Research in implant dentistry has predominantly focused on bone regeneration, osseous volume maintenance, and successful osseointegration. However, soft tissue healing, which influences implant functional sealing, long-term stability, and esthetic integration, remains underexplored. This study investigated the effects of three xenogenic bone substitutes on gingival healing in vitro. Three experimental groups were established using extracts from bone substitutes diffusing through the OsteoBiol® Evolution collagen membrane: two collagenated substitutes, OsteoBiol® Gen-Os® (Gen-Os) and OsteoBiol® GTO® (GTO), and one inorganic substitute, Bio-Oss® (Bio-Oss). The substitutes were prepared in test tubes, and the extracts diffusing through the collagen membrane were used to evaluate human gingival cell (hGC) proliferation (MTT assay), migration (scratch assay), and growth factor release (ELISA). Angiogenic potential was assessed by endothelial cell proliferation, recruitment (Boyden chambers), and organization (Matrigel® assays). The indirect interaction between stimulated gingival cells and human bone marrow mesenchymal stem cells (hMSC) was investigated by analyzing hMSC recruitment and osteogenic BMP-2 secretion. Collagenated GTO and Gen-Os significantly enhanced hGC proliferation and migration in the scratch assay, with 1.8-fold and 1.6-fold increases, respectively, compared to control. All three substitutes enhanced neoangiogenesis in vitro. VEGF and FGF-2 secretion was significantly higher with GTO, showing 5-fold and 5.7-fold increases, respectively, resulting in a 3.7-fold increase in tube formation compared to control. Collagenated materials promoted hMSC recruitment, whereas BMP-2 secretion was not affected by any material. The observed effects were higher with the collagenated Gen-Os and GTO, with 2.5-fold and 2.8-fold increases, respectively, than with the non-collagenated Bio-Oss, which showed a 1.5-fold increase. These findings demonstrate that collagenated bone substitutes enhance gingival healing and angiogenic potential through barrier membranes and confirm that stimulated gingival cells indirectly promote hMSC recruitment, indicating that bone substitute effects extend beyond bone regeneration to include soft tissue healing and inter-tissue communication.

OBJECTIVES:Mechanical and/or microbial stimuli can prompt extracellular ATP release in many cell types, where it acts as an inflammatory mediator to amplify pain signals and inflammation. This study aimed to investigate the synergistic effects of microbial and/or mechanical stimulation on ATP and cytokine release from dental pulp cells (DPCs) and to determine if the inflammatory signalling identified was dependent on ATP acting via the purinergic receptor, P2X3. METHODS:DPCs were prepared from sound third molars using explant culture method. P2X3 protein expression and functionality in DPCs were investigated by immunostaining and calcium mobilisation assays using the P2X3 agonist αβmeATP and antagonist AF-353. ATP or interleukin-6 (IL-6) release from DPCs treated with lipoteichoic acid (LTA) and fluid shear stress or stretch was measured using the ATPlite luciferase assay (PerkinElmer) or ELISA (R&D Systems), respectively. To assess the role of P2X3 activation in IL-6 release, the P2X3 antagonist AF-353 was included in ELISA experiments. Data were checked for normality and analysed by Mann-Whitney test for two-group comparisons and Kruskal-Wallis or ANOVA test with Dunn's or Dunnett's multiple comparisons test where appropriate for multiple-group comparisons. RESULTS:The DPCs expressed functional P2X3 receptors. The cells' response to αβmeATP was significantly inhibited by AF-353 (p < 0.0001). Increased ATP and IL-6 release were observed following co-stimulation with LTA and fluid shear stress (p < 0.01, p < 0.05) or LTA and stretch (p < 0.05). The addition of AF-353 reduced IL-6 release by 34% and 36% (p < 0.05) in DPCs co-stimulated with LTA and fluid shear stress or stretch, respectively. CONCLUSION:This study demonstrates that the combined bacterial mimetic and mechanical stimulation of DPCs significantly enhances ATP and IL-6 release. The inhibition of IL-6 release by the P2X3 antagonist AF-353 indicates the importance of P2X3 receptor activation in this process. These findings offer insights into the molecular mechanisms of dental pulp inflammation and potential therapeutic targets.

INTRODUCTION:Persistent inflammation is a critical factor influencing the outcome of endodontic treatment, particularly following removal of necrotic/infected tissues, root filling including a sealer application and a hermetic coronal seal. This study investigates the effect of BioRoot Flow (BRF), Pulp Canal Sealer, and AH Plus endodontic sealers on modulating inflammatory response of human periodontal ligament (hPDL) cells. To this end, we evaluated the effects of the sealers on proinflammatory cytokine secretion by hPDL cells. We also checked the effects on immune cell recruitment in vitro by investigating the sealers' extracts effects on key steps of the inflammatory reaction including monocyte cell adhesion to endothelial cells, their migration using Boyden chambers, and their activation (Figure 1). METHODS:Extracts were prepared by incubating the sealers in minimum essential medium. To mimic the bacterial infection, hPDL cells were stimulated with lipopolysaccharides and subsequently exposed to the extracts. Proinflammatory interleukin (IL)-6, IL-8, and tumor necrosis factor (TNF)-α cytokine secretion was quantified using enzyme-linked immunosorbent assay. Inflammatory cell recruitment was assessed in vitro using the THP-1 monocytics, focusing on their adhesion to endothelial cells, migration using Boyden chambers and activation by evaluating their adhesion on plastic petri dish surfaces. RESULTS:BRF significantly reduced TNF-α and IL-8 secretion levels by 65% and 52%, respectively. By contrast, AH Plus increased IL-8 secretion while Pulp Canal Sealer was associated with increased IL-6 and TNF-α secretion, with 100% and 500% increases, respectively. Furthermore, while all tested sealers decreased the monocyte activation, this decrease was more pronounced with BRF (50%) which was the only one to decrease THP-1 migration (20%). CONCLUSIONS:This study highlights the endodontic sealers' potential to modulate the inflammatory responses in vitro. BRF mitigated the inflammatory response more than the other materials. It exhibited significant anti-inflammatory properties, suggesting its potential for improving outcomes in endodontic therapy by minimizing the inflammatory response.

Vital pulp treatment (VPT) encompasses a range of biologically based procedures that aim to preserve the dental pulp, minimise intervention and improve long-term tooth survival. However, a lack of reliable, accurate diagnostic tests to assess the inflammatory status of the dental pulp coupled with use of outdated diagnostic nomenclature that does not support pulp preservation strategies, are two major limitations in effective implementation of VPT in clinical practice. This narrative review aims to discuss the limitations of the current diagnostic methods and the classification of pulp disease, while exploring emerging research on solutions designed to overcome these limitations. Contemporary evidence supports adoption of a pulp disease classification that best describes the disease as continuum, rather than dichotomous approach of reversible/irreversible pulpitis. The recent advances in understanding the pathophysiology of pulpitis at molecular level could be exploited for development of novel biomarker-based diagnostic chairside tests. Moving forward, a refinement of the nomenclatures describing pulpitis, together with integration of objective diagnostic technologies are crucial for improving the diagnostic accuracy and enabling more successful application of pulp preservation therapies and predictable treatment outcomes for patients.

Injured fibroblasts have been shown to play a significant role in controlling pulp inflammation and regeneration. Recent works have demonstrated that, depending on the stimulation type, fibroblasts induce macrophage differentiation into pro-inflammatory M1 or anti-inflammatory M2 phenotypes. This work was set to study macrophage effects on the initial steps of pulp regeneration, namely on pulp stem cell (DPSC) proliferation/migration as well as on neo-angiogenesis. Human pulp cells were isolated from third molars. DPSCs and pulp fibroblasts were obtained using cell sorting and characterisation. To mimic a deep carious lesion, fibroblasts were physically injured and incubated with Lipoteichoic Acid (LTA). Physically injured fibroblasts without adding LTA were used to simulate an inflammatory state without pathogen exposure. Undifferentiated macrophages (M0) were incubated with stimulated fibroblast supernatants to induce M1/M2 differentiation. After 24 h, the macrophage secretome was used to investigate the effects on DPSC viability with the MTT assay. Its effect on DPSC migration towards macrophages was performed using Boyden chambers. VEGF secretion by macrophages was quantified by ELISA. The macrophage secretome effect on endothelial cell viability was investigated using MTT assay, and that on neo-angiogenesis using endothelial cell organisation on Matrigel. The role played by macrophages in the initial steps of pulp inflammation and regeneration appears to be under the control of fibroblasts which determines macrophage differentiation into M1 or M2. Indeed, incubation of M0 with injured fibroblast supernatants induced a significant increase of DPSCs and endothelial cell viability, VEGF secretion, and endothelial cell organisation into tube-like structures. These effects are comparable to those of M2. However, incubating them with injured and LTA-stimulated fibroblasts induced similar effects to those of M1 macrophages including stem cell migration, VEGF secretion level and endothelial cell viability. This in vitro study shows that, depending on the type of stimulation, pulp fibroblasts induce macrophage differentiation into M1 or M2 which, in turn, modulate DPSC proliferation/migration and neo-angiogenesis. This highlights that interactions between different cell types play a significant role in the initial steps of pulp tissue regeneration.

Pulp tissue resides within rigid dentinal walls and depends on terminal circulation, making its healing capacity highly contingent on the degree of inflammation and suggesting the presence of intrinsic protective mechanisms to prevent necrosis. Multiple cell types including odontoblasts, fibroblasts, endothelial cells, and inflammatory cells, contribute to these mechanisms through the synthesis of bioactive molecules. Odontoblasts and pulp fibroblasts detect pathogen-associated molecular patterns via Toll-like receptors and release pro-inflammatory cytokines such as IL-6, IL-8, and TNF-α. These mediators, also produced during sterile inflammation, recruit phagocytic cells that clear pathogens and tissue debris.Pulp fibroblasts further contribute to innate defense by constitutively generating Complement components, including C3b, which opsonizes cariogenic bacteria, and the membrane attack complex, which lyses microbes. Physical injury or exposure to bacterial components enhances fibroblast production of these molecules, demonstrating potent local modulation during carious or traumatic insult.Beyond antimicrobial roles, fibroblasts influence repair by regulating stem cell recruitment and differentiation via Complement activation. They also participate in macrophage recruitment and polarization into M1 and M2 subsets. While M1 macrophages primarily mediate pathogen clearance, both phenotypes contribute to the early stages of pulp healing.This review examines key mechanisms governing local control of pulpal inflammation and repair, emphasizing emerging evidence that fibroblast-macrophage interactions and macrophage polarization are central regulators of both inflammatory resolution and initiation of tissue regeneration.

AIM:Injured fibroblasts have been shown to play a significant role in controlling pulp inflammation and regeneration. Recent works have demonstrated that, depending on the stimulation type, fibroblasts induce macrophage differentiation into pro-inflammatory M1 or anti-inflammatory M2 phenotypes. This work was set to study macrophage effects on the initial steps of pulp regeneration, namely on pulp stem cell (DPSC) proliferation/migration as well as on neo-angiogenesis. METHODOLOGY:Human pulp cells were isolated from third molars. DPSCs and pulp fibroblasts were obtained using cell sorting and characterisation. To mimic a deep carious lesion, fibroblasts were physically injured and incubated with Lipoteichoic Acid (LTA). Physically injured fibroblasts without adding LTA were used to simulate an inflammatory state without pathogen exposure. Undifferentiated macrophages (M0) were incubated with stimulated fibroblast supernatants to induce M1/M2 differentiation. After 24 h, the macrophage secretome was used to investigate the effects on DPSC viability with the MTT assay. Its effect on DPSC migration towards macrophages was performed using Boyden chambers. VEGF secretion by macrophages was quantified by ELISA. The macrophage secretome effect on endothelial cell viability was investigated using MTT assay, and that on neo-angiogenesis using endothelial cell organisation on Matrigel. RESULTS:The role played by macrophages in the initial steps of pulp inflammation and regeneration appears to be under the control of fibroblasts which determines macrophage differentiation into M1 or M2. Indeed, incubation of M0 with injured fibroblast supernatants induced a significant increase of DPSCs and endothelial cell viability, VEGF secretion, and endothelial cell organisation into tube-like structures. These effects are comparable to those of M2. However, incubating them with injured and LTA-stimulated fibroblasts induced similar effects to those of M1 macrophages including stem cell migration, VEGF secretion level and endothelial cell viability. CONCLUSION:This in vitro study shows that, depending on the type of stimulation, pulp fibroblasts induce macrophage differentiation into M1 or M2 which, in turn, modulate DPSC proliferation/migration and neo-angiogenesis. This highlights that interactions between different cell types play a significant role in the initial steps of pulp tissue regeneration.

Introduction Extracellular adenosine triphosphate (ATP) is a key alarmin in inflammatory responses and amplifies pain signalling through the activation of P2X3 receptors. This study aimed to validate a peripheral neuronal model, differentiated from human pulp stem cells, for the study of neuronal immunomodulation with relevance to ATP-P2X3 signalling. Methods Human dental pulp stem cells (DPSCs) were enriched from dental pulp cells obtained by explant culture from the pulp tissue of extracted third molar teeth. DPSCs were differentiated to peripheral neuronal equivalents (PNEs) in neurogenic media for 14 days. Characterisation of the neuronal transcriptome of PNEs was carried out by single-cell sequencing (ScRNA-seq). Transcriptomic and secretome changes in PNEs treated with the synthetic ATP analogue αβ-methylene ATP (αβmeATP) were analysed using ScRNA-seq and antibody microarray respectively. ATP release was measured using the ATPlite luciferase assay (Perkin Elmer) following mechanical stimulation by stretch or lipopolysaccharide (LPS) treatment of PNEs. The functional expression of P2X3 receptors was assessed by calcium mobilisation assays. Results PNEs were shown to express a neuronal gene signature, without the expression of genes typical of odontogenic cells. Transcriptomic analysis of PNEs following αβmeATP treatment demonstrated an enriched expression of pain and neuropathy pathways (P<0.05). Antibody microarray results indicated that the increased expression of 9 cytokines with αβmeATP treatment was attenuated by a P2X3 antagonist. Mechanical stretch or LPS treatment of PNEs resulted in increased ATP release (P<0.05). Responses to ATP were inhibited by a specific P2X3 antagonist indicating functional expression of P2X3 receptors in PNEs (P<0.001). Conclusions ScRNA-seq of the PNE model confirms a neuronal gene signature following differentiation, supporting its use as a relevant human model for studying neuronal immunomodulation and evaluating potential P2X3 receptor-targeted therapies for dental pain.

Rhinovirus (RV) is the leading cause of exacerbations of lung disease. A sensory neuronal model, derived from human dental pulp stem cells and differentiated into peripheral neuronal equivalents (PNEs), was used to examine RV’s effects on airway sensory nerves. We investigated whether RV can directly infect and alter PNEs or whether it exerts effects indirectly via the release of mediators from infected epithelial cells. PNEs or primary bronchial epithelial cells (PBECs) were infected with the RV-A16 strain. Viral replication was confirmed by viral titration assays, immunofluorescence (IF) for the double-stranded RNA (dsRNA) replication intermediate and western blotting (WB). RNA sequencing was used to determine transcriptomic changes in PNEs, and inflammatory responses were assessed by inflammatory microarray. Calcium mobilisation assays were used to investigate the effect of interleukin-1β (IL-1β) on PNE transient receptor potential (TRP) A1 channel responses. Viral titrations, WB and IF confirm RV-A16 entry and replication in PNEs and PBECs. Gene signatures associated with antiviral immune responses, sensory neuropathies and N-Methyl-D-aspartic acid (NMDA) receptor activity were upregulated in RV infected PNEs. Several cytokines were increased from PNEs and PBECs following RV infection, most notably IL-1β. Treatment of PNEs with IL-1β resulted in heightened TRPA1 channel sensitivity. We report the suitability of an airway neuronal model for the study of the direct effects of RV infection on nerves. RV-induced release of IL-1β from airway epithelium heightens neuronal TRPA1 responses suggesting a mechanism for virus-induced cough hypersensitivity.

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.

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.

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. 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 Ca 2+ imaging studies to assess the functionality of P2X3 receptors as well as membrane depolarization. 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. 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).

Complement C5a protein has been shown to play a major role in tissue regeneration through interaction with its receptor (C5aR) on target cells. Expression of this receptor has been reported in the nervous system which, upon injury, has no treatment to restore the lost functions. This work aimed at investigating the Complement C5a effect on axonal growth after axotomy in vitro. Primary hippocampal neurons were isolated from embryonic Wistar rats. Cell expression of C5aR mRNA was verified by RT-PCR while its membrane expression, localization, and phosphorylation were investigated by immunofluorescence. Then, the effects of C5a on injured axonal growth were investigated using a 3D-printed microfluidic device. Immunofluorescence demonstrated that the primary cultures contained only mature neurons (93%) and astrocytes (7%), but no oligodendrocytes or immature neurons. Immunofluorescence revealed a co-localization of NF-L and C5aR only in the mature neurons where C5a induced the phosphorylation of its receptor. C5a application on injured axons in the microfluidic devices significantly increased both the axonal growth speed and length. Our findings highlight a new role of C5a in regeneration demonstrating an enhancement of axonal growth after axotomy. This may provide a future therapeutic tool in the treatment of central nervous system injury.

AimsDental 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.MethodologyPolysialylated-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.ResultsPSA-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 (TM) in response to potassium chloride. Interestingly, only differentiated PSA-NCAM+ cells were capable of spontaneous membrane oscillations.ConclusionsIn 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).

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-β1 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-β1 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.

To evaluate the expression and function of the nod-like receptor pyrin domain containing 3 (NLRP3) inflammasome in caries induced pulpitis. 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. NLRP3 inflammasome was differentially expressed in dental pulp of sound and carious teeth. Treatment of DPCs with LTA significantly upregulates NLRP3 and IL-1 β-expression ( p  < .05) and in induces more ASC specks formation compared to LPS. IL-β 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-1β and IL-6 in an ex-vivo pulpitis model ( p  < .01) but had no effect on IL-8 or matrix metalloproteinase-9 (MMP-9). 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.

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 beta-expression (p < .05) and in induces more ASC specks formation compared to LPS. IL-beta 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-1 beta 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.

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.