In Vivo Regulation of Active Matrix Metalloproteinase-8 (aMMP-8) in Periodontitis: From Transcriptomics to Real-Time Online Diagnostics and Treatment Monitoring.

BACKGROUND: This study investigated in vivo regulation and levels of active matrix metalloproteinase-8 (aMMP-8), a major collagenolytic protease, in periodontitis. METHODS: Twenty-seven adults with chronic periodontitis (CP) and 30 periodontally healthy controls (HC) were enrolled in immunohistochemistry and transcriptomics analytics in order to assess Treponema denticola (Td) dentilisin and MMP-8 immunoexpression, mRNA expression of MMP-8 and its regulators (IL-1ß, MMP-2, MMP-7, TIMP-1). Furthermore, the periodontal anti-infective treatment effect was monitored by four different MMP-8 assays (aMMP-8-IFMA, aMMP-8-Oralyzer, MMP-8-activity [RFU/minute], and total MMP-8 by ELISA) among 12 CP (compared to 25 HC). RESULTS: Immunohistochemistry revealed significantly more Td-dentilisin and MMP-8 immunoreactivities in CP vs. HC. Transcriptomics revealed significantly elevated IL-1ß and MMP-7 RNA expressions, and MMP-2 RNA was slightly reduced. No significant differences were recorded in the relatively low or barely detectable levels of MMP-8 mRNAs. Periodontal treatment significantly decreased all MMP-8 assay levels accompanied by the assessed clinical indices (periodontal probing depths, bleeding-on-probing, and visual plaque levels). However, active but not total MMP-8 levels persisted higher in CP than in periodontally healthy controls. CONCLUSION: In periodontal health, there are low aMMP-8 levels. The presence of Td-dentilisin in CP gingivae is associated with elevated aMMP-8 levels, potentially contributing to a higher risk of active periodontal tissue collagenolysis and progression of periodontitis. This can be detected by aMMP-8-specific assays and online/real-time aMMP-8 chair-side testing.

Local and systemic responses in matrix metalloproteinase 8-deficient mice during Porphyromonas gingivalis-induced periodontitis.

Periodontitis is a bacterium-induced chronic inflammation that destroys tissues that attach teeth to jaw bone. Pathologically excessive matrix metalloproteinase 8 (MMP-8) is among the key players in periodontal destruction by initiating type I collagen degradation. We studied MMP-8 in Porphyromonas gingivalis-induced periodontitis by using MMP-8-deficient (MMP8(-/-)) and wild-type (WT) mice. Alveolar bone loss, inflammatory mediator expression, serum immunoglobulin, and lipoprotein responses were investigated to clarify the role of MMP-8 in periodontitis and systemic inflammatory responses. P. gingivalis infection induced accelerated site-specific alveolar bone loss in both MMP8(-/-) and WT mice relative to uninfected mice. The most extensive bone degradation took place in the P. gingivalis-infected MMP8(-/-) group. Surprisingly, MMP-8 significantly attenuated (P < 0.05) P. gingivalis-induced site-specific alveolar bone loss. Increased alveolar bone loss in P. gingivalis-infected MMP8(-/-) and WT mice was associated with increase in gingival neutrophil elastase production. Serum lipoprotein analysis demonstrated changes in the distribution of high-density lipoprotein (HDL) and very-low-density lipoprotein (VLDL) particles; unlike the WT mice, the MMP8(-/-) mice underwent a shift toward a smaller HDL/VLDL particle sizes. P. gingivalis infection increased the HDL/VLDL particle size in the MMP8(-/-) mice, which is an indicator of lipoprotein responses during systemic inflammation. Serum total lipopolysaccharide activity and the immunoglobulin G-class antibody level in response to P. gingivalis were significantly elevated in both infected mice groups. Thus, MMP-8 appears to act in a protective manner inhibiting the development of bacterium-induced periodontal tissue destruction, possibly through the processing anti-inflammatory cytokines and chemokines. Bacterium-induced periodontitis, especially in MMP8(-/-) mice, is associated with systemic inflammatory and lipoprotein changes that are likely involved in early atherosclerosis.

Human beta-defensin-1 and -2 and matrix metalloproteinase-25 and -26 expression in chronic and aggressive periodontitis and in peri-implantitis.

OBJECTIVE: Aberrant matrix metalloproteinase (MMP) and human beta-defensin (HBD) functions have been found in inflammatory diseases. The objectives of this study were to investigate the immunolocalisation, mRNA expression and molecular forms of MMP-25, MMP-26, HBD-1 and HBD-2 in chronic and aggressive periodontitis and in peri-implantitis. The expression of MMP-25 by cultured human plasmacytoma cells and macrophages, and the effects of MMP-26 and Porphyromonas gingivalis trypsin-like proteinase on HBD-1 and -2 were also studied. DESIGN: Immunohistochemistry, immunofluorescent analysis, reverse transcriptase polymerase chain reaction and immunoblotting were used to assess localisation, mRNA expression and molecular forms of MMP-25, MMP-26, HBD-1 and HBD-2. HBD degradation by MMP-26 and P. gingivalis proteinase was studied by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. RESULTS: MMP-25 was present in plasma cells and polymorphonuclear leucocytes, and MMP-26 was present in oral and sulcular basement membrane zones. HBD-1 was distributed perivasculary in gingival connective tissue and in oral and sulcular epithelium, and HBD-2 was found to a lesser extent in the perivascular space. Low MMP-25, MMP-26, HBD-1 and HBD-2 mRNA expression was found. Immunoblot revealed 29-57-kDa MMP-25 in myeloma cell lysates, but not in macrophages, and partly activated MMP-25 and -26 in diseased gingival crevicular fluid and peri-implant sulcular fluid. P. gingivalis trypsin-like proteinase degraded HBD-1 and -2. CONCLUSIONS: Both MMP-25 and -26 were expressed more strongly in extensively inflamed gingiva compared with healthy gingiva. The expression of HBD-1 was stronger than that of HBD-2 in periodontitis and peri-implantitis. De-novo expression of MMP-25 and -26 is associated with periodontal and peri-implant inflammation. Furthermore, P. gingivalis trypsin-like proteinase, but not MMP-26, can degrade HBD-1 and -2, which could lead to a weakened innate immune response.

Characteristics of collagenase-2 from gingival crevicular fluid and peri-implant sulcular fluid in periodontitis and peri-implantitis patients: pilot study.

OBJECTIVE: To compare collagenase activity and collagenolytic matrix metalloproteinase (MMP) levels in gingival crevicular fluid (GCF) and in peri-implant sulcular fluid (PISF) in gingivitis (G), chronic periodontitis (CP), and peri-implantitis (PI) human subjects. MATERIAL AND METHODS: GCF and PISF were collected on filter paper strips, volume was determined, and samples were extracted in buffer containing general proteinase but not MMP inhibitors. Collagenase activity was measured using a DNP-synthetic octapeptide, and molecular and activation forms of collagenase-2 by Western immunoblotting. RESULTS: GCF from CP and G sites exhibited elevated collagenase activity and flow, but collagenase concentrations expressed per microl were not significantly different between the healthy and G sites. Minimal fluid was obtained from healthy PISF, and collagenase concentration was the same or lower than in healthy GCF. Although PISF flow was 34% lower than GCF flow in CP subjects, collagenase concentration in CP and in PI sites was 78% and 971% greater, respectively, than in the appropriate healthy sites. Western immunoblot revealed MMP-8 in both PISF and GCF; fibroblast-type MMP-8 was not detected in healthy GCF and PISF. Immunoreactivity level and inactive and activated forms of PMN-type MMP-8 in GCF and PISF increased with the severity of periodontitis and peri-implantitis. Enhanced levels of fibroblast-type MMP-8 in active form were detected only in severe CP GCF and PI PISF. CONCLUSIONS: Peri-implantitis PISF contained higher collagenase-2 levels and activity than GCF from similar deep CP sites. GCF and PISF from severe CP and PI exhibited the highest activation of MMP-8 isoenzymes species (PMN and fibroblast-type).

Gingival crevicular fluid matrix metalloproteinase-25 and -26 levels in periodontal disease.

BACKGROUND: Tissue destruction associated with the progression of periodontal disease is caused by a cascade of host and microbial proteolytic enzymes. Host-derived matrix metalloproteinases (MMPs) play an important role in the degradation of the extracellular matrix. Leukolysin/membrane-type 6 (MT-6)/MMP-25, the latest member of the MT-MMP subgroup of the MMP family, is primarily expressed by neutrophils and involved in extracellular matrix turnover. Matrilysin-2/MMP-26 (endometase), a novel member of the matrilysin subgroup of the MMP family, can degrade the extracellular matrix, alpha1-antitrypsin, and activate pro-MMP-9. Our study aimed to examine the levels, molecular forms, and degrees of activation of MMP-25 and MMP-26 in gingival crevicular fluid (GCF) from patients with different periodontal diseases. METHODS: A total of 105 subjects, 35 with generalized aggressive periodontitis (GAgP), 29 with chronic periodontitis (CP), 20 with gingivitis, and 21 periodontally healthy subjects, were included in this study. Periodontal status was evaluated by measuring probing depth, clinical attachment loss, presence of bleeding on probing, and plaque. GCF MMP-25 and MMP-26 levels were analyzed by computer-quantitated Western immunoblotting using specific antibodies. RESULTS: The 57-kDa soluble pro-MMP-25 and 45- to 47-kDa active form of MMP-25 were detected by Western immunoblots in CP and GAgP GCF, and lesser levels of these soluble MMP-25 immunoreactive bands were detected in gingivitis GCF. An enhanced and similar degree of MMP-25 activation was found in GAgP, CP, and gingivitis groups. There were no detectable MMP-25 immunoreactivities in the healthy subjects' GCF. GAgP and CP groups had elevated GCF MMP-26 levels and degrees of activation compared to the gingivitis and healthy groups (P <0.008). The gingivitis group had higher GCF MMP-26 levels and degree of activation compared to the healthy group (P <0.008). CONCLUSIONS: The present study demonstrated the presence of soluble or shed forms of MMP-25 and MMP-26 in GCF of patients with different periodontal diseases. Increased levels and activation of MMP-25 and MMP-26 in GCF are associated with an enhanced severity of periodontal inflammation, suggesting that these novel MMPs can participate in the progression of periodontal diseases. They may prove to be diagnostically useful and could be targets of medication in the future.

Attachment of oral gram-negative anaerobic rods to a smooth titanium surface: an electron microscopy study.

PURPOSE: Attachment of bacteria to titanium may differ not only between bacterial species but also between strains within a species. The aim of the present in vitro study was to examine differences in bacterial attachment using 4 gram-negative anaerobic species of bacteria that are considered potential periodontal pathogens. MATERIALS AND METHODS: The attachment of clinical and laboratory strains (n = 23) representing 2 Fusobacterium nucleatum subspecies, Porphyromonas gingivalis, and Prevotella intermedia to smooth, commercially pure titanium was examined using scanning electron microscopy. RESULTS: All bacterial strains were attached to the smooth titanium surface by their outer membrane. F nucleatum cells were poorly attached to the titanium, unlike P gingivalis or P intermedia cells, but only slight differences were observed in the quantity of attached cells between the strains within each bacterial group. DISCUSSION: In favorable conditions, some anaerobes can attach directly to an inert titanium surface. Microbial adhesion and subsequent colonization on the dental implant surface can lead to infection of the peri-implant tissue. CONCLUSION: The results indicated that the avidity of bacterial attachment to a smooth titanium surface varies between species of oral gram-negative anaerobes but not between strains.

Gingival crevicular fluid laminin-5 gamma2-chain levels in periodontal disease.

AIM: Our study aimed to examine the molecular forms and gingival crevicular fluid (GCF) levels of laminin-5 gamma2-chain in patients with different periodontal disease, and compare the effects of P.gingivalis trypsin-like proteinase on intact laminin-5 gamma2-chain species. METHODS: Eighteen patients with generalized aggressive periodontitis (G-AgP), 29 patients with chronic periodontitis (CP), 20 with gingivitis and 20 periodontally healthy subjects were included. Probing depth, clinical attachment loss, presence of bleeding on probing and plaque were recorded. Molecular forms and GCF laminin-5 gamma2-chain levels and the effects of P. gingivalis trypsin-like proteinase on intact laminin-5 gamma2-chain were analysed by computer-quantitated Western immunoblotting. RESULTS: Laminin-5 gamma2-chain 40 and 70 kDa fragments could be detected in all groups, in varying levels. The CP group had elevated GCF laminin-5 gamma2-chain fragment levels compared with the gingivitis and healthy groups (p<0.008). The G-AgP group had GCF laminin-5 gamma2-chain fragment levels similar to the gingivitis and healthy groups (p>0.008). GCF laminin-5 gamma2-chain fragments differed clearly from the multiple lower molecular size fragments of P.gingivalis trypsin-laminin-5 gamma2-chain proteinases. CONCLUSION: Increased GCF laminin-5 gamma2-chain fragments in periodontitis sites with deep periodontal pocket suggest that these cleaved 40 and 70 kDa fragments could reflect the extent of the inflammatory reaction in CP.

Actinobacillus actinomycetemcomitans proportion of subgingival bacterial flora in relation to its clonal type.

We investigated whether certain Actinobacillus actinomycetemcomitans clones occur in elevated proportions in subgingival flora, and if the proportions relate to other bacteria in the samples. A total of 121 A. actinomycetemcomitans strains from 121 patients with periodontitis were serotyped and 60 strains were also genotyped. The 121 strains were divided into three groups and the 60 strains into two groups according proportion of A. actinomycetemcomitans. The samples from the 60 patients with genotyped strains were cultured for five other species. Among the 121 strains, serotype b occurred significantly more frequently in the high- (n = 14, proportions > 5%, mean = 18.09, SD = 20.07%) than low- (n = 49, proportions < or = 0.1%), mean = 0.04, SD = 0.03%) or intermediate-proportion groups (n = 58, proportions > 0.5%, mean = 1.31, SD = 1.24%). Genotype 3 occurred significantly more frequently in samples with low A. actinomycetemcomitans proportions (n = 28, < or = 0.1%, mean = 0.04, SD = 0.03%) than in those with high proportions (n = 32, > 0.1%, mean = 5.70, SD = 14.60%). No differences were seen in the detection frequencies or proportions of the five bacterial species between the samples with low or high A. actinomycetemcomitans proportions. The results indicate that certain clonotypes of A. actinomycetemcomitans may preferentially occur as low proportions, suggesting their controlled growth. Conversely, some serotype b clones may have a competitive advantage in subgingival flora.