Responses of BrdU label-retaining dental pulp cells to allogenic tooth transplantation into mouse maxilla

Recently, we demonstrated that a pulse of BrdU given to prenatal animals reveals the existence of slow-cycling long-term label-retaining cells (LRCs), putative adult stem or progenitor cells, which reside in the dental pulp. This study aims to clarify responses of LRCs to allogenic tooth transplantation into mouse maxilla using prenatal BrdU-labeling, in situ hybridization for osteopontin and periostin, and immunohistochemistry for BrdU, nestin, and osteopontin. The upper-right first molars were allografted in the original socket between BrdU-labeled and non-labeled mice or between GFP transgenic and wild-type mice. Tooth transplantation caused degeneration of the odontoblast layer, resulting in the disappearance of nestin-positive reactions in the dental pulp. On postoperative days 5–7, tertiary dentin formation commenced next to the preexisting dentin where nestin-positive odontoblast-like cells were arranged in the successful cases. In BrdU-labeled transplanted teeth, dense LRCs were maintained in the center of the dental pulp beneath the odontoblast-like cells including LRCs, whereas LRCs disappeared in the area surrounding the bone-like tissue. In contrast, LRCs were not recognized in the pulp chamber of non-labeled transplants through the experimental period. Tooth transplantation using GFP mice demonstrated that the donor cells constituted the dental pulp of the transplant except for endothelial cells and some migrated cells, and the periodontal tissue was replaced by host-derived cells except for epithelial cell rests of Malassez. These results suggest that the maintenance of BrdU label-retaining dental pulp cells play a role in the regeneration of odontoblast-like cells in the process of pulpal healing following tooth transplantation.     

Induction of transforming growth factor-beta 1 on dentine pulp cells in different culture patterns

Recent studies have documented that TGF-beta1 takes part in dental pulp tissue repair. Moreover, dental pulp cells have the potential to differentiate into odontoblast-like cells and produce reparative dentine in this process. However, the molecular mechanisms and potential interactions between TGF-beta1 and dental pulp cells are not clear due to the complexity of the pulp/dentine microenvironment. In this study, we investigated the induction of TGF-beta1 on the dental pulp cells in cell culture, tissue culture and three-dimensional culture patterns. These results demonstrated that TGF-beta1 significantly increased the proliferation of cells and activity of ALPase. Dental pulp cells cultured in the presence of TGF-beta1 formed mineralization nodules. In the organ culture, dental pulp cells treated with TGF-beta1 differentiated into odontoblast-like cells and formed a pulp-dentinal complex; and TGF-beta1 significantly induced synthesis of dentine relative proteins DSPP, DMP-1. The dental pulp cells share some characteristics of the odontoblast, such as a parallel arrangement with columnar form and a unilateral cell process. Together, these data indicate that TGF-beta1 can make dental pulp cells differentiated into odontoblast-like cells and form the pulp-dentinal complex. Moreover, these results suggest that TGF-beta1 is an important regulatory factor in odontoblast differentiation during tooth development and pulp repair.     

Synthesis of Oligonucleotide Prodrugs Bearing N-Acetyl Nucleobases

Abstract

N-Acetyl oligonucleotides and their prodrugs were synthesized on photolabile solid support. Tm studies showed a decrease of hydridization for N-acetyl A and G and an increase for N-acetyl C. In cells extract, acetyl groups were hydrolysed.     

Mitochondrial dysfunction mediated by quinone oxidation products of dopamine: Implications in dopamine cytotoxicity and pathogenesis of Parkinson's disease

The study has demonstrated that dopamine induces membrane depolarization and a loss of phosphorylation capacity in dose-dependent manner in isolated rat brain mitochondria during extended in vitro incubation and the phenomena are not prevented by oxyradical scavengers or metal chelators. Dopamine effects on brain mitochondria are, however, markedly prevented by reduced glutathione and N-acetyl cysteine and promoted by tyrosinase present in the incubation medium. The results imply that quinone oxidation products of dopamine are involved in mitochondrial damage under this condition. When PC12 cells are exposed to dopamine in varying concentrations (100-400 μM) for up to 24 h, a pronounced impairment of mitochondrial bio-energetic functions at several levels is observed along with a significant (nearly 40%) loss of cell viability with features of apoptotic nuclear changes and increased activities of caspase 3 and caspase 9 and all these effects of dopamine are remarkably prevented by N-acetyl cysteine. N-acetyl cysteine also blocks nearly completely the dopamine induced increase in reactive oxygen species production and the formation of quinoprotein adducts in mitochondrial fraction within PC12 cells and also the accumulation of quinone products in the culture medium. Clorgyline, an inhibitor of MAO-A, markedly decreases the formation of reactive oxygen species in PC12 cells upon dopamine exposure but has only mild protective actions against quinoprotein adduct formation, mitochondrial dysfunctions, cell death and caspase activation induced by dopamine. The results have indicated that quinone oxidation products and not reactive oxygen species are primarily involved in cytotoxic effects of dopamine and the mitochondrial impairment plays a central role in the latter process. The data have clear implications in the pathogenesis of Parkinson's disease.     

Establishment of in vitro culture system for evaluating dentin–pulp complex regeneration with special reference to the differentiation capacity of BrdU label-retaining dental pulp cells

We have proposed the new hypothesis that dental pulp stem cells play crucial roles in the pulpal healing process following exogenous stimuli in cooperation with progenitors. This study aimed to establish an in vitro culture system for evaluating dentin–pulp complex regeneration with special reference to the differentiation capacity of slow-cycling long-term label-retaining cells (LRCs). Three intraperitoneal injections of BrdU were given to pregnant ICR mice to map LRCs in the mature tissues of born animals. The upper bilateral first molars of 3-week-old mice were extracted and divided into two pieces and cultured for 0, 1, 3, 5 and 7 days using the Trowel’s method. We succeeded in establishing an in vitro culture system for evaluating dentin–pulp complex regeneration, where most odontoblasts were occasionally degenerated and lost nestin immunoreactivity because of the separation of cell bodies from cellular processes in the dentin matrix by the beginning of in vitro culture. Numerous dense LRCs mainly resided in the center of the dental pulp associating with blood vessels throughout the experimental periods. On postoperative days 1–3, the periphery of the pulp tissue including the odontoblast layer showed degenerative features. By Day 7, nestin-positive odontoblast-like cells were arranged along the pulp–dentin border and dense LRCs were committed in the odontoblast-like cells. These results suggest that dense LRCs in the center of the dental pulp associating with blood vessels were supposed to be dental pulp stem/progenitor cells possessing regenerative capacity for forming newly differentiated odontoblast-like cells.     

β-Carotene-induced apoptosis is mediated with loss of Ku proteins in gastric cancer AGS cells

High dietary intakes and high blood levels of β-carotene are associated with a decreased incidence of various cancers. The anticancer effect of β-carotene is related to its pro-oxidant activity. DNA repair Ku proteins, as a heterodimer of Ku70 and Ku80, play a crucial role in DNA double-strand break repair. Reductions in Ku70/80 contribute to apoptosis. Previously, we showed that reactive oxygen species (ROS) activate caspase-3 which induces degradation of Ku proteins. In the present study, we investigated the mechanism of β-carotene-induced apoptosis of gastric cancer AGS cells by determining cell viability, DNA fragmentation, apoptotic indices (increases in cytochrome c and Bax, decrease in Bcl-2), ROS levels, mitochondrial membrane potential, caspase-3 activity, Ku70/80 levels, and Ku-DNA-binding activity of the cells treated with or without antioxidant N-acetyl cysteine and caspase-3 inhibitor z-DEVED-fmk. As a result, β-carotene induced apoptosis (decrease in cell viability, increases in DNA fragmentation and apoptotic indices) and caspase-3 activation, but decreased Ku70/80 levels and Ku-DNA-binding activity. β-Carotene-induced alterations (increase in caspase-3 activity, decrease in Ku proteins) and apoptosis were inhibited by N-acetyl cysteine and z-DEVED-fmk. Increment of intracellular and mitochondrial ROS levels and loss of mitochondrial membrane potential were suppressed by N-acetyl cysteine, but not by z-DEVED-fmk in β-carotene-treated cells. Therefore, β-carotene-induced increases in ROS and caspase-3 activity may lead to reduction of Ku70/80 levels, which results in apoptosis in gastric cancer cells. Loss of Ku proteins might be the underlying mechanism for β-carotene-induced apoptosis in gastric cancer cells.     

Mapping of BrdU label-retaining dental pulp cells in growing teeth and their regenerative capacity after injuries

Recent studies have demonstrated that human dental pulp contains adult stem cells. A pulse of the thymidine analog BrdU given to young animals at the optimal time could clarify where slow-cycling long-term label-retaining cells (LRCs), putative adult stem cells, reside in the pulp tissue. This study focuses on the mapping of LRCs in growing teeth and their regenerative capacity after tooth injuries. Two to seven peritoneal injections of BrdU into pregnant Wistar rats revealed slow-cycling long-term dense LRCs in the mature tissues of born animals. Numerous dense LRCs were postnatally decreased in number and reached a plateau at 4 weeks after birth when they mainly resided in the center of the dental pulp, associating with blood vessels. Mature dental pulp cells were stained with Hoechst 33342 and sorted into (<0.76%) side population cells using FACS, which included dense LRCs. Some dense LRCs co-expressed mesenchymal stem cell markers such as STRO-1 or CD146. Tooth injuries caused degeneration of the odontoblast layer, and newly differentiated odontoblast-like cells contained LRCs. Thus, dense LRCs in mature pulp tissues were supposed to be dental pulp stem cells possessing regenerative capacity for forming newly differentiated odontoblast-like cells. The present study proposes the new hypothesis that both granular and dense LRCs are equipped in the dental pulp and that the dense LRCs with proliferative capacity play crucial roles in the pulpal healing process following exogenous stimuli in cooperation with the granular LRCs.     

Allogenic tooth transplantation inhibits the maintenance of dental pulp stem/progenitor cells in mice

Our recent study suggested that allogenic tooth transplantation may affect the maintenance of dental pulp stem/progenitor cells. This study aims to elucidate the influence of allograft on the maintenance of dental pulp stem/progenitor cells following tooth replantation and allo- or auto-genic tooth transplantation in mice using BrdU chasing, immunohistochemistry for BrdU, nestin and Ki67, in situ hybridization for Dspp, transmission electron microscopy and TUNEL assay. Following extraction of the maxillary first molar in BrdU-labeled animals, the tooth was immediately repositioned in the original socket, or the roots were resected and immediately allo- or auto-grafted into the sublingual region in non-labeled or the same animals. In the control group, two types of BrdU label-retaining cells (LRCs) were distributed throughout the dental pulp: those with dense or those with granular reaction for BrdU. In the replants and autogenic transplants, dense LRCs remained in the center of dental pulp associating with the perivascular environment throughout the experimental period and possessed a proliferative capacity and maintained the differentiation capacity into the odontoblast-like cells or fibroblasts. In contrast, LRCs disappeared in the center of the pulp tissue by postoperative week 4 in the allografts. The disappearance of LRCs was attributed to the extensive apoptosis occurring significantly in LRCs except for the newly-differentiated odontoblast-like cells even in cases without immunological rejection. The results suggest that the host and recipient interaction in the allografts disturbs the maintenance of dense LRCs, presumably stem/progenitor cells, resulting in the disappearance of these cell types.     

N-Acetyl Cysteine Depletes Reactive Oxygen Species and Prevents Dental Monomer-Induced Intrinsic Mitochondrial Apoptosis In Vitro in Human Dental Pulp Cells

Purpose

To investigate the involvement of intrinsic mitochondrial apoptosis in dental monomer-induced cytotoxicity and the influences of N-acetyl cysteine (NAC) on this process.

Methods

Human dental pulp cells (hDPCs) were exposed to several dental monomers in the absence or presence of NAC, and cell viability, intracellular redox balance, morphology and function of mitochondria and key indicators of intrinsic mitochondrial apoptosis were evaluated using various commercial kits.

Results

Dental monomers exerted dose-dependent cytotoxic effects on hDPCs. Concomitant to the over-production of reactive oxygen species (ROS) and depletion of glutathione (GSH), differential changes in activities of superoxide dismutase, glutathione peroxidase, and catalase were detected. Apoptosis, as indicated by positive Annexin V/propidium iodide (PI) staining and activation of caspase-3, was observed after dental monomer treatment. Dental monomers impaired the morphology and function of mitochondria, and induced intrinsic mitochondrial apoptosis in hDPCs via up-regulation of p53, Bax and cleaved caspase-3, and down-regulation of Bcl-2. NAC restored cell viability, relieved oxidative stress and blocked the apoptotic effects of dental monomers.

Conclusions

Dental monomers induced oxidative stress and mitochondrial intrinsic apoptosis in hDPCs. NAC could reduce the oxidative stress and thus protect hDPCs against dental monomer-induced apoptosis.     

Glycosylation of the Male Genital Ducts and Spermatozeugmata Formation in the Clearnose Skate Raja eglanteria

Genital ducts of three male Raja eglanteria were fixed and embedded in epoxy and methacrylate resin. Epoxy resin sections from the Leydig gland, upper and lower epididymis, ductus deferens and seminal vesicle were stained with 20 labelled lectins to examine their glycosylation. The Leydig gland consisted of columnar epithelial cells expressing N-linked glycans, N-acetyl galactosamine, glucosamine and lactosamine residues and sialic acid. Interspersed were ciliated cells of a different glycotype. The upper epididymis of cuboidal epithelium had a strongly glycosylated, ciliated apical surface and cytoplasmic granules that stained heavily with many lectins, with increased glycosylation compared to the Leydig gland. In the lower epididymis, tall, vacuolated cells showed some differences and a slight reduction in lectin staining. The ductus deferens contained two cell types and showed increased terminal N-acetyl galactosamine. The ciliated cuboidal epithelium of the seminal vesicle had marked differences from the ductus epithelium, with decreased N-acetyl galactosamine and lactosamine expression but increased subterminal N-acetyl lactosamine and galactosamine expression and sialylation. Spermatozoa were suspended in a glycosylated matrix and, in the seminal vesicle, were embedded in solid masses of matrix forming spermatozeugmata. These data show changes in glycan expression along the male genital tract, probably related to the nurture and maturation of the spermatozoa as they travel towards the seminal vesicle.     

Determination of DNA Damage in Experimental Liver Intoxication and Role of N-Acetyl Cysteine

The present study aimed at detecting DNA damage and fragmentation as well as histone acetylation depending on oxidative stress caused by CCl₄ intoxication. Also, the protective role of N-acetyl cysteine, a precursor for GSH, in DNA damage is investigated. Sixty rats were used in this study. In order to induce liver toxicity, CCl₄ in was dissolved in olive oil (1/1) and injected intraperitoneally as a single dose (2 ml/kg). N-acetyl cysteine application (intraperitoneal, 50 mg/kg/day) was started 3 days prior to CCl₄ injection and continued during the experimental period. Control groups were given olive oil and N-acetyl cysteine. After 6 and 72 h of CCl₄ injection, blood and liver tissue were taken under ether anesthesia. Nuclear extracts were prepared from liver. Changes in serum AST and ALT activities as well as MDA, TAS, and TOS levels showed that CCl₄ caused lipid peroxidation and liver damage. However, lipid peroxidation and liver damage were reduced in the N-acetyl cysteine group. Increased levels in 8-hydroxy-2-deoxy guanosine and histone acetyltransferase activities, decreased histone deacetylase activities, and DNA breakage detected in nuclear extracts showed that CCl₄ intoxication induces oxidative stress and apoptosis in rat liver. The results of the present study indicate that N-acetyl cysteine has a protective effect on CCl₄-induced DNA damage.     

In Vitro Reparative Dentin: a Biochemical and Morphological Study

In this study, starting from human dental pulp cells cultured in vitro, we simulated reparative dentinogenesis using a medium supplemented with different odontogenic inductors. The differentiation of dental pulp cells in odontoblast-like cells was evaluated by means of staining, and ultramorphological, biochemical and biomolecular methods. Alizarin red staining showed mineral deposition while transmission electron microscopy revealed a synthesis of extracellular matrix fibers during the differentiation process. Biochemical assays demonstrated that the differentiated phenotype expressed odontoblast markers, such as Dentin Matrix Protein 1 (DMP1) and Dentin Sialoprotein (DSP), as well as type I collagen. Quantitative data regarding the mRNA expression of DMP1, DSP and type I collagen were obtained by Real Time PCR. Immunofluorescence data demonstrated the various localizations of DSP and DMP1 during odontoblast differentiation. Based on our results, we obtained odontoblast-like cells which simulated the reparative dentin processes in order to better investigate the mechanism of odontoblast differentiation, and dentin extracellular matrix deposition and mineralization.Key words: dental tissue, in vitro differentiation, DMP1, DSP, type I collagen     

Capacity of Dental Pulp Differentiation in Mouse Molars as Demonstrated by Allogenic Tooth Transplantation

Dental pulp elaborates both bone and dentin under pathological conditions such as tooth replantation/transplantation. This study aims to clarify the capability of dental pulp to elaborate bone tissue in addition to dentin by allogenic tooth transplantation using immunohistochemistry and histochemistry. After extraction of the molars of 3-week-old mice, the roots and pulp floor were resected and immediately allografted into the sublingual region in a littermate. In addition, we studied the contribution of donor and host cells to the regenerated pulp tissue using a combination of allogenic tooth transplantation and lacZ transgenic ROSA26 mice. On Days 5–7, tubular dentin formation started next to the preexisting dentin at the pulp horn where nestin-positive odontoblast-like cells were arranged. Until Day 14, bone-like tissue formation occurred in the pulp chamber, where intense tartrate-resistant acid phosphatase–positive cells appeared. Furthermore, allogenic transplantation using ROSA26 mice clearly showed that both donor and host cells differentiated into osteoblast-like cells with the assistance of osteoclast-lineage cells, whereas newly differentiated odontoblasts were exclusively derived from donor cells. These results suggest that the odontoblast and osteoblast lineage cells reside in the dental pulp and that both donor and host cells contribute to bone-like tissue formation in the regenerated pulp tissue. (J Histochem Cytochem 56:1075–1086, 2008)     

STABILITY OF CHLAMYDOMONAS REINHARDI IN LIQUID NITROGEN STORAGE1

The procedure described is applicable to 11 mutant strains of C. reinhardi Dangeard. In general, freezing resulted in 3-log loss of viable cells. Nevertheless, the phenotypic response of the recovered viable cells to selected media was the same as before freezing. There was no further reduction in viability after 6–17 months storage at liquid N₂ temperatures.     

Bardoxolone-methyl inhibits migration and metabolism in MCF7 cells

Bardoxolone-methyl (BAR) is reported to have anti-inflammatory, anti-proliferative and anti-fibrotic effects. BAR activates Nrf2 and may ameliorate oxidative stress through induction of antioxidant genes. However, off-target effects, probably concentration and NFkB-dependent, have limited the clinical use of BAR. Nrf2 regulates expression of antioxidant and mitochondrial genes and has been proposed as a target for both obesity and breast cancer. Therefore, we explored whether BAR can alter migration and proliferation in the MCF7 cell line and whether metabolic function is affected by BAR. Incubation with BAR caused a time-dependent migratory inhibition and an associated decrease in mitochondrial respiration. Both migratory and mitochondrial inhibition by BAR were further enhanced in the presence of fatty acids. In addition to the activation of Nrf2, BAR altered the expression of target mRNA GCLC and UCP1. After 24?h, BAR inhibited both glycolytic capacity, reserve (p?p?N-acetyl cysteine. The fatty acid, palmitate, increased mitochondrial ROS, impaired migration and oxidative phosphorylation but palmitate toxicity towards MCF7 could not be inhibited by N-acetyl cysteine suggesting that they exert effects through different pathways. BAR-activated AKT, induced DNA damage and inhibited cell proliferation. When the proteasome was inhibited, there was loss of BAR-mediated changes in p65 phosphorylation and SOD2 expression suggesting non-canonical NFkB signaling effects. These data suggest that BAR-induced ROS are important in inhibiting MCF7 migration and metabolism by negatively affecting glycolytic capacity and mitochondrial function.