Characterization of cytokines, matrix metalloproteinases and toll-like receptors in human periodontal tissue destruction

Periodontal Disease affects the supporting structures of the teeth and is initiated by a microbial biofilm called dental plaque. Severity ranges from superficial inflammation of the gingiva (gingivitis) to extensive destruction of connective tissue and bone leading to tooth loss (periodontitis). In periodontitis the destruction of tissue is caused by a cascade of microbial and host factors together with proteolytic enzymes. Matrix metalloproteinases (MMPs) are known to be central mediators of the pathologic destruction in periodontitis. Initially plaque bacteria provide pathogen-associated molecular patterns (PAMPs) which are sensed by Toll-like receptors (TLRs), and initiate intracellular signaling cascades leading to host inflammation. Our aim was to characterize TNF-α (tumor necrosis factor-alpha) and its type I and II receptors in periodontal tissues, as well as, the effects of TNF-α, IL-1β (interleukin-1beta) and IL-17 on the production and/or activation of MMP-3, MMP-8 and MMP-9. Furthermore we mapped the TLRs in periodontal tissues and assessed how some of the PAMPs binding to the key TLRs found in periodontal tissues affect production of TNF-α and IL-1β by gingival epithelial cells with or without combination of IL-17. TNF-α and its receptors were detected in pericoronitis. Furthermore, increased expression of interleukin-1β and vascular cell adhesion molecule-1 was found as a biological indicator of TNF-α ligand-receptor interaction. MMP-3, -8, and 9 were investigated in periodontitis affected human gingival crevicular fluid and gingival fibroblasts produced pro-MMP-3. Following that, the effect of IL-17 was studied on MMP and pro-inflammatory cytokine production. IL-17 was increased in periodontitis and up-regulated IL-1β, TNF-α, MMP-1 and MMP-3. We continued by demonstrating TLRs in gingival tissues, in which significant differences between patients with periodontitis and healthy controls were found. Finally, enzyme-linked immunosorbent assays were performed to show that the gingival cells response to inflammatory responses in a TLR-dependent manner. Briefly, this thesis demonstrates that TLRs are present in periodontal tissues and present differences in periodontitis compared to healthy controls. The cells of gingival tissues respond to inflammatory process in a TLR-dependent manner by producing pro-inflammatory cytokines. During the destruction of periodontal tissues, the release (IL-1β and TNF-α) and co-operation with other pro-inflammatory cytokines (IL-17), which in turn increase the inflammation and thus be more harmful to the host with the increased presence of MMPs (MMP-1, MMP-3, MMP-8, MMP-9) in diseased over healthy sites.

Periodontitis and peri-implantitis biomarkers in human oral fluids and the null-allele mouse model

Tissue destruction associated with the periodontal disease progression is caused by a cascade of host and microbial factors and proteolytic enzymes. Aberrant laminin-332 (Ln-332), human beta defensin (hBD), and matrix metalloproteinase (MMP) functions have been found in oral inflammatory diseases. The null-allele mouse model appears as the next step in oral disease research. The MMP-8 knock-out mouse model allowed us to clarify the involvement of MMP-8 in vivo in oral and related inflammatory diseases where MMP-8 is suggested to play a key role in tissue destruction. The cleaved Ln-332 γ2-chain species has been implicated in the apical migration of sulcular epithelial cells during the formation of periodontal pockets. We demonstrated that increased Ln-332 fragment levels in gingival crevicular fluid (GCF) are strongly associated with the severity of inflammation in periodontitis. Porphyromonas gingivalis trypsin-like proteinase can cleave an intact Ln-332 γ2-chain into smaller fragments and eventually promote the formation of periodontal pockets. hBDs are components of an innate mucosal defense against pathogenic microbes. Our results suggest that P. gingivalis trypsin-like proteinase can degrade hBD and thus reduce the innate immune response. Elevated levels and the increased activity of MMPs have been detected in several pathological tissue-destructive conditions where MMPs are shown to cleave extracellular matrix (ECM) and basement membrane (BM) molecules and to facilitate tissue destruction. Elevated levels of MMP-8 have been reported in many inflammatory diseases. In periodontitis, MMP-8 levels in gingival crevicular fluid (GCF) and in peri-implant sulcular fluid (PISF) are elevated at sites of active inflammation, and the increased levels of MMP-8 are mainly responsible for collagenase activity, which leads to tissue destruction. MMP-25, expressed by neutrophils, is involved in inflammatory diseases and in ECM turnover. MMP-26 can degrade ECM components and serve as an activator of other MMP enzymes. We further confirmed that increased levels and activation of MMP-8, -25, and -26 in GCF, PISF, and inflamed gingival tissue are associated with the severity of periodontal/peri-implant inflammation. We evaluated the role of MMP-8 in P. gingivalis-induced periodontitis by comparing MMP-8 knock-out (MMP8-/-) and wild-type mice. Surprisingly, MMP-8 significantly attenuated P. gingivalis-induced site-specific alveolar bone loss. We also evaluated systemic changes in serum immunoglobulin and lipoprotein profiles among these mouse groups. P. gingivalis infection increased HDL/VLDL particle size in the MMP-8-/- mice, which is an indicator of lipoprotein responses during systemic inflammation. Serum total LPS and IgG antibody levels were enhanced in both mice groups. P. gingivalis-induced periodontitis, especially in MMP-8-/- mice, is associated with severe alveolar bone loss and with systemic inflammatory and lipoprotein changes that are likely to be involved in early atherosclerosis.

Arresting occlusal enamel caries lesions with pit and fissure sealants

The aim of this study was to evaluate the feasibility of pit and fissure sealants and the effectiveness of the two sealant methods applied in every-day practice in public dental health care in Finland. Two sealant methods were evaluated according to their effectiveness in preventing dentin caries and sealant retention. Application time with these sealant methods was compared. The survival rate of sealed first and second molars was followed for nine and 13 year periods, respectively. Caries risk evaluation and observed increased caries risk were the basis for considering sealant application. A questionnaire, sent to all public dental health centers in Finland, monitored the attitudes of the dental profession towards sealant application and explored the current policies used as well as changes noted in the sealant application protocol. DMFT (Decayed, Missing or Filled Teeth) index values collected from the health centers were evaluated. The difference in caries rate between two methods investigated was highly significant. When compared to the glass ionomer sealant method (GIC), the effectiveness of the resin-based method (RB) in preventing dentin caries was 74% and the rate difference 3%. The relative risk for RB-sealed surfaces vs. GIC-sealed surfaces of having detectable dentin caries was 0.3 (95% CI 0.12, 0.57). The retention rate of sealants was higher with RB than GIC (P<0.001). Application of RB sealant material was less time-consuming than application of GIC sealant. Occlusal dentin caries lesions were found in 4% and proximal caries in less than 2% of sealed teeth. The majority of respondents reported application of sealants on a systematic basis along with caries-risk evaluation. Those health centers sealing over suspected or detected enamel caries had lower average DMFT index values (1.0) when compared to DMFT values (1.2) of health centers applying sealants by alternative criteria. It is concluded that the RB sealant method is more effective than the GIC sealant method in preventing dentin caries. Sealant maintenance may increase the costs of a sealant program. Occlusal caries management may be improved if the applied sealant policies are changed towards an interceptive approach i.e. applying the sealants over detected or suspected enamel caries lesions instead of sealing sound teeth in a preventive manner.