Odontoblast cell death induces NLRP3 inflammasome-dependent sterile inflammation and regulates dental pulp cell migration, proliferation and differentiation.

AIM: To investigate the ability of dead odontoblasts to initiate NLRP3 inflammasome-dependent sterile inflammation and to explore the effect on dental pulp cell (DPCs) migration, proliferation and odontogenic differentiation. METHODS: Odontoblast-like cells were subjected to freezing-thawing cycles to produce odontoblast necrotic cell lysate (ONCL). DPCs were treated with ONCL to assess proliferation and migration. THP-1 differentiated macrophages stimulated with ONCL and live cell imaging and western blotting were used to assess NLRP3 inflammasome activation. Cytokines were measured with multiplex arrays and ELISA. qPCR, alkaline phosphatase and Alizarin red assays were used to assess odontogenic differentiation of DPCs. Data were analysed using the t-test or anova followed by a Bonferroni post hoc test with the level of significance set at P ≤ 0.05. RESULTS: ONCL induced migration and proliferation of DPCs. Treatment of THP-1 macrophages with ONCL resulted in the release of the inflammatory cytokines IL-1ß, IL-6, IL-8, TNFα, IFN-γ, CCL2 and angiogenic growth factors, angiogenin and angiopoietin. This inflammatory response was associated with activation of NFκB, p38MAPK and NLRP3 inflammasome. To confirm that ONCL induced inflammatory response is NLRP3 inflammasome-dependent, treatment with a caspase-1 inhibitor and a specific NLRP3 inhibitor significantly reduced IL-1ß release in THP-1 macrophages (P = 0.01 and 0.001). Inflammasome activation product, IL-1ß, induced odontogenic differentiation of DPCS as evident by the increase in odontogenic genes expression DMP-1, RUNX-2, DSPP and SPP, alkaline phosphatase activity and mineralization. CONCLUSION: Dead odontoblasts induced NLRP3 inflammasome-dependent sterile inflammation and activated the migration, proliferation and differentiation of DPCs.

A connectivity mapping approach predicted acetylsalicylic acid (aspirin) to induce osteo/odontogenic differentiation of dental pulp cells.

AIM: To use connectivity mapping, a bioinformatics approach, to identify compounds that could induce odontogenic differentiation of dental pulp cells (DPCs) and to experimentally validate this effect. A subsidiary aim was to investigate the anti-inflammatory effect of any identified compound. METHODOLOGY: The Gene Expression Omnibus (GEO) database was searched for microarray data sets assessing odontogenic differentiation of human DPCs. An odontogenic gene expression signature was generated by differential expression analysis. The statistical significant connectivity map (ssCMap) method was used to identify compounds with a highly correlating gene expression pattern. DPCs were treated with the compound identified, and osteo/odontogenic differentiation was assessed by Alizarin red staining, alkaline phosphatase activity and expression of osteo/odontogenic genes ALPL, RUNX2, COL1A1, DSPP, DMP1 and SPP1 by RT-PCR. The anti-inflammatory effect of the compound was assessed using an ex vivo pulpitis model, and cytokine levels were measured with multiplex assay. Means were compared using the t-test or ANOVA followed by a Bonferroni post hoc test with the level of significance set at P ≤ 0.05. RESULTS: The GEO database search identified a specific gene expression signature for osteo/odontogenic differentiation. Analysis using ssCMap found that acetylsalicylic acid [(ASA)/aspirin] was the drug with the strongest correlation with that gene signature. The treatment of DPCs with 0.05 mmol L-1 ASA showed increased alkaline phosphatase activity (P < 0.001), mineralization (P < 0.05), and increased the expression of the osteo/odontogenic genes, DMP1 and DSPP (P < 0.05). Low concentration (0.05 mmol L-1 ) ASA reduced inflammatory cytokines IL-6 (P < 0.001), CCL21 (P < 0.05) and MMP-9 (P < 0.05) in an ex vivo pulpitis model. CONCLUSIONS: Connectivity mapping, a web-based informatics method, was successfully used to identify aspirin as a candidate drug that could modulate the differentiation of DPCs. Aspirin was shown to induce odontogenic differentiation in DPCs in vitro and this, together with its anti-inflammatory effects, makes it a potential candidate for vital pulp therapies.

An international survey on the use of calcium silicate-based sealers in non-surgical endodontic treatment.

OBJECTIVES: To gain insight on the current clinical usage of bioceramic root canal sealers (BRCS) by general dental practitioners (GDPs) and endodontic practitioners (EPs) and to determine if BRCS clinical application is in accordance with the best available evidence. MATERIAL AND METHODS: An online questionnaire of 18 questions addressing BRCS was proposed to 2335 dentists via a web-based educational forum. Participants were asked about socio-demographic data, clinical practice with BRCS, and their motivation for using BRCS. Statistical analysis (chi-squared test or Fisher's exact test) was applied, as appropriate, to assess the association between the variable categories (p value < 0.05). RESULTS: The response rate was 28.91%. Among respondents, 94.8% knew BRCS (EPs more than GDPs, p < 0.05) and 51.70% were using BRCS. The primary reason for using BRCS was their belief of its improved properties (87.7%). Among BRCS users, single-cone technique (SCT) was the most employed obturation method (63.3%) which was more applied by GDPs (p < 0.05); EPs utilized more of the thermoplasticized obturation techniques (p < 0.05). A proportion of 38.4% of BRCS users indicated the usage of SCT with BRCS regardless of the root canal anatomy (GDPs more than EPs p < 0.05) and 55.6% considered that BRCS may influence their ability to re-establish apical patency during retreatment (GDPs more than EPs p < 0.05). CONCLUSIONS: This study highlights wide variation in the clinical use of BRCS which is not in accordance with the current literature. CLINICAL RELEVANCE: This inconsistency among EPs and GDPs on BRCS clinical application requires further clarifications to better standardize their use and improve their future evaluation.

Short-term treatment outcome of pulpotomies in primary molars using mineral trioxide aggregate and Biodentine: a randomized clinical trial.

INTRODUCTION: An ideal pulpotomy agent for primary molars has been sought for many years. Recently, new materials that allow regeneration of residual pulp tissue have been developed. In this study, we compared the preliminary clinical results obtained using Biodentine and mineral trioxide aggregate (MTA) as pulp-dressing agents in pulpotomies of primary molars. METHODS: A randomized clinical study was performed in children aged 4-9 years with at least one primary tooth with decay or caries requiring pulp treatment. A total of 90 primary molars requiring pulpotomy were randomly allocated to the MTA or Biodentine group, and 84 pulpotomies were performed. Clinical and radiographic evaluations were undertaken 6 and 12 months after treatment. All teeth were restored with a reinforced zinc oxide-eugenol base and stainless steel crowns. Statistical analysis using Fisher's exact test was performed to determine the significant differences between the groups. RESULTS: A total of four clinical failures were observed; all involved gingival inflammation. The clinical success rate in the MTA Group after 12 months was 92 % (36/39), whereas the Biodentine Group obtained 97 % (38/39) (p = 0.346). All radiographic failures were observed at the 12-month follow-up evaluation. One molar from MTA Group showed internal resorption obtaining a radiographic success rate of 97 % (38/39). Two molars from the Biodentine Group showed radiographic failure (1 internal resorption and 1 periradicular radiolucency) obtaining a radiographic success rate of 95 % (37/39). CONCLUSIONS: Biodentine showed similar clinical results as MTA with comparable success rates when used for pulpotomies of primary molars. However, longer follow-up studies are required to confirm our findings. CLINICAL RELEVANCE: This article demonstrates the effectiveness of Biodentine as a primary teeth pulpotomy material, performing similar results as MTA at 12-months evaluation.

Endoplasmic reticulum stress and mineralization inhibition mechanism by the resinous monomer HEMA.

AIM: To investigate the expression of two endoplasmic reticulum (ER)-resident key chaperone proteins, ERdj5 and BiP, under the influence of resinous monomers and its relationship with the inhibition of mineralization caused by the monomer 2-hydroxyethyl methacrylate (HEMA). METHODOLOGY: The ERdj5 and BiP expression was studied in vitro, in primary human pulp cell cultures after treatment with three different HEMA concentrations at different time periods. Subsequently, the expression of both the odontoblast markers dentine sialoprotein (DSP) and osteonectin (OSN) was studied in human pulp cells under the same conditions. RESULTS: The ERdj5 and BiP expression was upregulated in the pulp cells. DSP and OSN were largely dispersed in the cytoplasm in control cell cultures but accumulated in a perinuclear area after exposure to HEMA. Their expression levels were not affected. CONCLUSIONS: The increased expression of ERdj5 and BiP may reflect activation of ER stress. DSP and OSN accumulation into the cells may lead to their secretion arrest and inhibition of dentine matrix formation. These events may elucidate the mechanism by which HEMA inhibits the mineralization process.

Biodentine(TM) induces TGF-ß1 release from human pulp cells and early dental pulp mineralization.

AIM: To assess the ability of a recently developed tricalcium silicate-based cement (Biodentine™) to induce reparative dentine synthesis and to investigate its capacity to modulate pulp cells TGF-ß1 secretion. METHODOLOGY: Biodentine™ was directly applied onto the dental pulp in an entire human tooth culture model. After various culture periods, the interaction of the material with dental pulp tissue was analysed on tissue sections. The effect of increasing surface area of this material on TGF-ß1 secretion was investigated on pulp cell cultures and compared with that of MTA, calcium hydroxide and Xeno(®) III adhesive resin. After performing artificial injuries on pulp cell cultures, the materials eluates were added for 24 h and then TGF-ß1 secretion was quantified by ELISA. Controls were performed by incubating intact cells with the culture medium, while injured cells TGF-ß1 level was used as the baseline value. RESULTS: Biodentine™ induced mineralized foci formation early after its application. The mineralization appeared under the form of osteodentine and expressed markers of odontoblasts. Biodentine™ significantly increased TGF-ß1 secretion from pulp cells (P < 0.03) independently of the contact surface increase. This increase was also observed with calcium hydroxide and MTA, but not with the resinous Xeno(®) III. The statistical analysis showed statistically significant differences between capping materials and the resinous Xeno(®) III (P < 0.001). CONCLUSIONS: When Biodentine™ was applied directly onto the pulp, it induced an early form of reparative dentine synthesis, probably due to a modulation of pulp cell TGF-ß1 secretion.

Dentin regeneration in vitro: the pivotal role of supportive cells.

The elaboration of dentin-pulp engineering strategies requires the investigation of not only progenitor cell potentials but also their interactions with other non-progenitor "supportive" cells. Under severe caries lesions, progenitor cells may be activated by growth factors released after the acidic dissolution of carious dentin. However, dentin regeneration has also been observed after traumatic injuries without any significant dentin dissolution. This raises questions about the origin of signals involved in progenitor cell activation, migration, and differentiation. Study models such as the entire tooth culture and co-cultures of pulp and endothelial cells highlighted the role of interactions between the different pulp cell types and the pivotal role they play in dentin regeneration. Injured pulp fibroblasts secrete growth factors involved in progenitor cell activation and differentiation as well as neoangiogenesis which may pave the pathways for progenitor cell migration. This appears to be the first paper to focus on this very important field in dental pulp biology.

Effect of dual cure composite as dentin substitute on the marginal integrity of Class II open-sandwich restorations.

This study compared the marginal adaptation of Class II open-sandwich restorations with an RMGIC versus a dual-cure composite as dentin substitute. Class II cavities were prepared on 50 extracted human third molars. The teeth were randomly assigned to two groups of 25 teeth to compare one dual cure composite (Multicore Flow) with one resin-modified glass-ionomer cement (Fuji II LC) in open-sandwich restorations covered with a light cure composite. The teeth were thermomechanically cycled (2000 cycles, 5 degrees C to 55 degrees C; 100,000 cycles, 50 N/cm2). The specimens were then sealed with a 1 mm window around the cervical margin interface. Samples were immersed in a 50% w/v ammoniacal silver nitrate solution for two hours and exposed to a photodeveloping solution for six hours. The specimens were sectioned longitudinally and silver penetration was directly measured using a light microscope. The results were expressed as a score ranging from 0 to 3. The data were analyzed with a non-parametric Kruskall and Wallis test. The degree of leakage significantly increased with Multicore Flow (median = 2) compared to Fuji II LC (median = 1). The resin-modified glassionomer cements remain the best intermediate materials when open-sandwich restorations are indicated. A comparison of the degradation of these materials over time remains a topic to be investigated by future studies.

Antibacterial efficiency of passive ultrasonic versus sonic irrigation. Ultrasonic root canal irrigation.

OBJECTIVES: To compare the antibacterial effect of the passive ultrasonic to passive sonic irrigation with a 5.25% NaOCI solution. METHODS: Sixty human teeth were used. The crowns were removed and the roots were instrumented prior to sterilization and incubation with Enterococcus faecalis. Five mL of 5.25% NaOCI were used during a 3 min final rinsing with: syringe irrigation (n = 20), passive ultrasonic irrigation with the Irrisafe (n = 20) and passive sonic irrigation with the Endo Activator (n = 20). A sterile file was used to remove dentine shavings at the apex and placed in BHI. After 72 h, this infusion was used to inoculate blood agar plates. The presence or absence of Enterococcus faecalis colonies was determined and statistically analyzed. RESULTS: No statistically significant difference was found among the 3 groups. None of the irrigation regimen permitted to obtain 100% of bacteria free teeth. CONCLUSION: The passive agitation of the irrigation solution did not increase its antibacterial efficiency. Longer irrigation times may be required.

Effect of dual cure composite as dentin substitute on marginal integrity of class II open-sandwich restorations.

The current study compared the marginal adaptation of Class II open-sandwich restorations with a RMGIC versus a dual-cure composite as dentin substitute. Class II cavities were prepared on 50 extracted human third molars. The teeth were randomly assigned to two groups of 25 teeth to compare one dual cure composite (MultiCore Flow) with one resin-modified glass-ionomer cement (Fuji II LC) in open-sandwich restorations recovered with a light cure composite. The teeth were thermomechanocycled (2000 cycles, 5 degrees C to 55 degrees C; 100,000 cycles, 50 N/cm2). The specimens were then sealed with a 1 mm window around the cervical margin interface. Samples were immersed in a 50% w/v ammoniacal silver nitrate solution for two hours and exposed to a photo-developing solution for six hours. The specimens were sectioned longitudinally and silver penetration was directly measured using a light microscope. The results were expressed as a score from 0 to 3. The data were analyzed with a non-parametric Kruskal and Wallis test. The degree of leakage significantly increased with MultiCore Flow (median 2) compared to Fuji II LC (median 1). Resin-modified glassionomer cements remain the best intermediate material when open-sandwich restorations are indicated. A comparison of the degradation of these materials over time remains a topic to be investigated by future studies.

Clinical efficiency of a natural resin fluoride varnish (Shellac F) in reducing dentin hypersensitivity.

Previous in vitro permeability and scanning electron microscopic studies have demonstrated the effectiveness of a new natural based-resin varnish (Shellac F) in dentin permeability reduction and effective tubule occlusion. The aim of this randomized double-blind, controlled, split mouth 8-week clinical study was to evaluate the efficiency of Shellac F in reducing dentin hypersensitivity. Ten patients (eight women: two men) completed the study. A quadrant including at least one hypersensitive tooth (Visual Analog Scale - VAS = 15 mm to air blast) was considered as a unit and randomly assigned to different groups for Shellac F, Duraphat, Isodan. Three applications of each material were completed at days 0, 1 and 7. The subjective response was assessed by tactile and thermal/evaporative methods. Data were collected at baseline and after the first application, at 15 min, 1, 7, 14, 28 and 56 days. Analysis was based on Kruskall-Wallis test, Wilcoxon signed rank test and the method of the least square means. No statistically significant difference was noted between Shellac F and the two control materials. Regardless of the type of stimulus, Shellac F showed significant immediate and progressive continuous efficiency in reducing dentin hypersensitivity until 56 days (VAS of 14 +/- 12 mm and provoking pain force of 89 +/- 12 cN, respectively, compared with 38 +/- 23 mm and 41 +/- 10 cN at baseline), corresponding to a highly effective relief dentin hypersensitivity. Shellac F reduced dentin hypersensitivity and did not differ from the two desensitizing agents used as controls.

C5L2 Regulates DMP1 Expression during Odontoblastic Differentiation.

The presence of stem cells within the dental-pulp tissue as well as their differentiation into a new generation of functional odontoblast-like cells constitutes an important step of the dentin-pulp regeneration. Recent investigations demonstrated that the complement system activation participates in 2 critical steps of dentin-pulp regeneration: pulp progenitor's recruitment and pulp nerve sprouting. Surprisingly, its implication in odontoblastic differentiation has not been addressed yet. Since the complement receptor C5a receptor-like 2 (C5L2) is expressed by different stem cells, the aim of this study is to investigate if the dental pulp stem cells express C5L2 and if this receptor participates in odontoblastic differentiation. Immunohistochemistry performed on human third molar pulp sections showed a perivascular co-localization of the mesenchymal stem cell markers STRO1 and C5L2. In vitro immunofluorescent staining confirmed that hDPSCs express C5L2. Furthermore, we determined by real-time polymerase chain reaction that the expression of C5L2 is highly modulated in human dental pulp stem cells (hDPSCs) undergoing odontoblastic differentiation. Moreover, we showed that this odontogenesis-regulated expression of C5L2 is specifically potentiated by the proinflammatory cytokine TNFα. Using a C5L2-siRNA silencing strategy, we provide direct evidence that C5L2 constitutes a negative regulator of the dentinogenic marker DMP1 (dentin matrix protein 1) expression by hDPSCs. Our findings suggest a direct correlation between the odontoblastic differentiation and the level of C5L2 expression in hDPSCs and identify C5L2 as a negative regulator of DMP1 expression by hDPSCs during the odontoblastic differentiation and inflammation processes. This work is the first to demonstrate the involvement of C5L2 in the biological function of stem cells, provides an important knowledge in understanding odontoblastic differentiation of dental pulp stem cells, and may be useful in future dentin-pulp engineering strategies.

Quantification of angiogenic growth factors released by human dental cells after injury.

OBJECTIVE: Angiogenesis is a key step in the dental pulp healing sequence which involves the dentine bridge formation. In a previous work, we showed that dental pulp cells secrete soluble factors which interact with endothelial cells and affect the process of angiogenesis. The objective of this work was to quantify the angiogenic growth factors released by mechanically injured human dental pulp cells and the effect of 2-hydroxyethyl methacrylate (HEMA) on this secretion. DESIGN: Pulp cells were prepared from immature third molars explants by the outgrowth method. Cell monolayers were either subjected to mechanical injuries or treated with increasing concentrations of HEMA. ELISA was used to quantify the secreted angiogenic growth factors in the culture media after different time periods of injury and after incubation with different concentrations of HEMA. RESULTS: Pulp cells secreted significant levels of PDGF-AB, VEGF and FGF-2. The concentration of these factors increased shortly (5h) after injury and returned to initial values after 1 day. HEMA treatment increased VEGF secretion but decreased that of FGF-2 in a dose-dependent manner while it did not affect PDGF-AB level. CONCLUSIONS: Dental pulp cells secrete angiogenic growth factors which play a pivotal role in angiogenesis which precedes the reparative dentine formation. PDGF-AB seems to play a major role because its level showed the highest increase in mechanically injured cells. The presence of HEMA affects both FGF-2 and VEGF levels and may partially explain the lack of dentine bridging after direct pulp capping with an adhesive system.

C5L2 Receptor Represses Brain-Derived Neurotrophic Factor Secretion in Lipoteichoic Acid-Stimulated Pulp Fibroblasts.

The anaphylatoxin C5a constitutes a powerful fragment generated by complement system activation. Interestingly, this complement active fragment is also an important mediator of tissue regeneration. Recent findings suggest that C5a could be an initial signal orchestrating pulp nerve sprouting beneath carious injury, a critical step in dentin-pulp regeneration. Indeed, the expression and activation of the C5a active receptor (C5aR/CD88) by injured pulp fibroblasts controls the direction of neurite outgrowth toward carious injuries by modulating the secretion of brain-derived neurotrophic factor (BDNF) by pulp fibroblasts. A second C5a receptor, C5L2, has also been cloned but has received much less attention because its interaction with the ligand induces no signaling. This work aims to investigate the role of C5L2 in pulp nerve regeneration in the secretion of BDNF by pulp fibroblasts under sites of carious injury. Using fluorescence immunostaining on human tooth sections in vivo and on primary human pulp fibroblasts in vitro, the authors reveal that C5L2 and C5aR are co-expressed by pulp fibroblasts under lipoteichoic acid (LTA) stimulation. Moreover, silencing C5L2 significantly increases BDNF secretion by LTA-stimulated pulp fibroblasts. Finally, an analysis of the subcellular distribution of C5aR and C5L2 indicates that the negative regulation of BDNF secretion by C5L2 correlates with C5aR activation and its subsequent intracellular co-localization with C5L2. Overall, the current study sheds light on the mechanism of pulp nerve regeneration by identifying C5L2 as a negative regulator of BDNF secretion by pulp fibroblasts under carious teeth. This knowledge significantly increases the understanding of the functional mechanism linking C5aR and C5L2 in pulp nerve regeneration, which may be useful in future dentin-pulp engineering strategies that target fibroblast C5L2 to induce pulp innervation.

Pulp Vascularization during Tooth Development, Regeneration, and Therapy.

The pulp is a highly vascularized tissue situated in an inextensible environment surrounded by rigid dentin walls, with the apical foramina being the only access. The pulp vascular system is not only responsible for nutrient supply and waste removal but also contributes actively to the pulp inflammatory response and subsequent regeneration. This review discusses the underlying mechanisms of pulp vascularization during tooth development, regeneration, and therapeutic procedures, such as tissue engineering and tooth transplantation. Whereas the pulp vascular system is established by vasculogenesis during embryonic development, sprouting angiogenesis is the predominant process during regeneration and therapeutic processes. Hypoxia can be considered a common driving force. Dental pulp cells under hypoxic stress release proangiogenic factors, with vascular endothelial growth factor being one of the most potent. The benefit of exogenous vascular endothelial growth factor application in tissue engineering has been well demonstrated. Interestingly, dental pulp stem cells have an important role in pulp revascularization. Indeed, recent studies show that dental pulp stem cell secretome possesses angiogenic potential that actively contributes to the angiogenic process by guiding endothelial cells and even by differentiating themselves into the endothelial lineage. Although considerable insight has been obtained in the processes underlying pulp vascularization, many questions remain relating to the signaling pathways, timing, and influence of various stress conditions.

Role of human pulp fibroblasts in angiogenesis.

After pulp amputation, complete pulp healing requires not only reparative dentin production but also fibroblast proliferation, nerve fiber growth, and neoangiogenesis. This study was designed to investigate the role of pulp fibroblasts in angiogenesis. Human pulp fibroblasts from third molars co-cultured with human umbilical vein endothelial cells induced the organization of endothelial cells and the formation of tubular structures corresponding to capillaries in vivo. The direct contact between both cells was not necessary to induce angiogenesis, and the observed effect was due to soluble factors. This was confirmed with neutralizing antibodies against FGF-2 and VEGF, which decreased the angiogenic effects of these soluble factors. Immunohistochemistry showed that both FGF-2 and VEGF were expressed in human dental pulp fibroblasts, and this expression increased after injury. These results suggest that the pulp fibroblasts secrete angiogenic factors, which are necessary for complete pulp healing, particularly at the pulp injury site.

Pulp Fibroblasts Control Nerve Regeneration through Complement Activation.

Dentin-pulp regeneration is closely linked to the presence of nerve fibers in the pulp and to the healing mechanism by sprouting of the nerve fiber's terminal branches beneath the carious injury site. However, little is known about the initial mechanisms regulating this process in carious teeth. It has been recently demonstrated that the complement system activation, which is one of the first immune responses, contributes to tissue regeneration through the local production of anaphylatoxins such as C5a. While few pulp fibroblasts in intact teeth and in untreated fibroblast cultures express the C5a receptor (C5aR), here we show that all dental pulp fibroblasts, localized beneath the carious injury site, do express this receptor. This observation is consistent with our in vitro results, which showed expression of C5aR in lipoteichoic acid-stimulated pulp fibroblasts. The interaction of C5a, produced after complement synthesis and activation from pulp fibroblasts, with the C5aR of these cells mediated the local brain-derived neurotropic factor (BDNF) secretion. Overall, this activation guided the neuronal growth toward the lipoteichoic acid-stimulated fibroblasts. Thus, our findings highlight a new mechanism in one of the initial steps of the dentin-pulp regeneration process, linking pulp fibroblasts to the nerve sprouting through the complement system activation. This may provide a useful future therapeutic tool in targeting the fibroblasts in the dentin-pulp regeneration process.

LPS induces pulp progenitor cell recruitment via complement activation.

Complement system, a major component of the natural immunity, has been recently identified as an important mediator of the dentin-pulp regeneration process through STRO-1 pulp cell recruitment by the C5a active fragment. Moreover, it has been shown recently that under stimulation with lipoteichoic acid, a complex component of the Gram-positive bacteria cell wall, human pulp fibroblasts are able to synthesize all proteins required for complement activation. However, Gram-negative bacteria, which are also involved in tooth decay, are known as powerful activators of complement system and inflammation. Here, we investigated the role of Gram-negative bacteria-induced complement activation on the pulp progenitor cell recruitment using lipopolysaccharide (LPS), a major component of all Gram-negative bacteria. Our results show that incubating pulp fibroblasts with LPS induced membrane attack complex formation and C5a release in serum-free fibroblast cultures. The produced C5a binds to the pulp progenitor cells' membrane and induces their migration toward the LPS stimulation chamber, as revealed by the dynamic transwell migration assays. The inhibition of this migration by the C5aR-specific antagonist W54011 indicates that the pulp progenitor migration is mediated by the interaction between C5a and C5aR. Our findings demonstrate, for the first time, a direct interaction between the recruitment of progenitor pulp cells and the activation of complement system generated by pulp fibroblast stimulation with LPS.

Can Pulp Fibroblasts Kill Cariogenic Bacteria? Role of Complement Activation.

Complement system activation has been shown to be involved in inflammation and regeneration processes that can be observed within the dental pulp after moderate carious decay. Studies simulating carious injuries in vitro have shown that when human pulp fibroblasts are stimulated by lipoteichoic acid (LTA), they synthetize all complement components. Complement activation leads to the formation of the membrane attack complex (MAC), which is known for its bacterial lytic effect. This work was designed to find out whether human pulp fibroblasts can kill Streptococcus mutans and Streptococcus sanguinis via complement activation. First, histological staining of carious tooth sections showed that the presence of S. mutans correlated with an intense MAC staining. Next, to simulate bacterial infection in vitro, human pulp fibroblasts were incubated in serum-free medium with LTA. Quantification by an enzymatic assay showed a significant increase of MAC formation on bacteria grown in this LTA-conditioned medium. To determine whether the MAC produced by pulp fibroblasts was functional, bacteria sensitivity to LTA-conditioned medium was evaluated using agar well diffusion assay and succinyl dehydrogenase (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide [MTT]) assay. Both assays showed that S. mutans and S. sanguinis were sensitive to LTA-conditioned medium. Finally, to evaluate whether MAC formation on cariogenic bacteria, by pulp fibroblasts, can be directly induced by the presence of these bacteria, a specific coculture model of human pulp fibroblasts and bacteria was developed. Immunofluorescence revealed an intense MAC labeling on bacteria after direct contact with pulp fibroblasts. The observed MAC formation and its lethal effects were significantly reduced when CD59, an inhibitor of MAC formation, was added. Our findings demonstrate that the MAC produced by LTA-stimulated pulp fibroblasts is functional and can kill S. mutans and S. sanguinis. Taken together, these data clearly highlight the function of pulp fibroblasts in destroying cariogenic bacteria.