Regulation of CCl4-induced liver cirrhosis by hepatically differentiated human dental pulp stem cells.

Liver transplantation is the most effective treatment for treating liver cirrhosis. However, a limited number of donors, graft rejection, and other complications can undermine transplant success. It is considered that cell transplantation is an alternative approach of liver transplantation. We previously developed a protocol for hepatic differentiation of cluster of differentiation 117+ stem cells isolated from human exfoliated deciduous tooth pulp (SHEDs) under hydrogen sulfide exposure. These cells showed excellent hepatic function. Here, we investigated whether hepatocyte-like cell transplantation is effective for treating carbon tetrachloride (CCl4)-induced liver cirrhosis. SHEDs were hepatically differentiated, which was confirmed via immunological analyses and albumin concentration determination in the medium. Rats were intraperitoneally injected with CCl4 for and the differentiated cells were injected into rat spleen. Histopathological and immunohistochemical analyses were performed. Liver functions were serologically and pathologically determined. Quantitative real-time-polymerase chain reaction was implemented to clarify the treatment procedure of liver cirrhosis. In vitro-differentiated hepatocyte-like cells were positive for all examined hepatic markers. SHED-derived hepatocyte transplantation eliminated liver fibrosis and restored liver structure in rats. Liver immunohistochemical analyses showed the presence of human-specific hepatic markers, i.e., a large amount of human hepatic cells were very active in the liver and spleen. Serological tests revealed significant liver function recovery in the transplantation group. Expression of genes promoting fibrosis increased after cirrhosis induction but was suppressed after transplantation. Our results suggest that xenotransplantation of hepatocyte-like cells of human origin can treat cirrhosis. Moreover, cell-based therapy of chronic liver conditions may be an effective option.

Regeneration of insulin-producing islets from dental pulp stem cells using a 3D culture system.

AIM: In this study, we aimed to establish the differentiation protocol of dental pulp stem cells (DPSCs) into pancreatic islets using a 3D structure. MATERIALS & METHODS: DPSCs were differentiated in a 3D culture system using a stepwise protocol. Expression of ß-cell markers, glucose-stimulated insulin secretion, and PI3K/AKT and WNT pathways were compared between monolayer-cultured pancreatic cells and islets. RESULTS: Islet formation increased insulin and C-peptide production, and enhanced the expression of pancreatic markers. Glucose-dependent secretion of insulin was increased by islets. Pancreatic endocrine markers, transcriptional factors, and the PI3K/AKT and WNT pathways were also upregulated. CONCLUSION: Pancreatic islets were generated from DPSCs in a 3D culture system. This system could provide novel strategies for controlling diabetes through regenerative medicine.

Hydrogen sulfide enhances pancreatic ß-cell differentiation from human tooth under normal and glucotoxic conditions.

AIM: Glucotoxicity obstructs pancreatic differentiation from adult stem cells. The aim was to develop a novel protocol for differentiation of dental pulp stem cells (DPSCs) into pancreatic ß cells and determine the effect of H2S on glucotoxicity. MATERIALS & METHODS: DPSCs were differentiated with media containing 5.5 or 25.0 mM glucose, exposed to 1 ng/ml H2S. Glucotoxicity, expression of ß-cell markers, INS, PDX1 and GLUT2, and PI3K/AKT pathway were assessed. RESULTS: H2S exposure increased insulin and C-peptide, and protected DPSC-derived pancreatic ß-like cells from glucotoxicity and upregulated INS, PDX1 and GLUT2, and genes of PI3K/AKT pathway. CONCLUSION: H2S improved effects of glucotoxicity on ß-like cells via PI3K/AKT pathway. The protocol for pancreatic ß-cell differentiation might have applications in regenerative medicine rather than swine pancreas transplantation.

Disruption of biomineralization pathways in spinal tissues of a mouse model of diffuse idiopathic skeletal hyperostosis.

Equilibrative nucleoside transporter 1 (ENT1) mediates passage of adenosine across the plasma membrane. We reported previously that mice lacking ENT1 (ENT1(-/-)) exhibit progressive ectopic mineralization of spinal tissues resembling diffuse idiopathic skeletal hyperostosis (DISH) in humans. Here, we investigated mechanisms underlying aberrant mineralization in ENT1(-/-) mice. Micro-CT revealed ectopic mineralization of spinal tissues in both male and female ENT1(-/-) mice, involving the annulus fibrosus of the intervertebral discs (IVDs) of older mice. IVDs were isolated from wild-type and ENT1(-/-) mice at 2months of age (prior to disc mineralization), 4, and 6months of age (disc mineralization present) and processed for real-time PCR, cell isolation, or histology. Relative to the expression of ENTs in other tissues, ENT1 was the primary nucleoside transporter expressed in wild-type IVDs and mediated the functional uptake of [(3)H]2-chloroadenosine by annulus fibrosus cells. No differences in candidate gene expression were detected in IVDs from ENT1(-/-) and wild-type mice at 2 or 4months of age. However, at 6months of age, expression of genes that inhibit biomineralization Mgp, Enpp1, Ank, and Spp1 were reduced in IVDs from ENT1(-/-) mice. To assess whether changes detected in ENT1(-/-) mice were cell autonomous, annulus fibrosus cell cultures were established. Compared to wild-type cells, cells isolated from ENT1(-/-) IVDs at 2 or 6months of age demonstrated greater activity of alkaline phosphatase, a promoter of biomineralization. Cells from 2-month-old ENT1(-/-) mice also showed greater mineralization than wild-type. Interestingly, altered localization of alkaline phosphatase activity was detected in the inner annulus fibrosus of ENT1(-/-) mice in vivo. Alkaline phosphatase activity, together with the marked reduction in mineralization inhibitors, is consistent with the mineralization of IVDs seen in ENT1(-/-) mice at older ages. These findings establish that both cell-autonomous and systemic mechanisms contribute to ectopic mineralization in ENT1(-/-) mice.

Loss of equilibrative nucleoside transporter 1 in mice leads to progressive ectopic mineralization of spinal tissues resembling diffuse idiopathic skeletal hyperostosis in humans.

Diffuse idiopathic skeletal hyperostosis (DISH) is a noninflammatory spondyloarthropathy, characterized by ectopic calcification of spinal tissues. Symptoms include spine pain and stiffness, and in severe cases dysphagia and spinal cord compression. The etiology of DISH is unknown and there are no specific treatments. Recent studies have suggested a role for purine metabolism in the regulation of biomineralization. Equilibrative nucleoside transporter 1 (ENT1) transfers hydrophilic nucleosides, such as adenosine, across the plasma membrane. In mice lacking ENT1, we observed the development of calcified lesions resembling DISH. By 12 months of age, ENT1(-/-) mice exhibited signs of spine stiffness, hind limb dysfunction, and paralysis. Micro-computed tomography (µCT) revealed ectopic mineralization of paraspinal tissues in the cervical-thoracic region at 2 months of age, which extended to the lumbar and caudal regions with advancing age. Energy-dispersive X-ray microanalysis of lesions revealed a high content of calcium and phosphorus with a ratio similar to that of cortical bone. At 12 months of age, histological examination of ENT1(-/-) mice revealed large, irregular accumulations of eosinophilic material in paraspinal ligaments and entheses, intervertebral discs, and sternocostal articulations. There was no evidence of mineralization in appendicular joints or blood vessels, indicating specificity for the axial skeleton. Plasma adenosine levels were significantly greater in ENT1(-/-) mice than in wild-type, consistent with loss of ENT1--a primary adenosine uptake pathway. There was a significant reduction in the expression of Enpp1, Ank, and Alpl in intervertebral discs from ENT1(-/-) mice compared to wild-type mice. Elevated plasma levels of inorganic pyrophosphate in ENT1(-/-) mice indicated generalized disruption of pyrophosphate homeostasis. This is the first report of a role for ENT1 in regulating the calcification of soft tissues. Moreover, ENT1(-/-) mice may be a useful model for investigating pathogenesis and evaluating therapeutics for the prevention of mineralization in DISH and related disorders.

Standardization of clinical protocols in oral malodor research.

The objective of this study is to standardize protocols for clinical research into oral malodor caused by volatile sulfur compounds (VSCs). To detect VSCs, a gas chromatograph (GC) using a flame photometric detector equipped with a bandpass filter (at 393 nm) is the gold standard (sensitivity: 5 × 10(-11) gS s(-1)). The baselines of VSC concentrations in mouth air varied considerably over a week. When the subjects refrained from eating, drinking and oral hygiene including mouth rinsing, the VSC concentrations remained constant until eating. Over a 6 h period after a meal, VSC concentrations decreased dramatically (p < 0.01). These results point to optimal times and conditions for sampling subjects. Several portable devices were compared with the measurements by the GCs. Portable GCs demonstrated capabilities similar to those of the GCs. We also applied the recommended protocols described below to clinical research testing the efficacy of ZnCl(2) products, and confirmed that using the recommended protocols in a randomized crossover design would provide very clear results. Proposed protocols include: (a) a short-term study rather than a long-term study is strongly recommended, since the VSC concentrations are constant in the short term; (b) a crossover study would be the best design to avoid the effects of individual specificities on each clinical intervention; (c) measurements of VSCs should preferably be carried out using either a GC or portable GCs.

Hydrogen sulfide increases hepatic differentiation in tooth-pulp stem cells.

The toxicity of hydrogen sulfide (H(2)S), an oral malodorous compound, is well reported. We have recently established an experimental model of hepatic differentiation from human tooth-pulp stem cells (HTPC) using serum-free medium. The objective of the present study is to determine the effect of H(2)S on hepatic differentiation. The CD117 positive cell fraction was obtained from deciduous HTPC using magnetic cell sorting. After 3-4 passages, cells were grown in Dulbecco's modified Eagle's medium supplemented with insulin-transferrin-selenium-x (ITS-x), embryotrophic factor (ETF) and hepatocyte growth factor (HGF) for hepatic commitment (five days). For hepatic differentiation the cells were cultured in Iscove's modified Dulbecco's medium supplemented with ITS-x, ETF, oncostatin, HGF and dexamethasone for 15 days in air containing 5% CO(2), with or without H(2)S at 0.05 ng ml(-1). Cells were assayed for the expression of hepatic markers α-fetoprotein, albumin or carbamoyl phosphate synthetase, and urea concentrations and glycogen synthesis were also determined. The panel of hepatic markers was expressed more in the test groups exposed to H(2)S than in the control groups. Urea and glycogen production were also increased, especially glycogen which was approximately five times greater compared to the control (p < 0.01). We concluded that H(2)S at physiological concentrations increased the ability of HTPC to undergo hepatogenic differentiation.

The role of p53 in an apoptotic process caused by an oral malodorous compound in periodontal tissues: a review.

Oral malodor is caused by volatile sulfur compounds (VSCs) composed mainly of hydrogen sulfide (H(2)S) and methyl mercaptan. In particular, H(2)S is an important compound, since it is a major component of physiologic halitosis. The toxicity of VSCs is similar to that of hydrogen cyanide, and is well investigated. The role of VSCs in reducing collagen in human gingival fibroblasts is one of the main sources of their toxicity to human oral tissues. It has been reported recently that H(2)S may cause apoptosis in several periodontal tissues. In human gingival fibroblasts, H(2)S inhibits not only cytochrome c oxidase activity but also superoxide dismutase activity. The levels of reactive oxygen species are markedly increased, which causes the release of cytochrome c into the cytoplasm, resulting in caspase-9 activation; finally, the executor caspase, caspase-3, is activated. This pathway is commonly observed in cells from all periodontal tissues. Moreover, p53, an apoptotic factor, and phosphorlylated p53, which is the activated form, are increased by H(2)S in keratinocyte stem cells and osteoblasts. H(2)S also increases the expression of Bax, a primary response gene playing an important role in p53-mediated apoptosis, but maintains a lower expression of Bcl-2, an anti-apoptotic factor, in osteoblasts. It is concluded that the Bax apoptotic pathway and the mitochondrial pathway are activated by H(2)S.

Hydrogen sulfide causes apoptosis in human pulp stem cells.

INTRODUCTION: Untreated dental caries will eventually lead to pulpal inflammation. Although much is known regarding the interaction of microbial antigens and the immunologic defense systems of pulp, many aspects of the pathogenesis of pulpitis are not fully understood. The relationship between human pulp stem cells (HPSCs) and the pathogenesis of pulpitis remains among the poorly understood areas. Many of the invading bacteria are known to produce considerable amounts of hydrogen sulfide (H(2)S), which causes apoptosis in some tissues. The aims of this study were to determine whether H(2)S causes apoptosis in HPSCs and to examine its signaling pathway. METHODS: Stem cells were isolated from human dental pulp and incubated with 50 ng/mL H(2)S for 48 hours. To detect apoptosis, the cells were analyzed by using flow cytometry. The mitochondrial signaling pathway was examined by determining mitochondrial membrane depolarization. Activation of the key apoptotic enzymes caspase-9, caspase-8, and caspase-3 was assessed by using enzyme-linked immunosorbent assay. Release of cytochrome C from mitochondria was also determined. RESULTS: The number of apoptotic cells increased significantly with H(2)S treatment from 1.6% to 16.3% (P < .01). Significant increases were also measured in the amounts of caspase-9 and caspase-3 and in cytochrome C release (all P < .01) and in mitochondrial membrane depolarization (P < .05), whereas caspase-8 activity was not found. CONCLUSIONS: H(2)S causes apoptosis in HPSCs by activating the mitochondrial pathway. It is suggested that H(2)S might be one of the factors modifying the pathogenesis of pulpitis by causing loss of viability of HPSCs through apoptosis.

Effects of a composition containing lactoferrin and lactoperoxidase on oral malodor and salivary bacteria: a randomized, double-blind, crossover, placebo-controlled clinical trial.

We report a clinical trial of the effects of test tablets containing bovine lactoferrin and lactoperoxidase on oral malodor and salivary bacteria. Fifteen subjects with volatile sulfur compounds (VSCs) in mouth air above the olfactory threshold (H(2)S >1.5 or CH(3)SH >0.5 ng/10 ml) as detected by gas chromatography were enrolled in the trial. Either a test or a placebo tablet was ingested twice at 1-h intervals in two crossover phases. Mouth air was monitored for VSC levels at the baseline before ingestion of a tablet, 10 min after the first ingestion, 1 h (just before the second ingestion), and 2 h after the first ingestion. Whole saliva was analyzed at the baseline and at 2 h for bacterial numbers. At 10 min, the level of CH(3)SH was significantly lower in the test group (median [interquartile range] = 0.28 [0.00-0.68] ng/10 ml) compared to that in the placebo group (0.73 [0.47-1.00] ng/10 ml; P = 0.011). The median concentration of CH(3)SH in the test group was below the olfactory threshold after 10 min until 2 h, whereas the level in the placebo group was above the threshold during the experimental period. No difference in the numbers of salivary bacteria was detected by culturing or quantitative PCR, but terminal restriction fragment length polymorphism detected one fragment with a significantly lower copy number at 2 h in the test group (mean ± standard error, 4.89 ± 0.11 log(10) copies/10 µl) compared to that in the placebo group (5.38 ± 0.15 log(10) copies/10 µl; P = 0.033). These results indicate a suppressive effect of the test composition on oral malodor and suggest an influence on oral bacteria.

Oral malodorous compound induces osteoclast differentiation without receptor activator of nuclear factor κB ligand.

BACKGROUND: Hydrogen sulfide (H(2)S), the main component of halitosis, is one of the etiologic factors for periodontitis. We recently reported that H(2)S may induce pathologic changes in rat alveolar bone. The objective of this study is to determine the effect of H(2)S on osteoclast differentiation. METHODS: Murine macrophage cells RAW264 were cultured in medium lacking nuclear factor κB ligand (receptor activator of nuclear factor κB ligand) in 5% CO(2) with air at 37°C for 24 hours; then 0.05, 0.5, or 5 ng/ml H(2)S was added to the CO(2)-air mix for 4 days. The controls received the CO(2)-air mix with no H(2)S. Cell differentiation was evaluated by counting the tartrate-resistant acid-phosphatase (TRAP)-positive cells. Extracellular signaling-regulated kinase1/2 (ERK1/2) and mitogen-activated protein kinase p38 phosphorylation were examined by Western blotting. The bone-resorption activity was determined with the resorption assay of calcium phosphate. RESULTS: There were significantly more TRAP-positive cells at a concentration of 0.05 ng/ml H(2)S than at the other concentrations (P <0.001). Cathepsin K protein, a specific marker for osteoclasts, was expressed in the H(2)S-induced multinuclear cells. Resorption of calcium phosphate significantly increased in the H(2)S-induced TRAP-positive cells cultured on plates coated with calcium phosphate apatite (P <0.01). The phosphorylation of ERK1/2 and p38 were accelerated by H(2)S, and increased with time. PD98059 and SB203580, specific inhibitors of ERK1/2 and p38, suppressed the activation of these enzymes and osteoclast differentiation by H(2)S. CONCLUSION: Results demonstrate that H(2)S at physiologic concentrations in mouth air induces osteoclasts from RAW264 cells.

Oral malodorous compound activates mitochondrial pathway inducing apoptosis in human gingival fibroblasts.

Hydrogen sulfide (H(2)S) is a main cause of physiologic halitosis. H(2)S induces apoptosis in human gingival cells, which may play an important role in periodontal pathology. Recently, it has been reported that H(2)S induced apoptosis and DNA damage in human gingival fibroblasts (HGFs) by increasing the levels of reactive oxygen species. However, the mechanisms of H(2)S-induced apoptosis have not been clarified in HGFs. The objective of this study was to determine the apoptotic pathway activated by H(2)S in HGFs. The HGFs were exposed to 50 ng/mL H(2)S, resulting in 18 ng/mL in the culture medium, which is lower than the concentration in periodontal pockets. The number of apoptotic cells after 24 and 48 h incubation was significantly higher than that in the control cultures (p < 0.05). Mitochondrial membrane depolarization and the release of cytochrome c, and caspase-3, and caspase-9 were also significantly increased after both 24- and 48-h incubation (p < 0.05), whereas caspase-8, a key enzyme in the receptor ligand-mediated pathway causing apoptosis, was not activated. The present study shows that H(2)S triggered the mitochondrial pathway causing apoptosis in HGFs but did not activate the receptor ligand-mediated pathway.

Oral malodorous compound inhibits osteoblast proliferation.

BACKGROUND: Oral malodorous compounds including hydrogen sulfide (H2S) are causative agents of periodontitis because the toxicities are similar to that of cyanate. Previous studies demonstrated that volatile sulfur compounds (VSCs) were highly toxic to periodontal tissues, causing a large reduction in the amount of collagen in human gingival fibroblasts and extracellular matrix as well as, for example, apoptosis, immunologic responses, and matrix metalloproteinase production. The objective of this study was to determine the effect of H2S on the proliferation of osteoblasts and a signaling transduction pathway through the mitogen-activated protein kinase (MAPK). METHODS: Normal human osteoblasts (NHOst) and murine osteoblasts (cell line MC3T3-E1) were incubated with H2S. Cell proliferation was assessed by measuring [3H]thymidine incorporation. The effects of H2S on the signal transduction pathways, the MAPK cascade, that control cell proliferation were evaluated in NHOst by determining extracellular signal-regulated kinase (ERK)1/2 and p38 phosphorylation with a Western blot analysis. RESULTS: After incubating NHOst with H2S for 24 hours, [3H]thymidine incorporation into the DNA significantly decreased dose-dependently with H(2)S. At a concentration of 100 ng/ml H2S, [3H]thymidine incorporation decreased 79% compared to the control. Similar results were obtained from MC3T3-E1. The phosphorylation of ERK1/2 and p38 was increased by H2S at 10 minutes after starting the treatment and then decreased time dependently. The activation of ERK1/2 and p38 induced by H2S was inhibited by the specific inhibitor of MAPK/ERK kinase ([MEK]; U0126) or p38 (SB203580). CONCLUSION: H2S inhibited the proliferation of human osteoblastic cells through the MAPK pathway.

A single application of hydrogen sulphide induces a transient osteoclast differentiation with RANKL expression in the rat model.

OBJECTIVE: Oral malodor is mainly attributed to volatile sulphur compounds (VSCs) such as hydrogen sulphide (H(2)S), methyl mercaptan and dimethyl sulphide. VSC accelerate periodontal soft tissue destruction. However, there is little information about the potential role of H(2)S in alveolar bone loss. The purpose of this animal study was to examine the effects of sodium hydrogen sulphide (NaHS), H(2)S donor drug, on osteoclast differentiation in rat periodontal tissue. DESIGN: Twenty-four male Wistar rats (8 weeks old) were divided into four groups: a control group and three experimental groups, which were examined at 3h, 1 day, and 3 days after topical application of 3microl NaHS (lM in physiological saline) into the gingival sulcus of rat first molar. Expression of tumour necrosis factor (TNF)-alpha, RANKL, NF-kappaB and tartrate-resistant acid phosphatase (TRAP) was evaluated in the periodontal tissue. RESULTS: Three hours after NaHS application, TNF-alpha expression increased in the periodontal ligament. The numbers of RANKL-positive osteoblasts and TRAP-positive osteoclasts significantly increased progressively with time and reached a maximum level after 1 day. Significant up-regulation of RANKL and NF-kappaB mRNA was observed at 3h after NaHS application. CONCLUSIONS: H(2)S application caused a transient increase of osteoclast differentiation with up-regulation of RANKL expression in osteoblasts. H(2)S, which is primarily responsible for halitosis, may also contribute to alveolar bone resorption through RANKL expression.