Secreted products of oral bacteria and biofilms impede mineralization of apical papilla stem cells in TLR-, species-, and culture-dependent fashion.

Regenerative endodontics exploits the mineralization potential of stem cells from the apical papilla (SCAPs) in order to promote root maturation of permanent immature teeth. SCAPs may encounter post-disinfection residual bacteria either in planktonic or in biofilm growth mode. Bacterial components bind to Toll-like receptors (TLRs) and trigger pro-inflammatory responses. We hypothesized that biofilm-triggered TLR activation affects the mineralization potential of human SCAPs. SCAPs were challenged with conditioned media derived from standardized dual-species biofilms and planktonic bacterial cultures and their inflammatory status and mineralization capacity were studied. Bacterial products from both growth modes (planktonic vs. biofilm) compromised cell viability, proliferation and mineralization capacity of SCAPs, but in a species- and growth mode-dependent fashion. While TLR4 expression remained unaffected, TLR2 expression was upregulated coinciding with a pro-inflammatory activation of SCAPs. Moreover, TLR and its downstream TGF-ß-associated kinase (TAK1) appeared to be blocking mineralization, as inhibition of these factors restored it. In conclusion, bacterial products promoted the pro-inflammatory status and inhibited mineralization of human SCAPs in a TLR-, species-, and culture-dependent fashion. TLR2 emerged as the pivotal mediator of these responses and further research is warranted towards the judicious manipulation of SCAPs in order to modify the untoward events of TLR-priming and signaling.

Host-Bacterial Interactions in Post-treatment Apical Periodontitis: A Metaproteome Analysis.

INTRODUCTION: This study evaluated the bacterial and human metaproteome of root apexes and the matched inflammatory lesions from teeth with post-treatment apical periodontitis. METHODS: Root apexes and matched inflammatory lesions from root canal-treated teeth with apical periodontitis were obtained during periradicular surgery. All root canal fillings were rated as adequate on the basis of radiographs and cone-beam computed tomography. The specimens were cryopulverized and subjected to metaproteomic analysis for human and bacterial proteins by using a mass spectrometry platform that is based on nanoflow liquid chromatography coupled with linear ion trap quadrupole Velos Orbitrap. RESULTS: The metaproteome analyses revealed the presence of viable and metabolically active human and bacterial cells in both apexes and lesions. Several bacterial proteins of interest for pathogenicity and therapeutics were identified in both apexes and lesions, including proteins related to antibiotic resistance, proteolytic function, stress response, adhesion, and virulence. Many human proteins related to immune defense mechanisms were also detected in both root apex and lesion specimens, including immunoglobulins, complement system, and proteins linked to T-cell and B-cell activation, neutrophil microbicidal processes, antigen recognition/presentation, bone resorption, and protection against tissue damage. CONCLUSIONS: Occurrence of host defense factors from the innate and adaptive immune responses and bacterial virulence, survival, and resistance proteins in matched root apexes/periradicular inflammatory lesions indicates a complex and dynamic host-pathogen interaction in teeth with post-treatment apical periodontitis.

The expression of macrophage migration inhibitory factor is correlated with receptor activator of nuclear factor kappa B ligand in induced rat periapical lesions.

INTRODUCTION: Macrophage migration inhibitory factor (MIF) has been defined as a key cytokine in regulation of innate and adaptive immunity. The purpose of this study was to investigate the immunohistochemical localization of MIF and its relationship with receptor activator of nuclear factor kappa B ligand (RANKL) protein during the development of periapical lesions in rats. METHODS: Apical periodontitis was induced in Wistar rats by occlusal pulp exposure in mandibular first molar teeth. The animals were randomly killed at 0, 7, 14, 21, 28, and 35 days after pulp exposure. The jaws that contained the first molar were obtained and were prepared for histologic analysis, enzyme histochemistry, immunohistochemistry, and double immunofluorescence staining. RESULTS: From day 0 to day 35, the areas of periapical bone loss increased and seemed to be stabilized on day 35. A few MIF-positive and RANKL-positive cells and osteoclasts could be observed on day 7, and all climaxed on day 14. From day 21 to day 35, the expression of MIF and RANKL protein decreased, and fewer osteoclasts could be observed. CONCLUSIONS: These findings showed that MIF might be associated with the differentiation of osteoclasts in the periapical lesions. MIF contributes to the pathogenesis of the periapical lesions through the induction of RANKL protein.

Cytokine analysis in lesions refractory to endodontic treatment.

INTRODUCTION: Failure in endodontic treatment is often caused by the persistence of microorganisms in the root canal after therapy. When treatment fails, an immune response develops that is characterized by an extensive network of immunologic mechanisms that lead to the production of cytokines and chemokines. METHODS: The objective of this study was to determine the relative messenger RNA (mRNA) expression of IFN-γ, TNF-α, IL-1ß, IL-17A, IL-10, and MCP-1 in periapical dental lesions refractory to treatment. Clinical samples were taken from teeth presenting periapical lesions refractory to endodontic treatment (the experimental group) or from healthy teeth with pulp vitality (the control group). Three paper points passing through the root apex (2 mm) were used to collect the samples. The total RNA was extracted from each sample, complementary DNA was synthesized, and quantitative polymerase chain reaction analysis was performed. The Mann-Whitney U test was used to determine the statistical significance of our findings (P < .05). RESULTS: Significant differences in the levels of IFN-γ, TNF-α, IL-17A, and MCP-1 mRNA expression were observed in cases refractory to endodontic treatment as compared with the control group. The expression of IL-1ß mRNA was not significantly different between the groups. The expression of IL-10 mRNA was insignificant in both the experimental and control groups. CONCLUSIONS: A significantly increased expression of TNF-α, IFN-γ, IL-17A, and MCP-1 mRNA was observed in the periapical immune response in cases of endodontic failure. These results suggest that a proinflammatory cytokine profile predominates in these types of dental lesions.

Increased gene expression of Toll-like receptors and antigen-presenting cell-related molecules in the onset of experimentally induced furcation lesions of endodontic origin in rat molars.

INTRODUCTION: Early immunopathogenic mechanisms behind pulp infection-induced furcal inflammation have not been well understood. To address the immunopathology of the pulp infection-induced furcal region of the periodontal ligament (PDL), we performed immunohistochemical and quantitative gene expression analyses for toll-like receptors (TLRs) in the furcal PDL of rat molars subjected to unsealed or sealed pulpotomy. METHODS: Furcal inflammation in rat molars was generated by making unsealed pulpotomies that were exposed to the oral environment for 24 hours. Pulpotomized teeth sealed with a temporary filling material and untreated normal teeth served as controls. Gene expression was analyzed with laser capture real-time polymerase chain reaction for TLR-2, TLR-4, and antigen presenting cell (APC)-related molecules (class II MHC, CD83, and CD86). Immunohistochemistry for TLR-2 and TLR-4 was also performed. RESULTS: Messenger RNA expression levels of TLRs and the APC-related molecules in the furcal periodontal ligament were significantly up-regulated in teeth with unsealed pulpotomy. Immunohistochemistry for unsealed pulpotomized teeth revealed that TLRs-expressing cells were predominantly distributed within the PDL beneath the furcal dentin. CONCLUSIONS: These results suggested the involvement of innate immune mechanisms involving TLRs and resulting activation of APCs in the early pathogenesis of pulp infection-induced furcal inflammation.

Suppression of T cell proliferation by root apical papilla stem cells in vitro.

The use of allogeneic stem cells strongly extends the range of stem cell applications in dentistry; however, immunological rejection remains a major concern. There is little information about the immunological features of dental-related stem cells in the literature. Therefore, we investigated the immunological characteristics of stem cells from the root apical papilla (SCAP) of swine in vitro by measuring T cell immunomodulation and apoptosis. We found that SCAP expressed a low level of swine leukocyte antigen (SLA) class I molecules and were negative for SLA class II DR molecules. Moreover, SCAP could inhibit autologous T cell proliferation stimulated by phytohemagglutinin (PHA) and a one-way mixed lymphocyte reaction in a dose-dependent manner. In addition, SCAP could suppress proliferation of allogeneic T cells in a dose-dependent manner, with or without mitomycin C pretreatment. Moreover, soluble factor(s) may be involved in the SCAP-mediated immune suppression. After a 5-day coculture of SCAP, allogeneic T cells, and PHA, only 1.22% of T cells were apoptotic. These data indicated that SCAP were weakly immunogenic and suppressed T cell proliferation in vitro through an apoptosis-independent mechanism.