Theofilos Koutouzis

ATP-dependent activation of an inflammasome in primary gingival epithelial cells infected by Porphyromonas gingivalis

Production of IL‐1β typically requires two‐separate signals. The first signal, from a pathogen‐associated molecular pattern, promotes intracellular production of immature cytokine. The second signal, derived from a danger signal such as extracellular ATP, results in assembly of an inflammasome, activation of caspase‐1 and secretion of mature cytokine. The inflammasome component, Nalp3, plays a non‐redundant role in caspase‐1 activation in response to ATP binding to P2X7 in macrophages. Gingival epithelial cells (GECs) are an important component of the innate‐immune response to periodontal bacteria. We had shown that GECs express a functional P2X7 receptor, but the ability of GECs to secrete IL‐1β during infection remained unknown. We find that GECs express a functional Nalp3 inflammasome. Treatment of GECs with LPS or infection with the periodontal pathogen, Porphyromonas gingivalis, induced expression of the il‐1β gene and intracellular accumulation of IL‐1β protein. However, IL‐1β was not secreted unless LPS‐treated or infected cells were subsequently stimulated with ATP. Conversely, caspase‐1 is activated in GECs following ATP treatment but not P. gingivalis infection. Furthermore, depletion of Nalp3 by siRNA abrogated the ability of ATP to induce IL‐1β secretion in infected cells. The Nalp3 inflammasome is therefore likely to be an important mediator of the inflammatory response in gingival epithelium.

Bacterial Colonization of the Dental Implant Fixture-Abutment Interface: An In Vitro Study

BACKGROUND The geometry of the fixture-abutment interface (FAI) might influence the risk of bacterial invasion of the internal part of the implant. The aim of this study was to use an in vitro model to assess the potential risk for invasion of oral microorganisms into the FAI microgap of dental implants with different characteristics of the connection between the fixture and abutment. METHODS Thirty implants were divided into three groups (n = 10 per group) based on their microgap dynamics. Groups 1 and 2 were comprised of fixtures with internal Morse-taper connections that connected to standard abutments and the same abutments with a 0.5-mm groove modification, respectively. Group 3 was comprised of implants with a tri-channel internal connection. Fixtures and abutments were assembled and allowed to incubate in a bacterial solution of Aggregatibacter actinomycetemcomitans (previously Actinobacillus actinomycetemcomitans) and Porphyromonas gingivalis. Two standard abutments were either exposed to bacterial culture or left sterile to serve as positive and negative controls. After disconnection of fixtures and abutments, microbial samples were taken from the threaded portion of the abutment, plated, and allowed to culture under appropriate conditions. RESULTS Three of the 10 samples in group 1 developed one colony forming unit (CFU) for A. actinomycetemcomitans, whereas zero of 10 samples developed CFUs for P. gingivalis. Ten of 10 and nine of 10 samples from groups 2 and 3, respectively, developed multiple CFUs for A. actinomycetemcomitans and P. gingivalis. CONCLUSION This study indicated that differences in implant designs may affect the potential risk for invasion of oral microorganisms into the FAI microgap.

Bacterial Colonization of the Implant–Abutment Interface Using an In Vitro Dynamic Loading Model

BACKGROUND Previously, we demonstrated that the geometry of the fixture-abutment interface influences the risk of bacterial invasion into the internal part of the implant, although the contribution of loading on this invasion was not evaluated. The aim of the present study is to use an in vitro dynamic-loading model to assess the potential risk for invasion of oral microorganisms into the fixture-abutment interface microgap of dental implants with different fixture-abutment connection characteristics. METHODS Twenty-eight implants were divided into two groups (n = 14 per group) based on their microgap dynamics. Group 1 was comprised of fixtures with internal Morse-taper connection that connected to standard abutments. Group 2 was comprised of implants with a four-groove conical internal connection that connected to multibase abutments. The specimens were immersed in a bacterial solution of Escherichia coli and loaded with 500,000 cycles of 15 N in a wear simulator. After disconnection of fixtures and abutments, microbial samples were taken from the threaded portion of the abutment, plated, and cultured under appropriate conditions. The difference between loosening and tightening torque value was also measured. RESULTS One of the 14 samples in Group 1 and 12 of the 14 of samples in Group 2 developed multiple colony forming units for E. coli. Implants in Group 1 exhibited an increase in torque value in contrast to implants in Group 2, which exhibited a decrease. CONCLUSION This study indicates that differences in implant design may affect the potential risk for invasion of oral microorganisms into the fixture-abutment interface microgap under dynamic-loading conditions.

Porphyromonas gingivalis-nucleoside-diphosphate-kinase inhibits ATP-induced reactive-oxygen-species via P2X7 receptor/NADPH-oxidase signalling and contributes to persistence

Ligation of P2X7 receptors with a ‘danger signal’, extracellular ATP (eATP), has recently been shown to result in production of intracellular reactive‐oxygen‐species (ROS) in macrophages. We show that primary gingival epithelial cells (GECs) produce sustained, robust cellular ROS upon stimulation by eATP. The induction of ROS was mediated by P2X7 receptor signalling coupled with NADPH‐oxidase activation, as determined by pharmacological inhibition and RNA interference. Furthermore, Porphyromonas gingivalis, an oral opportunistic pathogen, upregulated the antioxidant glutathione response, modulated eATP‐induced cytosolic and mitochondrial ROS generated through P2X7/NADPH‐oxidase interactome, and subsequently blocked oxidative stress in GECs via temporal secretion of a P. gingivalis effector, nucleoside‐diphosphate‐kinase (Ndk). An ndk‐deficient P. gingivalis mutant lacked the ability to inhibit ROS production and persist intracellularly following eATP stimulation. Treatment with recombinant Ndk significantly diminished eATP‐evoked ROS production. P. gingivalis infection elicited a strong, time‐dependent increase in anti‐oxidativemitochondrial UCP2 levels, whereas ndk‐deficient mutant did not cause any change. The results reveal a novel signalling cascade that is tightly coupled with eATP signalling and ROS regulation. Ndk by P. gingivalis counteracts these antimicrobial signalling activities by secreting Ndk, thus contributing to successful persistence of the pathogen.

Crestal Bone Changes Around Implants With Reduced Abutment Diameter Placed Non-Submerged and at Subcrestal Positions: A 1-Year Radiographic Evaluation

BACKGROUND There is limited information about two-part implants placed in subcrestal positions. The aim of this study is to retrospectively evaluate the 1-year outcome of implants placed non-submerged and in a subcrestal position. METHODS Records of 50 consecutive partially edentulous patients restored with dental implants were reviewed. For each implant, radiographs from the surgical appointment were compared to those from the 1-year follow-up visit and evaluated regarding: 1) the degree of subcrestal positioning of the implant, 2) changes of marginal hard tissue height over time, and 3) if the marginal hard tissue could be detected on the implant platform at the follow-up visit. RESULTS The overall survival rate from baseline to the last recorded follow-up visit was 100%. At the surgery, the implants were placed, on average, 1.37 mm (mesial) and 1.28 mm (distal) subcrestally. The mean marginal loss of hard tissues was 0.11 +/- 0.30 mm. There were no statistical significant differences regarding the loss of marginal hard tissues between mesial and distal surfaces or the maxilla versus the mandible. There was no statistical significant correlation regarding the degree of subcrestal implant position and loss of marginal mineralized hard tissue (r = 0.15; P >0.05). The mineralized hard tissue on the implant shoulder was found in 69% of the implants at the 1-year follow-up visit. CONCLUSIONS The present study reported a minimal loss of mineralized hard tissue around dental implants placed non-submerged and at subcrestal positions. In addition, these implants had hard tissue healing that extended onto the implant shoulders on most of the observed implants.

Autoreactivity of Serum Immunoglobulin to Periodontal Tissue Components: A Pilot Study

BACKGROUND Periodontal diseases are inflammatory diseases resulting in the destruction of tissues of the periodontium. Although bacteria must be present for periodontal disease to occur, a susceptible host is also required, which is determined by genetic, environmental, and acquired factors. One such factor, autoimmunity, may play a role in the tissue destruction. Data indicate that some antibodies that occur in the gingival lesion are directed to host tissue components, such as type I collagen, although investigations of other periodontal autoimmune targets are limited. METHODS Histologic sections and extracts from periodontally healthy teeth and the associated soft tissues were probed with serum from localized aggressive periodontitis (LAgP), chronic periodontitis (CP), and periodontally healthy subjects to determine autoreactivity to components of the periodontium. Any autoreactivity observed was characterized further by mass spectrometry and enzyme-linked immunosorbent assay. RESULTS Autoreactivity to components of the periodontium was observed in CP and LAgP. Known autoimmune targets, such as collagen and heat shock protein, were identified along with multiple potential autoimmune targets, including members of the extracellular matrix, such as vimentin, spectrin, filamin, actin, lamin, keratin, and tubulin. Finally, it was determined that the autoreactivity observed in LAgP was more severe and diverse than that observed in CP. CONCLUSION These data demonstrated that autoimmune reactivity can play a role in the tissue destruction of periodontal disease but that the nature of the autoreactivity may differ based on the type and/or stage of periodontal disease.

The Role of Hyperglycemia in Mechanisms of Exacerbated Inflammatory Responses within the Oral Cavity

Immune modulating factors are necessary for pathogen clearance, but also contribute to host tissues damage, as those seen in periodontal diseases. Many of these responses can be exacerbated by host conditions including type 2 diabetes [T2D], where toll-like receptor 4 [TLR4] and the receptor for advanced glycated end products [RAGE] play a significant role. Here we investigate causality associated with the increase in inflammatory markers observed in periodontally diseased patients with T2D using multi-variant correlation analysis. Inflammation associated with periodontal diseases, characterized by elevated pro-inflammatory cytokines, innate immune receptor expression, and cellular infiltrate was exacerbated in patients with T2D. In addition, a feed forward loop regulated by poor glycemic control was associated with a loss of mucosal barrier integrity and accumulation of innate immune receptor ligands resulting in an exacerbation of ongoing inflammation, where RAGE and TLR4 cooperated to induce responses in oral epithelial cells, which were exacerbated by hyperglycemia.

Crestal Bone–Level Changes Around Implants Placed in Post-Extraction Sockets Augmented With Demineralized Freeze-Dried Bone Allograft: A Retrospective Radiographic Study

BACKGROUND There is limited information regarding marginal crestal bone–level changes around implants placed in post-extraction sockets augmented with demineralized freeze-dried bone allograft (DFDBA). The aim of this study was to retrospectively compare bone-level changes around implants placed in post-extraction sockets augmented with DFDBA to implants placed in native bone. METHODS Records of 30 consecutive patients treated with dental implants placed in post-extraction sockets augmented with DFDBA and 30 consecutive patients with implants placed in native bone were reviewed. For each implant, the radiographs from the surgical appointment were compared to those from the last follow-up visit and evaluated regarding changes of marginal bone level over time. RESULTS The overall survival rate from baseline to the last follow-up visit was 100% for both groups. The mean marginal bone loss was 0.15 mm for both groups for a mean follow-up time of 12 months. There were no significant differences regarding the percentage of implants and implant surfaces demonstrating marginal bone loss. CONCLUSION The present study indicates implants placed in post-extraction sockets augmented with DFDBA exhibited minimal marginal bone loss similar to implants placed in native bone.

The effect of healing abutment reconnection and disconnection on soft and hard peri-implant tissues: a short-term randomized controlled clinical trial.

PURPOSE It has been reported that multiple abutment disconnections and reconnections following implant placement may compromise the peri-implant mucosal seal and may lead to increased marginal bone loss. Thus, the aim of this study was to evaluate the effect of healing abutment disconnection and reconnection on soft and hard peri-implant tissues. MATERIALS AND METHODS Sixteen patients were included in this prospective randomized controlled clinical trial. Following one-stage implant placement, test group implants (n = 10) received a permanent abutment and control group implants (n = 11) received a healing abutment. After 2 months of healing, control group implants underwent a prosthetic protocol involving implant-level impressions and a two-time abutment disconnection and reconnection process prior to delivery of the definitive prosthesis. Test group implants underwent a prosthetic protocol involving abutment-level impressions without any abutment disconnection. Clinical parameters were recorded at 2 weeks, 2 months, 3 months, and 6 months, and marginal bone levels were assessed radiographically at implant placement, 3 months, and 6 months. RESULTS The overall survival rate from implant placement to the last follow-up visit was 100% for both groups. The mean marginal bone loss at the 6-month examination was 0.13 mm for test group implants and 0.28 mm for control group implants. There were no significant differences regarding changes in peri-implant mucosal dimensions between the two groups. CONCLUSION The present study indicates that implants receiving a final abutment at the time of implant placement exhibited minimal marginal bone loss and were similar to implants subjected to abutment disconnection and reconnection two times. Disconnection and reconnection of the abutment two times did not cause negative dimensional changes in the peri-implant mucosa.

The Effect of Dynamic Loading on Bacterial Colonization of the Dental Implant Fixture–Abutment Interface: An In Vitro Study

Bacterial colonization of the fixture-abutment interface (FAI) microgap may contribute to increased marginal bone loss. The contribution of loading on bacterial colonization has not been thoroughly evaluated with in vitro experiments. The aim of this study was to evaluate the effect of dynamic loading on the colonization of oral microorganisms in the FAI microgap of dental implants with internal Morse-taper connection. Forty implants were divided into two groups (n = 20/group) based on subjection to dynamic loading conditions. Both Group 1 and 2 were comprised of fixtures that connected to standard abutments and allowed to incubate in a bacterial solution of Escherichia coli . The specimens of Group 2 were loaded with 500 000 cycles of 50 N using a chewing simulator. Following disconnection of fixtures and abutments, microbial samples were taken from the threaded portion of the abutment, plated and cultured under appropriate conditions. One of the 20 implants of Group 1 and 4 of the 20 implants of Group 2 had FAI microgaps colonized by E coli . With the limits of this study, it indicates that implants with internal Morse-taper connection exhibited minimal bacterial penetration down to the threaded part of the FAI and that dynamic loading increases the potential for such bacterial penetration.