Catalase activity in healthy and inflamed pulp tissues of permanent teeth in young people.

AIM: To evaluate catalase (CAT, EC 1.11.1.6) activity in healthy and inflamed dental pulp of young patient's teeth and to investigate if an active defense system oxidizing agents is present as a response to bacterial invasion. MATERIALS AND METHODS: Twenty young patients between 15 and 25 ages, who were diagnosed to be healthy, were the source of the pulp tissue. The situation of the dental pulps was evaluated using clinical and radiographic assessments. The patients were divided two groups from healthy, and inflamed pulp tissues were obtained; each participant provided one pulp tissue specimens. The specimens were collected during endodontic treatment or by longitudinally grooving and splitting the teeth (if extracted). Catalase activity was determined through spectrophotometric methods and an independent sample t-test assessed the significance of differences between the groups. RESULTS: There was statistically a difference between healthy pulp tissue and inflamed pulp tissue (P < 0.005, independent sample t-test). The catalase activity of healthy group was significantly lower than inflamed pulp groups. CONCLUSION: The present study has shown that a significant increase in catalase activity is determined in inflamed dental pulps, which is due to pulpitis in comparison to healthy dental pulp.

Investigation of functional activity human dental pulp stem cells at acute and chronic pulpitis.

UNLABELLED: It is already recognized that together with the other connective tissues organ-specific progenic stem cells are also found in postnatal dental pulp. This group of undifferentiated cells is only 1% of total cell population of the pulp. The aim of the study was the identification of stem cells in human dental pulp, detection of their localization and assessment of functional activity during inflammation process and/or at norm. The obtained results showed that at acute pulpitis the pulp stroma is hypocellular in comparison with the norm but cells proliferative activity is low. CD 133 and NCAM (CD 56) positive stem cells were found in perivascularl space of the pulp stroma and in Hohle layer. At process prolongation and transition to the chronic phase pulp stroma is hypercellular, the cells with large, rounded or oval-shaped nuclei with clear chromatin appear together with fibroblasts. They are distributed as about entire thickness of the stroma as especially Hohle layer. In such cells higher proliferative activity (Ki67 expression) was observed. The cells in the mentioned proliferation phase are intensively marked by CD133, the rate of which is high in Hohle layer and along it. A large number of NCAM (CD 56) positive cells appear in pulp stroma. CONCLUSIONS: During pulpitis an involvement of stem cells into the process of reparative dentinogenesis should be conducted stepwise. In acute cases of the disease, stem cell perivascularl mobilization and proliferation and its migration to Hohle layer occur in response to irritation /stimulation. Chronification of the process leads not only to the migration of stem cells to the periphery of the pulp but also s their В«maturationВ¼ (increase of NCAM expression in the stem cells), which causes an increase the number of dentin producing active odontoblasts and initiation of reparative dentinogenesis.

Differential cholinoceptor modulation of nitric oxide isoforms in experimentally-induced inflammation of dental pulp tissue.

AIM: The aim of the study was to investigate the role of muscarinic acetylcholine receptor (mAChR) activity in the regulation of endothelial (e), neuronal (n) and inducible (i) nitric oxide synthase (NOS) activity and expression in experimentally induced inflammation of rat dental pulp tissue. METHODOLOGY: Inflammation was induced by application of bacterial lipopolysaccharide (LPS) to the pulp. Extirpated pulp-tissue samples were incubated in saline solution until the various experiments were performed. Saline-treated pulp and healthy pulp tissues were used as controls. NOS activity was measured by the production of [U-(14)C]-citrulline from [U-(14)C]-arginine. Nitrite/nitrate assay was evaluated by the conversion of nitrate to nitrite in the presence of nicotinamide adenine dinucleotide phosphate. i-nos, e-nos and n-nos mRNA levels were measured using reverse-transcriptase polymerase chain reaction by co-amplification of target cDNA with a single set of primers. RESULTS: Application of LPS to the pulp increased NOS activity and nitrate production (P < 0.001), generated by iNOS over-activity and expression. Pilocarpine acting on mAChRs triggered a biphasic action on NOS activity and NO accumulation. At low concentrations, pilocarpine induced a negative effect associated with a decrease in i-nos mRNA level, whilst at high concentration, it produced a positive effect associated with increased e-nos and n-nos mRNA levels. In control pulp tissue, only the positive effect of pilocarpine was observed. CONCLUSIONS: Irreversible pulpitis changes mAChR conformation increasing its efficiency of coupling to transducing molecules that in turn induce activate iNOS. The capacity of pilocarpine to prevent NO accumulation and iNOS activity, by acting on mAChR mutation induced by pulpitis, might be useful therapeutically as a local treatment.

Extracellular proteolytic activities expressed by Bacillus pumilus isolated from endodontic and periodontal lesions.

The purpose of the present study was to identify 12 Bacillus isolates that had been obtained from root canals of teeth requiring endodontic therapy and from periodontal pockets in severe marginal periodontitis, and to determine whether these isolates exhibited extracellular proteolytic activity and, using in vitro assays, whether any such activity could degrade substrates that would be pathophysiologically relevant with regard to the production of endodontic and periodontal lesions. Biochemical and carbohydrate fermentation patterns were used in the identification of all strains, which was confirmed by determination of the16S rRNA gene sequence for strain BJ0055. Screening for production of extracellular proteolytic activity by all strains was done with a general proteinase substrate. All isolates were identified as representing Bacillus pumilus and all exhibited extracellular proteolytic activity. The putative pathophysiological relevance of extracellular proteinase production in strain BJ0055 was assessed using fluorophore-labelled elastin and collagen and several chromogenic peptides. Probable classes of proteinases acting on each substrate were investigated using class-specific inhibitors. Activity-pH profiles were determined in buffers at different pH values. Extracellular activities that were caseinolytic, elastinolytic, collagenolytic, glutamyl endopeptidase-like, and alanyl tripeptidyl peptidase-like were observed. No trypsin-like activities were detected. Serine- and chymotrypsin-like serine proteinase activities were detected, with activity observed at neutral and alkaline, but not acidic, pH. B. pumilus strains isolated from endodontic and periodontal lesions exhibited extracellular activities that degrade elastin, collagen and other substrates. These activities may be virulence factors that contribute to tissue damage in apical periodontitis and severe marginal periodontitis.

Quantification of neural protein in extirpated tooth pulp.

Because the pulp tissue extirpated during root canal procedures might serve as a valuable resource with which to assess underlying mechanisms of persistent pain, we sought to determine whether standard Western blotting techniques could be used to quantify neural proteins in pulp extirpated from teeth with irreversible pulpitis. Pulp harvested from healthy intact teeth extracted for orthodontic reasons was used for comparison. The neural marker PGP9.5 was detectable in all samples tested. A membrane enrichment protocol enabled detection of even low abundance, high molecular weight proteins such as the sodium channel alpha-subunit NaV1.8. Importantly, it was possible to quantify a approximately 6-fold increase in the relative density of NaV1.8 in inflamed pulp compared with control pulp. Our results suggest that it should be possible to use extirpated tooth pulp to validate mechanisms of persistent pain implicated in preclinical studies as well as evaluate the therapeutic efficacy of novel antinociceptive interventions.

Age-related Changes in the Alkaline Phosphatase Activity of Healthy and Inflamed Human Dental Pulp.

INTRODUCTION: Alkaline phosphatase (ALP) plays an important role in inducing mineralization events in the dental pulp. This study investigated and compared the ALP levels in healthy and inflamed pulp in young and old human pulp. METHODS: Tissue samples were collected from young (<30 years) and old (>60 years) donors. In both age groups, healthy human pulp (n = 18) were collected from extracted wisdom teeth. For reversible and irreversible pulpitis, pulp samples (n = 18 each) were obtained during endodontic treatment. ALP activity was assessed by spectrophotometry and immunhistochemistry. RESULTS: Regardless of age, reversible pulpitis group samples showed a slight elevation in ALP activity compared with normal healthy pulp. In elderly patients, ALP expression with irreversible pulpitis was significantly higher than those with a healthy pulp (P < .05). CONCLUSIONS: In the hyperemic state, both the young and old pulp shows a slight increase in ALP activity, whereas in irreversible pulpitis, only the old pulp shows significantly elevated ALP levels. Such an increase may trigger calcification events, which may eventually cause difficulties in endodontic treatment procedures in elderly individuals.

Immune Cells and Molecular Networks in Experimentally Induced Pulpitis.

Dental pulp is a dynamic tissue able to resist external irritation during tooth decay by using immunocompetent cells involved in innate and adaptive responses. To better understand the immune response of pulp toward gram-negative bacteria, we analyzed biological mediators and immunocompetent cells in rat incisor pulp experimentally inflamed by either lipopolysaccharide (LPS) or saline solution (phosphate-buffered saline [PBS]). Untreated teeth were used as control. Expression of pro- and anti-inflammatory cytokines, chemokine ligands, growth factors, and enzymes were evaluated at the transcript level, and the recruitment of the different leukocytes in pulp was measured by fluorescence-activated cell-sorting analysis after 3 h, 9 h, and 3 d post-PBS or post-LPS treatment. After 3 d, injured rat incisors showed pulp wound healing and production of reparative dentin in both LPS and PBS conditions, testifying to the reversible pulpitis status of this model. IL6, IL1-ß, TNF-α, CCL2, CXCL1, CXCL2, MMP9, and iNOS gene expression were significantly upregulated after 3 h of LPS stimulation as compared with PBS. The immunoregulatory cytokine IL10 was also upregulated after 3 h, suggesting that LPS stimulates not only inflammation but also immunoregulation. Fluorescence-activated cell-sorting analysis revealed a significant, rapid, and transient increase in leukocyte levels 9 h after PBS and LPS stimulation. The quantity of dendritic cells was significantly upregulated with LPS versus PBS. Interestingly, we identified a myeloid-derived suppressor cell-enriched cell population in noninjured rodent incisor dental pulp. The percentage of this population, known to regulate immune response, was higher 9 h after inflammation triggered with PBS and LPS as compared with the control. Taken together, these data offer a better understanding of the mechanisms involved in the regulation of dental pulp immunity that may be elicited by gram-negative bacteria.

Effect of NOS inhibitor on cytokine and COX2 expression in rat pulpitis.

Various kinds of chemical mediators are synthesized in the course of pulpitis; thus, control of their production would assist in inducing a reduction in pulpal inflammation. We hypothesized that nitric oxide (NO) would be an important mediator of pulpal inflammation. Pulpal inflammation was induced by the application of LPS in rat incisor pulp, and inducible nitric oxide synthase (iNOS) expression was evaluated by reverse-transcription/polymerase chain-reaction and immunohistochemical staining. After LPS application, iNOS mRNA was first detected after 3 hrs, peaked at 6 hrs, and decreased thereafter. iNOS-positive cells were macrophages and neutrophils. An NOS inhibitor caused drastic decreases in the expression of pro-inflammatory cytokines and COX2 mRNA, which was highly induced in the LPS-induced pulpitis. These results indicate that NO synthesis is related to the initiation of mediator production, and that its down-regulation should contribute to the prevention of pro-inflammatory mediator synthesis.

A real time quantitative PCR analysis and correlation of COX-1 and COX-2 enzymes in inflamed dental pulps following administration of three different NSAIDs.

Dental pain is encountered daily by clinicians. Nonsteroidal anti-inflammatory drugs (NSAIDs) commonly used for pain management are traditionally cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) inhibitors, and more recently selective COX-2 inhibitors. This study was designed to identify and quantify COX-1 and COX-2 gene expression level in inflamed rat molar pulps after administration of three NSAIDs: Celebrex, Vioxx, and Advil. Fifty male Wistar rats had their first and second molar pulps exposed and sealed with Cavit for 4 days. Rats were randomly divided into the three drug groups and two control groups. RNA was isolated from the rat pulps. Real Time Quantitative Reverse Transcriptase-Polymerase Chain Reaction assay, a relatively new PCR technique, was used to quantify COX-1 and COX-2 mRNA. Statistical analysis demonstrated no significant differences in COX-1 and COX-2 levels among the drug groups. However, Vioxx and Advil significantly reduced COX-2 expression levels compared to inflamed (positive control) pulps (p < 0.05).

The role of endothelial nitric oxide in the Substance P induced vasodilation in bovine dental pulp.

Vasodilation, an important response in neurogenic inflammation, involves release of Substance P (SP) from the sensory nerve endings. It is now well known that SP causes edema formation and vascular relaxation in nondental tissues, however, the SP vasodilatory mechanism in the dental pulp is not completely understood. Endothelium-dependent relaxation is mediated by nitric oxide (NO) release with consecutive intracellular cyclic-GMP elevation in many vascular preparations. Recently, it has been shown in different vascular systems that SP-induced vasodilation is mediated by cyclic-GMP production through different pathways involving endothelial NO or direct endothelial-independent pathways. In the present study, the role of endothelial NO in SP induced vasodilation in the dental pulp was investigated to better understand the inflammatory mechanisms. Freshly extracted bovine dental pulp was used to measure NO production. Sodium nitroprusside (SNP), L-NAME and SP were utilized to induce and to inhibit NO production in endothelial cells. Released NO byproducts were measured with chemiluminescence assay technique. The present data demonstrate that SP induces NO production by activating NOsynthase (NOS) in endothelial cells. The NOS inhibitor L-NAME blocks NO production completely. In conclusion, in the bovine dental pulp, SP-induced vascular relaxation can be mediated by inducing NOS, and subsequently NO production in endothelial cells.

Ca2+- and Mg2+-activated ATP hydrolysis in human tooth pulp.

ATP-degrading enzyme activities in pulps of healthy and carious human teeth were quantitatively analyzed using the bioluminescence method. The patterns of both Ca2+- and MG2+-activated ATP hydrolysis resembled each other. Increased enzyme activities were observed during injured states.

Localization and changes in NADPH-diaphorase reactivity and nitric oxide synthase immunoreactivity in rat pulp following tooth preparation.

Inflammatory changes in the dental pulp are accompanied by release of a wide variety of chemical mediators. Nitric oxide, an oxidative free radical produced by the enzyme nitric oxide synthase (NOS), has been implicated in multiple inflammatory processes, which makes it a suitable marker for changes which likely occur following tooth pulp insult. Since limited information on nitric oxide in the pulp is available, it is necessary first to examine relative distributions of NOS in uninflamed and inflamed rat pulp. We accomplished this by characterizing regions of nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) activity and the distribution of both macrophage NOS (macNOS) and neuronal NOS (nNOS) immunoreactivity in normal and inflamed rat molar pulp at multiple time points. The results showed that: (1) deep cavity preparation on the mesial surface of the molar produced a time-dependent inflammation, with acute inflammation early progressing to chronic, granulomatous inflammation with necrosis later that spread preferentially down the mesial root; (2) control (non-prepared) teeth showed a relatively faint and homogeneous distribution of NADPH-d and macNOS reactivity but no discernible nNOS reactivity; (3) inflamed teeth displayed localized increased intensity of NADPH-d and macNOS reactivity surrounding the inflamed area of pulp, but no increased nNOS activity; (4) pulp vessels supplying the inflamed area showed increased NADPH-d reactivity, but no increased macNOS or nNOS reactivity; and (5) neither NADPH-d, macNOS, nor nNOS reactivity was observed in pulpal nerves. Therefore, nitric oxide may mediate the pulpal inflammatory response through its effects on the paralesional pulp tissue and surrounding endothelial/vascular structures.

Nitric oxide synthase in healthy and inflamed human dental pulp.

Nitric oxide synthase (NOS) plays a significant role in the pathogenesis of pulpitis. In this study, we hypothesized the existence of endothelial (eNOS) and inducible (iNOS) enzyme isoforms in human dental pulp. Extracted third molar pulps were divided into groups based on clinical diagnosis: healthy, hyperemic, and irreversible pulpitis. We have localized the eNOS and iNOS by immunohistochemistry and have tested their mRNA expression by RT-PCR and protein levels by Western blots. eNOS is present in the endothelial cells and odontoblasts of the healthy pulp, but an elevation of eNOS mRNA and protein levels with a concomitant dilation of vessels was characteristic under pathological conditions. Healthy pulp tissue failed to exhibit any iNOS; however, acute inflammation enhanced the mRNA and protein levels of iNOS, mainly in the leukocytes. There are differences in localization and expression between eNOS and iNOS in healthy and inflamed dental pulp.

Cyclic Amp phosphodiesterase activity in normal and inflamed human dental pulp.

Cyclic AMP phosphodiesterase (cAMP PDE) seems to be important in pulp tissues. High levels of cAMP PDE have been demonstrated to be in dental pulp cells. In the present study cAMP PDE activity was analyzed in normal healthy human dental pulps, in reversible pulpitis and in irreversible pulpitis. Enzymatic cAMP PDE control values for normal healthy pulps were 12.14 +/- 3.74 nmols/mg of proteins. In reversible pulpitis the cAMP PDE activity increased almost 2.5 times. In irreversible pulpitis specimens the values increased 4.5 times compared with normal healthy pulps activity. The differences between the groups (control vs. reversible pulpitis and vs. irreversible pulpitis) were statistically significant. These results could point to a role of cAMP PDE in the initial pulp response after injury.

Cyclic GMP phosphodiesterase activity role in normal and inflamed human dental pulp.

Cyclic GMP phosphodiesterase (cGMP PDE) plays an important role in pulp tissues. High levels of cGMP PDE are found in dental pulp cells. In the present study cGMP PDE activity was analyzed in normal healthy human dental pulps, in reversible pulpitis and in irreversible pulpitis. Enzymatic cGMP PDE control values for normal healthy pulps were 4.74+/-0.32 nmol/mg of proteins. In reversible pulpitis the cGMP PDE activity increased almost 3 times. In irreversible pulpitis specimens the values increased 4.5 times compared with the normal healthy pulps activity. The differences between the groups (control vs. reversible pulpitis and vs. irreversible pulpitis) were statistically significant. These results point to a role of cGMP PDE in the initial pulp response after injury.

Copper-zinc superoxide dismutase activity in normal and inflamed human dental pulp tissue.

Information regarding the presence of the free radical scavenging (inactivating, dismutating) enzyme superoxide dismutase in human dental pulp was sought. Free radicals, such as the superoxide anion radical (O2-) and the hydroxyl anion radical (OH.), are powerful biological oxidants produced by phagocytes during the normal tissue response to injury and infection. Also produced is hydrogen peroxide (H2O2), an aggressive oxygen species formed by the reaction of superoxide with itself, i.e., a dismutation in which one molecule of O2- is oxidized by the other. These three reactive oxygen intermediates serve as part of the normal host biological defense mechanism for the inactivation of microorganisms and the breakdown of their toxic products. Both normal and inflamed dental pulps were assayed for the presence of this enzyme. Superoxide dismutase activity was identified in the normal pulpal tissues. There was a slight decrease in activity with age. In the inflamed pulpal tissues, enzyme activity was markedly and significantly increased in comparison to that in the normal tissues. These observations indicate that human dental pulp possesses an endogenous defense mechanism designed to protect the tissue components (cells and matrix) from the toxic effects of the reactive oxygen intermediates. In this regard, the inflammatory response of this specialized and somewhat isolated (compartmentalized) tissue is not unlike that seen in other connective tissues.

Aspartate aminotransferase activity in human healthy and inflamed dental pulps.

Aspartate aminotransferase (AST) seems to be an important mediator of inflammatory processes. Its role in the progression and detection of inflammatory periodontal disease has been increasingly recognized in recent years. In the present study AST activity was analyzed in normal healthy human dental pulps, in reversible pulpitis, and in irreversible pulpitis. Enzymatic AST activity showed that the control values for the healthy pulps were 4.8 +/- 0.7 units/mg of pulp tissue. In reversible pulpitis specimens the AST activity increased to 7.98 +/- 2.1 units/mg of pulp tissue. In irreversible pulpitis specimens the values decreased to 2.28 +/- 1.7 units/mg of pulp tissue. Differences between the groups (control versus reversible pulpitis and reversible pulpitis versus irreversible pulpitis) were statistically significant (p = 0.0015). These results could point to a role of AST in the early events that lead to development of pulpal inflammation.

Alkaline phosphatase activity in normal and inflamed dental pulps.

Alkaline phosphatase (ALP) seems to be important in the formation of mineralized tissues. High levels of ALP have been demonstrated in dental pulp cells. In the present study ALP activity was analyzed in normal healthy human dental pulps, in reversible pulpitis, and in irreversible pulpitis. Enzymatic ALP control values for the normal healthy pulps were 110.96+/-20.93. In the reversible pulpitis specimens the ALP activity increased almost eight times to 853.6+/-148.27. In the irreversible pulpitis specimens the values decreased sharply to 137.15+/-21.28 and were roughly equivalent to those seen in normal healthy pulps. The differences between the groups (control vs. reversible pulpitis and reversible pulpitis vs. irreversible pulpitis) were statistically significant. These results could point to a role of ALP in the initial pulp response after injury.

Comparison of human polymorphonuclear neutrophil elastase, polymorphonuclear neutrophil cathepsin-G, and alpha 2-macroglobulin levels in healthy and inflamed dental pulps.

Polymorphonuclear neutrophils (PMNs) are found in dental pulp secondary to carious exposures, periodontal disease, or trauma. Lysosomal degranulation of these cells liberates cellular proteases, including elastase (PMN-E) and cathepsin-G (PMN-CG), which produce connective tissue degradation. However, nonspecific pulpal tissue destruction can be modified by a naturally occurring serum protease inhibitor alpha 2-macroglobulin (A2-M). This study relates the concentrations of human PMN-E, PMN-CG, and A2-M in healthy and inflamed pulpal samples. Evaluation of 21 specimens yielded statistically significant differences between healthy and moderate to severely inflamed pulps for all groups (p < 0.05). No significant correlation was detected among human PMN-E, PMN-CG, and A2-M in the healthy tissues (P > 0.05). However, in the moderate to severely inflamed pulps, there was a significant correlation between PMN-CG and A2-M (p < 0.05).

Human polymorphonuclear granule components: relative levels detected by a modified enzyme-linked immunosorbent assay in normal and inflamed dental pulps.

Lysosomal granules of polymorphonuclear leukocytes (PMN's) contain proteolytic enzymes and other components important in the regulation of inflammation and the elimination of bacteria or debris associated with pulp disease. However, PMN lysosomal degranulation is nonspecific and can result in destruction of healthy connective tissue adjacent to the areas of damaged or infected tissue. For this study a modified enzyme-linked immunosorbent assay was used to detect the human PMN lysosomal granule products: elastase, cathepsin G, and lactoferrin. Evaluation of 55 pulp samples yielded a statistically significant difference (p less than 0.05) among the levels of elastase and lactoferrin in normal and moderate to severely inflamed pulps. Although cathepsin G levels were increased, there was no statistical significance (p greater than 0.05) among groups. The results indicate that a modified enzyme-linked immunosorbent assay technique can be used to measure PMN lysosomal granule components in dental pulp tissues. Additionally, elastase and lactoferrin levels appear to be valid diagnostic markers of advanced dental pulp disease.