Osteopontin interacts with dendritic cells and macrophages in pulp inflammation: Comprehensive transcriptomic analysis and laboratory investigations.

AIM: To investigate novel diagnostic markers for pulpitis and validate by clinical samples from normal and inflamed pulp. To explore the relationship between diagnostic markers and immune cells or their phenotypes during pulp inflammation. METHODOLOGY: Two microarray datasets, GSE77459 and GSE92681, and identified differential expression genes were integrated. To understand immune features, gene functions, Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), Disease Ontology (DO) and ImmuneSigDB Gene Set Enrichment Analysis (GSEA) were analysed. For predictive purposes, machine learning techniques were applied to detect diagnostic markers. Immune infiltration in inflamed pulp was studied using CIBERSORT. The relationship between diagnostic markers and immune cells was investigated and validated their gene expression in clinical samples from the normal or inflamed pulp by qRT-PCR. Finally, the correlation between one marker, secreted phosphoprotein 1 (SPP1), encoding osteopontin (OPN), and dendritic cells (DCs)/macrophages was identified via HE staining and multiplex immunohistochemistry. An in vitro inflammatory dental pulp microenvironment model of THP-1 macrophages cocultured with dental pulp cells derived conditioned media (DPCs-CM) to investigate OPN production and macrophage phenotypes was established. RESULTS: Analysis revealed unique immunologic features in inflamed pulp. Three diagnostic markers for pulpitis: endothelin-1 (EDN1), SPP1, and purine nucleoside phosphorylase (PNP), and validated them using qRT-PCR were predicted. Multiplex immunohistochemistry demonstrated OPN co-localized with activated DCs and M2 macrophages during pulp inflammation. In vitro experiments showed that THP-1 macrophages produced the highest levels of OPN when stimulated with DPCs-CM derived from the 20 µg/mL LPS pre-conditioned group, suggesting an M2b-like phenotype by increasing surface marker CD86 and expression of IL6, TNFα, IL10, and CCL1 but not CCL17 and MerTK. Levels of CCL1 and IL10 elevated significantly in the macrophages' supernatant from the 20 µg/mL LPS pre-conditioned CM group. OPN was proven co-localizing with CD86 in the inflamed pulp by immunofluorescence. CONCLUSIONS: The current findings suggest that OPN can serve as a promising biomarker for pulpitis, correlated with DCs and macrophages. OPN+ macrophages in the inflamed pulp are associated with M2b-like phenotypes. These insights offer the potential for improved diagnosis and targeted therapy.

Moving silicone oil particles in the ventricle: a case report and updated review.

BACKGROUND: The movement of intraventricular silicone oil observed in the supine position is extremely rare. Herein, we describe a patient who presented with dynamically moving silicone oil particles in the ventricle when changing position and provide an updated review of this phenomenon. CASE PRESENTATION: We report a case of a 70-year-old woman who presented with intraventricular hyperdensities that were occasionally found on brain computed tomography (CT). Initial nonenhanced brain CT demonstrated nondependent hyperdensities in the bilateral anterior horns of the lateral ventricles, the third ventricle, and the right suprasellar cistern, mimicking an intraventricular hemorrhage. Further brain magnetic resonance imaging (MRI) in the supine position revealed abnormal signals in the bilateral anterior horns of the lateral ventricles, the posterior horn of the right lateral ventricle, the third ventricle, the right suprasellar cistern, and the bilateral eyeballs, with isosignal intensities surrounded by low-signal chemical shift artifacts on T1-weighted imaging and variable signals (hypo- or hyperintensity) on T2-weighted imaging. The lesion in the anterior horn of the right ventricle largely moved to the posterior horn of the ipsilateral ventricle. The final craniocervical CT angiography showed that the lesion in the posterior horn had moved back to the anterior horn of the right lateral ventricle. These features were consistent with intraventricular silicone oil migration. The final spinal MRI did not demonstrate a migration of silicone oil into the spinal subarachnoid space. DISCUSSION AND CONCLUSIONS: This case report describes a dynamic process of silicone oil displacement in the supine position and provides a comprehensive imaging presentation. The moving pattern and a characteristic chemical shift artifact on MRI are key to the diagnosis and may help prevent unnecessary examinations or intervention.

A novel, complex RUNX2 gene mutation causes cleidocranial dysplasia.

BACKGROUND: Haploinsufficiency of the runt-related transcription factor 2 (RUNX2) gene is known to cause cleidocranial dysplasia (CCD). Here, we investigated a complex, heterozygous RUNX2 gene mutation in a Chinese family with CCD and the pathogenesis associated with the variations. METHODS: Genomic DNA extracted from peripheral venous blood was taken from the proband, her parents and 3 siblings, and 150 normal controls. Analysis of their respective RUNX2 gene sequences was performed by PCR amplification and Sanger sequencing. Pathogenesis associated with RUNX2 mutations was investigated by performing bioinformatics, real-time PCR, western blot analysis, and subcellular localization studies. RESULTS: We identified 2 complex heterozygous mutations involving a c.398-399 insACAGCAGCAGCAGCA insertion and a c.411-412 insG frameshift mutation in exon 3 of the RUNX2 gene. The frameshift mutation changed the structure of the RUNX2 protein while did not affect its expression at the mRNA level. Transfection of HEK293T cells with a plasmid expressing the RUNX2 variant decreased the molecular weight of the variant RUNX2 protein, compared with that of the wild-type protein. Subcellular localization assays showed both nuclear and cytoplasmic localization for the mutant protein, while the wild-type protein localized to the nucleus. CONCLUSIONS: Our findings demonstrated that the novel c.398-399insACAGCAGCAGCAGCA mutation occurred alongside the c.411-412insG frameshift mutation, which resulted in RUNX2 truncation. RUNX2 haploinsufficiency was associated with CCD pathogenesis. These results extend the known mutational spectrum of the RUNX2 gene and suggest a functional role of the novel mutation in CCD pathogenesis.

lncRNA expression signatures in periodontitis revealed by microarray: the potential role of lncRNAs in periodontitis pathogenesis.

Periodontitis, a common chronic inflammatory disease of the periodontium, is caused by dental plaque formation induced by microorganisms. Recent studies have demonstrated that lncRNAs play a critical role in the regulation of gene expression and in the pathogenesis of diseases. To demonstrate that periodontitis is associated with lncRNAs, microarray analysis was used to detect differently expressed lncRNAs in chronic periodontitis and adjacent normal tissues. The results of some differently expressed lncRNAs were further confirmed using real-time PCR. A total of 8925 differentially expressed lncRNAs were detected, including 4313 upregulated lncRNAs and 4612 downregulated lncRNAs. Further lncRNA subgroup analysis showed there were 589 enhancer-like lncRNAs, 238 homeobox (HOX) cluster lncRNAs, and 1218 Rinn's lincRNAs, of which 656 lincRNAs were upregulated and 562 lincRNAs were downregulated. Therefore, we confirmed that lncRNAs were differently expressed in chronic periodontitis tissues compared with adjacent normal tissues, indicating that lncRNAs may exert partial or key roles in periodontitis pathogenesis and development. Taken together, this study may provide potential targets for future treatment of periodontitis and novel diagnostic biomarkers for periodontitis.

Sema3A Drives Alternative Macrophage Activation in the Resolution of Periodontitis via PI3K/AKT/mTOR Signaling.

Macrophages actively participate in immunomodulatory processes throughout periodontal inflammation. Regulation of M1/M2 polarization affects macrophage chemokine and cytokine secretion, resulting in a distinct immunological status that influences prognosis. Semaphorin 3A (Sema3A), a neurite growth factor, exerts anti-inflammatory effects. In this study, we investigated the immunomodulation of Sema3A on macrophage-related immune responses in vivo and in vitro. Topical medications of Sema3A in mice with periodontitis alleviated inflammatory cell infiltration into gingival tissue and reduced areas with positive IL-6 and TNFα expression. We observed that the positive area with the M2 macrophage marker CD206 increased and that of the M1 macrophage marker iNOS decreased in Sema3A-treated mice. It has been postulated that Sema3A alleviates periodontitis by regulating alternative macrophage activation. To understand the mechanism underlying Sema3A modulation of macrophage polarization, an in vitro macrophage research model was established with RAW264.7 cells, and we demonstrated that Sema3A promotes LPS/IFNγ-induced M1 macrophages to polarize into M2 macrophages and activates the PI3K/AKT/mTOR signaling pathways. Inhibition of the PI3K signaling pathway activation might reduce anti-inflammatory activity and boost the expression of the inflammatory cytokines, iNOS, IL-12, TNFα, and IL-6. This study indicated that Sema3A might be a feasible drug to regulate alternative macrophage activation in the inflammatory response and thus alleviate periodontitis.

Saccharibacteria (TM7), but not other bacterial taxa, are associated with childhood caries regardless of age in a South China population.

Background: Human microbiome dysbiosis is related to various human diseases, and identifying robust and consistent biomarkers that apply in different populations is a key challenge. This challenge arises when identifying key microbial markers of childhood caries. Methods: We analyzed unstimulated saliva and supragingival plaque samples from children of different ages and sexes, performed 16S rRNA gene sequencing, and sought to identify whether consistent markers exist among subpopulations by using a multivariate linear regression model. Results: We found that Acinetobacter and Clostridiales bacterial taxa were associated with caries in plaque and saliva, respectively, while Firmicutes and Clostridia were found in plaque isolated from children of different ages in preschool and school. These identified bacterial markers largely differ between different populations, leaving only Saccharibacteria as a significant caries-associated phylum in children. Saccharibacteria is a newly identified phylum, and our taxonomic assignment database could not be used to identify its specific genus. Conclusion: Our data indicated that, in a South China population, oral microbial signatures for dental caries show age and sex differences, but Saccharibacteria might be a consistent signal and worth further investigation, considering the lack of research on this microbe.

980 nm Photobiomodulation Promotes Osteo/Odontogenic Differentiation of the Stem Cells from Human Exfoliated Deciduous Teeth Via the Cross talk Between BMP/Smad and Wnt/ß-Catenin Signaling Pathways.

Increasing evidence suggests stem cells from human exfoliated deciduous teeth (SHEDs) serve as desirable sources of dentin regeneration. Photobiomodulation (PBM) has shown great potential in enhancing the proliferation and osteogenesis of human bone marrow mesenchymal stem cells (hBMMSCs). However, the specific role of PBM in odontogenic differentiation of SHEDs is little know, and we further investigated potential mechanism of PBM osteo/odontogenisis. A 980 nm diode laser with different energy densities of (0.5, 5, 10 J cm-2 ) in a 100-mW continuous wave was used for irradiation every 24 h. Osteo/odontogenic differentiation of SHEDs was achieved by performing alkaline phosphatase (ALP) and alizarin red staining (ARS) and osteo/odontogenic markers were also evaluated by qRT-PCR and western blotting. Additionally, western blot and immunohistochemical staining were performed to evaluate the levels of BMP/Smad and Wnt/ß-catenin signaling-related proteins. We found that PBM at 5 J cm-1 increased mineral deposition and upregulated the expression of related osteo/odontogenic markers along with the elevated expression of ß-catenin and phosphorylation level of Smad1/5/9. Furthermore, Wnt signaling inhibition using DKK1 and BMP signaling inhibition using noggin inhibited PBM-induced osteo/odontogenic marker expression when used individually or jointly. In conclusion, PBM induces the osteo/odontogenic differentiation of SHEDs through cross talk between BMP/Smad and Wnt/ß-catenin signaling pathways.

Effect of LncRNA-MALAT1 on mineralization of dental pulp cells in a high-glucose microenvironment.

Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) belongs to the long non-coding RNA (LncRNA) family. LncRNA-MALAT1 is expressed in a variety of tissues and is involved in a variety of diseases and biological processes. Although LncRNA-MALAT1 is upregulated in a high-glucose microenvironment and may participate in odontogenic differentiation, the underlying mechanism is not yet well elucidated. Here, we show that MALAT1 was mainly expressed in the cytoplasm of dental pulp cells (DPCs) in situ hybridization. In addition, high levels of mineralization-related factors, namely, tumor growth factors ß 1 and 2 (TGFß-1 and TGFß-2), bone morphogenetic proteins 2 and 4 (BMP2 and BMP4), bone morphogenetic protein receptor 1 (BMPR1), SMAD family member 2 (SMAD2), runt-related transcription factor 2 (RUNX2), Msh homeobox 2 (MSX2), transcription factor SP7 (SP7), alkaline phosphatase (ALP), dentin matrix acidic phosphoprotein 1 (DMP1), and dentin sialophosphoprotein (DSPP), were expressed, and MALAT1 was significantly overexpressed in DPCs 7 and 14 days after mineralization induction in a high-glucose microenvironment, but only TGFß-1, BMP2, MSX2, SP7, ALP, and DSPP were significantly downregulated in DPCs after MALAT1 inhibition. MALAT1 may participate in the mineralization process of DPCs by regulating multiple factors (TGFß-1, BMP2, MSX2, SP7, ALP, and DSPP).

Dental pulp stem cells from human teeth with deep caries displayed an enhanced angiogenesis potential in vitro.

BACKGROUND/PURPOSE: Dental pulp stem cells can be isolated from human teeth with deep caries (cDPSCs), but their biological characteristics are still unclear. The aim of this study was to investigate the angiogenic potential of cDPSCs and compare them to dental pulp stem cells from human normal teeth (nDPSCs). MATERIALS AND METHODS: Cells were isolated from human pulp tissue of normal and infected teeth with deep caries. Basic mesenchymal stem cell (MSC) characterization was conducted. Colony forming units and proliferation ability were evaluated in nDPSCs and cDPSCs. Expression of VEGF in both tissues and cells was examined by immunohistochemical staining. After stimulating nDPSCs and cDPSCs with an angiogenic medium, angiogenic markers were evaluated by qRT-PCR and western blotting. Finally, tube formation assays were used to evaluate the in vitro angiogenesis potential of both cell populations. RESULTS: Both nDPSCs and cDPSCs possessed typical MSC characteristics. cDPSCs had enhanced colony formation and proliferation capacities than nDPSCs did. The expression of VEGF was higher in pulp tissue from teeth with deep caries and cDPSCs than in normal tissue and nDPSCs. When both cell types were grown in vitro under angiogenic conditions, cDPSCs expressed a higher level of angiogenic markers and showed a stronger angiogenesis potential than nDPSCs did. CONCLUSION: cDPSCs maintained MSC traits and presented a higher angiogenesis potential than nDPSCs.

Exosomes from LPS-Stimulated hDPSCs Activated the Angiogenic Potential of HUVECs In Vitro.

BACKGROUND: Exosomes from human dental pulp stem cells (hDPSCs) were indicated to play a positive role in vascular regeneration processes. But the angiogenic capabilities of exosomes from inflammatory hDPSCs and the underlying mechanism remain unknown. In this study, the inflammatory factor lipopolysaccharide (LPS) was used to stimulate hDPSCs, and exosomes were extracted from these hDPSCs. The proangiogenic potential of exosomes was examined, and the underlying mechanism was studied. METHOD: Exosomes were isolated from hDPSCs with or without LPS stimulation (N-EXO and LPS-EXO) and cocultured with human umbilical vein endothelial cells (HUVECs). The proangiogenic potential of exosomes was evaluated by endothelial cell proliferation, migration, and tube formation abilities in vitro. To investigate the proangiogenic mechanism of LPS-EXO, microRNA sequencing was performed to explore the microRNA profile of N-EXO and LPS-EXO. Gene Ontology (GO) analysis was used to study the functions of the predicted target genes. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis was used to estimate the signaling pathways associated with the inflammation-induced angiogenesis process. RESULT: Compared to the uptake of N-EXO, uptake of LPS-EXO activated the angiogenic potential of HUVECs by promoting the proliferation, migration, and tube formation abilities in vitro. The mRNA expression levels of vascular endothelial growth factor (VEGF) and kinase-insert domain-containing receptor (KDR) in the LPS-EXO group were significantly higher than those in the N-EXO group. MicroRNA sequencing showed that 10 microRNAs were significantly changed in LPS-EXO. Pathway analysis showed that the genes targeted by differentially expressed microRNAs were involved in multiple angiogenesis-related pathways. CONCLUSION: This study revealed that exosomes derived from inflammatory hDPSCs possessed better proangiogenic potential in vitro. This is the first time to explore the role of exosomal microRNA from hDPSCs in inflammation-induced angiogenesis. This finding sheds new light on the effect of inflammation-stimulated hDPSCs on tissue regeneration.

Differential expression of long noncoding RNAs from dental pulp stem cells in the microenvironment of the angiogenesis.

INTRODUCTION: Angiogenesis is important in pulp-dentin formation. Among the regulatory factors, long noncoding RNA (LncRNA) is a class of functional RNA molecules that are not translated into protein and involved in regulating multiple physiological processes. The different expression of LncRNA and its target gene in dental pulp stem cells (DPSCs) were explored and may provide a theoretical basis for future regulation of dental pulp angiogenesis. METHODS: In this study, we cultured DPSCs from healthy dental pulp tissues and divided them into two groups: the normal DPSCs and the DPSCs cultured in vascular induction medium. In total, 40,173 LncRNA probes and 20,730 protein coding mRNAs were detected through microarray, which were then verified by the quantitative reverse transcription-polymerase chain reaction (qRT-PCR) method. RESULTS: The result of differential expressions measured in LncRNA through microarray showed that 376 LncRNAs increased significantly and 426 were downregulated among the two groups of cells. Moreover, the mRNA microarray in normal cultured DPSCs showed that 629 LncRNAs were significantly upregulated, while 529 of them were downregulated compared with the DPSCs that were cultured in vascular induction medium. Gene ontology (GO) analysis inferred the molecular function of mRNAs. Pathway analysis showed that 52 signaling pathways were involved in the differentiation process of DPSCs. qRT-PCR analysis, conducted for validation, showed results consistent with the microarray analysis. CONCLUSIONS: We found that a number of different regulators are involved in inducing vascular differentiation of DPSCs, which provides a foundation for subsequent experiments.

Establishment of an Immortalized Mouse Bmp2 Knockout Dental Papilla Mesenchymal Cell Line.

Bone morphogenetic protein 2 (Bmp2) is essential for dentin formation. Bmp2 cKO mice exhibited similar phenotype to dentinogenesis imperfecta (DGI), showing dental pulp exposure, hypomineralized dentin, and delayed odontoblast differentiation. As it is relatively difficult to obtain primary Bmp2 cKO dental papilla mesenchymal cells and to maintain a long-term culture of these primary cells, availability of immortalized deleted Bmp2 dental papilla mesenchymal cells is critical for studying the underlying mechanism of Bmp2 signal in odontogenesis. Here we describe the generation of an immortalized deleted Bmp2 dental papilla mesenchymal (iBmp2ko/ko-dp) cell line by introducing Cre fluorescent protein (GFP) into the immortalized mouse floxed Bmp2 dental papilla mesenchymal (iBmp2flox/flox-dp) cells.

Effects of vascular endothelial growth factor and insulin growth factor­1 on proliferation, migration, osteogenesis and vascularization of human carious dental pulp stem cells.

The present study aimed to investigate the effects of vascular endothelial growth factor (VEGF) and insulin­like growth factor­1 (IGF­1) on the proliferation, migration and differentiation of human carious dental pulp stem cells (hCDPSCs), and to elucidate the underlying mechanism(s). Cell counting kit­8 assay was used to detect the effect of different concentrations of IGF­1 and VEGF on the proliferation of hCDPSCs. Transwell assay was used to detect the migratory ability of the hCDPSCs. Alizarin red and alkaline phosphatase (ALP) staining were used to detect the osteogenic ability of hCDPSCs, whereas the angiogenic ability of the hCDPSCs was tested by tube formation assay. Reverse transcription­quantitative polymerase chain reaction (RT­qPCR) and western blotting were used to detect the expression levels of associated genes and proteins. IGF­1 (100 ng/ml) or VEGF (25 ng/ml) alone were revealed to be able to promote proliferation and migration of hCDPSCs; however, the combined use of IGF­1 and VEGF enhanced this effect when compared with the use of either agent in isolation. Alizarin red and ALP staining revealed that the use of either VEGF or IGF­1 alone did not result in any significant effects, whereas their use in combination promoted the osteogenic differentiation of hCDPSCs. In addition, the RT­qPCR and western blotting analyses revealed that the expression levels of Runt­related transcription factor 2 (RUNX2), bone sialoprotein (BSP) and ALP were increased upon combined treatment of the cells with VEGF and IGF­1. The expression levels of VEGF and plateletderived growth factor (PDGF) in hCDPSCs were enhanced upon treatment with either VEGF or IGF­1 in isolation, with greater effects observed when VEGF and IGF­1 were added in combination, indicating that VEGF and IGF­1 may exert a synergistic role in these events. Further experiments revealed that the combination of VEGF and IGF­1 led to an activation of the AKT signaling pathway. The proliferation and angiogenesis of hCDPSCs were also shown to be more effective compared with treatment with either VEGF or IGF­1 in isolation. Taken together, the present study has demonstrated that the combined use of VEGF and IGF­1 leads to an increase in the proliferation, migration, osteogenesis and angiogenesis of hCDPSCs and, furthermore, these signaling molecules may mediate their effects via activation of the AKT signaling pathway.

Systemically Transplanted Bone Marrow-derived Cells Contribute to Dental Pulp Regeneration in a Chimeric Mouse Model.

INTRODUCTION: Migratory cells via blood circulation or cells adjacent to the root apex may potentially participate in dental pulp tissue regeneration or renewal. This study investigated whether systemically transplanted bone marrow cells can contribute to pulp regeneration in a chimeric mouse model. METHODS: A chimeric mouse model was created through the injection of bone marrow cells from green fluorescent protein (GFP) transgenic C57BL/6 mice into the tail veins of recipient wild-type C57BL/6 mice that had been irradiated with a lethal dose of 8.5 Gy from a high-frequency linear accelerator. These mice were subjected to pulpectomy and pulp revascularization. At 1, 4, and 8 weeks after surgery, in vivo animal imaging and histologic analyses were conducted. RESULTS: In vivo animal imaging showed that the green biofluorescence signal from the transplanted GFP+ cells increased significantly and was maintained at a high level during the first 4 weeks after surgery. Immunofluorescence analyses of tooth specimens collected at 8 weeks postsurgery showed the presence of nestin+/GFP+, α smooth muscle actin (α-SMA)/GFP+, and NeuN/GFP+ cells within the regenerated pulplike tissue. CONCLUSIONS: These data confirm that transplanted bone marrow-derived cells can contribute to dental pulp regeneration.