Impact of Metal Particles Released during Ultrasonic Scaling of Titanium Surfaces on Human Gingival Fibroblasts.

PURPOSE: Metal particles found in tissues around dental implants have been proposed to play a pathogenic role in peri-implantitis. Ultrasonic scaling has been suggested as a mechanism by which these particles can be inadvertently released into surrounding tissues. Furthermore, risk factors like diabetes can result in exacerbation of this inflammatory condition. The current study aimed to analyze metal particles released from titanium surfaces during ultrasonic scaling and their impact on pro-inflammatory cytokine production by human gingival fibroblasts. METHODS: Metal particles generated from ultrasonic scaling of titanium discs using two different tips (metal and poly-etheretherketone tips) were characterized using scanning electron microscopy and elemental analysis. Endotoxin levels and Human gingival fibroblast viability, in the presence commercial and ultrasonically generated particles were determined. Fibroblasts, cultured in high or low glucose growth medium, were incubated with commercial titanium particles or ultrasonically generated particles in the presence or absence of interluekin-1ß. Interleukin 6 and interleukin 8 production were then quantified using Enzyme linked immunosorbent assay. RESULTS: Analysis of particles after scaling of titanium discs showed significant levels of titanium particles. Commercial titanium particles and generated particles had no effect of fibroblast viability. Endotoxin levels of all particles were too low to stimulate HGF cells. IL-1ß significantly stimulated IL-6 and IL-8 production. However, commercial, and generated particles generally had no significant effect on IL- 6 and IL-8 production. CONCLUSION: Our study concluded that particles generated during ultrasonic scaling had no significant effect on viability of HGF cells and cytokine production.

Phytocannabinoids regulate inflammation in IL-1ß-stimulated human gingival fibroblasts.

OBJECTIVES: Billions of individuals worldwide suffer from periodontal disease, an inflammatory disease that results in hard-tissue and soft-tissue destruction. A viable therapeutic option to treat periodontal disease may be via cannabinoids that exert immunomodulatory effects, and the endocannabinoid system (ECS) is readily present in periodontal tissues that exhibit cannabinoid type 1 and 2 receptors (CB1R and CB2R). Phytocannabinoids (pCBs), which are a part of a heterogeneous group of molecules acting on cannabinoid receptors (CBR) derived from the cannabis plants, have been attributed to a wide variety of effects including anti-inflammatory activity and some pro-inflammatory effects depending on the cell type. Thus, this study aims to examine the effects of pCBs on primary human gingival fibroblasts (HGFs) in IL-1ß stimulated (simulated periodontal disease) HGFs. MATERIALS AND METHODS: Human gingival fibroblasts (HGFs) obtained from ATCC were cultured per the manufacturer's recommendation. The functional activity of cannabinoid receptors was measured using ACTOne (cAMP)-based CB1R and CB2R assay. The effects of three pCBs (0.1-10 µg/ml or 10-4.5 -10-6.5  M) on cell viability were assessed using the CCK-8 cellular dehydrogenase assay. IL-1ß (1 ng/ml) was added an hour before the treatment to stimulate inflammation in the HGFs before the addition of cannabinoid ligands. After 24-h incubation, the production of INF-γ, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p70, IL-13, and TNF-α was measured using Mesoscale Discovery (MSD) Human Pro-Inflammatory kit. To measure prostaglandin E 2 levels (PGE2), Cisbio HTRF PGE2 assay kit was used per the manufacturer's recommendation to measure after 24-h incubation. The data were analyzed using GraphPad Prism 6.0. The analytes for each group were compared using a one-way ANOVA test with Bonferroni's correction. RESULTS: Cannabidivarin (CBVN or CBDV) (EC50  = 12 nM) and cannabigerol (CBG) (EC50  = 30 nM) exhibited agonist activity on CB2R with intermediate efficacy. Cannabidiol (CBD) did not exhibit activation of the CB2R, and the CB1R activation was not observed with any of the pCBs. Cytotoxicity results showed that concentrations of 2.50 µg/ml or greater for the pCBs were toxic except for CBVN. Lower concentrations of CBD and CBG (0.1-0.75 µg/ml), and CBVN at 2.50 µg/ml exhibited significant effects on HGF proliferation. In IL-1ß-stimulated HGFs, prostaglandin E2 (PGE2) production was significantly suppressed only by CBG and CBVN. CBD and CBG treatment alone did, however, elevate PGE2 production significantly compared to control. IL-1ß stimulation resulted in a robust increase in the production of all cytokines tested. Treatment of IL-ß-stimulated HGF with the three pCBs (1 µg/ml) significantly reduced INF-É£, TNF-α, and IL-2. The significant suppression of IL-4 was seen with CBD and CBVN, while only CBVN exerted suppression of IL-13. The three pCBs significantly increased IL-6, IL-10, and IL-12 levels, while none of the pCBs reduced the expression of IL-8 in IL-1ß-stimulated HGF. CONCLUSION: The effective inhibition of IL-1ß-stimulated production of PGE2 and cytokines by the pCB in HGFs suggests that targeting the endocannabinoid system may lead to the development of therapeutic strategies for periodontal therapy. However, each pCB has its unique anti-inflammatory profile, in which certain pro-inflammatory activities are also exhibited. The pCBs alone or in combination may benefit and aid in improving public oral health.

Multiplexing of Immune Markers via Electrochemiluminescence Immunoassays for Systems Biology.

Electrochemiluminescence immunoassays are based on the principle of light emission in a chemical environment to detect and analyze different proteins and biomolecules. It has numerous advantages over traditional analytical methods including conservation of sample, high sensitivity, broad range, and relative ease of use. Herein, we describe the electrochemiluminescence methods by using Mesoscale Discovery System with recommendations and optimization of protocols to aid in discovery of biological relevant markers and also discuss avoidance of major pitfalls for accurate biomarker detection.

Use of Collagen Matrix Scaffolds as a Substitute for Soft Tissue Augmentation: Case Series.

INTRODUCTION: The presence of keratinized mucosa (KM) around natural teeth is believed to be beneficial in certain restorative, prosthetic, and orthodontic situations. Lack of adequate KM is common and predictably treated by autogenous gingival grafts (AGGs); however, AGGs have the disadvantages of harvest site morbidity, limited donor site availability, and compromised esthetics. CASE PRESENTATION: This case series presents the use of the xenogeneic porcine bilayer collagen matrix (BCM) in increasing the width of attached KM around natural teeth. Patients with a limited amount of KM, shallow vestibule, and aberrant frenum attachment were treated using this graft material. The patients were followed up to 4.5 years postoperatively and were evaluated regarding the amount of KM, gingival margin stability, and tissue esthetics. CONCLUSIONS: Within the limitations of the sample size of patients in this report, the BCM appears to be a viable alternative option to AGG for increasing the width of KM gingiva around teeth. This method resulted in gain of KM, gingival margin stability, vestibular deepening, aberrant frenum elimination, and favorable esthetics in terms of color matching, texture, and contour blending. This xenogeneic graft material could be used in cases where the autogenous graft supply is limited or in highly esthetically demanding cases. Additionally, it could be an alternative option when a second surgical site is not desired by the patient or a less invasive procedure is preferred by the clinician in certain medical conditions. Well-controlled long-term studies are required to validate our limited clinical observations.

Dataset on the chemokine and cytokine responses of multi-cell cultures treated with Porphyromonas gingivalis hemagglutinin B.

Chemokines and cytokines produced in gingival tissues exposed to microorganisms and microbial products in dental plaque lead to local inflammation and tissue damage seen in periodontal disease. Bates et al. 2018 [1] reported that Porphyromonas gingivalis hemagglutinin B (HagB)-induced matrix metalloproteinase (MMP) responses of single cell cultures containing dendritic cells, gingival epithelial (GE) keratinocytes, or T cells were significantly different from the MMP responses of these same cells grown in multi-cell cultures. Here we report the concentrations (pg/ml) of HagB-induced IL1α, IL6, IL8, IL12(p40), GM-CSF, MIP1α, MIP1ß, RANTES, TNFα, and VEGF produced by dendritic cells, GE keratinocytes, or T cells in single cell cultures, two-cell cultures, or three-cell cultures.

Matrix Metalloproteinase Response of Dendritic Cell, Gingival Epithelial Keratinocyte, and T-Cell Transwell Co-Cultures Treated with Porphyromonas gingivalis Hemagglutinin-B.

Matrix metalloproteinases (MMPs) are enzymes involved in periodontal tissue destruction. Hemagglutinin B (HagB) from the periodontal pathogen Porphyromonas gingivalis induces an elevated MMP response in dendritic cells, but responses from cultures of single-cell types do not reflect the local tissue environment. The objective of this study was to measure HagB-induced MMP responses in a transwell co-culture system containing dendritic cells, gingival epithelial (GE) keratinocytes, and CD4+ T-cells. Transwell co-cultures were assembled and treated with or without HagB. Immunoassays were used to determine production of MMP1, MMP7, MMP9, and MMP12 in response to HagB up to 64 h. Control responses were subtracted from HagB-induced responses. A two-way fixed effect ANOVA was fit to log-transformed concentrations and pairwise group comparisons were conducted (p < 0.05). At 64 h, dendritic cells produced elevated MMP1 and MMP9 responses, which were attenuated in the 3-cell co-culture (p < 0.05). There were also significant differences in MMP7 and MMP12 production between single-cell cultures and co-cultures. These results support the need to use multiple cell types in culture models to evaluate a more representative response to proinflammatory agonists. This three-cell transwell co-culture model may help us better understand the inflammatory process in periodontal disease and test novel therapeutic approaches.

The Role of Systems Biologic Approach in Cell Signaling and Drug Development Responses-A Mini Review.

The immune system is an integral aspect of the human defense system and is primarily responsible for and involved in the communication between the immune cells. It also plays an important role in the protection of the organism from foreign invaders. Recent studies in the literature have described its role in the process of hematopoiesis, lymphocyte recruitment, T cell subset differentiation and inflammation. However, the specific molecular mechanisms underlying these observations remain elusive, impeding the elaborate manipulation of cytokine sequential delivery in tissue repair. Previously, the discovery of new drugs and systems biology went hand in hand; although Systems biology as a term has only originated in the last century. Various new chemicals were tested on the human body, and studied through observation. Animal models replaced humans for initial trials, but the interactions, response, dose and effect between animals and humans could not be directly correlated. Therefore, there is a need to form disease models outside of human subjects to check the effectiveness and response of the newer natural or synthetic chemicals. These emulate human disease conditions wherein the behavior of the chemicals would be similar in the disease model and humans.