The Added Value of a Collagenated Thermosensitive Bone Substitute as a Scaffold for Bone Regeneration.

A pre-hydrated thermosensitive collagenated biomaterial which sets at body temperature and maintains the space of the missing alveolar bone volume, OsteoBiol GTO® (GTO), has been released as a bone substitute. This study was designed to check its angiogenic and osteogenic potentials compared to OsteoBiol Gen-Os® (Gen-Os) and Geistlich Bio-Oss® (Bio-Oss). Samples of materials were incubated in culture media to obtain the extracts. Collagen release was measured in the extracts, which were used to investigate human periodontal ligament (hPDL) cell proliferation (MTT), colonization (Scratch assays) and growth factor release (ELISA). The effects on endothelial cell proliferation (MTT) and organization (Matrigel® assays) were also studied. Finally, endothelial and mesenchymal Stem Cell (hMSC) recruitment (Boyden Chambers) were investigated, and hMSC Alkaline Phosphatase (ALP) activity was measured. A higher collagen concentration was found in GTO extract, which led to significantly higher hPDL cell proliferation/colonization. All materials increased VEGF/FGF-2 growth factor secretion, endothelial cell recruitment, proliferation, and organization, but the increase was highest with GTO. All materials increased hMSC recruitment and ALP activity. However, the increase was highest with collagenated GTO and Gen-Os, which enhanced C5a and BMP-2 secretion. Overall, GTO has higher angiogenic/osteogenic potentials than the collagenated Gen-Os and the anorganic Bio-Oss. It provides a suitable scaffold for endothelial and mesenchymal stem cell recruitment, which represent essential bone regeneration requirements.

Osteogenic Potential of a Polyethylene Glycol Hydrogel Functionalized with Poly-Lysine Dendrigrafts (DGL) for Bone Regeneration.

Resorbable hydrogels are widely used as scaffolds for tissue engineering. These hydrogels can be modified by grafting dendrimer-linked functionalized molecules (dendrigrafts). Our aim was to develop a tunable poly(L-lysine) dendrigrafts (DGL)/PEG-based hydrogel with an inverse porosity and to investigate its osteogenic potential. DGL/PEG hydrogels were emulsified in a surfactant-containing oil solution to form microspheres. The toxicity was evaluated on Human Vascular Endothelial Cells (HUVECs) and Bone Marrow Mesenchymal Stem Cells (hMSCs) with Live/Dead and MTT assays. The effects on HUVECs were investigated through C5 Complement expression by RT-PCR and C5a/TGF-ß1 secretion by ELISA. Recruitment of hMSCs was investigated using Boyden chambers and their osteogenic differentiation was studied by measuring Alkaline Phosphatase activity (ALP) and BMP-2 secretion by ELISA. Adjusting the stirring speed during the emulsification allowed to obtain spherical microspheres with tunable diameters (10-1600 µm). The cell viability rate with the hydrogel was 95 and 100% with HUVECs and hMSCs, respectively. Incubating HUVECs with the biomaterial induced a 5-fold increase in TGF-ß1 and a 3-fold increase in Complement C5a release. Furthermore, HUVEC supernatants obtained after incubation with the hydrogel induced a 2.5-fold increase in hMSC recruitment. The hydrogel induced a 3-fold increase both in hMSC ALP activity and BMP-2 secretion. Overall, the functionalized hydrogel enhanced the osteogenic potential by interacting with endothelial cells and hMSCs and represents a promising tool for bone tissue engineering.

Current strategies for conservative endodontic access cavity preparation techniques-systematic review, meta-analysis, and decision-making protocol.

OBJECTIVES: To assess related studies and discuss the clinical implications of endodontic access cavity (AC) designs. MATERIALS AND METHODS: A systematic review of studies comparing the fracture resistance and/or endodontic outcomes between different AC designs was conducted in two electronic search databases (PubMed and Web of Science) following the PRISMA guidelines. Study selection, data extraction, and quality assessment were performed. Meta-analyses were undertaken for fracture resistance and root canal detection, with the level of significance set at 0.05 (P = 0.05). RESULTS: A total of 33 articles were included in this systematic review. The global evaluation of the risk of bias in the included studies was assessed as moderate, and the level of evidence was rated as low. Four types of AC designs were categorized: traditional (TradAC), conservative (ConsAC), ultraconservative (UltraAC), and truss (TrussAC). Their impact on fracture resistance, cleaning/disinfection, procedural errors, root canal detection, treatment time, apical debris extrusion, and root canal filling was discussed. Meta-analysis showed that compared to TradAC, (i) there is a significant higher fracture resistance of teeth with ConsAC, TrussAC, or ConsAC/TrussAC when all marginal ridges are preserved (P < 0.05), (ii) there is no significant effect of the type of AC on the fracture resistance of teeth when one or two marginal ridges are lost (P > 0.05), and (iii) there is a significantly higher risk of undetected canals with ConsAC if not assisted by dental operating microscope and ultrasonic troughing (P < 0.05). CONCLUSIONS: Decreasing the AC extent does not necessarily present mechanical and biological advantages especially when one or more surfaces of the tooth structure are lost. To date, the evidence available does not support the application of TrussAC. UltraAC might be applied in limited occasions. CLINICAL RELEVANCE: Maintaining the extent of AC design as small as practical without jeopardizing the root canal treatment quality remains a pragmatic recommendation. Different criteria can guide the practitioner for the optimal extent of AC outline form which varies from case to case.

Calcium Silicate-Based Root Canal Sealers: A Narrative Review and Clinical Perspectives.

Over the last two decades, calcium silicate-based materials have grown in popularity. As root canal sealers, these formulations have been extensively investigated and compared with conventional sealers, such as zinc oxide-eugenol and epoxy resin-based sealers, in in vitro studies that showed their promising properties, especially their biocompatibility, antimicrobial properties, and certain bioactivity. However, the consequence of their higher solubility is a matter of debate and still needs to be clarified, because it may affect their long-term sealing ability. Unlike conventional sealers, those sealers are hydraulic, and their setting is conditioned by the presence of humidity. Current evidence reveals that the properties of calcium silicate-based sealers vary depending on their formulation. To date, only a few short-term investigations addressed the clinical outcome of calcium silicate-based root canal sealers. Their use has been showed to be mainly based on practitioners' clinical habits rather than manufacturers' recommendations or available evidence. However, their particular behavior implies modifications of the clinical protocol used for conventional sealers. This narrative review aimed to discuss the properties of calcium silicate-based sealers and their clinical implications, and to propose rational indications for these sealers based on the current knowledge.

Fibroblasts Control Macrophage Differentiation during Pulp Inflammation.

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.

Complement activation links inflammation to dental tissue regeneration.

OBJECTIVES: Complement is an efficient plasma immune surveillance system. It initiates inflammation by inducing vascular modifications and attracting immune cells expressing Complement receptors. Investigating Complement receptors in non-immune cells pointed out Complement implication in the regeneration of tissue such as liver, skin, or bone. This review will shed the light on Complement implication in the initial steps of dental tissue regeneration. MATERIALS AND METHODS: Review of literature was conducted on Complement local expression and implication in oral tissue regeneration in vivo and in vitro. RESULTS: Recent data reported expression of Complement receptors and soluble proteins in dental tissues. Cultured pulp fibroblasts secrete all Complement components. Complement C3b and MAC have been shown to control bacteria growth in the dental pulp while C3a and C5a are involved in the initial steps of pulp regeneration. Indeed, C3a induces pulp stem cell/fibroblast proliferation, and fibroblast recruitment, while C5a induces neurite growth, guides stem cell recruitment, and odontoblastic differentiation. Similarly, cultured periodontal ligament cells produce C5a which induces bone marrow mesenchymal stem cell recruitment. CONCLUSIONS: Overall, this review highlights that local Complement synthesis in dental tissues plays a major role, not only in eliminating bacteria but also in the initial steps of dental tissue regeneration, thus providing a link between dental tissue inflammation and regeneration. CLINICAL RELEVANCE: Complement provides an explanation for understanding why inflammation preceeds regeneration. This may also provide a biological rational for understanding the reported success conservative management of mature permanent teeth with carious pulp exposure.

Pulp Fibroblast Contribution to the Local Control of Pulp Inflammation via Complement Activation.

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.

Investigating unset endodontic sealers' eugenol and hydrocortisone roles in modulating the initial steps of inflammation.

INTRODUCTION: Endodontic treatment success is achieved not only when the cement provides a hermetic seal but also when the injured periapical tissue is regenerated. However, an exaggerated inflammatory reaction hinders tissue regeneration and it has been shown that dental materials affect the inflammatory response through modulation of cytokine secretion. This work was set to investigate the effects of the presence of hydrocortisone in zinc oxide eugenol sealers (Endomethasone N) on modulating the initial steps of inflammation in vitro. MATERIAL AND METHODS: Hydrocortisone and eugenol leaching from Endomethasone N and Pulp Canal Sealer (PCS) were quantified by ELISA and spectrofluorometry, respectively. The effects of Endomethasone N and Pulp Canal Sealer were studied on lipopolysaccharides (LPS)-stimulated human periodontal ligament (hPDL) cells. Cytokine (IL-6, TNF-α) secretion from cells was quantified by ELISA. Inflammatory cell (THP-1) adhesion to activated endothelial cells, their migration and activation were studied in vitro. RESULTS: Endomethasone N decreased secretion of IL-6 and TNF-α from hPDL cells. THP-1 adhesion to activated endothelial cells (HUVECs) and migration significantly decreased with Endomethasone N while no effect was observed with PCS. Activation of THP-1 decreased with both materials' extracts but was significantly lower with Endomethasone N than with PCS. CONCLUSION: These results performed in vitro show that Endomethasone N anti-inflammatory effects are due to the presence of hydrocortisone. CLINICAL RELEVANCE: Endomethasone N has potential local anti-inflammatory effects which appear to be due to its hydrocortisone rather than eugenol content. Decreasing the inflammatory response is a pre-requisite to initiate the periapical healing.

BioRoot RCS Extracts Modulate the Early Mechanisms of Periodontal Inflammation and Regeneration.

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.

Pulp capping materials modulate the balance between inflammation and regeneration.

The interrelations between inflammation and regeneration are of particular significance within the dental pulp tissue inextensible environment. Recent data have demonstrated the pulp capacity to respond to insults by initiating an inflammatory reaction and dentin pulp regeneration. Different study models have been developed in vitro and in vivo to investigate the initial steps of pulp inflammation and regeneration. These include endothelial cell interaction with inflammatory cells, stem cell interaction with pulp fibroblasts, migration chambers to study cell recruitment and entire human tooth culture model. Using these models, the pulp has been shown to possess an inherent anti-inflammatory potential and a high regeneration capacity in all teeth and at all ages. The same models were used to investigate the effects of tricalcium silicate-based pulp capping materials, which were found to modulate the pulp anti-inflammatory potential and regeneration capacity. Among these, resin-containing materials such as TheraCal® shift the pulp response towards the inflammatory reaction while altering the regeneration process. On the opposite, resin-free materials such as Biodentine™ have an anti-inflammatory potential and induce the pulp regeneration capacity. This knowledge contradicts the new tendency of developing resin-based calcium silicate hybrid materials for direct pulp capping. Additionally, it would allow investigating the modulatory effects of newly released pulp capping materials on the balance between tissue inflammation and regeneration. It would also set the basis for developing future capping materials targeting these processes.

Tricalcium Silicate Capping Materials Modulate Pulp Healing and Inflammatory Activity In Vitro.

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-ß1) 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-ß1 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.

Light-cured Tricalcium Silicate Toxicity to the Dental Pulp.

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.

Complement Activation by Pulp Capping Materials Plays a Significant Role in Both Inflammatory and Pulp Stem Cells' Recruitment.

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.

Biodentine Reduces Tumor Necrosis Factor Alpha-induced TRPA1 Expression in Odontoblastlike Cells.

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.