Date on Master's Thesis/Doctoral Dissertation

5-2017

Document Type

Master's Thesis

Degree Name

M.S.

Department

Oral Biology

Degree Program

Oral Biology, MS

Committee Chair

Liang, Shuang

Committee Co-Chair (if applicable)

Darling, Douglas

Committee Member

Darling, Douglas

Committee Member

Wang, Huizhi

Author's Keywords

NBD-nanoparticles; NF-ĸB pathway; periodontal disease; osteoclastogenesis; inflammation; alveolar bone loss

Abstract

Periodontitis is an infection-driven inflammatory disease characterized by gingival inflammation and bone loss. The NF-ĸB signaling pathway is pivotal in osteoclastogenesis and infection-induced pro-inflammatory responses. The use of nanoparticles as a vehicle to deliver drug increases stability, loading capacity, and facilitates transmembrane transportation. The hypothesis was that a novel nanoparticle carrying therapeutic NBD inhibitory peptides (NBD-nanoparticles) will inhibit measures of periodontal disease. In this project, we tested the nanoparticles for their ability to directly inhibit osteoclastogenesis and inflammation as an original strategy for periodontitis therapy. We also tested the capability of the nanoparticles to inhibit gingival inflammation and alveolar bone loss in an animal model. Methods: In vitro- In order to test the impact of NBD-nanoparticles on osteoclastogenesis, RAW 264.7 cells were stimulated using RANKL and treated with NBD-nanoparticles. Controls included treatment with empty nanoparticles and no treatment. Seven days later, the cultures were fixed and stained with TRAP, an osteoclast marker, and the number of multinucleated TRAP positive cells were counted. In order to test the impact of NBD-nanoparticles on pro-inflammatory responses, RAW or THP1 cells were stimulated with the periodontal pathogen P. gingivalis, treated with NBD-nanoparticles or empty nanoparticle and tested for expression of cytokines critical in periodontitis, such as IL-1β, IL-6, TNFα by ELISA. Differences were evaluated by ANOVA. In vivo - NBD-nanoparticles were tested in a murine ligature-induced periodontitis model where mice received a ligation around the second molar, P. gingivalis infection and microinjections of NBD-nanoparticles, empty nanoparticles, or PBS in the gingiva. Gingival tissue was tested for mRNA expression of pro-inflammatory cytokines by real-time PCR. Alveolar bone loss was determined by measuring the CEJ-ABC distance. Bacterial culture from oral swabs confirmed bacterial persistence. Differences were evaluated by ANOVA. Results: NBD-nanoparticles inhibit osteoclastogenesis directly and P. gingivalis-induced pro-inflammatory cytokine production. NBD-nanoparticle application inhibits the gingival expression of periodontitis-related cytokines and alveolar bone loss in a murine ligature model. Conclusions: NBD-nanoparticle is able to inhibit osteoclastogenesis directly and pro-inflammatory cytokines production in vitro. This nanoparticle prevents gingival inflammation and bone loss in a murine model for periodontitis.

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