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.

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.

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.

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.

A Computer-Assisted Training Approach for Performing and Charting Periodontal Examinations: A Retrospective Study.

The aim of this study was to retrospectively investigate the development of a model-based, computer-assisted training approach for performing and charting periodontal examinations in a dental clinic in Germany. The study was initiated in summer semester 2013 and repeated in two consecutive semesters (S1: 44 students, S2: 48 students, and S3: 61 students) because technical features were introduced (S2: feedback and time control; S3: input control). In each semester, new dental students who had never performed periodontal examinations participated. Students were divided into two groups and received intense training at different time points. Agreement levels were calculated at baseline, after the first group received training, and after the second group received training. Comparisons were also made among the semesters. All 153 enrolled students in the three semesters participated. The results showed that probing depth accuracy significantly decreased in S1 from baseline to training completion (79.9% to 74.5%), and the probing depth accuracy significantly increased in S2 (76.1% to 78.9%) and S3 (77.2% to 82.3%). The students who received intense training at a late stage of the tutorial showed greater improvement, especially in the case of S3. Small changes in accuracy were observed for recession (S1: 94.5% to 96.1%; S2: 93.8% to 93.9%; S3: 95.4% to 96.6%). Accuracy for furcation involvement improved significantly in S1 (46.1% to 52.0%), S2 (46.8% to 59.7%), and S3 (44.2% to 58.3%); the improvements occurred when the students received intense training. The time taken for periodontal examination decreased significantly for S2 (23.6 to 14.2 min) and S3 (25.7 to 13.9 min). This study found that when feedback was provided, the students' periodontal examinations improved in accuracy and duration.

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.

Full-length amelogenin influences the differentiation of human dental pulp stem cells.

BACKGROUND: Amelogenin is an extracellular matrix protein well known for its role in the organization and mineralization of enamel. Clinically, it is used for periodontal regeneration and, due to its finding also in predentin and intercellular spaces of dental pulp cells, it has recently been suggested for pulp capping procedures. The aim of this study was to analyse in vitro the effect of the recombinant human full-length amelogenin on the growth and differentiation of human dental pulp stem cells (hDPSCs). METHODS: Human DPSCs were treated with a supplement of amelogenin at a concentration of 10 ng/ml, 100 ng/ml and 1000 ng/ml. The groups were compared to the unstimulated control in terms of cell morphology and proliferation, mineralization and gene expression for ALP (alkaline phosphatase), DMP1 (dentin matrix protein-1) and DSPP (dentin sialophosphoprotein). RESULTS: Amelogenin affects hDPSCs differently than PDL (periodontal ligament) cells and other cell lines. The proliferation rate at two weeks is significantly reduced in presence of the highest concentration of amelogenin as compared to the unstimulated control. hDPSCs treated with low concentrations present a downregulation of DMP1 and DSPP, which is significant for DSPP (p = 0.011), but not for DMP1 (p = 0.395). CONCLUSIONS: These finding suggest that the role of full-length amelogenin is not restricted to participation in tooth structure. It influences the differentiation of hDPSC according to various concentrations and this might impair the clinical results of pulp capping.

A New Model for Training in Periodontal Examinations Using Manikins.

The aim of this study was to develop and test models for training dental students in periodontal examinations using manikins that had distinct anatomical designs but were indistinguishable in external appearance. After four models were tested for inter- and intra-examiner reliability by two experienced dentists, 26 additional models were produced. The models were tested by 35 dental students at a dental school in Germany in 2014. The testing involved completing a periodontal examination that included probing depths, gingival recessions, and furcation involvements. The primary purpose of the study was to determine whether the models could be used as a tool for periodontal examination training by the students. Levels of agreement (students and dentists) and Kappa statistics (dentists) were calculated using absolute (±0 mm) and tolerable difference (±1 mm). Over the span of two weeks, the dentists' reliability with preset values for probing depths, gingival recessions, and furcation involvements ranged from 0.29 to 0.38, 0.52 to 0.61, and 0.54 to 0.57, respectively, under absolute difference and from 0.86 to 0.90, 0.96 to 0.99, and 0.62 to 0.73, respectively, under tolerable difference. The students' proportions of agreement for probing depths and gingival recessions under absolute vs. tolerable difference were 34.8% vs. 79.9% and 71.9% vs. 94.4%, respectively. The students frequently scored values higher than the preset values, overestimated furcation involvements, and failed to differentiate the levels of furcations. The models used did not pose any systematic or technical difficulties in the pilot study. Students were unable to measure furcation involvements with acceptable agreement. Thus, these models could be used for student periodontal examination training.

Interactions of human endothelial and multipotent mesenchymal stem cells in cocultures.

Current strategies for tissue engineering of bone rely on the implantation of scaffolds, colonized with human mesenchymal stem cells (hMSC), into a recipient. A major limitation is the lack of blood vessels. One approach to enhance the scaffold vascularisation is to supply the scaffolds with endothelial cells (EC).The main goal of this study was to establish a coculture system of hMSC and EC for the purposes of bone tissue engineering. Therefore, the cell behaviour, proliferation and differentiation capacity in various cell culture media as well as cell interactions in the cocultures were evaluated.The differentiation capacity of hMSC along osteogenic, chondrogenic, and adipogenic lineage was impaired in EC medium while in a mixed EC and hMSC media, hMSC maintained osteogenic differentiation. In order to identify and trace EC in the cocultures, EC were transduced with eGFP. Using time-lapse imaging, we observed that hMSC and EC actively migrated towards cells of their own type and formed separate clusters in long term cocultures. The scarcity of hMSC and EC contacts in the cocultures suggest the influence of growth factor-mediated cell interactions and points to the necessity of further optimization of the coculture conditions.

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.