Inflamed Human Pulp Research Articles

Investigating In Situ Expression of Neurotrophic Factors and Partner Proteins in Irreversible Pulpitis

IntroductionIn situ assessments of neurotrophic factors and their associated molecular partners have not been explored to date, particularly in humans. The present investigation aimed to explore the expressional dysregulation of neurotrophic factors (nerve growth factor [NGF], brain derived neurotrophic factor [BDNF], and NT4/5), their receptors (TrkA and TrkB), and their modulators (USP36 and Nedd4-2) directly in irreversibly inflamed human pulp tissues. MethodsForty samples each of healthy and irreversibly inflamed pulp were extirpated for the study. Immunohistochemical examinations were carried out for the anatomic changes and expression of neurotrophic factors and partner proteins. Expression was digitally quantified using the IHC profiler module of ImageJ and deduced as optical density. Statistical analyses were carried out by GraphPad Prism. ResultsDecrease in nuclear and vessel diameters was observed in irreversibly inflamed pulp tissues. NGF and BDNF were found to be significantly upregulated in symptomatic irreversible pulpitis (SIP), whereas no significant difference was observed in the expression of TrkA and TrkB. Expression of Nedd4-2, USP36, and TrkA was found positively correlated with the NGF in healthy pulp tissues. However, in SIP, positive correlation was only observed between the expression of USP36 and NGF. Among the ligands, BDNF expression was found positively correlated with NGF in healthy pulp but not with NT4/5. In the case of SIP, no correlation was observed between any neurotrophic factors. ConclusionsUpregulation of NGF, BDNF, USP36 and Nedd4-2 in SIP indicates dysregulation in the molecular events underlying the disease biology and could be exploited as potential markers for the disease diagnosis.

MicroRNA-126 inhibits vascular cell adhesion molecule-1 and interleukin-1beta in human dental pulp cells.

BackgroundVascular cell adhesion molecule (VCAM‐1) mediates pulpitis via regulating interleukin (IL)‐1β. microRNA (miR)‐126 was reported to regulate the VCAM‐1 under many different pathophysiological circumstances. We investigated variations of miR‐126 and VCAM‐1 in inflamed patient pulp tissues and determined potential roles of miR‐126 in pulpitis using human dental pulp cells (hDPCs) in vitro.MethodsWe quantitatively measured the transcripts of miR‐126 and VCAM‐1 in inflamed human pulp tissues using qRT‐PCR and compared with those from healthy human pulp tissues. In addition, we transfected miR‐126 in hDPCs using plasmid DNA (pDNA)‐encoding miR‐126 delivered by polyethylenimine (PEI) nanoparticles.ResultsThe irreversible pulpitis significantly reduced miR‐126 and increased the transcript of VCAM‐1 in pulp tissues (p < 0.05). pDNA‐encoding miR‐126 delivered PEI nanoparticles and effectively upregulated the expression of miR‐126 in hDPCs (p < 0.05). The overexpression of miR‐126 could effectively suppress the transcripts and protein levels of VCAM‐1 and IL‐1β induced by Pg‐LPS at 100ng/mL in DPCs (p < 0.05).Conclusions miR‐126 is involved in pulpitis and downregulated the VCAM‐1 and IL‐1β in DPCs. miR‐126 may be a potential target to attenuate the inflammation of pulpitis.

Differential Expression of Long Noncoding RNAs in Normal and Inflamed Human Dental Pulp

IntroductionDental pulp inflammation is an excellent model for the interaction between tissue inflammation and regenerative processes. It is worthwhile to better understand molecular signaling of repair and regeneration in inflammatory processes. Emerging evidence suggests that long noncoding RNA (lncRNA) participates in immune system inflammatory processes. Here we investigate the expression of lncRNAs in pulpitis, the inflammation of dental pulp tissue, and identify lncRNAs that possibly participate in inflammation responses and odontogenesis. MethodsIntegrated comparative lncRNA microarray was used to examine lncRNA and mRNA expression between inflamed and normal human pulp tissue. The differential expression of lncRNAs and mRNAs was then validated by quantitative real-time polymerase chain reaction. A bioinformatics analysis was performed to predict the function of differentially expressed lncRNAs and mRNAs. ResultsOur data indicated 752 lncRNAs were significantly differentially expressed (fold change > 2.0; P < .05) in inflamed pulp tissues compared with normal pulp tissues, including 338 upregulated and 414 downregulated lncRNAs. Among the 646 differentially expressed mRNAs (fold change > 2.0; P < .05), 460 were upregulated, and 186 were downregulated. The differentially downregulated genes are enriched for gene ontology terms related to odontogenesis and cell development in biological processes, whereas the differentially upregulated genes are related to immune and inflammation processes. ConclusionsLncRNAs are differentially expressed in inflamed human pulp tissue compared with normal pulp tissue, indicating that lncRNAs may play key roles in pulpitis pathogenesis and development.

Viral MicroRNAs Identified in Human Dental Pulp

IntroductionMicroRNAs (miRs) are a family of noncoding RNAs that regulate gene expression. They are ubiquitous among multicellular eukaryotes and are also encoded by some viruses. Upon infection, viral miRs (vmiRs) can potentially target gene expression in the host and alter the immune response. Although prior studies have reported viral infections in human pulp, the role of vmiRs in pulpal disease is yet to be explored. The purpose of this study was to examine the expression of vmiRs in normal and diseased pulps and to identify potential target genes. MethodsTotal RNA was extracted and quantified from normal and inflamed human pulps (N = 28). Expression profiles of vmiRs were then interrogated using miRNA microarrays (V3) and the miRNA Complete Labeling and Hyb Kit (Agilent Technologies, Santa Clara, CA). To identify vmiRs that were differentially expressed, we applied a permutation test. ResultsOf the 12 vmiRs detected in the pulp, 4 vmiRs (including those from herpesvirus and human cytomegalovirus) were differentially expressed in inflamed pulp compared with normal pulp (P < .05). Using bioinformatics, we identified potential target genes for the differentially expressed vmiRs. They included key mediators involved in the detection of microbial ligands, chemotaxis, proteolysis, cytokines, and signal transduction molecules. ConclusionsThese data suggest that miRs may play a role in interspecies regulation of pulpal health and disease. Further research is needed to elucidate the mechanisms by which vmiRs can potentially modulate the host response in pulpal disease.

DPSCs from Inflamed Pulp Modulate Macrophage Function via the TNF-α/IDO Axis

Human dental pulp stem cells (DPSCs) can be isolated from inflamed pulp derived from carious teeth with symptomatic irreversible pulpitis (I-DPSCs), which possess stemness and multidifferentiation potentials similar to DPSCs from healthy pulp. Since macrophages—essential cell players of the pulpal innate immunity—can regulate pulpal inflammation and repair, the authors investigated the immunomodulatory effects of DPSCs/I-DPSCs on macrophage functions and their underlying mechanisms. Similar to DPSCs, I-DPSCs were capable of colony-forming efficiency and adipogenic and osteo/dentinogenic differentiation under in vitro induction conditions. I-DPSCs also expressed a similar phenotypic profile of mesenchymal stem cell markers, except a relatively higher level of CD146 as compared with DPSCs. Coculture of DPSCs or I-DPSCs with differentiated THP-1 cells, the human monocyte cell line, markedly suppressed tumor necrosis factor α (TNF-α) secretion in response to stimulation with lipopolysaccharides (LPS) and/or nigericin. However, unlike TNF-α, the secreted level of interleukin 1β was not affected by coculture with DPSCs or I-DPSCs. Furthermore, DPSC/I-DPSC-mediated inhibition of TNF-α secretion by macrophages was abolished by pretreatment with 1-methyl-D-tryptophan, a specific inhibitor of indoleamine-pyrrole 2,3-dioxygenase (IDO), but not by NSC-398, a specific inhibitor of COX-2, suggesting IDO as a mediator. Interestingly, IDO expression was significantly augmented in macrophages and mesenchymal stromal cells in inflamed human pulp tissues. Collectively, these findings show that I-DPSCs, similar to DPSCs, possess stem cell properties and suppress macrophage functions via the TNF-α/IDO axis, thereby providing a physiologically relevant context for their innate immunomodulatory activity in the dental pulp and their capability for pulp repair.

Differential Expression of MicroRNAs in Normal and Inflamed Human Pulps

IntroductionMicroRNAs (miRNAs) are small noncoding RNAs that regulate gene expression via posttranscriptional repression. They are critical to normal cellular function, and bioinformatic predictions indicate that at least one third of all messenger RNAs might be regulated by miRNAs. Although both the innate and adaptive immune responses are known to be regulated by miRNAs, their role in regulating endodontic disease has yet to be explored. The purpose of this study was to examine the differential expression of miRNAs in normal and inflamed human dental pulps and to explore their functional gene targets. MethodsAfter obtaining informed consent, we collected normal and inflamed human pulps (N = 30). Microarray and molecular biology techniques were then used for gene profiling and identifying functional gene targets. ResultsOf the 335 human miRNAs identified in the pulp tissues, 3 miRNAs, miR-150∗, miR-584, and miR-766, were significantly up-regulated in inflamed pulps as compared with normal pulps (P < .003). Thirty-three miRNAs were down-regulated in the inflamed pulps (P < .003). The false discovery rate for these findings is estimated to be approximately 5%. The potential gene targets for these miRNAs include proinflammatory cytokines as well as other key mediators of the immune and inflammatory response to infection. ConclusionsOur data identify differential expression of miRNAs in healthy and diseased human dental pulps. These findings highlight the intricate and specific roles of miRNA in inflammation and immunity, both of which are key aspects of pulpal pathology.

Mature dendritic cells in inflamed human pulps beneath deep caries

Dendritic cells (DCs) in pulps have been identified with markers for immature DCs and the relationship of DC maturity to pulpal health has not been carefully examined. We sought to test the hypothesis that the frequency of CD83+ mature DCs would correlate with caries invasion. Pulps were collected from extracted teeth exhibiting (I) no caries (n = 9), (II) shallow dentinal caries (n = 5), and (III) deep caries (n = 9). Immature DCs (CD209+), mature DCs (CD83+), and monocytes/macrophages (CD14+) in three groups were enumerated immunohistochemically. Mature DCs were frequently found beneath deep caries (21.3 mature DCs/3 grids versus <1 in groups I and II) with increasing numbers of CD209+ DCs and CD14+ cells. Co-localization of CD4+ T cells with mature DCs and macrophages was observed in deep caries. Mature DCs were frequently found only beneath deep caries and these DCs were co-localized with CD4+ T cells suggesting antigen presentation.

Substance P receptor expression in healthy and inflamed human pulp tissue

To use radioreceptor analysis for comparing substance P (SP) receptor expression in human pulp tissue samples collected from teeth having a clinical diagnosis of acute irreversible pulpitis, healthy pulps and teeth with induced inflammation. Five pulp samples were obtained from teeth having a clinical diagnosis of acute irreversible pulpitis. Another 10 pulp samples were obtained from healthy premolars where extraction was indicated for orthodontic purposes. In five of these premolars inflammation was induced prior to pulp collection. All of the samples were processed and labelled with 125I-SP. Binding sites were identified by 125I-SP and standard SP competition assays. Kruskal-Wallis and Mann-Whitney (post-hoc) tests were used to establish statistically significant differences between the groups. Substance P receptor expression was found in all human pulp tissue samples. Most receptors were found in the group of pulps from teeth having a clinical diagnosis of acute irreversible pulpitis, followed by the group of pulps having induced inflammation. The least number of receptors was expressed in the group of healthy pulps. Statistical analysis revealed significant differences between the group of healthy pulp and both inflamed pulp groups (P < 0.01). Substance P receptor expression in human pulp tissue is significantly increased during inflammatory phenomena such as acute irreversible pulpitis.

Co-Expression of Cyclooxygenase-2 and Vascular Endothelial Growth Factor in Inflamed Human Pulp: An Immunohistochemical Study

Recent data from the medical literature indicates that cyclooxygenase-2 (COX-2) plays a key role in the production of vascular endothelial growth factor (VEGF), a glycoprotein that has the ability to increase the permeability of blood vessels and to induce angiogenesis. This study was undertaken to investigate the immunohistological co-expression of COX-2 and VEGF in inflamed human pulp, in conjunction with the expression of CD34, a transmembrane glycoprotein expressed in endothelial cells. Pulp tissue of extracted carious human third molars with a recent history of spontaneous pain were collected and processed for immunostaining of COX-2, VEGF, and CD34 using the biotin-streptoavidin method. Healthy pulp samples served as controls. COX-2 expression was not observed in healthy pulps, whereas all inflamed pulps demonstrated COX-2-expressing cells. Similarly, VEGF was not expressed in normal pulp tissue, but was strongly positive in inflamed pulps. CD34 was expressed in the endothelium of both normal and inflamed pulp tissues. Co-expression of COX-2 and VEGF in all consecutive sections of inflamed pulps could be suggestive of a possible release of VEGF via a COX-2-dependent pathway.

Quantification of neuropeptides (calcitonin gene‐related peptide, substance P, neurokinin A, neuropeptide Y and vasoactive intestinal polypeptide) expressed in healthy and inflamed human dental pulp

To quantify the expression of calcitonin gene-related peptide (CGRP), substance P (SP), neurokinin A (NKA), neuropeptide Y (NPY) and vasoactive intestinal polypeptide (VIP) in healthy and inflamed human dental pulp tissue. Six pulp samples were obtained from teeth having a clinical diagnosis of acute irreversible pulpitis. Another 12 pulp samples were obtained from premolars where extraction was indicated for orthodontic purposes. In six of these premolar teeth inflammation was induced by mechanical pulp exposure prior to sample collection. All samples were processed and 125I-labelled; neuropeptides were quantified by competition assays. ANOVA and Mann-Whitney's (post hoc) tests were used to establish statistically significant differences between the groups. Expression of five neuropeptides was found in all human pulp samples. Statistical analysis revealed a significantly higher (P < 0.05) expression of CGRP, SP, NKA and NPY in both inflammatory conditions compared with healthy pulp control values. VIP expression remained stable during the inflammatory conditions. Expression of CGRP, SP and NKA released from C-fibres and NPY released from sympathetic fibres is significantly higher in the inflamed human pulp compared with healthy pulp. Expression of VIP released from parasympathetic fibres is not increased during the inflammatory conditions of human dental pulp.

The Upregulation of Matrix Metalloproteinase-9 in Inflamed Human Dental Pulps

Abstract The matrix metalloproteinases (MMPs) participate in a wide variety of extracellular matrix degradation. Detailed knowledge of MMPs may be important for understanding the pathogenesis of pulpal inflammation. The purpose of this study was to compare MMP-9 expression in clinically healthy human pulp and inflamed human pulp tissue specimens. We compared the levels of MMP-9 between clinically healthy pulp and inflamed pulp tissues by using the semi-quantitative reverse-transcriptase polymerase chain reaction analysis. In addition, immunohistochemistry was used to identify the in situ localization of MMP-9 expression in pulp specimens. MMP-9 mRNA gene was found to be increased in inflamed pulps as compared with clinically healthy pulp tissues (p < 0.05). The results from immunohistochemistry demonstrated that MMP-9 expression was significantly higher in the inflamed pulps than clinically healthy pulps (p < 0.05). MMP-9 stain was detected in the odontoblasts, fibroblasts, inflammatory infiltrates, and endothelial cells. Taken together, MMP-9 may play an important role in the pathogenesis of pulpal inflammation.

Calcitonin gene‐related peptide receptor expression in healthy and inflamed human pulp tissue

To use radioreceptor analysis for comparing calcitonin gene-related peptide (CGRP) receptor expression in human pulp tissue samples collected from teeth having a clinical diagnosis of acute irreversible pulpitis, healthy pulps and teeth with induced inflammation. Six pulp samples were obtained from teeth having a clinical diagnosis of acute irreversible pulpitis. Another eight pulp samples were obtained from healthy premolars where extraction was indicated for orthodontic purposes. In four of these premolars, inflammation was induced prior to pulp collection. All the samples were processed and labelled with 125I-CGRP. Binding sites were identified by 125I-CGRP and standard CGRP competition assays. CGRP receptor expression was found in all human pulp tissue samples. Most receptors were found in the group of pulps from teeth having a clinical diagnosis of acute irreversible pulpitis, followed by the group of pulps having induced inflammation. The least number of receptors was expressed in the group of healthy pulps. The Kruskal-Wallis and Mann-Whitney (post-hoc) tests showed statistically significant differences between the groups (P < 0.05). CGRP receptor expression in human pulp tissue is significantly increased during inflammatory phenomena such as acute irreversible pulpitis.

Upregulation of tissue‐type plasminogen activator in inflamed human dental pulps

To compare tissue-type plasminogen activator (t-PA) expression in normal human pulp and inflamed human pulp tissue specimens. Thirty pulpal tissue specimens (13 normal and 17 inflamed pulps) were obtained from extracted third molars. The levels of t-PA between normal pulp and inflamed pulp tissues were compared using the quantitative reverse-transcriptase polymerase chain reaction analysis. In addition, immunohistochemistry was used to identify the in situ localization of t-PA expression in pulp specimens. Wilcoxon-Mann-Whitney rank sum test was applied for the statistical analysis of the results. t-PA mRNA gene was found more in inflamed pulps when compared with normal pulp tissue (P<0.05). The results from immunohistochemistry demonstrated that t-PA expression was significantly higher in the inflamed pulp (P=0.025). t-PA stain was detected in the fibroblasts, inflammatory infiltrates and endothelial cells. t-PA expression was significantly higher in inflamed pulp tissue. t-PA may play an important role in the pathogenesis of pulpal inflammation.

Increased interleukin-8 expression in inflamed human dental pulps

Objective. Elevated levels of interleukin-8, a potent chemoattractant and activator of neutrophils, are associated with infectious and inflammatory diseases. However, little is known about interleukin-8 expression in human dental pulp. The purpose of this study was to determine whether tissue levels of interleukin-8 are elevated in irreversibly inflamed human pulps. Study design. Experimental samples were from teeth clinically diagnosed with irreversible pulpitis (diseased pulps). Controls were from freshly extracted, caries-free third molars (normal pulps). Samples were subjected to enzyme-linked immunosorbent assay and/or immunohistochemical analysis with specific antibodies to interleukin-8. Results. The enzyme-linked immunosorbent assay studies showed elevated levels of interleukin-8 in diseased pulps (mean, 1.82 ± 0.79 pg/mL/μg protein), as compared to detectable interleukin-8 levels in samples from normal pulps (mean, 0.08 ± 0.04 pg/mL/μg protein; P < .05). Immunohistochemical analyses demonstrated that diseased samples exhibited a higher density of localized interleukin-8 staining in areas with heavy infiltration of inflammatory cells. In contrast, normal pulps showed negative or weak interleukin-8 staining. Conclusions. Interleukin-8 concentration was higher in pulps diagnosed with irreversible pulpitis; only negligible amounts of interleukin-8 were present in normal pulps. (Oral Surg Oral Med Oral Pathol Oral Radiol Endod 1999;88:214-20)

Detection of interleukin‐6 in human dental pulp and periapical lesions

Pulpal and periapical diseases are characterized by inflammation. The cytokine IL-6 is a major mediator of the host response to tissue injury and infection. This study examined the level of interleukin-6 (IL-6) in six inflamed human pulps and six human periapical lesions of endodontic origin using ELISA. Pulp samples from eight clinically impacted teeth were used as controls. The periapical samples exhibited significant levels of IL-6 (mean = 78.1 +/- 9 pg/mg protein) as did inflamed pulpal tissues mean = 36 +/- 3.9 pg/mg protein) compared to healthy pulp mean = 0.01 +/- 0.02 pg/mg protein). These data indicated that IL-6 was produced and released locally in the inflamed pulpal and periapical lesions.

Natural modifiers of the inflammatory process in the human dental pulp

Concentrations of the protease inhibitors alpha 1-antitrypsin and alpha 2-macroglobulin were determined in normal and inflamed human dental pulps. Carious pulpal exposure which is associated with polymorphonuclear leukocyte infiltration and release of lysosomal enzymes was chosen as the point of verifiable inflammatory activity in the pulp. Normal samples were collected from nondiseased third molar teeth treatment planned for extraction and inflamed human pulps were collected from teeth with deep carious lesions. One half of each sample was assayed for concentration of protease inhibitors by enzyme-linked immunosorbent assay and the remaining half was examined histologically to verify the clinical diagnosis and categorize the extent of the inflammatory process. alpha 1-Antitrypsin and alpha 2-macroglobulin were detected in normal and inflamed human dental pulps in the nanogram per milliliter range. Statistically significant differences were found in the concentrations of alpha 2-macroglobulin (p less than 0.01) in moderate to severe inflammation versus normal pulp categories and between mildly inflamed pulps and moderate to severely inflamed pulps (p less than 0.05). Although differences in concentrations of alpha 1-antitrypsin were seen between inflamed and normal pulps, the differences were not statistically significant. The presence of these two protease inhibitors in the human dental pulp tissue and the increase in their concentration in acute inflammation indicates that these proteins play a role in the pathogenesis of pulpal inflammatory disease.