2 and subsets G3 and G4 were compared independently. Among T2DM subjects, GIP, GLP-1 and glucagon were significantly up-regulated in G1 compared to G2. Moreover, there were no statistical differences between the two groups regarding C-peptide, insulin, ghrelin, leptin and PAI-1. Comparisons among individuals without T2DM revealed significantly lower amounts of C-peptide and ghrelin in G3 than in G4. The number of sites with pocket depth ≥ 4mm correlated negatively with C-peptide (Spearman’s correlation co-efficient: -0.240, P < 0.01) and positively with GIP and visfatin (Spearman’s correlation co-efficient: 0.255 and 0.241, respectively, P < 0.01). The results demonstrate that chronic periodontitis adversely influences the GCF levels of glucoregulatory biomarkers, as it is associated with disturbed levels of biomarkers related to the onset of T2DM and its medical complications.

Subgingival microbial profiles of Sudanese patients with aggressive periodontitis

Background and Objective Aggressive periodontitis (AgP) is prevalent and shows a rapid course in African individuals. Although a strong focus has been placed on Aggregatibacter actinomycetemcomitans, new methods support the existence of a complex subgingival microflora in AgP. The purpose of the present study was to map the subgingival microbiota as well as explore the presence of A. actinomycetemcomitans and the JP2 clone in a group of Sudanese individuals with AgP, using different analytical methods. Material and Methods A study population consisting of 19 patients with AgP was recruited from patients seeking treatment at University of Science and Technology (UST) in Khartoum. Fifteen healthy subjects were included as controls. Plaque samples were analyzed for 272 taxa using human oral microbe identification microarrays and for 26 periodontal taxa using DNA-DNA hybridization checkerboard. Conventional polymerase chain reaction (PCR) was applied for the detection of A. actinomycetemcomitans and the JP2 clone in plaque. Saliva from patients with AgP was analyzed using quantitative PCR (qPCR) for the detection of A. actinomycetemcomitans. Results Eubacterium yurii was detected more frequently in patients with AgP than in controls, and E. nodatum was found in patients with AgP only. A. actinomycetemcomitans was found in plaque samples of two (12%) patients by human oral microbe identification microarrays and in five (29%) patients with AgP by conventional PCR, as well as in six (32%) of the AgP saliva samples by qPCR. The JP2 clone was identified in only one patient. Conclusion The classical periodontal pathogens were not present in high amounts in AgP in the population studied here. Species of Eubacterium, which are not typically associated with AgP, were often detected in individuals with disease. Using laboratory methods with different sensitivities and detection levels allowed identification of variances in microbial communities. The findings reported in this study provide a basis for the further understanding of AgP.

Initial responses of osteoblasts derived from human alveolar bone to various compressive forces

Mechanical stress generated by orthodontic force is recognized as a major factor in the modulation of alveolar bone remodeling. During this process, osteoblasts play a crucial role, not only by participating in bone formation but also by promoting osteoclastogenesis. The aim of this study was to investigate how continuous compressive force (CF) affects human primary osteoblasts (HOBs) in terms of cell proliferation, apoptosis, and expression of interleukin-6 (IL-6) and chemokine CXC ligand 8 (CXCL8). Human primary osteoblasts, isolated from human mandibular bone pieces, were cultured with or without CF (1-4 g cm(-2)) for up to 72 h. Cell viability and proliferation were evaluated using the MTT assay. RT-PCR was used to determine the levels of expression of KI67 (a proliferation marker), BAX (a pro-apoptotic marker), BCL2 (an apoptotic inhibitor), IL6, and CXCL8 mRNAs, while a multiplexed bead immunoassay was used to measure the release of IL-6 and CXCL8. The results revealed that CF decreased cell viability and proliferation in a time- and force-dependent manner. After applying CF for 24 h, the mRNA expression of KI67 was markedly inhibited, whereas the mRNA expression of BAX and BCL2 was unaltered. In addition, CF enhanced the levels of IL6 and CXCL8 mRNAs in a force-dependent manner, whereas the levels of the corresponding proteins were reduced in the compressed HOBs.

Influence of type 2 diabetes on local production of inflammatory molecules in adults with and without chronic periodontitis: a cross-sectional study

BackgroundPathological changes in periodontal tissues are mediated by the interaction between microorganisms and the host immune-inflammatory response. Hyperglycemia may interfere with this process. The aim of this study was to compare the levels of 27 inflammatory molecules in the gingival crevicular fluid (GCF) of patients with type 2 diabetes, with and without chronic periodontitis, and of chronic periodontitis subjects without diabetes. A putative correlation between glycated haemoglobin (HbA1c) and levels of the inflammatory molecules was also investigated.MethodsThe study population comprised a total of 108 individuals, stratified into: 54 with type 2 diabetes and chronic periodontitis (DM + CP), 30 with chronic periodontitis (CP) and 24 with type 2 diabetes (DM). Participants were interviewed with the aid of structured questionnaire. Periodontal parameters (dental plaque, bleeding on probing and periodontal pocket depth) were recorded. The GCF levels of the 27 inflammatory molecules were measured using multiplex micro-bead immunoassay. A glycated haemoglobin (HbA1c) test was performed for patients with diabetes by boronate affinity chromatography.ResultsAfter adjustment for potential confounders, the DM + CP group had higher levels of IL-8 and MIP-1β, and lower levels of TNF-α, IL-4, INF-γ, RANTES and IL-7 compared to the CP group. Moreover, the DM + CP group had lower levels of IL-6, IL-7 and G-CSF compared to the DM group. The DM group had higher levels of IL-10, VEGF, and G-CSF compared to the CP group. The levels of MIP-1α and FGF were lower in diabetes patients (regardless of their periodontal status) than in chronic periodontitis subjects without diabetes. Diabetes patients (DM + CP and DM) had higher Th-2/Th-1 ratio compared to the CP group. HbA1c correlated positively with the pro-inflammatory cytokines (Pearson correlation coefficient = 0.27, P value: 0.02).ConclusionType 2 diabetes and chronic periodontitis may influence the GCF levels of inflammatory molecules synergistically as well as independently. Type 2 diabetes was associated with high Th-2/Th-1 ratio, and modulated the local expression of molecules involved in the anti-inflammatory and healing processes.

Influence of Type 2 Diabetes on Prevalence of Key Periodontal Pathogens, Salivary Matrix Metalloproteinases, and Bone Remodeling Markers in Sudanese Adults with and without Chronic Periodontitis

This study compared the influence of type 2 diabetes on the occurrence of six periodontal pathogens in plaque samples of patients with and without chronic periodontitis. Levels of salivary MMP-8, MMP-9, RANKL, and OPG were also investigated. The study enrolled 31 patients with type 2 diabetes and chronic periodontitis (DM + CP), 29 with chronic periodontitis (CP), and 20 with type 2 diabetes (DM). Questionnaire-guided interviews were conducted and plaque index, bleeding on probing, and pocket depth were recorded. Polymerase chain reaction (PCR) was utilized to determine the prevalence of the bacteria. The levels of salivary molecules were determined by enzyme immunosorbent assay (ELISA). The CP group had the highest prevalence of P. gingivalis (81.5%), followed by the DM + CP (59.3%) and DM (55.0%) groups (P > 0.05). Similar trends were observed for P. intermedia and T. denticola. The prevalence of T. forsythia was 100% in both periodontitis groups compared to 90% in the DM group. There were no significant differences between the groups regarding the concentrations of MMP-8, MMP-9, or OPG. RANKL concentrations were below the detection limit. Our data show that type 2 diabetes has no significant influence on the prevalence of the investigated periodontal pathogens, or the levels of salivary MMP-8, MMP-9, and OPG.

The effect of smoking on inflammatory and bone remodeling markers in gingival crevicular fluid and subgingival microbiota following periodontal therapy

BACKGROUND AND OBJECTIVE Periodontal health is mediated by suppressing microorganisms inducing a local inflammatory host response. Smoking may impair this process. This study compares gingival crevicular fluid levels of inflammatory and bone remodeling markers in heavy smokers and non-smokers following active and supportive periodontal therapy in patients with chronic periodontitis. MATERIAL AND METHODS Gingival crevicular fluid and subgingival plaque were collected from the deepest periodontal pocket in 50 patients, 25 smokers and 25 non-smokers, at baseline (T0), following active (T1) and 12 mo of supportive periodontal therapy (T2). Smoking status was validated measuring serum cotinine levels. Gingival crevicular fluid levels of 27 inflammatory and two bone remodeling markers were analyzed using multiplex and singleplex micro-bed immunoassays, and subgingival plaque samples using checkerboard DNA-DNA hybridization. Amounts of markers in smokers and non-smokers were compared calculating the effect size. RESULTS Expression of inflammatory and bone-remodeling markers in smokers demonstrated an overall reduced effect size at T0 and T2 (p < 0.001). In particular, proinflammatory markers (p < 0.001), chemokines (p = 0.007) and growth factors (p = 0.003) at T0, osteoprotegerin (p = 0.003) at T1, proinflammatory markers (p = 0.019) and chemokines (p = 0.005) at T2. At T2, interleukin-8 was detected in significantly higher levels in smokers. Ten different markers in non-smokers and none in smokers responded to periodontal therapy (p < 0.05). An overall negative association was revealed between smoking and subgroups of markers at sites presenting ≥ 105 red complex periodontal microbial species. CONCLUSION Except for an upregulation of interleukin-8, smokers exhibited reduced gingival crevicular fluid levels of several inflammatory markers at baseline and following active and supportive periodontal therapy. Only inflammatory responses in non-smokers adapted to periodontal therapy. Apparently, there seems to be an immunosuppressant effect of smoking regulating the local inflammatory response and bone remodeling markers captured in gingival crevicular fluid following periodontal therapy.

Dental plaque microbial profiles of children from Khartoum, Sudan, with congenital heart defects

ABSTRACT Few studies have focused on the bacterial species associated with the deterioration of the dental and gingival health of children with congenital heart defects (CHD). The aims of this study were (1) to examine the dental plaque of children with CHD in order to quantify bacterial load and altered bacterial composition compared with children without CHD; and (2) to investigate the correlation between the level of caries and gingivitis and dental biofilm bacteria among those children. In this cross-sectional study, participants were children (3–12 years) recruited in Khartoum State, Sudan. A total of 80 CHD cases from the Ahmed Gasim Cardiac Centre and 80 healthy controls from randomly selected schools and kindergartens were included. Participants underwent clinical oral examinations for caries (decayed, missing, and filled teeth indices [DMFT] for primary dentition, and DMFT for permanent dentition), and gingivitis (simplified gingival index [GI]). Pooled dental biofilm samples were obtained from four posterior teeth using paper points. Real-time quantitative polymerase chain reaction was used for the detection and quantification of Streptococcus mutans, Streptococcussanguinis, and Lactobacillus acidophilus. Checkerboard DNA–DNA hybridization was used for the detection of 40 additional bacterial species. CHD cases had a significantly higher caries experience (DMFT = 4.1 vs. 2.3, p < 0.05; DMFT = 1.4 vs. 0.7, p < 0.05) and a higher mean number of examined teeth with gingivitis (4.2 vs. 2.0; p < 0.05) compared with controls. S. mutans counts were significantly higher among the CHD cases (p < 0.05). Checkerboard results revealed that 18/40 bacterial species exhibited significantly higher mean counts among CHD cases (p < 0.01). Correlation analyses revealed that among CHD cases, the detection levels of Tannerella forsythia, Campylobacter rectus, Fusobacterium nucleatum subsp. vincentii, F. nucleatum subsp. nucleatum, and F. nucleatum subsp. polymorphum were highly positively correlated with GI. CHD cases harbor more cariogenic and periodontopathogenic bacterial species in their dental plaque, which correlated with higher levels of caries and gingivitis.

Cytokine profile in gingival crevicular fluid and plasma of patients with aggressive periodontitis

Abstract Objective: This study aimed to determine the content of cytokines in gingival crevicular fluid (GCF) as well as in plasma of Sudanese patients with aggressive periodontitis (AgP) and healthy controls (HC). Materials and methods: Nineteen AgP patients and 19 HC were included. The mean probing pocket depth and clinical attachment level of the GCF sampled sites in patients were both ≥5 mm. The GCF and plasma levels of 27 cytokines were determined using 27-multiplex fluorescent bead-based immunoassays. Ratios were calculated among cytokines of the T-helper cell subsets Th1 and Th2. Descriptive statistics, the Mann–Whitney U-test and Spearman’s rho rank correlation coefficient analysis were used. Results: Interferon-γ was the only cytokine found in significantly lower levels in GCF of patients compared with HC. Levels of interleukin (IL)-10, IL-13, IL-1Ra, monocyte chemoattractant protein-1 (MCP-1), regulated on activation normal T-cell expressed and secreted (RANTES), granulocyte-colony-stimulating factor (G-CSF), and granulocyte-macrophage-CSF (GM-CSF) were significantly lower in plasma of AgP compared with HC. The ratios of Th1:Th2 in GCF and Treg:Th17 in plasma were significantly lower in AgP. Conclusions: The lower levels of cytokines detected systemically in plasma of AgP patients may have an impact on the immune response. The lower ratio of Th1:Th2 cytokines in GCF samples of AgP patients suggests a role for Th2 at the local site of disease.