The association between periodontal microbial biomarkers and primary therapy outcome.

OBJECTIVE: This study aims to analyse the association between the baseline microbial load of selected periodontopathogenic bacteria collected from gingival crevicular fluid (GCF) and the primary outcome of steps I and II therapy. MATERIALS AND METHODS: 222 patients with stage III periodontitis were included into this retrospective analysis that received steps 1 and 2 periodontal therapy without adjunctive systemic antibiotics. Baseline GCF samples were quantitatively analysed using ELISA-based kits for levels of periodontopathogens (Porphyromonas gingivalis (Pg), Aggregatibacter actinomycetemcomitans (Aa), Prevotella intermedia (Pi), Fusobacterium nucleatum (Fn), Treponema denticola (Td), and Tannerella forsythia (Tf)) and associated with the primary therapy outcome using a "treat-to-target" therapy endpoint (TE) defined as ≤ 4 sites with PD ≥ 5 mm six months after therapy. RESULTS: 38.2% of the patients achieved TE. Patients failing to achieve TE revealed significantly increased levels of Pg, Fn, and Tf at baseline (Pg: p = 0.010, Fn: p = 0.008 Tf: p = 0.004). Multivariate binary logistic regression adjusted for sex, mean probing depth, diabetes, and current smoking status showed an independent relationship between Tf and the TE (aOR 2.570, p = 0.023). CONCLUSION: Increased microbial load is associated with decreased responsiveness to therapy. The findings suggest that specifically baseline Tf levels are associated with poorer treatment outcomes and might improve the accuracy of periodontal diagnosis. CLINICAL RELEVANCE: The findings of this study support the concept of a critical biomass that is sufficient to induce and maintain an immune response within the periodontal pocket, which ultimately leads to irreversible tissue destruction. However, calculating this level in advance may serve as an early indicator for intervention. KEY FINDING: Baseline Tannerella forsythia levels are associated with poorer treatment outcome.

Periodontal grading-estimation of responsiveness to therapy and progression of disease.

OBJECTIVE: To investigate the capability of periodontal grading to estimate the progression of periodontal disease and the responsiveness to therapy. MATERIALS AND METHODS: Eighty-four patients who underwent non-surgical therapy (NST) were included. Direct and indirect evidence of progression were determined according to the current classification. Responsiveness to therapy was examined using mean pocket probing depths reduction (PPDRed), reduction of bleeding on probing (BOPRed), and the rate of pocket closure (%PC) after six months. RESULTS: Statistical analysis revealed no agreement between direct and indirect evidence in grading periodontitis (κ = 0.070). The actual rate of progression as determined by longitudinal data was underestimated in 13% (n = 11), overestimated in 51% (n = 43) and correctly estimated in 30% (n = 36) by indirect evidence. No significant differences in responsiveness to therapy were observed in patients graded according to direct evidence. Using indirect evidence, patients assigned grade C showed more PPDRed but less BOPRed and lower %PC compared to grade B. CONCLUSION: The present data indicate that indirect evidence may lead to inaccuracies compared to direct evidence regarding the estimation of periodontal progression. However, indirect evidence seems to be more suitable in the estimation of responsiveness to therapy than direct evidence, helping to identify cases that are more likely to require additional therapies such as re-instrumentation or periodontal surgery. CLINICAL RELEVANCE: Regarding the estimation of disease progression and responsiveness to periodontal therapy, accuracy and reliability of both direct and indirect evidence are limited when grading periodontitis.

Probing pocket depth reduction after non-surgical periodontal therapy: Tooth-related factors.

BACKGROUND: To investigate tooth-related factors that influence the reduction of probing pocket depths (PPD) after non-surgical periodontal therapy (NST). METHODS: Seven hundred forty-six patients with a total of 16,825 teeth were included and retrospectively analyzed. PPD reduction after NST was correlated with the tooth-related factors; tooth type, number of roots, furcation involvement, vitality, mobility, and type of restoration; using logistic multilevel regression for statistical analysis. RESULTS: NST was able to reduce probing depth overall stratified probing depths (1.20 ± 1.51 mm, p ≤ 0.001). The reduction was significantly higher at teeth with higher probing depths at baseline. At pockets with PPD ≥ 6 mm, PPD remains high after NST. Tooth type, number of roots, furcation involvement, vitality, mobility, and type of restoration are significantly and independently associated with the rate of pocket closure. CONCLUSIONS: The tooth-related factors: tooth type, number of roots, furcation involvement, vitality, mobility, and type of restoration had a significant and clinically relevant influence on phase I and II therapy. Considering these factors in advance may enhance the prediction of sites not responding adequately and the potential need for additional treatment, such as re-instrumentation or periodontal surgery, to ultimately achieve the therapy end points.

Cytotoxicity of 3D printed resin materials for temporary restorations on human periodontal ligament (PDL-hTERT) cells.

OBJECTIVES: Various dental resin materials are available for the fabrication of temporary restorations using modern additive printing methods. Albeit these materials are placed for several months in intimate contact with dental hard and soft tissues, including the gingival crevice, there exists only insufficient evidence on the biocompatibility of these materials. This in vitro study aimed to delineate the biocompatibility of 3D printable materials on periodontal ligament cells (PDL-hTERTs). METHODS: Samples of four dental resin materials for additive fabrication of temporary restorations using 3D printing (MFH, Nextdent; GC Temp, GC; Freeprint temp, Detax; 3Delta temp, Deltamed), one material for subtractive fabrication (Grandio disc, Voco) and one conventional temporary material (Luxatemp, DMG) were prepared with a standardized size according to the manufacturer's instructions. Human PDL-hTERTs were exposed to resin specimens or eluates of the material for 1, 2, 3, 6 and 9 days. For determination of cell viability, XTT assays were performed. In addition, the expression of the proinflammatory cytokines interleukin 6 and 8 (IL-6 and 8) was assessed in the supernatants with ELISA. Cell viability and the expression of IL-6 and 8 in presence of the resin material or their eluates was compared with untreated controls. Immunofluorescence staining for IL-6 and IL-8, as well as scanning electron microscopy of the discs after culturing, were performed. Differences between groups were analyzed with Student´s t-test for unpaired samples. RESULTS: Compared to untreated control samples, the exposure against the resin specimen induced strong reduction of cell viability in case of the conventional material Luxatemp (p < 0.001) and the additive material 3Delta temp (p < 0.001) irrespective of the observation period. On the contrary, the presence of eluates of the various materials induced only minor changes in cell viability. Considering IL-6 (day 2: p = 0.001; day 6 and 9: p < 0.001) and IL-8 (day 1: p = 0.001; day 2, 3, 6, 9: p < 0.001) their expression was strongly reduced in presence of the eluate of Luxatemp. Except for IL-6 at day 1 and 6 also the material 3Delta temp caused significant reduction of both proinflammatory mediators at any time point. SIGNIFICANCE: The conventional material Luxatemp and the additive material 3Delta temp appear to severely affect cell viability when in direct contact with PDL-hTERTs. The other tested materials of this new category of additive materials and the subtractive material Grandio seem to induce only minor changes in direct contact with these cells. Therefore, they could serve as a viable alternative in the fabrication of temporary restorations.

In-vitro cytocompatibility of self-adhesive dual-curing resin cements on human mesenchymal stem cells (hMSC) and periodontal ligament cells (PDL-hTERT).

OBJECTIVES: Self-adhesive dual cured resin cements provide easier clinical application than conventional resin cements but release higher amounts of unreacted monomers, potentially affecting their biocompatibility. This study aimed to compare the cytotoxic effects of self-adhesive dual cured resin cements with two conventional resin cements. METHODS: Samples of four resin cements, two self-adhesive dual cured cements (group A: RelyX Unicem, group B: SmartCem), and two conventional resin cements (group C: Panavia 2.0, group D: Variolink Esthetic DC) were prepared with a similar dimension under standardized polymerization conditions and stored in water. For each material 18 samples were used and cell cultures of human mesenchymal stem cells (hMSCs) or periodontal ligament cells (PDL-hTERT) were added under appropriate conditions. One experimental group (group E) was left untreated as control. A cell viability WST test, was performed in each experimental group at day 1, 7, 14 and 21. Moreover, microscopic examination of cells was performed using cell viability staining. RESULTS: Viability of both cell types as determined by WST test was significantly impaired at all time periods by the four different cement materials compared to the untreated control. Comparison between the four materials revealed different inhibition of the viability of both, PDL-hTERT and hMSC cells (group C > group B > group A > group D; p < 0.0001). SIGNIFICANCE: All resin-based cements caused significant impairment of cell viability, reflecting considerable cytotoxicity. Variolink caused significantly smaller changes of viability than the other tested materials.

Salivary and gingival CXCL8 correlation with periodontal status, periodontal pathogens, and smoking.

OBJECTIVES: Neutrophil granulocytes have been proposed to play a major role in the mediation of periodontitis-associated tissue destruction. Their recruitment and activation are regulated by the chemokine CXCL8. This study aimed to delineate the dependency of CXCL8 expression in gingival crevicular fluid (GCF) and saliva on periodontal status, bacterial infection, and smoking, in patients with periodontitis. METHODS: The study cohort comprised 279 subjects with untreated periodontitis. Probing pocket depth (PPD), gingival recession, bleeding on probing (BOP), plaque index, and bone loss were evaluated. CXCL8 was determined in saliva and GCF using flow cytometry. RESULTS: Considering the entire study sample, CXCL8 levels were correlated with the mean PPD (ρ = 0.131; p = 0.029), severity of periodontitis (ρ = 0.121; p = 0.043), BOP (ρ = 0.204; p = 0.001), and smoking (ρ = -0.219; p < 0.0001) in GCF; and, in whole saliva, with mean PPD (ρ = 0.154; p = 0.010) severity of periodontitis (ρ = 0.140; p = 0.020), gender (ρ = 0.178; p = 0.003), and smoking (ρ = -0.156; p = 0.010). Subgroup analysis among non-smokers revealed significantly higher amounts of CXCL8 in GCF (p = 0.012) and saliva (p = 0.026) comparing subjects with mean PPD ≤3mm and >3mm. CONCLUSION: The current study revealed a strong dependency of CXCL8 expression in GCF on the severity and activity of periodontal disease. Smoking causes a significant reduction in CXCL8 expression in saliva and GCF.

Differentiation of hMSC and hPDLSC induced by PGE2 or BMP-7 in 3D models.

Regenerative therapies of pathogenic tissue defects are gaining increasing importance in periodontology. Among others, the osteogenic effect of BMP-7 seems to play a major role in the development of teeth and alveolar bone. Human periodontal ligament stem cells (hPDLSC), as well as human mesenchymal stem cells (hMSC), show the ability to differentiate into various types of tissues. Regarding prostaglandin E2, many studies have confirmed that it is involved in the inflammation associated to periodontitis stimulating osteoclasts, which ultimately leads to resorption of tooth supporting bone. Herein, we aimed to investigate how PGE2 influences regenerative processes. The influence of PGE2 and BMP-7 on the osteogenic differentiation of hMSC and hPDLSC was determined in a 3D cell culture model using qRT-PCR, immunocytochemistry and REM. BMP-7 enhanced the expression of osteogenic markers in hMSC and lowered it in hPDLSC-TERT. BMP-7 had a lower osteogenic effect on hPDLSC-hTERT than on hMSC, while PGE2 decreases the osteogenic differentiation in both cell types, thus, inhibiting anabolic processes. Both cell types presented good proliferation and adhesion onto the scaffolds. The well-developed structural morphology and the support of osteogenic differentiation suggest that the scaffolds are potential candidate materials for bone regeneration. The positivity for Cap in hPDLSC and more in hMSC immunostaining samples indicates the initiation of neocementogenesis as part of periodontal regeneration. In conclusion, BMP7, in particular combined with MSC, seems to have a favourable application also in periodontal regeneration. Our results show that inflammation plays an important role in periodontal regeneration. PGE2 is a key mediator, which stimulates bone resorption also via a mechanism involving the inhibition of osteogenic differentiation of MSC as well as PDLSC. Therefore, regenerative approaches should always be conducted in combination with anti-inflammatory measures oriented to control inflammation.

Influence of osteogenic stimulation and VEGF treatment on in vivo bone formation in hMSC-seeded cancellous bone scaffolds.

BACKGROUND: Tissue engineering approaches for reconstruction of large bone defects are still technically immature, especially in regard to sufficient blood supply. Therefore, the aim of the present study was to investigate the influence of osteogenic stimulation and treatment with VEGF on new bone formation and neovascularization in hMSC-loaded cancellous bone scaffolds in vivo. METHODS: Cubic scaffolds were seeded with hMSC and either cultured in stem cell medium or osteogenic stimulation medium. One osteogenically stimulated group was additionally treated with 0.8 µg VEGF prior to subcutaneous implantation in athymic mice. After 2 and 12 weeks in vivo, constructs and selected organs were harvested for histological and molecular analysis. RESULTS: Histological analysis revealed similar vascularization of the constructs with and without VEGF treatment and absence of new bone formation in any group. Human DNA was detected in all inoculated scaffolds, but a significant decrease in cells was observed after 2 weeks with no further decrease after 12 weeks in vivo. CONCLUSION: Under the chosen conditions, osteogenic stimulation and treatment with VEGF does not have any influence on the new bone formation and neovascularization in hMSC-seeded cancellous bone scaffolds.

Differentiation of individual human mesenchymal stem cells probed by FTIR microscopic imaging.

Objective of this study is the novel application of Fourier transform infrared (FTIR) microscopic imaging to identify the differentiation state of individual human mesenchymal stem cells with or without osteogenic stimulation. IR spectra of several hundred single cells with lateral resolution of 5-10 microm were recorded using a FTIR imaging spectrometer coupled to a microscope with a focal plane array detector. A classification model based on linear discriminant analysis was trained to distinguish four cell types by their IR spectroscopic fingerprint. Without stimulation two cell types dominated, showing low or high levels of glycogen accumulation at the cell periphery. After stimulation, the protein composition in the cells changed and some cells started expressing calcium phosphate salts such as octacalciumphosphate, a precursor of the bone constituent hydroxyapatite. Few cells were identified which remained in their non-stimulated state. This study demonstrated for the first time that FTIR microscopic imaging can probe stem cell differentiation at the single cell level rapidly, non-destructively and with minimal preparation.