Immunological isolation and characterization of neuronal progenitors from human dental pulp: A laboratory-based investigation.

AIMS: Dental pulp stem cells (DPSCs) contain a population of stem cells with a broad range of differentiation potentials, as well as more lineage-committed progenitors. Such heterogeneity is a significant obstacle to experimental and clinical applications. The aim of this study is to isolate and characterize a homogenous neuronal progenitor cell population from human DPSCs. METHODOLOGY: Polysialylated-neural cell adhesion molecule (PSA-NCAM+) neural progenitors were isolated from the dental pulp of three independent donors using magnetic-activated cell sorting (MACS) technology. Immunofluorescent staining with a panel of neural and non-neural markers was used to characterize the magnetically isolated PSA-NCAM+ fraction. PSA-NCAM+ cells were then cultured in Neurobasal A supplemented with neurotrophic factors: dibutyryl cyclic-AMP, neurotrophin-3, B27 and N2 supplements to induce neuronal differentiation. Both PSA-NCAM+ and differentiated PSA-NCAM+ cells were used in Ca2+ imaging studies to assess the functionality of P2X3 receptors as well as membrane depolarization. RESULTS: PSA-NCAM+ neural progenitors were isolated from a heterogeneous population of hDPSCs using magnetic-activated cell sorting and anti-PSA-NCAM MicroBeads. Flow cytometry analysis demonstrated that immunomagnetic sorting significantly increased the purity of PSA-NCAM+ cells. Immunofluorescent staining revealed expression of pan-neuronal and mature neuronal markers, PGP9.5 and MAP2, respectively, as well as weak expression of the mature sensory markers, peripherin and islet1. ATP-induced response was mediated predominately by P2X3 receptors in both undifferentiated and differentiated cells, with a greater magnitude observed in the latter. In addition, membrane depolarizations were also detected in cells before and after differentiation when loaded with fast-voltage-responding fluorescent molecule, FluoVolt™ in response to potassium chloride. Interestingly, only differentiated PSA-NCAM+ cells were capable of spontaneous membrane oscillations. CONCLUSIONS: In summary, DPSCs contain a population of neuronal progenitors with enhanced neural differentiation and functional neural-like properties that can be effectively isolated with magnetic-activated cell sorting (MACS).

Novel Antibacterial Properties of the Human Dental Pulp Multipotent Mesenchymal Stromal Cell Secretome.

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.

Regulation of caries-induced pulp inflammation by NLRP3 inflammasome: A laboratory-based investigation.

AIM: To evaluate the expression and function of the nod-like receptor pyrin domain containing 3 (NLRP3) inflammasome in caries induced pulpitis. METHODOLOGY: NLRP3 expression was determined with immunohistochemistry in the dental pulp and qPCR in dental pulp cells (DPCs). THP-1 macrophages expressing the apoptosis-related speck-like protein (ASC) and green fluorescent protein (GFP) fusion protein were used to assess NLRP3 inflammasome activation by live cell imaging, following treatment with lipopolysaccharide (LPS) and lipoteichoic acid (LTA). Caspase I inhibitor was used to confirm inflammasome activation. An ex-vivo pulpitis model in which the DPCs were co-cultured with THP-1 macrophages was used to study the effect of the NLRP3 inflammasome inhibitor (MCC950), and cytokines were measured using ELISA and multiplex array. Data were analysed using the t-test or anova followed by a Bonferroni post hoc test with the level of significance set at p ≤ .05. RESULTS: NLRP3 inflammasome was differentially expressed in dental pulp of sound and carious teeth. Treatment of DPCs with LTA significantly upregulates NLRP3 and IL-1 ß-expression (p < .05) and in induces more ASC specks formation compared to LPS. IL-ß release in response to LTA treatment is significantly reduced with Caspase I inhibitor suggesting inflammasome dependent mechanism (p < .01). NLRP3-specific inhibitor, MCC950, significantly reduced IL-1ß and IL-6 in an ex-vivo pulpitis model (p < .01) but had no effect on IL-8 or matrix metalloproteinase-9 (MMP-9). CONCLUSIONS: Expression and upregulation of NLRP3 inflammasome with caries and LTA treatment suggest a role in caries-induced pulpitis. NLRP3 inhibitor attenuated the release of selective inflammatory cytokines and could be a potential treatment target that merit further investigation.

Endogenous Mas-related G-protein-coupled receptor X1 activates and sensitizes TRPA1 in a human model of peripheral nerves.

Mas-related G-protein-coupled receptor X1 (MrgprX1) is a human-specific Mrgpr and its expression is restricted to primary sensory neurons. However, its role in nociception and pain signaling pathways is largely unknown. This study aims to investigate a role for MrgprX1 in nociception via interaction with the pain receptor, Transient Receptor Potential Ankyrin 1 (TRPA1), using in-vitro and in-vivo human neuronal models. MrgprX1 protein expression in human trigeminal nociceptors was investigated by the immunolabeling of the dental pulp and cultured peripheral neuronal equivalent (PNE) cells. MrgprX1 receptor signaling was monitored by Fura-2-based Ca2+ imaging using PNEs and membrane potential responses were measured using FluoVoltTM . Immunofluorescent staining revealed MrgprX1 expression in-vivo in dental afferents, which was more intense in inflamed compared to healthy dental pulps. Endogenous MrgprX1 protein expression was confirmed in the in-vitro human PNE model. MrgprX1 receptor signaling and the mechanisms through which it couples to TRPA1 were studied by Ca2+ imaging. Results showed that MrgprX1 activates TRPA1 and induces membrane depolarization in a TRPA1 dependent manner. In addition, MrgprX1 sensitizes TRPA1 to agonist stimulation via Protein Kinase C (PKC). The activation and sensitization of TRPA1 by MrgprX1 in a model of human nerves suggests an important role for this receptor in the modulation of nociception.

Nanoarchitectonics of Electrically Activable Phosphonium Self-Assembled Monolayers to Efficiently Kill and Tackle Bacterial Infections on Demand.

Prosthetic implants are widely used in dentistry and orthopedics and, as a result, infections can occur which cause their removal. Therefore, it is essential to propose methods of eradicating the bacteria that remain on the prosthesis during treatment. For this purpose, it is necessary to develop surfaces whose antibacterial activity can be controlled. Herein, we designed innovative and smart phosphonium self-assembled monolayer (SAM) interfaces that can be electrically activated on demand for controlling bacterial contaminations on solid surfaces. Upon electroactivation with a low potential (0.2 V for 60 min., conditions determined through a DOE), a successful stamping out of Gram-positive and Gram-negative bacterial strains was obtained with SAM-modified titanium surfaces, effectively killing 95% of Staphylococcus aureus and 90% Klebsiellapneumoniae. More importantly, no toxicity towards eukaryotic cells was observed which further enhances the biocompatible character of these novel surfaces for further implementation.

Identification and validation of novel biomarkers and therapeutics for pulpitis using connectivity mapping.

AIM: To create an irreversible pulpitis gene signature from microarray data of healthy and inflamed dental pulps, followed by a bioinformatics approach using connectivity mapping to identify therapeutic compounds that could potentially treat pulpitis. METHODOLOGY: The Gene Expression Omnibus (GEO) database, an international public repository of genomics data sets, was searched for human microarray datasets assessing pulpitis. An irreversible pulpitis gene expression signature was generated by differential expression analysis. The statistically significant connectivity map (ssCMap) method was used to identify compounds with a highly correlating gene expression pattern. qPCR was used to validate novel pulpitis genes. An ex vivo pulpitis model was used to test the effects of the compounds identified, and the level of inflammatory cytokines was measured with qPCR, ELISA and multiplex array. Means were compared using the t-test or ANOVA with the level of significance set at p ≤ .05. RESULTS: Pulpitis gene signatures were created using differential gene expression analysis at cutoff points p = .0001 and .000018. Top upregulated genes were selected as potential pulpitis biomarkers. Among these, IL8, IL6 and MMP9 were previously identified as pulpitis biomarkers. Novel upregulated genes, chemokine (C-C motif) ligand 21 (CCL21), metallothionein 1H (MT1H) and aquaporin 9 (AQP9) were validated in the pulp tissue of teeth clinically diagnosed with irreversible pulpitis using qPCR. ssCMap analysis identified fluvastatin (Statin) and dequalinium chloride (Quaternary ammonium) as compounds with the strongest correlation to the gene signatures (p = .0001). Fluvastatin reduced IL8, IL6, CCL21, AQP9 (p < .001) and MMP9 (p < .05) in the ex vivo pulpitis model, while dequalinium chloride reduced AQP9 (p < .001) but had no significant effect on the other biomarkers. CONCLUSIONS: AQP9, MT1H and CCL21 were identified and validated as novel biomarkers for pulpitis. Fluvastatin and dequalinium chloride identified by the ssCMap as potential therapeutics for pulpitis reduced selected pulpitis biomarkers in an ex vivo pulpitis model. In vivo testing of these licenced drugs is warranted.

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.

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.

TRPA1 activation in a human sensory neuronal model: relevance to cough hypersensitivity?

The cough reflex becomes hyperresponsive in acute and chronic respiratory diseases, but understanding the underlying mechanism is hampered by difficulty accessing human tissue containing both nerve endings and neuronal cell bodies. We refined an adult stem cell sensory neuronal model to overcome the limited availability of human neurones and applied the model to study transient receptor potential ankyrin 1 (TRPA1) channel expression and activation.Human dental pulp stem cells (hDPSCs) were differentiated towards a neuronal phenotype, termed peripheral neuronal equivalents (PNEs). Using molecular and immunohistochemical techniques, together with Ca2+ microfluorimetry and whole cell patch clamping, we investigated roles for nerve growth factor (NGF) and the viral mimic poly I:C in TRPA1 activation.PNEs exhibited morphological, molecular and functional characteristics of sensory neurons and expressed functional TRPA1 channels. PNE treatment with NGF for 20 min generated significantly larger inward and outward currents compared to untreated PNEs in response to the TRPA1 agonist cinnamaldehyde (p<0.05). PNE treatment with poly I:C caused similar transient heightened responses to TRPA1 activation compared to untreated cells.Using the PNE neuronal model we observed both NGF and poly I:C mediated sensory neuronal hyperresponsiveness, representing potential neuro-inflammatory mechanisms associated with heightened nociceptive responses recognised in cough hypersensitivity syndrome.

TNF-α-induced p38MAPK activation regulates TRPA1 and TRPV4 activity in odontoblast-like cells.

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.

Pulp fibroblasts synthesize functional complement proteins involved in initiating dentin-pulp regeneration.

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.

The tick tock of odontogenesis.

Although a big deal of dental research is being focused to the understanding of early stages of tooth development, a huge gap exist on our knowledge on how the dental hard tissues are formed and how this process is controlled daily in order to produce very complex and diverse tooth shapes adapted for specific functions. Emerging evidence suggests that clock genes, a family of genes that controls circadian functions within our bodies, regulate also dental mineralized tissues formation. Enamel formation, for example, is subjected to rhythmical molecular signals that occur on short (24h) periods and control the secretion and maturation of the enamel matrix. Accordingly, gene expression and ameloblast functions are also tightly modulated in regular daily intervals. This review summarizes the current knowledge on the circadian controls of dental mineralized tissues development with a special emphasis on amelogenesis.

Tissue-Engineered Oral Epithelium for Dental Material Testing: Toward In Vitro Biomimetic Models.

Tissue-engineered oral epithelium (ΤΕΟΕ) was developed after comparing various culture conditions, including submerged (SUB) and air-liquid interface (ALI) human cell expansion options. Barrier formation was evaluated via transepithelial electrical resistance (TEER) and calcein permeation via spectrofluorometry. TEOE was further assessed for long-term viability via live/dead staining and development of intercellular connections via transmission electron microscopy. Tissue architecture was evaluated via histochemistry and the expression of pancytokeratin (pCK) via immunohistochemistry. The effect of two commonly used dental resinous monomers on TEOE was evaluated for alterations in cell viability and barrier permeability. ALI/keratinocyte growth factor-supplemented (ALI-KGS) culture conditions led to the formation of an 8-20-layer thick, intercellularly connected epithelial barrier. TEER values of ALI-KGS-developed TEOE decreased compared with all other tested conditions, and the established epithelium intensively expressed pCK. Exposure to dental monomers affected the integrity and architecture of TEOE and induced cellular vacuolation, implicating hydropic degeneration. Despite structural modifications, the permeability of TEOE was not substantially affected after exposure to the monomers. In conclusion, the biological properties of the TEOE mimicking the physiological functional conditions and its value as biocompatibility assessment tool for dental materials were characterized.

In vitro microleakage of Biodentine as a dentin substitute compared to Fuji II LC in cervical lining restorations.

PURPOSE: 1) To evaluate the marginal sealing efficacy of Biodentine at the cervical margins of approximal cavities placed in molars; 2) to evaluate and compare the use of Biodentine in combination with resin-based adhesives and a resin composite, compared with a resin-modified glass-ionomer cement (Fuji II LC). MATERIALS AND METHODS: Sixty approximal cavities were prepared on mesial and distal surfaces of 30 extracted human third molars. The teeth were randomly assigned into 6 groups of 10 cavities each: (G1) Biodentine, (G2) Fuji II LC as a filling material, (G3) Biodentine as a base + Optibond Solo Plus + silane + Filtek Z250, (G4) as in G3 without silane, (G5) Biodentine as a base + Septobond SE + Filtek Z250, (G6) Fuji II LC as a base + Optibond Solo Plus + Filtek Z250. The materials were applied according to the manufacturers' instructions. Biodentine required no dentin or enamel surface conditioning treatment. The teeth were thermocycled 2500x (5°C to 55°C). The specimens were then sealed with a 1-mm window around the marginal interface. Samples were immersed in a 50% w/v silver nitrate solution and exposed to a photo developing solution. The teeth were embedded in resin (Sody 33) and sectioned through the restorations. The silver penetration was directly measured using a light microscope. The results were expressed as ordinal scores from 0 to 3 at cervical, interfacial, and enamel margins. The data were analyzed with the nonparametric Kruskal-Wallis, Games Howell, and Wilcoxon signed rank tests (p < 0.05). RESULTS: No statistically significant differences were found between the 6 groups, neither for the dentin cervical margins nor for cervical lining (Biodentine or Fuji II LC)/resin composite interfaces. Statistically significant differences were observed between G5 (median score = 2.0) and the other groups (median score = 1.0) for the enamel margins. Statistically significant differences were found between enamel and dentin cervical margins in G2 (enamel median score = 1.0; dentin median score = 1.5) and G5 (enamel median score = 2.0; dentin median score = 1.0). CONCLUSION: Within the limits of this in vitro study, Biodentine as dentin substitute in cervical lining restorations or as a restorative material in approximal cavities when the cervical extent is under the CEJ seems to perform well without any conditioning treatment. However, the operating time is longer than when a RMGIC (Fuji II LC) is used.

Influence of in-office whitening gel pH on hydrogen peroxide diffusion through enamel and color changes in bovine teeth.

PURPOSE: To assess the influence of in-office whitening gel pH on whitening efficiency. METHODS: Hydrogen peroxide diffusion and color changes on bovine teeth were assessed. Three gels with close hydrogen peroxide concentrations but with various pH levels were tested: Zoom 2 (Discus Dental), Opalescence Endo and Opalescence Boost (Ultradent). The pH levels were respectively: 3.0, 5.0 and 7.0. Thirty enamel slices and tooth crowns were used for both studies (n = 10 per group per study). Hydrogen peroxide diffusion through the enamel slices and the tooth crowns was spectrophotometrically recorded every 10 minutes for 1 hour to calculate the diffusion coefficients. Color changes were spectrophotometrically recorded every 10 minutes for 1 hour and quantified in term of CIE-Lab. RESULTS: The hydrogen peroxide diffusion coefficient through enamel ranged from 5.12 +/- 0.82 x 10(-9) cm2 s(-1) for pH 3 to 5.19 +/- 0.92 x 10(-9) cm2 S(-1) for pH 7. Through tooth crowns it ranged from 4.80 +/- 1.75 x 10(-10) cm2 s(-1) for pH 5 to 4.85 +/- 1.82 x 10(-10) cm2 s(-1) for pH 3. After 1 hour, the deltaE varied from 5.6 +/- 4.0 for pH 7 to 7.0 +/- 5.0 for pH 3 on enamel slices and from 3.9 +/- 2.5 for pH 5 to 4.9 +/- 3.5 for pH 7 on tooth crowns. There was no statistically significant difference between groups for both parameters.

Advanced in Vitro Experimental Models for Tissue Engineering-based Reconstruction of a 3D Dentin/pulp Complex: a Literature Review.

OBJECTIVE: Experimental procedures have been used to monitor cellular responses at the dentin/pulp interface. Aiming to divert from in vivo studies and oversimplified two-dimensional assays, three-dimensional (3D) models have been developed. This review provides an overview of existing literature, regarding 3D in vitro dentin/pulp reconstruction. MATERIAL & METHODS: PubMed, Scopus, Cochrane Library and Web of Science- were systematically searched for attributes between 1998 and 2020. The search focused on articles on the development of three-dimensional tools for the reconstruction of a dentin/pulp complex under in vitro conditions, which were then screened and qualitatively assessed. Article grouping according to mode of implementation, resulted in five categories: the customised cell perfusion chamber (CPC) (n = 8), the tooth bud model (TBM) (n = 3), the 3D dentin/pulp complex manufactured by tissue engineering (DPC) (n = 6), the entire tooth culture (ETC) (n = 4) and the tooth slice culture model (TSC) (n = 5). RESULTS: A total of 26 publications, applying nine and eight substances for pulp and dentin representation respectively, were included. Natural materials and dentin components were the most widely utilized. The most diverse category was the DPC, while the CPC group was the test with the highest longevity. The most consistent categories were the ETC and TSC models, while the TBM presented as the most complete de novo approach. CONCLUSIONS: All studies presented with experimental protocols with potential upgrades. Solving the limitations of each category will provide a complete in vitro testing and monitoring tool of dental responses to exogenous inputs. CLINICAL RELEVANCE: The 3D dentin/pulp complexes are valid supplementary tools for in vivo studies and clinical testing. Graphical Abstract.

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.

Biocompatibility assessment of resin-based cements on vascularized dentin/pulp tissue-engineered analogues.

OBJECTIVES: A three-dimensional (3D) dentin/pulp tissue analogue, resembling the human natural tissue has been engineered in an in vitro setup, aiming to assess the cytocompatibility of resin-based dental restorative cements. METHODS: Stem Cells from Apical Papilla (SCAP) and Human Umbilical Vein Endothelial Cells (HUVEC) were embedded in Collagen-I/Fibrin hydrogels at 1:3 ratio within 24-well plates. Hanging culture inserts were placed over the hydrogels, housing an odontoblast-like cell layer and a human treated-dentin barrier. Shear modulus of the hydrogels at 3.5 and 5 mg/ml was evaluated by dynamic mechanical analysis. Eluates of two resin-based cements, a dual-cure- (Breeze™, Pentron: Cement-1/C1), and a self-adhesive cement (SpeedCEMplus™, Ivoclar-Vivadent: Cement-2/C2) were applied into the dentin/pulp tissue analogue after pre-stimulation with LPS. Cytocompatibility was assessed by MTT assay, live/dead staining and real-time PCR analysis. RESULTS: Both hydrogel concentrations showed similar shear moduli to the natural pulp until day (D) 7, while the 5 mg/ml-hydrogel substantially increased stiffness by D14. Both cements caused no significant toxicity to the dentin/pulp tissue analogue. C1 induced stimulation (p < 0.01) of cell viability (158 ± 3%, 72 h), while pre-stimulation with LPS attenuated this effect. C2 (±LPS) caused minor reduction of viability (15-20%, 24 h) that recovered at 72 h for the LPS+ group. Both cements caused upregulation of VEGF, ANGP-1, and downregulation of the respective receptors VEGFR-2 and Tie-1. SIGNIFICANCE: Both resin-based cements showed good cytocompatibility and triggered angiogenic response within the dentin/pulp tissue analogue, indicating initiation of pulp repair responses to the released xenobiotics.

Multicomponent Peptide Hydrogels as an Innovative Platform for Cell-Based Tissue Engineering in the Dental Pulp.

In light of the increasing levels of antibiotic resistance, nanomaterials and novel biologics are urgently required to manage bacterial infections. To date, commercially available self-assembling peptide hydrogels have not been studied extensively for their ability to inhibit micro-organisms relevant to tissue engineering sites such as dental root canals. In this work, we assess the biocompatibility of dental pulp stem/stromal cells with commercially available multicomponent peptide hydrogels. We also determine the effects of dental pulp stem/stromal cell (DPSC) culture in hydrogels on growth factor/cytokine expression. Furthermore, to investigate novel aspects of self-assembling peptide hydrogels, we determine their antimicrobial activity against the oral pathogens Staphylococcus aureus, Enterococcus faecalis, and Fusobacterium nucleatum. We show that self-assembling peptide hydrogels and hydrogels functionalized with the adhesion motif Arg-Gly-Asp (RGD) are biocompatible with DPSCs, and that cells grown in 3D hydrogel cultures produce a discrete secretome compared with 2D-cultured cells. Furthermore, we show that soluble peptides and assembled hydrogels have antimicrobial effects against oral pathogens. Given their antibacterial activity against oral pathogens, biocompatibility with dental pulp stem/stromal cells and enhancement of an angiogenic secretome, multicomponent peptide hydrogels hold promise for translational use.

Ultrashort Peptide Hydrogels Display Antimicrobial Activity and Enhance Angiogenic Growth Factor Release by Dental Pulp Stem/Stromal Cells.

Recent studies on peptide hydrogels have shown that ultrashort peptides (<8 amino acids) can self-assemble into hydrogels. Ultrashort peptides can be designed to incorporate antimicrobial motifs, such as positively charged lysine residues, so that the peptides have inherent antimicrobial characteristics. Antimicrobial hydrogels represent a step change in tissue engineering and merit further investigation, particularly in applications where microbial infection could compromise healing. Herein, we studied the biocompatibility of dental pulp stem/stromal cells (DPSCs) with an ultrashort peptide hydrogel, (naphthalene-2-ly)-acetyl-diphenylalanine-dilysine-OH (NapFFεKεK-OH), where the epsilon (ε) amino group forms part of the peptide bond rather than the standard amino grouping. We tested the antimicrobial properties of NapFFεKεK-OH in both solution and hydrogel form against Staphylococcus aureus, Enterococcus faecalis and Fusobacterium nucleatum and investigated the DPSC secretome in hydrogel culture. Our results showed NapFFεKεK-OH hydrogels were biocompatible with DPSCs. Peptides in solution form were efficacious against biofilms of S. aureus and E. faecalis, whereas hydrogels demonstrated antimicrobial activity against E. faecalis and F. nucleatum. Using an angiogenic array we showed that DPSCs encapsulated within NapFFεKεK-OH hydrogels produced an angiogenic secretome. These results suggest that NapFFεKεK-OH hydrogels have potential to serve as novel hydrogels in tissue engineering for cell-based pulp regeneration.