Objective: The goal of this project was to determine the impact of local inflammation on changes in the subgingival biofilm composition in ligature-induced periodontitis in rats using the specialized pro-resolving mediator (SPM), resolvin E1 (RvE1).
Materials and Methods: The impact of RvE1 on the microbiota of ligature-induced periodontitis was assessed in two separate experiments; treatment of established periodontitis and prevention of ligature-induced periodontitis. In the treatment study, eighteen rats were separated into four groups comprising no ligature, ligature alone (no treatment), ligature with topical RvE1 treatment (ligature+RvE1) and, ligature with topical vehicle treatment (ligature + Vehicle). 3-0 silk ligatures were tied around maxillary second molars bilaterally for three weeks to induce disease. After three weeks, the treatment phase began with the application of RvE1 or vehicle (ethanol) every other day for an additional three weeks. Subgingival plaque samples were collected every four days throughout the experiment. The composition of the subgingival microbiota was initially screened by checkerboard DNA-DNA hybridization using probes on 40 subgingival species. Definitive, unbiased characterization of the subgingival microbiota was accomplished with next-generation sequencing using the Illumina MiSeq® platform. Six rats were sacrificed on Days 1, 21 and 42 and maxillae were dissected to collect samples for gingival RNA extraction, bone morphometric measurements, and histomorphometric analysis. Local tissue gene expression (Cxcl-1, Ptgs2, Nos2) was detected using qRT-PCR. Tissue specimens were prepared for histology and stained with H&E and tartrate resistant acid phosphatase (TRAP). In the prevention study, sixteen rats were separated into four groups (no ligature, ligature + RvE1 (0.1µg/µl), ligature + RvE1 (0.5 µg/µl), ligature + Vehicle). 5-0 silk ligatures were placed around maxillary second molars bilaterally to induce disease. At the time of ligature placement, animals received assigned treatment thrice weekly (M, W, F) for four weeks. Subgingival plaque samples were collected every four days (M and F). Four rats were sacrificed at baseline (Day 1) and the vehicle and two treatment groups (four each) were sacrificed at day 28 and samples processed as described above. The two-group comparisons were assessed by Student’s t-test. The multiple-group comparison was assessed by one-way ANOVA and post hoc tests.
Results: In the first study (treatment), topical application of RvE1 significantly reversed the bone loss associated with periodontitis compared to the vehicle. RvE1 application significantly reduced the expression of Cxcl1 and osteoclast density compared to the vehicle application. In the prevention study, RvE1 treatment significantly prevented the bone loss during the disease progression. RvE1 application significantly reduced the expression of Ptgs2, Nos2 compared to the vehicle application. Osteoclast density and inflammatory cell infiltration in the RvE1 groups were significantly lower than these in the Vehicle group.
The cell counts of bacterial species gradually increased and the subgingival microbiota shifted during the disease progression. In the treatment study, RvE1 treatment significantly reduced cell counts compared to the vehicle application at the end of treatment phase. The shift of subgingival microbiota was limited by the RvE1 treatment. In the prevention study, the taxonomic composition and diversity of subgingival microbiota was controlled by the RvE1 application. The change of subgingival microbiota appeared to be associated with the state of inflammation in the periodontal environment.
Conclusion: Resolvin E1 treatment of existing ligature-induced periodontitis significantly regenerates lost alveolar bone and prevents alveolar bone loss. Resolvin E1 treatment limits microbial shifts and reduces total bacterial load by inhibiting inflammation of local environment in experimental periodontitis.
| Identifer | oai:union.ndltd.org:harvard.edu/oai:dash.harvard.edu:1/17331953 |
| Date | 02 May 2016 |
| Creators | Lee, Chun-Teh |
| Publisher | Harvard University |
| Source Sets | Harvard University |
| Language | English |
| Detected Language | English |
| Type | Thesis or Dissertation, text |
| Format | application/pdf |
| Rights | open |
Page generated in 0.0014 seconds