Homeostatic maintenance is essential for pulp function. Disrupting pulp homeostasis may lead to pulp degeneration, such as fibrosis and calcifications. Sensory nerves constitute a crucial component of the dental pulp. However, the precise involvement of sensory nerves in pulp homeostasis remains uncertain. In this study, we observed the short-term and long-term histological changes in the dental pulp after inferior alveolar nerve transection. Additionally, we cultured primary dental pulp cells (DPCs) from the innervated and denervated groups and compared indicators of cellular senescence and cellular function. The results revealed that pulp fibrosis occurred at 2 w after the operation. Furthermore, the pulp area, as well as the height and width of the pulp cavity, showed accelerated reductions after sensory denervation. Notably, the pulp area at 16 w after the operation was comparable to that of 56 w old rats. Sensory denervation induced excessive extracellular matrix (ECM) deposition and increased predisposition to mineralization. Furthermore, sensory denervation promoted the senescence of DPCs. Denervated DPCs exhibited decelerated cell proliferation, arrest in the G2/M phase of the cell cycle, imbalance in the synthesis and degradation of ECM, and enhanced mineralization. These findings indicate that sensory nerves play an essential role in pulp homeostasis maintenance and dental pulp cell fate decisions, which may provide novel insights into the prevention of pulp degeneration.

Ideal regeneration of hard tissue and dental pulp has been reported with the use of a combination of bioactive glass and basic fibroblast growth factor (bFGF). However, no previous study has investigated the molecular mechanisms underlying the processes induced by this combination in dental pulp cells. This study aimed to examine the cellular phenotype and transcriptional changes induced by the combination of bioactive glass solution (BG) and bFGF in dental pulp cells using phase-contrast microscopy, a cell counting kit-8 assay, alkaline phosphatase staining, and RNA sequence analysis. bFGF induced elongation of the cell process and increased the number of cells. Whereas BG did not increase ALP activity, it induced extracellular matrix-related genes in the dental pulp. In addition, the combination of BG and bFGF induces gliogenesis-related genes in the nervous system. This is to say, bFGF increased the viability of dental pulp cells, bioactive glass induced odontogenesis, and a dual stimulation with bioactive glass and bFGF induced the wound healing of the nerve system in the dental pulp. Taken together, bioactive glass and bFGF may be useful for the regeneration of the dentin-pulp complex.

UNASSIGNED: Nanoparticulate Ca(OH)2 had greater antibacterial effect than conventional Ca(OH)2. Conversely, a study reported that nanoparticulate Ca(OH)2 had toxicity against murine fibroblast. However, the study of nanoparticulate Ca(OH)2, involving human dental pulp cells (DPCs) and apical papilla cells (APCs) is lacking. The aim of this study is to compare the effects of conventional Ca(OH)2 and nanoparticulate Ca(OH)2 on the viability of DPCs and APCs.
UNASSIGNED: Primary human DPCs/APCs from the 3rd to 5th passage were divided into control and experimental groups. In the control group, cells were cultured in complete media. In the experimental group, cells were cultured in complete media containing 10, 100, or 1000 μg/mL of either conventional Ca(OH)2 or nanoparticulate Ca(OH)2 for 1, 3, 5, and 7 days. After the treatment period, the cells were tested for viability using MTT assay.
UNASSIGNED: DPCs treated with conventional Ca(OH)2 in all concentrations at day 5 revealed significantly higher proliferation compared to nanoparticulate Ca(OH)2 treated groups. In additions, DPCs treated with 1000 µg/ml nanoparticulate Ca(OH)2 at day7 were significantly lower proliferation compared to DPCs treated with conventional Ca(OH)2. In contrast, APCs treated with 1000 µg/ml nanoparticulated Ca(OH)2 were significantly higher proliferation than APCs treated with 1000 µg/ml conventional Ca(OH)2 at day7.
UNASSIGNED: Nanoparticulate Ca(OH)2 increased the viability of APCs and can be an alternative choice of intracanal medication for regenerative endodontic procedures. However, Nanoparticulate Ca(OH)2 exerted some effects on DPCs. The use of nanoparticulate Ca(OH)2 has no advantages over the conventional Ca(OH)2 for vital pulp therapy.

OBJECTIVE: The primary goal of this study was to investigate the potential effects of A5G81 in inducing reparative dentine (RD) formation both in vitro and in vivo.
METHODS: Cell adhesion was observed by crystal violet staining and quantified by Sodium Dodecyl Sulphate (SDS) extraction. Cell proliferation was investigated using Cell Counting Kit-8 (CCK-8) assay. Spreading of cytoskeleton was visualized using immunofluorescence staining. Protein expression level of Akt signalling pathway was compared in a human Akt pathway phosphorylation array. Genes that were up or downregulated by A5G81 were identified by RNA sequencing. The mRNA expression of odontoblastic markers was detected by quantitative real-time polymerase chain reaction (qPCR). Moreover, mineralization of human dental pulp cells (hDPCs) was visualized by alizarin red staining and quantified using cetylpyridinium chloride (CPC). A direct pulp-capping model was established in SD rats and the RD formation at 2 weeks after operation was observed using HE staining.
RESULTS: A5G81 (optimal coating concentration: 0.5 mg/mL) promoted hDPCs adhesion and proliferation to a level that was similar to Type I collagen (COL-1). Meanwhile, A5G81 activated Akt signalling pathway, albeit to a lesser extent than COL-1. An inhibition test indicated that A5G81 induced hDPCs adhesion by activating PI3K pathway. A5G81 induced the expression of ECM remodelling genes and odontoblastic genes, which were demonstrated by RNA-seq and qPCR, respectively. In addition, A5G81 efficiently accelerated the mineralization of hDPCs in both immobilized and soluble forms, a property that makes it more applicable in dental clinic. Finally, the pulp-capping study in rats suggested that use of A5G81 could successfully induce the formation of RD within 2 weeks.
CONCLUSIONS: Coating of A5G81 to non-tissue culture-treated polystyrene facilitates spreading, proliferation and differentiation of hDPCs, resulting in rapid RD formation in artificially exposed pulp.

Concentrated growth factors (CGF) is the newest generation platelet concentrate product, which has been reported to promote the proliferation and differentiation of human dental pulp cells (hDPCs). However, the effect of liquid phase of CGF (LPCGF) has not been reported. This study was aimed to evaluate the influence of LPCGF on the biological properties of hDPCs, and to explore the in vivo mechanism of dental pulp regeneration based on the hDPCs-LPCGF complex transplantation. It was found that LPCGF could promote the proliferation, migration and odontogenic differentiation of hDPCs, and 25% LPCGF induced the most mineralization nodule formation and the highest DSPP gene expression. The heterotopic transplantation of the hDPCs-LPCGF complex resulted in the formation of regenerative pulp tissue with newly formed dentin, neovascularization and nerve-like tissue. Together, these findings provide key data on the effect of LPCGF on the proliferation, migration, odontogenic/osteogenic differentiation of hDPCs, and the in vivo mechanism of hDPCs-LPCGF complex autologous transplantation in pulp regeneration therapy.

OBJECTIVE: Pyroptosis is a type of inflammatory cell death and is related to pulpitis and apical periodontitis. In this study, the aim was to investigate how periodontal ligament fibroblasts (PDLFs) and dental pulp cells (DPCs) respond to pyroptotic stimuli and explore whether dimethyl fumarate (DMF) could block pyroptosis in PDLFs and DPCs.
METHODS: Three methods (stimulation with lipopolysaccharide [LPS] plus nigericin, poly(dA:dT) transfection and LPS transfection) were used to induce pyroptosis in PDLFs and DPCs, two types of fibroblasts related to pulpitis and apical periodontitis. THP-1 cell was used as a positive control. Afterwards, PDLFs and DPCs were treated with or without DMF before inducing pyroptosis to examine the inhibitory effect of DMF. Pyroptotic cell death was measured by lactic dehydrogenase (LDH) release assays, cell viability assays, propidium iodide (PI) staining and flow cytometry. The expression levels of cleaved gasdermin D N-terminal (GSDMD NT), caspase-1 p20, caspase-4 p31 and cleaved PARP were examined by immunoblotting. Immunofluorescence analysis was used to detect the cellular distribution of GSDMD NT.
RESULTS: Periodontal ligament fibroblasts and DPCs were more sensitive to cytoplasmic LPS-induced noncanonical pyroptosis than to canonical pyroptosis induced by stimulation with LPS priming plus nigericin or by poly(dA:dT) transfection. In addition, treatment with DMF attenuated cytoplasmic LPS-induced pyroptotic cell death in PDLFs and DPCs. Mechanistically, it was shown that the expression and plasma membrane translocation of GSDMD NT were inhibited in DMF-treated PDLFs and DPCs.
CONCLUSIONS: This study indicates that PDLFs and DPCs are more sensitive to cytoplasmic LPS-induced noncanonical pyroptosis and that DMF treatment blocks pyroptosis in LPS-transfected PDLFs and DPCs by targeting GSDMD, suggesting DMF might be a promising drug for the management of pulpitis and apical periodontitis.

Dental caries or trauma can expose human dental pulp cells (DPCs) to various oral microorganisms, which play an important role in the development of an innate immune response. In the present study, we examined the expression of Toll-like receptors (TLRs) for sensing microbe-associated molecular patterns in human DPCs. Interestingly, real-time PCR analysis demonstrated that TLR3 is the most highly expressed among 10 different TLRs in human DPCs. Poly(I:C), a representative TLR3 ligand mimicking viral double-stranded RNA, potently induced IL-8 expression in a time- and dose-dependent manner. Concordantly, poly(I:C) treatment substantially increased the expression of pro-inflammatory cytokines and chemokines such as IL-6, CCL2, and CXCL10. Human DPCs transfected with TLR3 siRNA exhibited decreased IL-8 production compared with non-targeting siRNA-transfected cells, suggesting that the expression of poly(I:C)-induced inflammatory cytokines is dependent on TLR3. IL-8 secretion induced by poly(I:C) was down-regulated by MAP kinase inhibitors, indicating that the MAP kinase pathway contributes to IL-8 production. Furthermore, C/EBPβ and NF-κB were essential transcriptional factors for poly(I:C)-induced IL-8 expression, as demonstrated by the transient transfection and reporter gene assay. Since lipoproteins are known as major immunostimulatory components of bacteria, human DPCs were treated with poly(I:C) together with Pam2CSK4, a synthetic lipopeptide mimicking bacterial lipoproteins. Pam2CSK4 and poly(I:C) co-treatment synergistically increased IL-8 production in comparison to Pam2CSK4 or poly(I:C) alone, implying that co-infection of viruses and bacteria can synergistically induce inflammatory responses in the dental pulp. Taken together, these results suggest that human DPCs potentially sense and respond to viral double-stranded RNAs, leading to effective induction of innate immune responses.