Characterization of an ester-based core-multishell (CMS) nanocarrier for the topical application at the oral mucosa

Objectives Topical drug administration is commonly applied to control oral inflammation. However, it requires sufficient drug adherence and a high degree of bioavailability. Here, we tested the hypothesis whether an ester-based core-multishell (CMS) nanocarrier is a suitable nontoxic drug-delivery system that penetrates efficiently to oral mucosal tissues, and thereby, increase the bioavailability of topically applied drugs. Material and methods To evaluate adhesion and penetration, the fluorescence-labeled CMS 10-E-15-350 nanocarrier was applied to ex vivo porcine masticatory and lining mucosa in a Franz cell diffusion assay and to an in vitro 3D model. In gingival epithelial cells, potential cytotoxicity and proliferative effects of the nanocarrier were determined by MTT and sulphorhodamine B assays, respectively. Transepithelial electrical resistance (TEER) was measured in presence and absence of CMS 10-E-15-350 using an Endohm-12 chamber and a volt-ohm-meter. Cellular nanocarrier uptake was analyzed by laser scanning microscopy. Inflammatory responses were determined by monitoring pro-inflammatory cytokines using real-time PCR and ELISA. Results CMS nanocarrier adhered to mucosal tissues within 5 min in an in vitro model and in ex vivo porcine tissues. The CMS nanocarrier exhibited no cytotoxic effects and induced no inflammatory responses. Furthermore, the physical barrier expressed by the TEER remained unaffected by the nanocarrier. Conclusions CMS 10-E-15-350 adhered to the oral mucosa and adhesion increased over time which is a prerequisite for an efficient drug release. Since TEER is unaffected, CMS nanocarrier may enter the oral mucosa transcellularly. Clinical relevance Nanocarrier technology is a novel and innovative approach for efficient topical drug delivery at the oral mucosa

miRNAs from inflamed gingiva link gene signaling to increased MET expression

Several array-based microRNA (miRNA) expression studies independently showed increased expression of miRNAs hsa-miR-130a-3p, -142-3p, -144-3p, -144-5p, -223-3p, -17-5p, and -30e-5p in gingiva affected by periodontal inflammation. We aimed to determine direct target genes and signaling pathways regulated by these miRNAs to identify processes relevant to gingival inflammatory responses and tissue homeostasis. We transfected miRNA mimics (mirVana) for each of the 7 miRNAs separately into human primary gingival fibroblasts cultured from 3 different donors. Following RNA sequencing, differential gene expression and second-generation gene set enrichment analyses were performed. miRNA inhibition and upregulation was validated at the transcript and protein levels using quantitative reverse transcriptase polymerase chain reaction, Western blotting, and reporter gene assays. All 7 miRNAs significantly increased expression of the gene MET proto-oncogene, receptor tyrosine kinase (MET). Expression of known periodontitis risk genes CPEB1, ABCA1, and ATP6V1C1 was significantly repressed by hsa-miR-130a-3p, -144-3p, and -144-5p, respectively. The genes WASL, ENPP5, ARL6IP1, and IDH1 showed the most significant and strongest downregulation after hsa-miR-142-3p, -17-5p, -223-3p, and -30e-5p transfection, respectively. The most significantly regulated gene set of each miRNA related to cell cycle (hsa-miRNA-144-3p and -5p [P(adj) = 4 × 10(-40) and P(adj) = 4 × 10(-6)], -miR-17-5p [P(adj) = 9.5 × 10(-23)], -miR-30e-5p [P(adj) = 8.2 × 10(-18)], -miR-130a-3p [P(adj) = 5 × 10(-15)]), integrin cell surface interaction (-miR-223-3p [P(adj) = 2.4 × 10(-7)]), and interferon signaling (-miR-142-3p [P(adj) = 5 × 10(-11)]). At the end of acute inflammation, gingival miRNAs bring together complex regulatory networks that lead to increased expression of the gene MET. This underscores the importance of mesenchymal cell migration and invasion during gingival tissue remodeling and proliferation in restoring periodontal tissue homeostasis after active inflammation. MET, a receptor of the mitogenic hepatocyte growth factor fibroblast secreted, is a core gene of this process

Increased periodontal attachment loss in patients with systemic sclerosis

Background: Patients with inflammatory rheumatic diseases and periodontitis share common pathogenetic characteristics, such as pro-inflammatory traits causative for tissue degradation and loss of function. Aim of the present case control study was to investigate the association between systemic sclerosis (SSc) and periodontitis. Methods: The association between SSc and periodontitis was examined in 58 SSc patients and 52 control patients, matched for age and gender. Periodontal examination included periodontal attachment loss, probing pocket depth, bleeding on probing, plaque index and gingival index. Potential risk factors of periodontitis were assessed through patients' questionnaires. Results: In unadjusted analyses, patients with SSc had a significant 0.61 mm higher periodontal attachment loss (95 % confidence interval (CI), 0.24 - 0.97; p = 0.002) when compared to controls. In a stepwise logistic regression, including SSc status, age, gender, education, smoking, alcohol consumption and BMI, only SSc status, age, and gender remained significantly associated with periodontitis. Adjusted for age and gender, patients with SSc had 0.52 mm higher periodontal attachment loss compared to controls (95 % CI, 0.16 - 0.88; p = 0.005). The strength of the association of SSc with periodontal attachment loss remained statistically significant after further adjustment for plaque index (0.44 mm; 95 % CI 0.02 - 0.86; p = 0.038) or gingival index (0.61 mm; 95 % CI, 0.24 - 0.97 p = 0.001). Conclusions: The study demonstrates higher periodontal clinical attachment loss in SSc patients, which remained significant following adjustment. The study indicates a possible relationship between SSc and periodontitis

Biomaterials and regenerative technologies used in bone regeneration in the craniomaxillofacial region: Consensus report of group 2 of the 15th European Workshop on Periodontology on Bone Regeneration

Geistlich Pharma A

Role of microbial biofilms in the maintenance of oral health and in the development of dental caries and periodontal diseases. Consensus report of group 1 of the Joint EFP/ORCA workshop on the boundaries between caries and periodontal disease.

BACKGROUND AND AIMS: The scope of this working group was to review (1) ecological interactions at the dental biofilm in health and disease, (2) the role of microbial communities in the pathogenesis of periodontitis and caries, and (3) the innate host response in caries and periodontal diseases. RESULTS AND CONCLUSIONS: A health-associated biofilm includes genera such as Neisseria, Streptococcus, Actinomyces, Veillonella and Granulicatella. Microorganisms associated with both caries and periodontal diseases are metabolically highly specialized and organized as multispecies microbial biofilms. Progression of these diseases involves multiple microbial interactions driven by different stressors. In caries, the exposure of dental biofilms to dietary sugars and their fermentation to organic acids results in increasing proportions of acidogenic and aciduric species. In gingivitis, plaque accumulation at the gingival margin leads to inflammation and increasing proportions of proteolytic and often obligately anaerobic species. The natural mucosal barriers and saliva are the main innate defence mechanisms against soft tissue bacterial invasion. Similarly, enamel and dentin are important hard tissue barriers to the caries process. Given that the present state of knowledge suggests that the aetiologies of caries and periodontal diseases are mutually independent, the elements of innate immunity that appear to contribute to resistance to both are somewhat coincidental

Recombinant human osteopontin expressed in Nicotiana benthamiana stimulates osteogenesis related genes in human periodontal ligament cells.

Tissue engineering aims to utilise biologic mediators to facilitate tissue regeneration. Several recombinant proteins have potential to mediate induction of bone production, however, the high production cost of mammalian cell expression impedes patient access to such treatments. The aim of this study is to produce recombinant human osteopontin (hOPN) in plants for inducing dental bone regeneration. The expression host was Nicotiana benthamiana using a geminiviral vector for transient expression. OPN expression was confirmed by Western blot and ELISA, and OPN was purified using Ni affinity chromatography. Structural analysis indicated that plant-produced hOPN had a structure similar to commercial HEK cell-produced hOPN. Biological function of the plant-produced hOPN was also examined. Human periodontal ligament stem cells were seeded on an OPN-coated surface. The results indicated that cells could grow normally on plant-produced hOPN as compared to commercial HEK cell-produced hOPN determined by MTT assay. Interestingly, increased expression of osteogenic differentiation-related genes, including OSX, DMP1, and Wnt3a, was observed by realtime PCR. These results show the potential of plant-produced OPN to induce osteogenic differentiation of stem cells from periodontal ligament in vitro, and suggest a therapeutic strategy for bone regeneration in the future

Translation of mouse model to human gives insights into periodontitis etiology

To suggest candidate genes involved in periodontitis, we combined gene expression data of periodontal biopsies from Collaborative Cross (CC) mouse lines, with previous reported quantitative trait loci (QTL) in mouse and with human genome-wide association studies (GWAS) associated with periodontitis. Periodontal samples from two susceptible, two resistant and two lines that showed bone formation after periodontal infection were collected during infection and naïve status. Differential expressed genes (DEGs) were analyzed in a case-control and case-only design. After infection, eleven protein-coding genes were significantly stronger expressed in resistant CC lines compared to susceptible ones. Of these, the most upregulated genes were MMP20 (P = 0.001), RSPO4 (P = 0.032), CALB1 (P = 1.06×10-4), and AMTN (P = 0.05). In addition, human orthologous of candidate genes were tested for their association in a case-controls samples of aggressive (AgP) and chronic (CP) periodontitis (5,095 cases, 9,908 controls). In this analysis, variants at two loci, TTLL11/PTGS1 (rs9695213, P = 5.77×10-5) and RNASE2 (rs2771342, P = 2.84×10-5) suggested association with both AgP and CP. In the association analysis with AgP only, the most significant associations were located at the HLA loci HLA-DQH1 (rs9271850, P = 2.52×10-14) and HLA-DPA1 (rs17214512, P = 5.14×10-5). This study demonstrates the utility of the CC RIL populations as a suitable model to investigate the mechanism of periodontal disease

In Vitro Effect of Porphyromonas gingivalis Methionine Gamma Lyase on Biofilm Composition and Oral Inflammatory Response

Methanethiol (methyl mercaptan) is an important contributor to oral malodour and periodontal tissue destruction. Porphyromonas gingivalis, Prevotella intermedia and Fusobacterium nucleatum are key oral microbial species that produce methanethiol via methionine gamma lyase (mgl) activity. The aim of this study was to compare an mgl knockout strain of P. gingivalis with its wild type using a 10-species biofilm co-culture model with oral keratinocytes and its effect on biofilm composition and inflammatory cytokine production. A P. gingivalis mgl knockout strain was constructed using insertion mutagenesis from wild type W50 with gas chromatographic head space analysis confirming lack of methanethiol production. 10-species biofilms consisting of Streptococcus mitis, Streptococcus oralis, Streptococcus intermedius, Fusobacterium nucleatum ssp polymorphum, Fusobacterium nucleatum ssp vincentii, Veillonella dispar, Actinomyces naeslundii, Prevotella intermedia and Aggregatibacter actinomycetemcomitans with either the wild type or mutant P. gingivalis were grown on Thermanox cover slips and used to stimulate oral keratinocytes (OKF6-TERT2), under anaerobic conditions for 4 and 24 hours. Biofilms were analysed by quantitative PCR with SYBR Green for changes in microbial ecology. Keratinocyte culture supernatants were analysed using a multiplex bead immunoassay for cytokines. Significant population differences were observed between mutant and wild type biofilms; V. dispar proportions increased (p<0.001), whilst A. naeslundii (p<0.01) and Streptococcus spp. (p<0.05) decreased in mutant biofilms. Keratinocytes produced less IL-8, IL-6 and IL-1α when stimulated with the mutant biofilms compared to wild type. Lack of mgl in P. gingivalis has been shown to affect microbial ecology in vitro, giving rise to a markedly different biofilm composition, with a more pro-inflammatory cytokine response from the keratinocytes observed. A possible role for methanethiol in biofilm formation and cytokine response with subsequent effects on oral malodor and periodontitis is suggested

Protease-activated receptor-2 mediates the expression of inflammatory cytokines, antimicrobial peptides, and matrix metalloproteinases in keratinocytes in response to Propionibacterium acnes

Propionibacterium acnes (P. acnes) has been known to produce various exogenous proteases, however, their role in acne pathogenesis remains largely unknown. Proteases elicit cellular responses, at least in part, via proteinase-activated receptor-2 (PAR-2), which is known to mediate inflammation and immune response. In this study, we investigated whether proteases from P. acnes could activate PAR-2 on keratinocytes and induce pro-inflammatory cytokines, antimicrobial peptides (AMPs), and matrix metalloproteinases (MMPs) via PAR-2 signaling. We examined PAR-2 expression and protease activity in acne lesions using immunofluorescence staining and in situ zymography. The effect of the culture supernatant of P. acnes on Ca2+ signaling in immortalized keratinocytes (HaCaT) was measured using a fluorescence method. HaCaT cells were treated with P. acnes strain ATCC 6919 culture supernatant, with or without pretreatment with serine protease inhibitor or selective PAR-2 antagonist and the gene expression of pro-inflammatory cytokines, AMPs, and MMPs was detected using real-time reverse transcription-polymerase chain reaction. We found that the protease activity and PAR-2 expression were increased in acne lesions. The P. acnes culture supernatant induced calcium signaling in keratinocytes via PAR-2 and stimulated the mRNA expression of interleukin (IL)-1α, -8, tumor necrosis factor (TNF)-α, human beta defensin (hBD)-2, LL-37, MMP-1, -2, -3, -9, and -13 in keratinocytes, which was significantly inhibited by serine protease inhibitor as well as selective PAR-2 specific antagonist. These results indicate that PAR-2 plays an important role in the pathogenesis of acne by inducing inflammatory mediators in response to proteases secreted from P. acnes