The scientific basis and development of a matrix metalloproteinase (MMP) -8 specific chair-side test for monitoring of periodontal health and disease from gingival crevicular fluid

Matrix metalloproteinase (MMP) -8, collagenase-2, is a key mediator of irreversible tissue destruction in chronic periodontitis and detectable in gingival crevicular fluid (GCF). MMP-8 mostly originates from neutrophil leukocytes, the first line of defence cells which exist abundantly in GCF, especially in inflammation. MMP-8 is capable of degrading almost all extra-cellular matrix and basement membrane components and is especially efficient against type I collagen. Thus the expression of MMP-8 in GCF could be valuable in monitoring the activity of periodontitis and possibly offers a diagnostic means to predict progression of periodontitis. In this study the value of MMP-8 detection from GCF in monitoring of periodontal health and disease was evaluated with special reference to its ability to differentiate periodontal health and different disease states of the periodontium and to recognise the progression of periodontitis, i.e. active sites. For chair-side detection of MMP-8 from the GCF or peri-implant sulcus fluid (PISF) samples, a dip-stick test based on immunochromatography involving two monoclonal antibodies was developed. The immunoassay for the detection of MMP-8 from GCF was found to be more suitable for monitoring of periodontitis than detection of GCF elastase concentration or activity. Periodontally healthy subjects and individuals suffering of gingivitis or of periodontitis could be differentiated by means of GCF MMP-8 levels and dipstick testing when the positive threshold value of the MMP-8 chair-side test was set at 1000 µg/l. MMP-8 dipstick test results from periodontally healthy and from subjects with gingivitis were mainly negative while periodontitis patients sites with deep pockets ( 5 mm) and which were bleeding on probing were most often test positive. Periodontitis patients GCF MMP-8 levels decreased with hygiene phase periodontal treatment (scaling and root planing, SRP) and even reduced during the three month maintenance phase. A decrease in GCF MMP-8 levels could be monitored with the MMP-8 test. Agreement between the test stick and the quantitative assay was very good (κ = 0.81) and the test provided a baseline sensitivity of 0.83 and specificity of 0.96. During the 12-month longitudinal maintenance phase, periodontitis patients progressing sites (sites with an increase in attachment loss ≥ 2 mm during the maintenance phase) had elevated GCF MMP-8 levels compared with stable sites. General mean MMP-8 concentrations in smokers (S) sites were lower than in non-smokers (NS) sites but in progressing S and NS sites concentrations were at an equal level. Sites with exceptionally and repeatedly elevated MMP-8 concentrations during the maintenance phase were clustered in smoking patients with poor response to SRP (refractory patients). These sites especially were identified by the MMP-8 test. Subgingival plaque samples from periodontitis patients deep periodontal pockets were examined by polymerase chain reaction (PCR) to find out if periodontal lesions may serve as a niche for Chlamydia pneumoniae. Findings were compared with the clinical periodontal parameters and GCF MMP-8 levels to determine the correlation with periodontal status. Traces of C. pneumoniae were identified from one periodontitis patient s pooled subgingival plaque sample by means of PCR. After periodontal treatment (SRP) the sample was negative for C. pneumoniae. Clinical parameters or biomarkers (MMP-8) of the patient with the positive C. pneumoniae finding did not differ from other study patients. In this study it was concluded that MMP-8 concentrations in GCF of sites from periodontally healthy individuals, subjects with gingivitis or with periodontitis are at different levels. The cut-off value of the developed MMP-8 test is at an optimal level to differentiate between these conditions and can possibly be utilised in identification of individuals at the risk of the transition of gingivitis to periodontitis. In periodontitis patients, repeatedly elevated GCF MMP-8 concentrations may indicate sites at risk of progression of periodontitis as well as patients with poor response to conventional periodontal treatment (SRP). This can be monitored by MMP-8 testing. Despite the lower mean GCF MMP-8 concentrations in smokers, a fraction of smokers sites expressed very high MMP-8 concentrations together with enhanced periodontal activity and could be identified with MMP-8 specific chair-side test. Deep periodontal lesions may be niches for non-periodontopathogenic micro-organisms with systemic effects like C. pneumoniae and possibly play a role in the transmission from one subject to another.

Experimental peri-implant mucositis at different implant surfaces

OBJECTIVES To histologically and immunologically assess experimental peri-implant mucositis at surface enhanced modified (mod) hydrophilic titanium implants. MATERIALS AND METHODS In a split-mouth design (n = 6 foxhounds), four different implants were inserted on each side of the maxilla: three titanium-zirconium alloy implants (TiZr) with either modSLA (sand-blasted, acid etched and chemically mod), modMA (machined, acid etched and chemically mod), or M (machined) surfaces in the transmucosal portion, and one titanium implant with a machined transmucosal portion (TiM). Experimental mucositis was induced at one randomly assigned side (NPC), whereas the contra-lateral maxillary side received mechanical plaque removal three times per week (PC). At 16 weeks, tissue biopsies were processed for histological (primary outcome: apical extension of the inflammatory cell infiltrate measured from the mucosal margin - PM-aICT) and immunohistochemical (CD68 antigen reactivity) analyses. Peri-implant sulcus fluid was analysed for interleukin (IL)-1β, IL-8, matrix metalloproteinase (MMP)-8 and myeloperoxidase (MPO). RESULTS Mean PM-aICT values varied between 1.86 (TiZrmodSLA) and 3.40 mm (TiM) in the UPC group, and between 0.88 (TiZrmodSLA) and 2.08 mm (TiZrM) in the PC group. Mean CD68, IL-1β, IL-8, MMP-8 and MPO values were equally distributed between mod- and control implants in both NPC and PC groups. CONCLUSIONS The progression of experimental mucositis was comparable at all implant surfaces investigated.

Remodelação alveolar e perfil bacteriano em implantes instalados em diferentes níveis relacionados à crista óssea

A substituição clínica de dentes naturais perdidos por implantes osteointegrados tem representado uma das primeiras opções terapêuticas para a reabilitação de pacientes total ou parcialmente edêntulos. Apesar dos excelentes índices de sucesso demonstrados pelas restaurações implanto-suportadas, alguns fatores permanecem não esclarecidos, principalmente no que diz respeito à remodelação óssea ao redor dos implantes osteointegrados. Desta forma, o objetivo deste estudo foi avaliar a relação do nível de instalação dos implantes dentários com os parâmetros clínicos, com a remodelação óssea peri-implantar e com a colonização bacteriana, em implantes de plataforma regular, submetidos à carga imediata. Implantes de plataforma regular foram instalados em dois diferentes níveis em relação à crista óssea ao nível ósseo e supra ósseo (1 mm). No total, trinta e cinco implantes em 9 pacientes (idade média de 62,4 11,2 anos) foram avaliados radiograficamente no momento da instalação dos implantes (T1) e 6 meses após (T2), momento no qual também foram feitas análises clínicas e coleta de amostras para o teste microbiológico. Nos exames radiográficos foram analisadas a perda óssea, a partir de mensurações lineares da distância entre um ponto fixo do componente protético e o ponto mais coronário do contato osso-implante, e a densidade óptica alveolar obtida a partir de regiões ósseas de interesse (ROIs). As análises clínicas consistiram na avaliação da profundidade de sondagem e na mensuração do volume do fluido gengival peri-implantar. O perfil bacteriano dos sítios avaliados foi caracterizado por meio do método de análise de checkerboard DNA-DNA hybridization. Os testes estatísticos realizados mostraram não haver relação entre o nível de instalação dos implantes em relação à crista óssea e a remodelação óssea alveolar, tanto com relação à perda óssea (p = 0,725), como com relação à densidade óptica alveolar (p = 0,975). Também não foi possível estabelecer uma correlação entre a remodelação óssea e parâmetros clínicos como profundidade de sondagem e volume do fluido gengival peri-implantar. Com relação ao perfil bacteriano, não foram encontradas diferenças estatisticamente significativas entre os grupos avaliados para nenhuma das 40 bactérias analisadas.

Release of Bone Markers in Immediately Loaded and Nonloaded Dental Implants: A Randomized Clinical Trial

The aim of this study was to compare the release of bone markers during osseointegration of immediately loaded and nonloaded implants. Forty patients who were indicated for rehabilitation with dental implants randomly received either implant and prosthesis placement within 72 hours (group IM) or implant insertion and no prosthesis placement (group NL). Peri-implant crevicular fluid was collected immediately after implant insertion and 7, 15, 30, 60, 90, and 120 days after surgery and levels of osteoprotegerin, transforming growth factors, osteocalcin, osteopontin, and parathyroid hormone were evaluated using Luminex assay. Bleeding index and peri-implantar sulcus depth were also evaluated. The data were compared using statistical tests ( = 5%). No statistical difference was found regarding demographic and clinical parameters (p > .05). Transforming growth factors, osteoprotegerin, osteopontin, and parathyroid hormone presented an earlier release peak in group IM than in NL group (p < .05). Osteocalcin achieved higher levels in group IM versus group NL between 7 and 30 days of evaluation (p < .05). It may be concluded that earlier loading positively modulates bone mediators release around immediately loaded implants when compared with nonloaded dental implants (ClinicalTrials.gov NCT01909999).

Interleukin-1 beta and Interleukin-6 Expression and Gene Polymorphisms in Subjects with Peri-Implant Disease

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Tenascin-C and matrix metalloproteinase-9 levels in crevicular fluid of teeth and implants

The role of and interaction between bacterial infection and biomechanical impact in the development of peri-implant inflammatory processes is not clear.

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.

Osteocyte Density in the Peri-Implant Bone of Immediately Loaded and Submerged Dental Implants

Background: The role of osteocytes in bone structure and function remains partially unresolved. Their participation in mechanotransduction, i.e., the conversion of a physical stimulus into a cellular response, has been hypothesized. The present study was an evaluation of the osteocyte density in the peri-implant bone of immediately loaded and submerged dental implants. Methods: Fourteen male patients were included in the study; all of them were partially edentulous and needed a posterior mandibular restoration. Implants were inserted in these areas; half of the sample was loaded immediately (included in a fixed provisional prosthesis on the same day as implant surgery), whereas the other half was left to heal submerged. Fourteen implants (seven immediately loaded and seven unloaded) were retrieved with a trephine after a healing period of 8 weeks. The specimens were treated to obtain thin ground sections, and histomorphometry was used to evaluate the osteocyte index in the peri-implant bone. Results: A higher and statistically significant number of osteocytes was found in the peri-implant bone around immediately loaded implants (P=0.0081). A correlation between the percentage of bone-implant contact and osteocyte density was found for immediately loaded implants (P=0.0480) but not for submerged implants (P=0.2667). Conclusion: The higher number of osteocytes in the peri-implant bone around immediately loaded implants could be related to the functional adaptation required by the loading stimulus, which also explains the hypothesized involvement of the osteocytes in the maintenance of the bone matrix. J Periodontol 2009;80:499-504.

The Peri-Implant Sulcus Compared with Internal Implant and Suprastructure Components: A Microbiological Analysis

Purpose: A recent in vivo study has shown considerable contamination of internal implant and suprastructure components with great biodiversity, indicating bacterial leakage along the implant-abutment interface, abutment-prosthesis interface, and restorative margins. The goal of the present study was to compare microbiologically the peri-implant sulcus to these internal components on implants with no clinical signs of peri-implantitis and in function for many years. Checkerboard DNA-DNA hybridization was used to identify and quantify 40 species. Material and Methods: Fifty-eight turned titanium Brånemark implants in eight systemically healthy patients (seven women, one man) under regular supportive care were examined. All implants had been placed in the maxilla and loaded with a screw-retained full-arch bridge for an average of 9.6 years. Gingival fluid samples were collected from the deepest sulcus per implant for microbiological analysis. As all fixed restorations were removed, the cotton pellet enclosed in the intra-coronal compartment and the abutment screw were retrieved and microbiologically evaluated. Results: The pellet enclosed in the suprastructure was very similar to the peri-implant sulcus in terms of bacterial detection frequencies and levels for practically all the species included in the panel. Yet, there was virtually no microbial link between these compartments. When comparing the abutment screw to the peri-implant sulcus, the majority of the species were less frequently found, and in lower numbers at the former. However, a relevant link in counts for a lot of bacteria was described between these compartments. Even though all implants in the present study showed no clinical signs of peri-implantitis, the high prevalence of numerous species associated with pathology was striking. Conclusions: Intra-coronal compartments of screw-retained fixed restorations were heavily contaminated. The restorative margin may have been the principal pathway for bacterial leakage. Contamination of abutment screws most likely occurred from the peri-implant sulcus via the implant-abutment interface and abutment-prosthesis interface.

Detection of tissue plasminogen activator (t-PA) and plasminogen activator inhibitor 2(PAI-2) in gingival crevicular fluid from healthy, gingivitis and periodontitis patients

Background: The regulation of plasminogen activation is a key element in controlling proteolytic events in the extracellular matrix. Our previous studies had demonstrated that in inflamed gingival tissues, tissue-type plasminogen activator (t-PA) is significantly increased in the extracellular matrix of the connective tissue and that interleukin 1β (IL-1β) can up regulate the level of t-PA and plasminogen activator inhibitor-2 (PAI-2) synthesis by human gingival fibroblasts. Method: In the present study, the levels of t-PA and PAI-2 in gingival crevicular fluid (GCF) were measured from healthy, gingivitis and periodontitis sites and compared before and after periodontal treatment. Crevicular fluid from106 periodontal sites in 33 patients were collected. 24 sites from 11 periodontitis patients received periodontal treatment after the first sample collection and post-treatment samples were collected 14 days after treatment. All samples were analyzed by enzyme-linked immunosorbent assay (ELISA) for t-PA and PAI-2. Results: The results showed that significantly high levels of t-PA and PAI-2 in GCF were found in the gingivitis and periodontitis sites. Periodontal treatment led to significant decreases of PAI-2, but not t-PA, after 14 days. A significant positive linear correlation was found between t-PA and PAI-2 in GCF (r=0.80, p<0.01). In the healthy group, different sites from within the same subject showed little variation of t-PA and PAI-2 in GCF. However, the gingivitis and periodontitis sites showed large variation. These results suggest a good correlation between t-PA and PAI-2 with the severity of periodontal conditions. Conclusion: This study indicates that t-PA and PAI-2 may play a significant rôle in the periodontal tissue destruction and tissue remodeling and that t-PA and PAI-2 in GCF may be used as clinical markers to evaluate the periodontal diseases and assess treatment.