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It is well recognized that clearance of bacterial infection within the dental pulp precedes pulpal regeneration. However, although the regenerative potential of the human dental pulp has been investigated extensively, its antimicrobial potential remains to be examined in detail. In the current study bactericidal assays were used to demonstrate that the secretome of dental pulp multipotent mesenchymal stromal cells (MSCs) has direct antibacterial activity against the archetypal Gram-positive and Gram-negative bacteria, Staphylococcus aureus and Escherichia coli, respectively, as well as the oral pathogens Streptococcus mutans, Lactobacillus acidophilus, and Fusobacterium nucleatum. Furthermore, a cytokine/growth factor array, enzyme-linked immunosorbent assays, and antibody blocking were used to show that cytokines and growth factors present in the dental pulp MSC secretome, including hepatocyte growth factor, angiopoietin-1, IL-6, and IL-8, contribute to this novel antibacterial activity. This study elucidated a novel and diverse antimicrobial secretome from human dental pulp MSCs, suggesting that these cells contribute to the antibacterial properties of the dental pulp. With this improved understanding of the secretome of dental pulp MSCs and its novel antibacterial activity, new evidence for the ability of the dental pulp to fight infection and restore functional competence is emerging, providing further support for the biological basis of pulpal repair and regeneration., (Copyright © 2022 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Introduction: During pulp inflammation, recruited macrophages can differentiate into 2 phenotypes: proinflammatory M1 and anti-inflammatory M2. Pulp fibroblasts have previously been shown to regulate pulp inflammation via cytokine and growth factor secretion. We hypothesized that upon carious injury, pulp fibroblasts interact with macrophages and modulate their differentiation., Methods: Cultures of pulp fibroblasts were physically injured and incubated with lipoteichoic acid (LTA) to mimic the pulp environment underlying a carious lesion. Physical injuries without LTA were performed on cultured fibroblasts to simulate the surrounding pulp tissue. Fibroblast supernatants were collected and added to undifferentiated macrophages to study their differentiation into M1 or M2 phenotypes by investigating cytokine secretion profiles and phagocytosis capacity. Histologic staining and immunofluorescence were performed on healthy and carious human tooth sections to localize the 2 macrophage phenotypes., Results: LTA-stimulated fibroblasts induced macrophage differentiation into the M1 phenotype with a significant increase both in tumor necrosis factor alpha secretion and phagocytosis capacity. By contrast, injured fibroblasts without LTA led to M2 differentiation with a significant increase in interleukin 10 secretion and low phagocytosis capacity. In carious teeth, M1 macrophages were detected mainly in the pulp zone underlying caries, whereas M2 macrophages were detected in the peripheral inflammatory zone., Conclusions: Fibroblasts induced macrophage differentiation to proinflammatory M1 with high bacteria phagocytosis capacity to control infection at the carious front. Fibroblasts located at the periphery of the inflammatory zone induced macrophage differentiation to anti-inflammatory M2. The fine balance between the 2 phenotypes may represent a prerequisite for initiating the healing process., (Copyright © 2021 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)

Objectives: To investigate the pulpal repair potential of an experimental zirconium-oxide containing tricalcium-silicate cement, referred to as 'TCS 50'., Materials and Methods: The effect of TCS 50 on viability, proliferation, migration, and odontoblastic differentiation of human dental pulp cells (HDPCs) was assessed using XTT assay, in-vitro wound healing assay and RT-PCR, respectively. Additionally, the pulp-capping potential was evaluated using a vital human tooth model. Statistical analysis was performed using non-parametric Kruskal-Wallis test and post-hoc test (Mann-Whitney U test). The tests were performed at a significance level of α = 0.05., Results: The effect of TCS 50 towards HDPCs was dose dependent. Undiluted TCS 50 extract showed no immediate adverse impact on cell viability (p > .05); however, it significantly inhibited proliferation and migration of HDPCs (p < .05). A 25% diluted TCS 50 extract showed no significant effect on cell viability, proliferation or migration (p > .05), and it significantly enhanced odontoblastic differentiation of HDPCs (p < .05). In pulps capped with TCS 50 for both 2 and 4 weeks, H&E staining revealed a normal morphology of pulp tissue; mineralized foci with cellular components entrapped in the matrix were formed underneath the exposure site. Collagen I expression was weak within the matrix of mineralized foci, while the expression of nestin was positive for entrapped cellular components within the mineralized foci, indicating that the formed mineralized foci corresponded to an initial form of reparative dentin formation., Conclusion: TCS 50 is capable of generating an early pulp-healing reaction and therefore could serve as a promising pulp-capping agent., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Upon traumatic injuries or carious lesions, the elimination of bacteria infiltrating the pulp is recognized as a prerequisite for initiating the regeneration process. Complement is a major system involved in initiating the inflammatory reaction and the subsequent bacteria elimination. This plasma system of above 35 proteins is synthesized by the liver and some immune cells. It is activated by 3 pathways: the classical, alternative, and lectin pathways that can be triggered by physical injuries, infection, and biomaterials. Recent data have shown that the pulp fibroblast represents a unique nonimmune cell type able to synthesize Complement proteins. Indeed, after physical injuries/bacteria stimulation, the pulp fibroblast has been shown to synthesize and to activate the complement system leading to the production of biologically active molecules such as C5a, C3b, and the membrane attack complex. This local secretion represents a rapid and efficient mechanism for eliminating bacteria invading the pulp, thus supporting complement activation from the plasma. Pulp fibroblast-secreted Complement proteins allow cariogenic bacteria direct lysis via membrane attack complex formation on their surface, phagocytic cell recruitment by producing C5a and cariogenic bacteria opsonization by C3b fixation on their surface, stimulating cariogenic bacteria phagocytosis. Overall, this review highlights that, in addition to initiating the inflammatory reaction, pulp fibroblasts also provide a powerful control of this inflammation via local Complement activation. The pathogen elimination capacity by fibroblast-produced complement demonstrates that this system is a strong local actor in arresting bacterial progression into the dental pulp., (Copyright © 2020 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)

Vital pulp therapy (VPT) in mature permanent teeth with carious pulp exposure has been a matter of debate, with root canal therapy being the conventional standard of care. Previously reported negative outcomes for VPT in these teeth were based on data from studies that have used calcium hydroxide in direct pulp capping and partial and full pulpotomy. The introduction of hydraulic calcium silicate-based materials with sealing and bioactive potentials have opened a new era in VPT with more favorable results. Understanding the histopathology and histobacteriology of the cariously exposed pulp and the healing potential of the inflamed pulp could guide the decision-making process toward an ultraconservative management of these teeth. However, proper case selection, strict aseptic condition, capping material, and good coronal seal are crucial for long-term success., (Copyright © 2020 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)

Introduction: The balance between periapical tissue inflammation and regeneration after the removal of necrotic/infected tissues is pivotal in determining the success of endodontic treatment. This study was designed to investigate the effect of silicate-based root canal sealer BioRoot RCS (BRCS; Septodont, Saint-Maur-des-Fossés, France) on modulating the inflammatory mechanisms and early steps of regeneration initiated by human periodontal ligament (PDL) fibroblasts., Methods: Samples of BRCS and Pulp Canal Sealer (PCS; SybronEndo, Orange, CA) were incubated in culture medium to obtain material extracts. To simulate bacterial infection and endodontic sealer use, PDL fibroblasts were stimulated with lipopolysaccharides and cultured with material extracts. The secretion of proinflammatory cytokine (interleukin 6) and growth factor (transforming growth factor beta 1) were quantified by enzyme-linked immunosorbent assay. Inflammatory cell recruitment sequence was investigated using a human inflammatory monocytic cell line (THP-1) that can be activated into macrophage-like cells. The adhesion of THP-1 to endothelial cells (human umbilical vein endothelial cells) was studied using fluorescent THP-1, their migration using Boyden chambers, and their activation into macrophage-like cells using a cell adhesion assay. The proliferation of PDL fibroblasts was quantified by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, whereas the migration of PDL stem cells was investigated using Boyden chambers after immunofluorescence and reverse transcription polymerase chain reaction characterization., Results: Interleukin 6 secretion decreased with BRCS, whereas it increased with PCS. Transforming growth factor beta 1 secretion significantly increased only with BRCS. The material extracts did not affect THP-1 adhesion to human umbilical vein endothelial cells, but only BRCS inhibited their migration. Moreover, activation of THP-1 decreased with BRCS and to a lesser extent with PCS. Finally, BRCS increased PDL fibroblast proliferation without affecting PDL stem cell migration. By contrast, PCS decreased PDL fibroblast proliferation and PDL stem cell migration., Conclusions: This work shows that the endodontic sealers modulate the PDL inflammatory and regeneration potentials in vitro. It demonstrates that BRCS has anti-inflammatory effects and the potential to promote tissue regeneration., (Copyright © 2019 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)

Objectives: This study aimed to validate the human tooth model by investigating the growth efficiency, expression of mesenchymal stem cell (MSC) markers and differentiation ability of human dental pulp cells (hDPCs) harvested from extracted immature third molars and cultured for different periods. Moreover, the effect of exposure and capping with a hydraulic calcium-silicate cement on pulp tissue after 4-week culture in the tooth model was investigated., Methods: Primary hDPCs were collected from 18 molars from six individuals (15-19 years). One tooth of each patient was immediately cultured (control), while the other teeth were exposed to culture medium for 1, 2 or 4 weeks. After different culture periods, cells were harvested using the explant method, upon which cells were evaluated for cell-doubling time, colony-forming efficiency and expression of cell surface markers. The osteogenic, adipogenic and chondrogenic differentiation efficacy was also determined. Two teeth from three different patients (n = 6) were used for the pulp-capping assay. Three teeth were capped with ProRoot MTA (Dentsply Sirona), while three other exposed teeth remained uncapped (control)., Results: Cells were found to grow, express MSC markers and showed osteogenic, adipogenic and chondrogenic differentiation potential at all time periods. Histology of the teeth subjected to the pulp-capping assay showed the formation of mineralized tissue after 4-week exposure to ProRoot MTA (Dentsply Sirona) and normal histological features in the control teeth., Conclusions: This study confirmed that hDPCs of teeth cultured for up to 4 weeks in a human tooth model are viable, express MSC markers and show differentiation ability., Clinical Significance: The human tooth model can be seen as an advanced cell-culture model that makes use of the original 3D pulp-chamber structure. It can serve as a screening tool to evaluate new pulp-capping formulations in a relatively cheap and fast manner., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Introduction: Previous works have shown that human pulp fibroblasts synthetize all complement components. Local complement activation in the dental pulp is known to be involved in inflammation and regeneration and also in pathogen destruction through membrane attack complex formation. Bacterial elimination by complement-mediated phagocytosis implies microorganism opsonization with the complement C3b protein, which is recognized by specific phagocytic cell CR1 receptors for subsequent intracellular destruction. This work was designed to find out whether pulp fibroblasts produce C3b and check its subsequent implication in bacteria phagocytosis., Methods: The expression of C3b was investigated in carious and healthy human pulp tissues. To simulate a bacterial infection in vitro, cultured human pulp fibroblasts were stimulated with lipoteichoic acid, and C3b secretion was quantified by an enzyme-linked immunosorbent assay. C3b fixation on bacteria (opsonization) and the inflammatory THP-1 cell complement receptor 1 was studied by immunofluorescence. A gentamycin protection assay was used to check the implication of C3b secretion by fibroblasts in bacteria phagocytosis., Results: Pulp cells constitutively express C3b in vivo, and cultured pulp fibroblasts produce C3b. We observed a fixation of this C3b protein on the bacterial surface (opsonization) and the THP-1 CR1 receptor. This recognition leads to a significant increase in bacteria phagocytosis., Conclusions: These results showed that pulp fibroblasts mediate the process of phagocytosis by producing the complement C3b protein and opsonizing bacteria. This highlights a significant role of fibroblasts in the dental pulp local regulation of inflammation., (Copyright © 2019 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)

Introduction: On stimulation by lipoteichoic acid or by a physical injury, fibroblasts have been shown to play a major role in the initiation of the pulp inflammatory reaction and healing through secretion of complement proteins and growth factors. The application of direct pulp-capping materials on these cells may interfere with the inflammatory and the healing processes within the pulp's inextensible environment. This work was designed to study in vitro the effects of silicate-based materials on pulp fibroblast modulation of the initial steps of pulp inflammation and healing., Methods: The effects of Biodentine, TheraCal, and Xeno III eluates were studied on lipoteichoic acid-stimulated and physically injured fibroblasts. Cytokine secretion (interleukin 6, vascular endothelial growth factor, fibroblast growth factor-2, and transforming growth factor-β1) was quantified by enzyme-linked immunosorbent assay. Inflammatory THP-1 adhesion to endothelial cells and their migration and activation were studied in vitro. Human pulp fibroblast proliferation was investigated with the MTT test, and their migration to the injury site was studied with the scratch healing assay., Results: Interleukin 6 and vascular endothelial growth factor secretion increased with all materials but to a lesser extent with Biodentine. Fibroblast growth factor-2 and transforming growth factor-β1 secretion was significantly higher with Biodentine than with all other materials. THP-1 cell adhesion to endothelial cells and their activation were reduced by Biodentine and TheraCal. However, their migration decreased only with Biodentine. Fibroblast proliferation significantly increased with Biodentine but significantly decreased with Xeno III after day 6. Finally, only Biodentine induced fibroblast migration to the injury site in the scratch assay., Conclusions: These results confirm that pulp-capping materials affect the early steps of pulp inflammation and healing. They show that Biodentine had the highest pulp healing and anti-inflammatory potential when compared with the resin-containing materials. This highlights the interest of the material choice for direct pulp-capping., (Copyright © 2018 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)

Introduction: A lack of information exists regarding the efficacy of RetroMTA (BioMTA, Seoul, Korea) directly applied on the pulp in vital pulp therapy. This study was designed to examine the clinical efficacy of RetroMTA compared with ProRoot mineral trioxide aggregate (MTA) (Dentsply Tulsa Dental, Tulsa, OK) for partial pulpotomy., Methods: Partial pulpotomy was performed in 22 healthy human maxillary and mandibular third molars planned for extraction. The teeth were randomly divided into 2 groups (n = 11) and underwent partial pulpotomy with RetroMTA and ProRoot MTA as the control. The teeth were then restored with glass ionomer cement. Clinical and electric pulp tests were performed after 1 and 8 weeks. The teeth were radiographed and extracted at 8 weeks. Histologic sections were prepared and analyzed for pulp inflammation and dentinal bridge formation. Data were analyzed using the Mann-Whitney U test., Results: Clinical examination after 1 and 8 weeks showed no sensitivity to heat, cold, or palpation in the ProRoot MTA and RetroMTA groups. Periapical radiographs taken before the extraction of teeth showed no evidence of periapical pathology. Electric pulp testing revealed no sensitivity. Data comparisons using the Mann-Whitney U test showed no significant difference between the materials with regard to the pulp inflammation type, intensity and extension (P = .3), or bridge continuity (P = .12). However, these data revealed a significant difference between the 2 materials in pulp morphology (P < .05) and bridge thickness (P < .01)., Conclusions: This is the first work to evaluate a RetroMTA histologic outcome in partial pulpotomy in human permanent teeth. It shows pulp disorganization, an absence of inflammation, and discontinuous mineralization, which may represent a potential drawback with RetroMTA in this indication., (Copyright © 2018 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)

Introduction: We recently reported that caries-associated C5a receptor (C5aR) expression and activation result in up-regulation of brain-derived neurotropic factor secretion by pulp fibroblasts inducing prominent neurite outgrowth toward the carious site. Our data further showed a negative regulation of this brain-derived neurotropic factor secretion by C5L2, another C5aR. C5L2 was considered a nonfunctional receptor and thus has received much less attention than C5aR. The aim of this study was to identify the role of C5L2 in pulp fibroblast-mediated neurite outgrowth., Methods: In this study, lipoteichoic acid (LTA) was used to mimic dental caries-like inflammation. To evaluate the role of C5L2 in pulp neurite outgrowth, human pulp fibroblasts were C5L2 small interfering RNA silenced and cocultured with human neurons in a nerve growth assay system., Results: C5L2 silencing drastically increased the neurite outgrowth toward the LTA-stimulated pulp fibroblasts. The number of neurites detected was increased in the LTA-treated pulp fibroblasts., Conclusions: Our results show that C5L2 constitutes a negative regulator of the neurite outgrowth under LTA stimulation. Of the events occurring during dentin-pulp regeneration, nerve regeneration is the key factor for maintaining tooth viability after infection or injury. Our study provides a foundation for creating therapeutic tools that target pulp fibroblasts during pulp/nerve regeneration., (Copyright © 2018 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)

Introduction: Numerous studies reported dentin bridge formation after pulp capping with tricalcium silicates. By contrast, pulp capping with resins leads to pulp toxicity and inflammation. Hybrid materials made up of tricalcium silicates and resins have also been developed to be used in direct pulp capping. This work was designed to study the consequences of adding resins to tricalcium silicates by investigating TheraCal (BISCO, Lançon De Provence, France) and Biodentine (Septodont, Saint Maur des Fosses, France) interactions with the dental pulp., Methods: Media conditioned with the biomaterials were used to analyze pulp fibroblast proliferation using the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) test and proinflammatory cytokine interleukin 8 (IL-8) secretion using the enzyme-linked immunosorbent assay. The effects of conditioned media on dentin sialoprotein (DSP) and nestin expression by dental pulp stem cells (DPSCs) were investigated by immunofluorescence. The materials' interactions with the vital pulp were investigated using the entire tooth culture model., Results: TheraCal-conditioned media significantly decreased pulp fibroblast proliferation, whereas no effect was observed with Biodentine. When DPSCs were cultured with Biodentine-conditioned media, immunofluorescence showed an increased expression of DSP and nestin. This expression was lower with TheraCal, which significantly induced proinflammatory IL-8 release both in cultured fibroblasts and entire tooth cultures. This IL-8 secretion increase was not observed with Biodentine. Entire tooth culture histology showed a higher mineralization with Biodentine, whereas significant tissue disorganization was observed with TheraCal., Conclusions: Within the limits of these preclinical results, resin-containing TheraCal cannot be recommended for direct pulp capping., (Copyright © 2017 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)

Introduction: Questions exist regarding the efficacy of resin-containing materials such as TheraCal directly applied on the pulp. This study sought to investigate the clinical efficacy of TheraCal as compared with Biodentine and ProRoot mineral trioxide aggregate (MTA) for partial pulpotomy., Methods: In this clinical trial, partial pulpotomy was performed for 27 sound human maxillary and mandibular third molars scheduled for extraction. The teeth were randomly divided into 3 groups (n = 9) and underwent partial pulpotomy with TheraCal, Biodentine, and ProRoot MTA. The teeth were then restored with glass ionomer cement. Clinical and electric pulp tests were performed after 1 and 8 weeks. The teeth were radiographed and extracted at 8 weeks. Histologic sections were prepared and analyzed for pulp inflammation and dentinal bridge formation. Data were analyzed by using one-way analysis of variance., Results: Clinical examination showed no sensitivity to heat, cold, or palpation in ProRoot MTA and Biodentine groups. Two patients in TheraCal group (20%) reported significant pain at 1 week. Periapical radiographs showed no periapical pathology, and electric pulp test revealed a normal pulp response with no hypersensitivity. Inflammation was absent with all materials at 8 weeks. Normal pulp organization was seen in 33.33% of the teeth in ProRoot MTA, 11.11% in TheraCal, and 66.67% in Biodentine group (P = .06). Biodentine group showed complete dentinal bridge formation in all teeth, whereas this rate was 11% and 56% in TheraCal and ProRoot MTA groups, respectively (P = .001)., Conclusions: Overall, Biodentine and MTA performed better than TheraCal when used as partial pulpotomy agent and presented the best clinical outcomes., (Copyright © 2017 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)

Fibroblasts represent the most abundant population within the dental pulp. Although other cell types such as odontoblasts and stem cells have been extensively investigated, very little attention was given to the fibroblasts, which have major roles in regulating the pulp biology and function under normal and pathologic conditions. Indeed, although pulp fibroblasts control the pulp vascularization and innervation under physiological conditions, these cells synthesize growth factors that enhance dentin-pulp regeneration, vascularization, and innervation. Pulp fibroblasts also represent a unique cell population because they are the only non-hepatic and non-immune cell type capable of synthesizing all complement proteins leading to production of biologically active fragments such as C3a, C5a, and membrane attack complex, which play major roles in the pulp regeneration processes. C3a fragment is involved in inducing the proliferation of both stem cells and pulp fibroblasts. It is also involved in stem cell mobilization and pulp fibroblast recruitment. C5a guides nerve sprouting and stem cell recruitment. The membrane attack complex fixes on cariogenic bacteria walls, leading to their direct destruction. These data demonstrate the central role played by pulp fibroblasts in regulating the dentin-pulp tissue by directly destroying cariogenic bacteria and by releasing bioactive fragments involved in nerve sprouting and stem cell recruitment and pulp regeneration. Taken together, this shows that targeting pulp fibroblasts represents a realistic strategy to induce complete dentin-pulp regeneration., (Copyright © 2017 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)

Introduction: The role of complement, especially through the C5a fragment, is well-known for the initiation of inflammation. Its involvement in regeneration has been shown more recently by the recruitment of mesenchymal stem cells. C5a can be produced locally by the pulp fibroblasts in response to injury or infection. This work aims to investigate the effect of different pulp capping biomaterials on complement activation and its possible influence on inflammatory and pulp stem cell recruitment., Methods: Conditioned media were prepared from 3 pulp capping biomaterials: Biodentine (Septodont, Saint-Maur-des-Fosses, France), TheraCal (BISCO, Lançon De Provence, France), and Xeno III (Dentsply Sirona, Versaille, France). Injured pulp fibroblasts were cultured with these conditioned media to analyze C5a secretion using an enzyme-linked immunosorbent assay. Dental pulp stem cells (DPSCs) were isolated from human third molar explants by magnetic cell sorting with STRO-1 antibodies. The expression of C5a receptor on DPSCs and inflammatory (THP-1) cells was investigated by immunofluorescence. The migration of both DPSCs and THP-1 cells was studied in Boyden chambers., Results: Pulp fibroblast production of C5a significantly increased when the cells were incubated with TheraCal- and Xeno III-conditioned media. The recruitment of cells involved in inflammation (THP-1 cells) was significantly reduced by Biodentine- and TheraCal-conditioned media, whereas the migration of DPSCs was reduced with TheraCal- and Xeno III-conditioned media but not with that of Biodentine. The involvement of C5a in cell recruitment is demonstrated with a C5a receptor-specific antagonist (W54011)., Conclusions: After pulp injury, the pulp capping material affects complement activation and the balance between inflammation and regeneration through a differential recruitment of DPSCs or inflammatory cells., (Copyright © 2017 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)

Introduction: Complement activation is considered a major mechanism in innate immunity. Although it is mainly involved in initiating inflammation, recent data reported its involvement in other processes such as tissue regeneration. In the dental pulp, complement C5a fragment has been shown to be involved in the recruitment of dental pulp stem cells (DPSCs). This study sought to investigate the possible role of C3a, another complement fragment, in the early steps of dentin-pulp regeneration., Methods: Expression of C3a receptor (C3aR) was investigated by immunofluorescence and reverse transcriptase polymerase chain reaction on cultured pulp fibroblasts, STRO-1-sorted DPSCs, as well as on human tooth sections in vivo. The effect of C3a on proliferation of both DPSCs and pulp fibroblasts was investigated by MTT assay. Cell migration under a C3a gradient was investigated by using microfluidic chemotaxis chambers., Results: C3aR was expressed in vivo as well as in cultured pulp fibroblasts co-expressing fibroblast surface protein and in DPSCs co-expressing STRO-1. Addition of recombinant C3a induced a significant proliferation of both cell types. When subjected to a C3a gradient, DPSCs were mobilized but not specifically recruited, whereas pulp fibroblasts were specifically recruited following a C3a gradient., Conclusions: These results provide the first demonstration of C3aR expression in the dental pulp and demonstrate that C3a is involved in increasing DPSCs and fibroblast proliferation, in mobilizing DPSCs, and in specifically guiding fibroblast recruitment. This provides an additional link to the tight correlation between inflammation and tissue regeneration., (Copyright © 2016. Published by Elsevier Inc.)

Introduction: The transient receptor potential (TRP) ion channels have emerged as important cellular sensors in both neuronal and non-neuronal cells, with TRPA1 playing a central role in nociception and neurogenic inflammation. The functionality of TRP channels has been shown to be modulated by inflammatory cytokines. The aim of this study was to investigate the effect of inflammation on odontoblast TRPA1 expression and to determine the effect of Biodentine (Septodent, Paris, France) on inflammatory-induced TRPA1 expression., Methods: Immunohistochemistry was used to study TRPA1 expression in pulp tissue from healthy and carious human teeth. Pulp cells were differentiated to odontoblastlike cells in the presence of 2 mmol/L beta-glycerophosphate, and these cells were used in quantitative polymerase chain reaction, Western blotting, calcium imaging, and patch clamp studies., Results: Immunofluorescent staining revealed TRPA1 expression in odontoblast cell bodies and odontoblast processes, which was more intense in carious versus healthy teeth. TRPA1 gene expression was induced in cultured odontoblastlike cells by tumor necrosis factor alpha, and this expression was significantly reduced in the presence of Biodentine. The functionality of the TRPA1 channel was shown by calcium microfluorimetry and patch clamp recording, and our results showed a significant reduction in tumor necrosis factor alpha-induced TRPA1 responses after Biodentine treatment., Conclusions: In conclusion, this study showed TRPA1 to be modulated by caries-induced inflammation and that Biodentine reduced TRPA1 expression and functional responses., (Copyright © 2016 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)

The transient receptor potential (TRP) channels are unique cellular sensors that are widely expressed in many neuronal and nonneuronal cells. Among the TRP family members, TRPA1 and TRPV4 are emerging as candidate mechanosensitive channels that play a pivotal role in inflammatory pain and mechanical hyperalgesia. Odontoblasts are nonneuronal cells that possess many of the features of mechanosensitive cells and mediate important defense and sensory functions. However, the effect of inflammation on the activity of the odontoblast's mechanosensitive channels remains unknown. By using immunohistochemistry and calcium microfluorimetry, we showed that odontoblast-like cells express TRPA1 and TRPV4 and that these channels were activated by hypotonicity-induced membrane stretch. Short treatment of odontoblast-like cells with tumor necrosis factor (TNF)-α enhanced TRPA1 and TRPV4 responses to their chemical agonists and membrane stretch. This enhanced channel activity was accompanied by phospho-p38 mitogen-activated protein kinase (MAPK) expression. Treatment of cells with the p38 inhibitor SB202190 reduced TNF-α effects, suggesting modulation of channel activity via p38 MAPK. In addition, TNF-α treatment also resulted in an up-regulation of TRPA1 expression but down-regulation of TRPV4. Unlike TRPV4, enhanced TRPA1 expression was also evident in dental pulp of carious compared with noncarious teeth. SB202190 treatment significantly reduced TNF-α-induced TRPA1 expression, suggesting a role for p38 MAPK signaling in modulating both the transcriptional and non-transcriptional regulation of TRP channels in odontoblasts., (Copyright © 2015 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Introduction: Tricalcium silicate-based materials are recognized as bioactive materials through their capacity to induce hard tissue formation both in the dental pulp and bone. Sealing the apex implies that the root canal filling materials interact with the periapical tissues. This work was designed to study the interactions of newly developed tricalcium silicate cement (BioRoot RCS; Septodont, Saint Maur Des Fosses, France) with apical tissue compared with a standard zinc oxide-eugenol sealer (Pulp Canal Sealer [PCS]; SybronEndo, Orange, CA)., Methods: Cell viability was assessed by direct contact between human periodontal ligament (PDL) cells and BioRoot RCS or PCS. In addition, an in vitro tooth model was used to study the interactions between these materials and PDL cells. For this purpose, human extracted incisors were sectioned at the enamel-cementum junction; root canals were prepared, sterilized, and filled with lateral condensation with both materials. The root apices were dipped in the culture medium for 24 hours. These conditioned media were used to investigate their effects on human PDL cells. Cell proliferation was investigated with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, and the secretion of angiogenic and osteogenic growth factors was quantified using an enzyme-linked immunosorbent assay., Results: BioRoot RCS has less toxic effects on PDL cells than PCS and induced a higher secretion of angiogenic and osteogenic growth factors than PCS., Conclusions: Taken together, these preclinical results suggest that the calcium silicate cement (BioRoot RCS) has a higher bioactivity than the zinc oxide-eugenol sealer (PCS) on human PDL cells., (Copyright © 2015 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)

Introduction: The calcium-releasing ability of pulp-capping materials induces pulp tissue regeneration. Tricalcium silicate-based materials produce calcium hydroxide as a by-product of hydration. Assessment of hydration and calcium ion leaching is usually performed on samples that have been aged in physiological solution for a predetermined period of time. The hydration and activity of the materials in vivo may not be similar to those displayed in vitro because of insufficient fluid available in contact with dentin. The aim of this research was the assessment of hydration of Biodentine, Theracal LC, and a prototype radiopacified tricalcium silicate-based material after pulp capping and to compare it with direct hydration in an aqueous solution., Methods: The extent of hydration of Biodentine, Theracal LC, and a prototype radiopacified tricalcium silicate-based material with a similar composition to Biodentine but not incorporating the additives was assessed by scanning electron microscopy and energy dispersive spectroscopy of polished specimens after being allowed to hydrate in Hank's balanced salt solution for 14 days. The extent of hydration was compared with material hydration when used as direct pulp capping materials by using a tooth culture model. Material activity was also assessed by x-ray diffraction analysis to investigate the deposition of calcium hydroxide by the materials, and calcium ion leaching in Hank's balanced salt solution was assessed by ion chromatography., Results: Biodentine and the prototype tricalcium silicate cement hydrated and reaction by-products were deposited in the cement matrix both after pulp capping and when incubated in an aqueous solution. Calcium hydroxide was formed, and calcium ions were leached in solution. Theracal LC hydration was incomplete because of the limited moisture diffusion within the material. Thus, no calcium hydroxide was produced, and a lower calcium ion leaching was recorded., Conclusions: Theracal LC had a heterogeneous structure with large unhydrated particles because not enough moisture was present to allow hydration to proceed. Biodentine composition was shown to be optimized, and the environmental conditions did not affect material microstructure. Biodentine exhibited formation of calcium hydroxide and calcium ion leaching, which are beneficial to the dental pulp., (Copyright © 2014 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)

The complement system is an efficient plasma immune surveillance system that controls tissue injury and infection. Although the liver constitutes the primary circulating complement protein synthesis site, extrahepatic synthesis is known to optimize local tissue inflammatory reaction. Because dentin-pulp regeneration is known to be regulated locally, we investigated activation of the local complement system within the dental pulp and its role in initiating the regeneration process. Membrane attack complex (C5b-9) formation and Gram's staining revealed that complement activation is correlated with the presence of Gram-positive bacteria in carious human teeth. RT-PCR analysis demonstrated that cultured human pulp fibroblasts stimulated with lipoteichoic acid produce all the proteins required for efficient complement activation. This was demonstrated in vitro by C5b-9 formation and C5a active fragment production in the absence of plasma proteins. Finally, the dynamic migration assays performed in μ-Slide chemotaxis chambers and use of a C5aR-specific antagonist (W54011) demonstrated that the activation of complement proteins synthesized by pulp fibroblasts and the subsequent release of C5a specifically induced pulp progenitor cell recruitment. Our study reveals human pulp fibroblasts as the first nonimmune cell type capable of synthesizing all complement proteins. These fibroblasts cells contribute significantly to tissue regeneration by recruiting pulp progenitors via complement activation, which suggests to a potential therapeutic strategy of targeting pulp fibroblasts in dentin-pulp regeneration., (Copyright © 2014 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Many aspects of dentin pulp tissue regeneration have been investigated, and it has been shown that dentin pulp has a high regeneration capacity. This seems to be because of the presence of progenitor cells and inductive regeneration signals from different origins. These signals can be liberated after the acidic dissolution of carious dentin as well as from pulp fibroblasts and endothelial cells in cases of traumatic injury. Thus, both carious lesions and pulp cells provide the required mediators for complete dentin-pulp regeneration including reparative dentin secretion, angiogenesis, and innervation. Additionally, all dentin pulp insults including carious "infection," traumatic injuries, application of restorative materials on the injured dentin pulp, and subsequent apoptosis are known activators of the complement system. This activation leads to the production of several biologically active fragments responsible for the vascular modifications and the attraction of immune cells to the inflammatory/injury site. Among these, C5a is involved in the recruitment of pulp progenitor cells, which express the C5a receptor. Thus, in addition to dentin and pulp cells, plasma should be considered as an additional source of regeneration signals., (Copyright © 2014 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)

Introduction: Little information is yet available on the signals involved in progenitor cell migration that precede reparative dentin synthesis. Our aim was to investigate the effect of the controlled release of fibroblast growth factor (FGF)-2 and transforming growth factor β1 (TGF-β1) on permanent teeth pulp cell proliferation and progenitor cell migration., Methods: FGF-2 and TGF-β1 were encapsulated into a biodegradable polymer matrix of lactide and glycolide. Human pulp cells were prepared from third molars, and progenitor cells were sorted by STRO-1. The synthesized microsphere toxicity was checked with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide test. The growth factor release kinetics were checked by an enzyme-linked immunosorbent assay while maintaining their biological activity and were evaluated by investigating their effects on pulp cell proliferation. Their chemotactic potential was investigated on STRO-1-sorted cells in a migration chamber on Matrigel (Cambrex Bio Science, Walkersville, MD)., Results: The cell viability was unaffected by the presence of microspheres. The released amount of FGF-2 and TGF-β1 from the microspheres was maintained after 21 days. Increasing the FGF-2-loaded microsphere concentration or the release period significantly increased dental pulp cell proliferation. TGF-β1 acted as a potent chemotactic factor of STRO-1-sorted cells., Conclusions: Encapsulating TGF-β1 and FGF-2 in a biodegradable polymer of lactide and glycolide microsphere allowed a sustained release of growth factors and provided a protection to their biological activities. Our results clearly show the usefulness of growth factor controlled release in investigating the early events of pulp/dentin regeneration. It provides additional data on the signals required for vital pulp therapy and future tissue engineering., (Copyright © 2013 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)

Introduction: The purposes of the present study were to (1) assess the effect of the addition of benzalkonium chloride to sodium hypochlorite on its wetting properties, contact angle, and surface energy; (2) determine the critical micellar concentration of benzalkonium chloride in sodium hypochlorite; and (3) investigate the influence of addition of benzalkonium chloride on the free chlorine level, cytotoxicity, and antiseptic properties of the mixture., Methods: Solutions of benzalkonium chloride, with concentrations ranging from 0%-1%, were mixed in 2.4% sodium hypochlorite and tested as follows. The wetting properties were investigated by measuring the contact angle of the solutions on a nondehydrated dentin surface by using the static sessile drop method. The pending drop technique was subsequently used to determine the surface energy of the solutions. The critical micellar concentration of benzalkonium chloride mixed in sodium hypochlorite was calculated from the data. When 2.4% NaOCl was mixed with benzalkonium chloride at the critical micellar concentration, 3 parameters were tested: free chloride content, cytotoxicity, and antibacterial effects against Enterococcus faecalis., Results: The contact angle (P < .001) as well as the surface energy (P < .001) significantly decreased with increasing benzalkonium chloride concentrations. The critical micellar concentration of benzalkonium chloride in sodium hypochlorite was 0.008%. At this concentration, the addition of benzalkonium chloride had no effect on the free chlorine content, cytotoxicity, or antibacterial efficiency of the mixture., Conclusions: The addition of benzalkonium chloride to sodium hypochlorite at the critical micellar concentration reduced the contact angle by 51.2% and the surface energy by 53.4%, without affecting the free chloride content, cytotoxicity, or antibacterial properties of the mixture., (Copyright © 2012 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)

Introduction: Transient receptor potential (TRP) channels comprise a group of nonselective calcium-permeable cationic channels, which are polymodal sensors of environmental stimuli such as thermal changes and chemicals. TRPM8 and TRPA1 are cold-sensing TRP channels activated by moderate cooling and noxious cold temperatures, respectively. Both receptors have been identified in trigeminal ganglion neurones, and their expression in nonneuronal cells is now the focus of much interest. The aim of this study was to investigate the molecular and functional expression of TRPA1 and TRPM8 in dental pulp fibroblasts., Methods: Human dental pulp fibroblasts were derived from healthy molar teeth. Gene and protein expression was determined by polymerase chain reaction and Western blotting. Cellular localization was investigated by immunohistochemistry, and TRP functionality was determined by Ca(2+) microfluorimetry., Results: Polymerase chain reaction and Western blotting showed gene and protein expression of both TRPA1 and TRPM8 in fibroblast cells in culture. Immunohistochemistry studies showed that TRPA1 and TRPM8 immunoreactivity co-localized with the human fibroblast surface protein. In Ca(2+) microfluorimetry studies designed to determine the functionality of TRPA1 and TRPM8 in pulp fibroblasts, we showed increased intracellular calcium ([Ca(2+)](i)) in response to the TRPM8 agonist menthol, the TRPA1 agonist cinnamaldehyde, and to cool and noxious cold stimuli, respectively. The responses to agonists and thermal stimuli were blocked in the presence of specific TRPA1 and TRPM8 antagonists., Conclusions: Human dental pulp fibroblasts express TRPA1 and TRPM8 at the molecular, protein, and functional levels, indicating a possible role for fibroblasts in mediating cold responses in human teeth., (Crown Copyright © 2011. Published by Elsevier Inc. All rights reserved.)

Objectives: The objective of this study was to evaluate the influence of neutralizing a 2.5% NaOCl solution on its cytotoxicity, genotoxicity, and tissue-dissolving potential., Study Design: The cytotoxicity and the genotoxicity of Dakin, a 2.5% NaOCl solution, and a neutralized 2.5% NaOCl solution were assessed according to ISO 10993 standards. The weight of palatal mucosa samples placed in neutralized 2.5% NaOCl, 2.5% NaOCl was recorded over time as well as the pH of the solutions., Results: The neutralized 2.5% NaOCl solution was 10-fold more cytotoxic than the 2.5% NaOCl solution. None of the solutions was genotoxic. The 2.5% NaOCl solution had a better tissue-dissolving capacity than the neutralized 2.5% NaOCl solution. The pH of the 2.5% NaOCl solution and neutralized 2.5% NaOCl solution decreased from 12 to 9 and from 7.5 to 5.6, respectively., Conclusion: Neutralizing a 2.5% NaOCl solution increased its cytotoxicity, did not induce any genotoxic effect, and reduced its tissue-dissolving ability., (Copyright (c) 2010 Mosby, Inc. All rights reserved.)

Objectives: The aim was to evaluate the shelf life and the dissolving and antibacterial properties of a neutralized 2.5% NaOCl solution., Study Design: The loss of available chlorine and the pH of the neutralized 2.5% NaOCl solution were recorded to determine its shelf life. The dissolving action on bovine dental pulp was assessed measuring weight loss, pH variation, and decrease in available chlorine content. The antibacterial activity was evaluated on artificially infected human teeth. The roots were endodontically prepared, sterilized, and inoculated with Enterococcus faecalis before irrigation with the neutralized solution. The presence of intracanal bacteria after irrigation was recorded., Results: The neutralized solution presented a shelf life of 2 hours, dissolving capacities equivalent to control for the first 5 minutes, and a better antibacterial efficiency., Conclusion: The neutralized 2.5% NaOCl solution must be used within 2 hours after mixing, should be frequently renewed to maintain its dissolving capacities, and presented enhanced antibacterial properties.

Introduction: Map-1B belongs to the family of proteins that govern the dynamic state and organization of microtubules within cells. MAP-1B is a microtubule-associated protein highly expressed during the development of the nervous system. Its expression, regulated by the fragile X mental retardation protein (FMRP), is essential to stabilize microtubules during the elongation of dendrites and neurites. Other microtubules-associated molecules such as tau or MAP2 seem to act similarly. The aim of this work was to identify the MAP-1B expression in in vitro and in vivo human odontoblasts during development and carious processes. The expression of MAP2 and tau was also studied., Materials and Methods: In cultured cells, MAP-1B expression was analyzed by real-time polymerase chain reaction, flow cytometry, and Western blot. Its distribution was visualized by in situ hybridization and immunochemistry both in vitro and in vivo. The expression of FMRP, MAP2, and tau was identified by real-time polymerase chain reaction and immunochemistry., Results: MAP-1B is specifically expressed in odontoblasts from adult third molars as well as incisor germs from human embryos. In adult carious teeth, it is also expressed in newly differentiated dentin-forming cells. In vitro, MAP-1B expression is related to the differentiation state of odontoblasts. MAP-1B clearly underlines the cellular architecture of cell bodies and processes of differentiated cells. FMRP, MAP2, and tau are also detected in vivo., Conclusion: On the basis of these data, MAP-1B could be considered as a new protein involved in the terminal differentiation of odontoblasts.

The purpose of this study was to record the time-course diffusion of hydrogen peroxide through human dentin from a peroxide carbamide gel designed for the walking bleach technique in order to determine its optimal renewal time. It was considered that the optimal renewal rate corresponded to the time necessary to achieve 80% of the maximal diffusion because a much longer time does not involve further significant diffusion. Thirty-six freshly extracted human premolars were used for this study. Eighteen were extracted for orthodontic reasons on patients under 20 years old (young-teeth group). Eighteen were extracted for periodontal reasons on patients between 40 and 60 years old (old-teeth group). The teeth were endodontically treated, and a flat defect was created at the enamel-cementum junction. The teeth were suspended in vials containing water, and the access cavities were filled with 20 microL of 20% hydrogen peroxide gel. The amount of diffusing hydrogen peroxide was assessed at 1 hour, 24 hours, 48 hours, and 120 hours. The diffusive flux and the maximal diffusion were calculated as well as the optimal renewal time. Hydrogen peroxide diffusion through young teeth lasted 352 hours but lasted 291 hours through old teeth. Diffusive flux and maximal diffusion were higher through young teeth than through old teeth. The optimal renewal time for young teeth was 33 hours and for old teeth was 18 hours.

Objective: The aim of this study was to determine if there is a significant correlation between the in vivo presence of periapical radiolucency and ex vivo apical dye penetration on the same human teeth., Study Design: Eighty-four endodontically filled teeth that were scheduled for extraction were classified into 2 groups according to the presence or absence of a periapical radiolucency and further divided into 2 subgroups according to the quality of the root canal filling. After extraction, the apical filling was evaluated by a dye penetration method., Results: The dye extraction evaluation showed no correlation between apical dye penetration and the presence of a periapical radiolucency (not significant), but a statistically significant correlation with the quality of the root canal filling (P = .03)., Conclusion: The results of the dye penetration study were correlated to the quality of the root canal filling but had no predictive value for the development of periapical radiolucency.

The purpose of this study was to compare ISO standards versus a new technique for in vitro evaluation of cytotoxicity of root canal sealers. The cytotoxicity of AH Plus, Cortisomol, and Sealapex was first recorded according to ISO standards on L 929 fibroblasts by the MTT assay. In parallel, 30 single-rooted teeth were cut at the cementum enamel junction (CEJ), and the roots were prepared and sterilized before filling with the lateral condensation using one of three sealers (n = 10). The apexes of the roots were dipped into 1 ml of minimum essential medium for 1, 2, and 30 days renewing the medium every other day. After 24-h contact between the medium and the filled roots, the medium was used to measure the cytotoxicity on L 929 with the MTT assay. ISO standards always gave a statistically higher cytotoxicity than the root-dipping technique (p < 0.0001), whatever the sealer and the exposure time. The ISO standards showed statistically significant differences among the sealers (p < 0.0001). AH Plus was noncytotoxic, Cortisomol showed a high cytotoxicity decreasing over time (p < 0.001), and Sealapex displayed a high cytotoxicity that did not decrease over time (NS). The new technique showed statistically significant differences among the sealers (p = 0.001), but the differences were so small that they were likely not clinically relevant. The high cytotoxicity of Sealapex decreased over time but the cytotoxicity of AH Plus and Cortisomol did not. The results show that the ISO standards may strongly over-evaluate the cytotoxicity of the endodontic sealers, emphasize the difference among the sealers, and may clinically correspond to a large overfilling. The new technique reduces the discrimination of the test and may clinically correspond to a classical filling. Therefore, both methods might be considered as clinically relevant, corresponding to classical and overfilling conditions.

The purpose of this study was to evaluate the sealing properties of four root canal sealers. Forty-eight maxillary central incisors were instrumented with Profile rotary instruments. They were randomly divided into four groups (n = 12) and filled using lateral condensation with one of the four sealers: Sealapex, Pulp Canal Sealer, AH 26, and Ketac-Endo. The apical leakage was measured with a fluid filtration method and expressed as L s(-1) KPa(-1). The teeth filled with Sealapex displayed a higher apical leakage (8.42 +/- 4.2 10(-11) L s(-1) KPa(-1)) than those filled with AH 26 (2.10 +/- 1.39 10(-11) L s(-1) KPa(-1)), Pulp Canal Sealer (0.17 +/- 0.09 10(-11) L s(-1) KPa(-1)) or Ketac-Endo (0.32 +/- 0.24 10(-1) L s(-1) KPa(-1)) (p < 0.01). No statistically significant difference was found among AH 26, Pulp Canal Sealer, and Ketac-Endo. No correlation was found between the sealing efficiency of the four sealers and their adhesive properties recorded in a previous study.

Cadherins are calcium-dependent cell adhesion molecules involved in the regulation of various biological processes such as cell recognition, intercellular communication, cell fate, cell polarity, boundary formation, and morphogenesis. Although previous studies have shown E-cadherin expression during rodent or human odontogenesis, there is no equivalent study available on N-cadherin expression in dental tissues. Here we examined and compared the expression patterns of E- and N-cadherins in both embryonic and adult (healthy, injured, carious) human teeth. Both proteins were expressed in the developing teeth during the cap and bell stages. E-cadherin expression in dental epithelium followed an apical-coronal gradient that was opposite to that observed for N-cadherin. E-cadherin was distributed in proliferating cells of the inner and outer enamel epithelia but not in differentiated cells such as ameloblasts, whereas N-cadherin expression was up-regulated in differentiated epithelial cells. By contrast to E-cadherin, N-cadherin was also expressed in mesenchymal cells that differentiate into odontoblasts and produce the hard tissue matrix of dentin. Although N-cadherin was not detected in permanent intact teeth, it was re-expressed during dentin repair processes in odontoblasts surrounding carious or traumatic sites. Similarly, N-cadherin re-expression was seen in vitro, in cultured primary pulp cells that differentiate into odontoblast-like cells. Taken together these results suggest that E- and N-cadherins may play a role during human tooth development and, moreover, indicate that N-cadherin is important for odontoblast function in normal development and under pathological conditions.

Objectives: The incidence of bacterial microleakage, pulp inflammation and necrosis associated with dentine etching treatments prior to restoration are not known. Consequently, to resolve some of the controversy surrounding the effects and importance of vital dentine etching, the authors investigated these factors., Methods: 110 standardised class V cavities were cut into buccal dentine, without exposing the pulp of teeth scheduled for extraction for orthodontic reasons. Cavities were either left unetched, or etched with the non-equivalent treatments of phosphoric acid gel for 60s or Ethylenediaminetetraacetic acid (EDTA) for 30s, prior to placement of composite resin. Teeth were collected and pulp responses were evaluated according to ISO guidelines, using pathohistomorphometric analysis and ANOVA statistics., Results: Etching was found to be correlated to bacterial microleakage (p=0.0001) and tertiary dentine formation (p=0.0023). Bacterial microleakage was correlated to inflammatory activity (p=0.0001). The frequency of bacterial microleakage was: no etching (65%), EDTA (51%) and phosphoric acid (PA) (20%)., Significance: Vital dentine etching treatment is of extreme importance for the placement of RC to minimise bacterial microleakage. PA etching proved to be more effective at preventing bacterial microleakage than non-etching, and etching with EDTA.