Clinical cell-based versus cell-free regenerative endodontics: clarification of concept and term.

There is no consensus on the true meaning of clinical regenerative endodontics, and there is confusion over the concept and the term. Commonly used terms include revitalization and revascularization. The clinical methods for endodontic revitalization procedures and the tissue engineering concept differ depending on whether there is exogenous delivery of cells - called cell therapy, or not. Here, in this review, the difference is clarified by emphasizing the correct terminology: cell-free versus cell-based regenerative endodontic therapy (CF-RET versus CB-RET). The revitalization procedures practised clinically do not fit into the modern tissue engineering concepts of pulp regeneration but can be categorized as CF-RET. The modern tissue engineering concept in pulp regeneration is a CB-RET, which so far is at the clinical trial stage. However, histological examination of teeth following regenerative endodontic treatments reveals healing with repair derived from stem cells that originate from the periodontal, bone and other tissues. The aim of regenerative endodontics is regeneration of the pulp-dentine complex. This review discusses why CF-RET is unlikely to regenerate a pulp-dentine complex with current protocols. The American Association of Endodontists and the European Society of Endodontology have not yet recommended autologous stem cell transplantation (CB-RERT) which aspires for regeneration. Therefore, an understanding of the concept, term, difficulties and differences in current protocols is important for the clinician. However, rather than being discouraged that ideal regeneration has not been achieved to date, repair can be an acceptable outcome in clinical regenerative endodontics as it has also been accepted in medicine. Repair should also be considered in the context that resolution of the clinical signs/symptoms of pulp necrosis/apical periodontitis is generally reliably obtained in clinical regenerative endodontics.

The Tennessee study: factors affecting treatment outcome and healing time following nonsurgical root canal treatment.

AIM: To determine factors that may influence treatment outcome and healing time following root canal treatment. METHODOLOGY: Root filled and restored teeth by pre-doctoral students were included in this study. Teeth/roots were followed-up regularly, and treatment outcome was evaluated at every follow-up appointment (healed, healing, uncertain or unsatisfactory). Host (age, immune condition, pulp/periapical diagnosis, tooth/root type, location and anatomy) and treatment factors (master apical file size, apical extension, voids and density of root filling) were recorded from patient dental records. Univariate, bivariate and multivariate analyses were performed to determine the impact of the factors on treatment outcomes and healing times. RESULTS: A total of 422 roots from 291 teeth met the inclusion criteria with a mean follow-up period of 2 years. The preoperative pulp condition, procedural errors during treatment, apical extension and density of root fillings significantly affected the treatment outcome. The average time required for a periapical lesion to heal was 11.78 months. The healing time increased in patients with compromised healing, patients older than 40 years, roots with Weine type II root canal systems, root canal systems prepared to a master apical file size <35, and roots with overextended fillings (P < 0.1). CONCLUSION: Multiple host and treatment factors affected the healing time and outcome of root canal treatment. Follow-up protocols should consider these factors before concluding the treatment outcome: patient's age, immune condition, as well as roots with overextended fillings, root canal systems with smaller apical preparations (size <35) or roots with complex canal systems. Intervention may be recommended if the treatment quality was inadequate or if patients became symptomatic.

Regeneration of the dentine-pulp complex with revitalization/revascularization therapy: challenges and hopes.

The concept of regenerative endodontics has gained much attention in clinical endodontics in the past decade. One aspect of this discipline is the application of revitalization/revascularization therapies for infected and/or necrotic immature pulps in permanent teeth. Following the publication of a case report (Iwaya et al. ), investigators have been rigorously examining the types of tissues formed in the canals as well as exploring strategies to regenerate the pulp-dentine complex in revitalized teeth. This review will provide an update on the types of tissues generated in the canals after revitalization/revascularization therapy in both animal and human studies. The understanding of the role of stem cells and microenvironment in the process of wound healing resulting in either regeneration or repair will be thoroughly discussed. Stem cells and microenvironmental cues introduced into the canal during revitalization/revascularization procedures will be examined. In addition, requirement of a sterile microenvironment in the canal and vital tissue generation in revitalization/revascularization therapy will be emphasized. The challenges that we face and the hopes that we have in revitalization/revascularization therapy for regenerative endodontics will be presented.

Neovascularization by DPSC-ECs in a Tube Model for Pulp Regeneration Study.

The process of neovascularization during cell-based pulp regeneration is difficult to study. Here we developed a tube model that simulates root canal space and allows direct visualization of the vascularization process in vitro. Endothelial-like cells (ECs) derived from guiding human dental pulp stem cells (DPSCs) into expressing endothelial cell markers CD144, vWF, VEGFR1, and VEGFR2 were used. Human microvascular endothelial cells (hMVECs) were used as a positive control. DPSC-ECs formed tubules on Matrigel similar to hMVECs. Cells were mixed in fibrinogen/thrombin or mouse blood and seeded into wells of 96-well plates or injected into a tapered plastic tube (14 mm in length and 1 or 2 mm diameter of the apex opening) with the larger end sealed with MTA to simulate root canal space. Cells/gels in wells or tubes were incubated for various times in vitro and observed under the microscope for morphological changes. Samples were then fixed and processed for histological analysis to determine vessel formation. Vessel-like networks were observed in culture from 1 to 3 d after cell seeding. Cells/gels in 96-well plates were maintained up to 25 d. Histologically, both hMVECs and DPSC-ECs in 96-well plates or tubes showed intracellular vacuole formation. Some cells showed merged large vacuoles indicating the lumenization. Tubular structures were also observed resembling blood vessels. Cells appeared healthy throughout the tube except some samples (1 mm apical diameter) in the coronal third. Histological analysis also showed pulp-like soft tissue throughout the tube samples with vascular-like structures. hMVECs formed larger vascular lumen size than DPSC-ECs while the latter tended to have more lumen and tubular structure counts. We conclude that DPSC-ECs can form vascular structures and sustained in the 3-dimensional fibrin gel system in vitro. The tube model appears to be a proper and simple system simulating the root canal space for vascular formation and pulp regeneration studies.

Regeneration of dental pulp tissue in immature teeth with apical periodontitis using platelet-rich plasma and dental pulp cells.

AIM: To investigate the new tissues growing into the pulp space of immature dog teeth that were infected, disinfected and filled with blood clot (BC), dental pulp cells (DPCs), platelet-rich plasma (PRP) or a combination of DPCs and PRP in immature dog teeth with apical periodontitis. METHODOLOGY: Fifty-six immature roots from mandibular premolars of four beagles were divided into four experimental groups (n = 40) and two control groups. After the induction of apical periodontitis, the root canals of experimental groups were disinfected with NaOCl irrigation and a tri-antibiotic paste medication. The canals were then filled with different materials according to the experimental group: BC group, DPCs group, PRP group or DPCs + PRP group. Access cavities were sealed with MTA and composite. Radiographs were taken after 90 days, and the jaws including the teeth were processed for histologic analysis. The data were statistically analysed using chi-square evaluation and Student's t-test. RESULTS: Radiographic analyses demonstrated no significant difference between experimental groups in periradicular bone healing (P > 0.05), whilst those groups that used DPCs produced a significantly greater root thickening (P < 0.01). The histologic evaluation showed that the groups with PRP formed more tissues in the canals (P = 0.01). The groups with DPCs had substantially more mineralized tissue formation in the canal than those without DPCs, especially in the apical third. In DPCs + PRP group, bone-like tissue grew into the canal space from the periapical tissue. CONCLUSIONS: A combination of DPCs + PRP increased vital tissue regeneration within the root canals of immature teeth associated with apical periodontitis.

Utility of PDL progenitors for in vivo tissue regeneration: a report of 3 cases.

OBJECTIVE: Periodontal disease is an inflammatory disorder with widespread morbidities involving both oral and systemic health. The primary goal of periodontal treatment is the regeneration of the lost or diseased periodontium. In this study, we retrospectively examined feasibility and safety of reconstructing the periodontal intrabony defects with autologous periodontal ligament progenitor (PDLP) implantation in three patients. MATERIALS AND METHODS: In this retrospective pilot study, we treated 16 teeth with at least one deep intrabony defect of probing depth (PD) > OR = 6 mm with PDLP transplantation and evaluated clinical outcome measures in terms of probing depth, gingival recession and attachment gain for a duration of 32-72 months. Furthermore, we compare PDLPs with standard PDL stem cells (PDLSCs) and confirmed that PDLPs possessed progenitor characters. RESULTS: Clinical examination indicated that transplantationof PDLPs may provide therapeutic benefit for the periodontal defects. All treated patients showed no adverse effects during the entire course of follow up. We also found that PDLPs were analogous to PDLSCs in terms of high proliferation, expression of mesenchymal surface molecules, multipotent differentiation, and in vivo tissue regain. However, PDLPs failed to express scleraxis, a marker of tendon, as seen in PDLSCs. CONCLUSIONS: This study demonstrated clinical and experimental evidences supporting a potential efficacy and safety of utilizing autologous PDL cells in the treatment of human periodontitis.

Expression of Normal or Mutated X-Linked BCOR Transcripts in OFCD iPSCs.

Reprogramming diseased cells with mutated genes into induced pluripotent stem cells (iPSCs) can allow studies of disease mechanism and correct the mutation. Oculofaciocardiodental (OFCD) syndrome is a developmental disorder caused by heterozygous mutations in the X-linked BCL-6 corepressor (BCOR) gene. In this present study, we aimed to reprogram stem cells from a tooth apical papilla (SCAP) of a patient with OFCD, termed SCAP-O, into iPSCs. The SCAP-O carry a copy of the BCOR gene having 1 nucleotide deletion in 1 of the alleles, therefore harboring a mixture of cells expressing either normal (SCAP-OBCOR-WT) or mutated (SCAP-OBCOR-mut) BCOR transcripts. We subcloned SCAP-O and separated SCAP-OBCOR-WT and SCAP-OBCOR-mut as verified by sequencing. The selected subclone SCAP-OBCOR-mut expressed only the mutated BCOR transcripts and remained in such condition after multiple passages. We reprogrammed SCAP-O and subclone SCAP-OBCOR-mut into transgene-free iPSCs using an excisable lentiviral vector system (hSTEMCCA-loxP) carrying 4 reprogramming factors in a single cassette, followed by removal of transgenes via Cre-mediated excision. We found that after reprogramming SCAP-O or subclone SCAP-OBCOR-mut into iPSCs, some of the iPSC clones expressed either solely the normal BCOR-WT or BCOR-mut transcripts, while other clones expressed both BCOR-WT and BCOR-mut transcripts. This is our first step toward establishing OFCD study models by generating isogenic control BCOR-WT iPSCs versus BCOR-mut iPSCs.

Apexification: the beginning of its end.

Apexification is a procedure for treating and preserving immature permanent teeth that have lost pulp vitality. It contrasts apexogenesis in terms of its outcome in that apical maturation and normal root thickness cannot be obtained. Apexification has been a routine practice for such teeth for many decades, and despite a literature replete with discussion, including recent artificial barrier methods with mineral trioxide aggregate, ultimately there has been no major breakthrough to improve this treatment. Recently, two new clinical concepts have emerged. One involves a revitalization approach to achieve tissue generation and regeneration. In this method, new living tissue is expected to form in the cleaned canal space, allowing continued root development in terms of both length and thickness. The other is the active pursuit of pulp/dentine regeneration via tissue engineering technology to implant or re-grow pulps. Although the technology is still at its infancy, it has the potential to benefit immature pulpless teeth by allowing continued growth and maturation. With this understanding, it may be predicted that apexification will become less needed in years to come. This study will overview the recent concept of pulp revitalization in the treatment of immature teeth with nonvital pulps and the emerging research on pulp tissue engineering and regeneration.

Gingival epithelial cells increase interleukin-8 secretion in response to Actinobacillus actinomycetemcomitans challenge.

Periodontal diseases result from the interaction of bacterial pathogens with the host gingival tissues. The role of gingival epithelial cells in the initiation of host defense mechanisms after encountering oral bacteria has not been investigated. Actinobacillus actinomycetemcomitans is a key periodontal pathogen that adheres to and invades oral epithelial cells. Thus, we examined whether gingival epithelial cells increase secretion of the potent neutrophil chemoattractant interleukin-8 (IL-8) following A. actinomycetemcomitans challenge. Normal human oral keratinocytes (NHOK), isolated from gingival tissue, and 3 oral epithelial cell lines (HOK-18A, HOK-16B-BaP-T1, and HEp-2) were co-cultured with A. actinomycetemcomitans for 2 hours to allow bacteria-epithelial cell interactions. The epithelial cells were then washed, and fresh medium with gentamicin was added to kill extracellular bacteria. Cell cultures were further incubated for 24 hours before the supernatant was collected for IL-8 detection with ELISA. The results showed that IL-8 secretion increased 2- to 7-fold 24 hours after bacterial challenge. The highest IL-8 secretion was at the multiplicity of infection (MOI) of 1,000:1 in bacterial dose response studies using HOK-16B-BaP-T1 cells. Time-course studies revealed that IL-8 secretion rapidly reached a maximum level 6 hours after bacterial challenge and subsequently decreased to basal levels. These data indicate that gingival epithelial cells are capable of upregulating IL-8 expression rapidly in response to A. actinomycetemcomitans challenge and thus may facilitate the recruitment of neutrophils as a host defense mechanism.

Increased interleukin-8 expression in inflamed human dental pulps.

OBJECTIVE: Elevated levels of interleukin-8, a potent chemoattractant and activator of neutrophils, are associated with infectious and inflammatory diseases. However, little is known about interleukin-8 expression in human dental pulp. The purpose of this study was to determine whether tissue levels of interleukin-8 are elevated in irreversibly inflamed human pulps. STUDY DESIGN: Experimental samples were from teeth clinically diagnosed with irreversible pulpitis (diseased pulps). Controls were from freshly extracted, caries-free third molars (normal pulps). Samples were subjected to enzyme-linked immunosorbent assay and/or immunohistochemical analysis with specific antibodies to interleukin-8. RESULTS: The enzyme-linked immunosorbent assay studies showed elevated levels of interleukin-8 in diseased pulps (mean, 1.82+/-0.79 pg/mL/microg protein), as compared to detectable interleukin-8 levels in samples from normal pulps (mean, 0.08+/-0.04 pg/mL/microg protein; P<.05). Immunohistochemical analyses demonstrated that diseased samples exhibited a higher density of localized interleukin-8 staining in areas with heavy infiltration of inflammatory cells. In contrast, normal pulps showed negative or weak interleukin-8 staining. CONCLUSIONS: Interleukin-8 concentration was higher in pulps diagnosed with irreversible pulpitis; only negligible amounts of interleukin-8 were present in normal pulps.

Effect of interleukin-6 deficiency on the formation of periapical lesions after pulp exposure in mice.

OBJECTIVE: The multifunctional cytokine interleukin-6 (IL-6) has actions on multiple cell types, including promotion of the development of immune cells and osteoclasts. Periapical inflammation as the result of root canal infection is characterized by the accumulation of inflammatory cells and bone resorption. The effect of IL-6 on periapical lesion formation after pulpal infection is unknown. We sought to determine whether deletion of IL-6 affects periapical lesion formation after pulp exposure. METHODS: Molar pulps of homozygous IL-6 knock-out mice (IL-6(-/-), strain B6, 129-Il6tm1Koe ) and wild-type mice (IL-6(+/+)) were exposed, and the mice were killed at 1, 2, 3, 5, and 8 weeks after the exposure. Mouse jaws were decalcified and prepared for histologic examination of periapical lesions. RESULTS: The IL-6(-/-) mice developed larger periapical lesions more rapidly than did the IL-6(+/+) mice. CONCLUSIONS: IL-6 deletion promoted periapical lesion development.

Missing Concepts in De Novo Pulp Regeneration.

Regenerative endodontics has gained much attention in the past decade because it offers an alternative approach in treating endodontically involved teeth. Instead of filling the canal space with artificial materials, it attempts to fill the canal with vital tissues. The objective of regeneration is to regain the tissue and restore its function to the original state. In terms of pulp regeneration, a clinical protocol that intends to reestablish pulp/dentin tissues in the canal space has been developed--termed revitalization or revascularization. Histologic studies from animal and human teeth receiving revitalization have shown that pulp regeneration is difficult to achieve. In tissue engineering, there are 2 approaches to regeneration tissues: cell based and cell free. The former involves transplanting exogenous cells into the host, and the latter does not. Revitalization belongs to the latter approach. A number of crucial concepts have not been well discussed, noted, or understood in the field of regenerative endodontics in terms of pulp/dentin regeneration: (1) critical size defect of dentin and pulp, (2) cell lineage commitment to odontoblasts, (3) regeneration vs. repair, and (4) hurdles of cell-based pulp regeneration for clinical applications. This review article elaborates on these missing concepts and analyzes them at their cellular and molecular levels, which will in part explain why the non-cell-based revitalization procedure is difficult to establish pulp/dentin regeneration. Although the cell-based approach has been proven to regenerate pulp/dentin, such an approach will face barriers--with the key hurdle being the shortage of the current good manufacturing practice facilities, discussed herein.

Mesenchymal stem cells derived from dental tissues vs. those from other sources: their biology and role in regenerative medicine.

To date, 5 different human dental stem/progenitor cells have been isolated and characterized: dental pulp stem cells (DPSCs), stem cells from exfoliated deciduous teeth (SHED), periodontal ligament stem cells (PDLSCs), stem cells from apical papilla (SCAP), and dental follicle progenitor cells (DFPCs). These postnatal populations have mesenchymal-stem-cell-like (MSC) qualities, including the capacity for self-renewal and multilineage differentiation potential. MSCs derived from bone marrow (BMMSCs) are capable of giving rise to various lineages of cells, such as osteogenic, chondrogenic, adipogenic, myogenic, and neurogenic cells. The dental-tissue-derived stem cells are isolated from specialized tissue with potent capacities to differentiate into odontogenic cells. However, they also have the ability to give rise to other cell lineages similar to, but different in potency from, that of BMMSCs. This article will review the isolation and characterization of the properties of different dental MSC-like populations in comparison with those of other MSCs, such as BMMSCs. Important issues in stem cell biology, such as stem cell niche, homing, and immunoregulation, will also be discussed.

Localization of substance P-induced upregulated interleukin-8 expression in human dental pulp explants.

AIM: To localize ex vivo expression of interleukin-8 (IL-8) induced by substance P (SP) in human dental pulps. METHODOLOGY: Intact caries-free, freshly extracted third molars (n = 20) were collected from patients (15-25 years old). The teeth were split and pulpal tissue was obtained and stored in Dulbecco's modified Eagle medium. Human dental pulp tissue explants were stimulated with SP. Expression of IL-8 in pulp explants was detected and localized by immunohistochemistry. RESULTS: Moderated IL-8 immunoreactivities were detected mainly in the cell-rich zone in pulp tissues 12 h after tumour necrosis factor alpha (TNF-alpha) stimulation (positive controls), whereas only weak IL-8 expression was observed in tissues stimulated with SP at the same time interval. These data did not differ from those in negative controls. Increased IL-8 expression in pulp explants after 24 h of SP stimulation was noted compared with negative controls and located in fibroblast-like cells, blood vessel-associated cells and extracellular matrix in the central zone and cell-rich zone of pulp explants. Tissues stimulated with TNF-alpha for 24 h (positive controls) revealed weak IL-8 immunoreactivities with altered cell morphology. CONCLUSIONS: Substance P induces IL-8 expression and was located in fibroblast-like pulp cells, blood vessel-associated cells and extracellular matrix of human dental explants. These data support the hypothesis that neuropeptide (SP) coordinates the modulation of pulpal inflammation via up-regulating chemokine IL-8.

In vitro investigation of shear bond strength of a phosphate based dentinal bonding agent.

The objective with this study was to investigate how the shear bond strength of a phosphate based dentinal bonding agent is influenced by storage in saline at 37 degrees C up to 6 months. The effect of dentin bonding agent on the leachability of calcium from dentin was also determined. The results showed that the shear bond strength decreased after saline storage. After half a year, the strength was ca. 60% of the initial dry strength. The bonding agent also accelerated the leaching of calcium from the dentin discs, a finding which was explained as being a result of acidic reactions occurring at the dentin-bonding agent interface. In addition to the increased calcium leachability, the dentin bonding agent peeled off with time from the dentin discs. SEM investigation of the dentin surfaces after either shear strength determination or the film had peeled off due to water storage showed that a residual film was left on the dentin. This film, based on morphologic appearance, is most likely composed of the bonding agent but may also contain residuals of the smear layer.

Role of substance P and calcitonin gene-related peptide in the regulation of interleukin-8 and monocyte chemotactic protein-1 expression in human dental pulp.

AIM: To determine whether leucocyte infiltration during neurogenic inflammation in the pulp is regulated by neuropeptides via inducing the release of proinflammatory chemokines interleukin-8 (IL-8) and monocyte chemotactic protein-1 (MCP-1) from human dental pulp. METHODOLOGY: Cultured primary pulp cells and pulp tissue explants were stimulated with substance P (SP) and/or calcitonin gene-related peptide (CGRP). IL-8 or MCP-1, secreted from cultured cells or produced in pulp explants, was analysed by enzyme-linked immunosorbent assay. RESULTS: Substance P induced IL-8 secretion from cultured pulp cells (approximately threefold increase over control, P < 0.05) and from pulp tissue explants (two- to three fold). SP only minimally to moderately induced MCP-1 (approximately two fold) in cultured pulp cells. While MCP-1 induction in cultured pulp cells was detected after 24 h of SP stimulation, no induction was observed in pulp tissue. CGRP did not induce IL-8, but moderately increased MCP-1 production (approximately three fold) in cultured pulp cells. There was no synergistic induction of MCP-1 by SP plus CGRP stimulation of pulp cells. CONCLUSIONS: Substance P is a stronger inducer of IL-8 production in dental pulp than CGRP. IL-8 is more strongly induced than MCP-1 by SP, suggesting a more important role for IL-8 than MCP-1 in leucocyte infiltration during neurogenic inflammation in dental pulp.

Differential expression of interleukin-8 and intercellular adhesion molecule-1 by human gingival epithelial cells in response to Actinobacillus actinomycetemcomitans or Porphyromonas gingivalis infection.

Little is known regarding the molecules expressed by gingival epithelial cells that are involved in initiating and maintaining inflammation following the interaction with periodontal pathogens. Thus, we investigated the effect of Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis infection on the expression of neutrophil chemoattractant interleukin 8 (IL-8) and the adhesion molecule intercellular adhesion molecule-1 by gingival epithelial cells. The data revealed that both IL-8 and intercellular adhesion molecule-1 expression increased after infection with A. actinomycetemcomitans (IL-8: 2- to 7-fold; intercellular adhesion molecule-1: 2.5- to 3.7-fold). IL-8 secretion reached a maximal level 6 h after the infection and the expression subsequently decreased to basal level. The increased cell surface intercellular adhesion molecule-1 expression started at 4 h after infection and reached a maximal level 14 h after the infection. In contrast, the expression of both molecules rapidly decreased 2 h after challenge with P. gingivalis. This opposite influence of A. actinomycetemcomitans and P. gingivalis infection on the expression of IL-8 and intercellular adhesion molecule-1 by gingival epithelial cells suggests that A. actinomycetemcomitans infection may initiate the recruitment of neutrophils, whereas the P. gingivalis infection may retard this process and therefore demonstrate a distinct perspective of virulence.

Interleukin-8 and intercellular adhesion molecule 1 regulation in oral epithelial cells by selected periodontal bacteria: multiple effects of Porphyromonas gingivalis via antagonistic mechanisms.

Interaction of bacteria with mucosal surfaces can modulate the production of proinflammatory cytokines and adhesion molecules produced by epithelial cells. Previously, we showed that expression of interleukin-8 (IL-8) and intercellular adhesion molecule 1 (ICAM-1) by gingival epithelial cells increases following interaction with several putative periodontal pathogens. In contrast, expression of IL-8 and ICAM-1 is reduced after Porphyromonas gingivalis ATCC 33277 challenge. In the present study, we investigated the mechanisms that govern the regulation of these two molecules in bacterially infected gingival epithelial cells. Experimental approaches included bacterial stimulation of gingival epithelial cells by either a brief challenge (1.5 to 2 h) or a continuous coculture throughout the incubation period. The kinetics of IL-8 and ICAM-1 expression following brief challenge were such that (i) secretion of IL-8 by gingival epithelial cells reached its peak 2 h following Fusobacterium nucleatum infection whereas it rapidly decreased within 2 h after P. gingivalis infection and remained decreased up to 30 h and (ii) IL-8 and ICAM-1 mRNA levels were up-regulated rapidly 2 to 4 h postinfection and then decreased to basal levels 8 to 20 h after infection with either Actinobacillus actinomycetemcomitans, F. nucleatum, or P. gingivalis. Attenuation of IL-8 secretion was facilitated by adherent P. gingivalis strains. The IL-8 secreted from epithelial cells after F. nucleatum stimulation could be down-regulated by subsequent infection with P. gingivalis or its culture supernatant. Although these results suggested that IL-8 attenuation at the protein level might be associated with P. gingivalis proteases, the Arg- and Lys-gingipain proteases did not appear to be solely responsible for IL-8 attenuation. In addition, while P. gingivalis up-regulated IL-8 mRNA expression, this effect was overridden when the bacteria were continuously cocultured with the epithelial cells. The IL-8 mRNA levels in epithelial cells following sequential challenge with P. gingivalis and F. nucleatum and vice versa were approximately identical and were lower than those following F. nucleatum challenge alone and higher than control levels or those following P. gingivalis challenge alone. Thus, together with the protease effect, P. gingivalis possesses a powerful strategy to ensure the down-regulation of IL-8 and ICAM-1.

Expression of MHC Class II, CD70, CD80, CD86 and pro-inflammatory cytokines is differentially regulated in oral epithelial cells following bacterial challenge.

Oral epithelium may play a regulatory role in local immune responses when interacting with bacteria. The present study was undertaken to investigate the effects of selected bacterial pathogens found in periodontal and endodontic infections on oral epithelial cells. Expression of cell surface molecules (major histocompatibility complex (MHC) Class II, CD54, CD70, CD80 and CD86) and secretion of inflammatory cytokines (interleukin (IL)-1beta, IL-6, and tumor necrosis factor (TNF)-alpha) in response to selected bacterial challenge were examined on an immortalized oral epithelial cell line, HOK-18A and a skin epithelial cell line, HaCaT. Actinomyces viscosus, Actinomyces israelii, Fusobacterium nucleatum lipopolysaccharide (LPS) or primary human periradicular exudate from a granuloma were co-cultured with epithelial cells for 4 or 24 h. Subsequently, cell surface expression of MHC Class II, CD54, CD70, CD80 and CD86, along with pro-inflammatory cytokine levels were determined using flow cytometry, ELISA and RT-PCR. Results indicated that the selected oral bacteria have greater effects on oral versus skin epithelial cells. F. nucleatum increased MHC Class II and CD54 (ICAM-1) cell surface expression on HOK-18A and HaCaT cells. A. israelii also had enhancing effects on the expression of CD54 and MHC Class II. A. israelii and LPS induced a 2.8-fold (P < 0.001) and 4.4-fold (P < 0.005) TNF-alpha secretion, respectively, while F. nucleatum and LPS induced a 10-fold (P < 0.0004) and 6-fold (P < 0.01) IL-1beta secretion, respectively by HOK-18A. Interestingly, CD70, CD80, and CD86 were generally decreased upon bacteria and LPS challenge on HOK-18A. The effects of increased MHC Class II and decreased CD70 were also evident with challenge of human periradicular exudate on HOK-18A. The implications of the study are unique in that oral epithelial cells may play both activating and inhibitory roles in the host immune response towards infection by oral bacteria. We introduce a concept of 'dormancy' where the differential expression of key cell surface antigens on oral epithelial cells may keep the recruited immune effector cells in a state of unresponsiveness, thus contributing to the long term quiescent period observed in many periodontal and endodontic lesions.

Interactions between periodontal bacteria and human oral epithelial cells: Fusobacterium nucleatum adheres to and invades epithelial cells.

Bacteria are causative agents of periodontal diseases. Interactions between oral bacteria and gingival epithelial cells are essential aspects of periodontal infections. Using an in vitro tissue culture model, a selected group of gram-negative anaerobic bacteria frequently associated with periodontal diseases, including Bacteroides forsythus, Campylobacter curvus, Eikenella corrodens, Fusobacterium nucleatum, Porphyromonas gingivalis, and Prevotella intermedia, were examined for their ability to adhere to and invade primary cultures of human gingival epithelial cells (HGEC). The effects of these bacteria on the production of interleukin-8 (IL-8), a proinflammatory chemokine, were also measured. These studies provided an initial demonstration that F. nucleatum adhered to and invaded HGEC and that this was accompanied by high levels of IL-8 secretion from the epithelial cells. The attachment and invasion characteristics of F. nucleatum were also tested using KB cells, an oral epithelial cell line. The invasion was verified by transmission electron microscopy and with metabolic inhibitors. Invasion appeared to occur via a "zipping" mechanism and required the involvement of actins, microtubules, signal transduction, protein synthesis, and energy metabolism of the epithelial cell, as well as protein synthesis by F. nucleatum. A spontaneous mutant, lam, of F. nucleatum, isolated as defective in autoagglutination, was unable to attach to or invade HGEC or KB cells, further indicating the requirement of bacterial components in these processes. Sugar inhibition assays indicated that lectin-like interactions were involved in the attachment of F. nucleatum to KB cells. Investigation of these new virulence phenotypes should improve our understanding of the role of F. nucleatum in periodontal infections.