Bogdan Calenic

Deciduous and Permanent Dental Pulp Mesenchymal Cells Acquire Hepatic Morphologic and Functional Features In Vitro

INTRODUCTION Mesenchymal stem cells display extensive proliferative capacity of multilineage differentiation. The stromal compartment of mesenchymal tissues is considered to harbor stem cells. We assessed the endodermal differentiation of mesenchymal cells from deciduous and wisdom tooth pulp. METHODS Dental mesenchymal cells were isolated and expanded in vitro. After cell cultures had been established, cells were characterized using known stem cell markers. For hepatic differentiation the media was supplemented with hepatic growth factor, dexamethasone, Insulin-Transferrin-Selenium-X, and oncostatin. RESULTS Both cultures showed a number of cells positive for specific hepatic markers including alpha-fetoprotein, albumin, and hepatic nuclear factor 4alpha after differentiation. Also, small clusters of cells positive for insulin-like growth factor 1 were found. The concentration of urea increased significantly in the media. Moreover, a significant amount of glycogen was found in the cells. CONCLUSION Because the cells proved to produce specific hepatic proteins and to start functions specific for hepatocytes, such as storing glycogen and urea production, we may state that the mesenchymal cell cultures from wisdom and deciduous tooth pulp acquired morphologic and functional characteristics of hepatocytes.

Salivary biomarkers: Relationship between oxidative stress and alveolar bone loss in chronic periodontitis

Abstract Objectives. Oxidative stress is implicated in the pathogenesis of many systemic and oral diseases such as periodontal disease. The main aim of this study is to explore a possible association between salivary markers of OS and alveolar bone loss. Materials and methods. The study included 20 patients with chronic periodontitis and 20 controls. Salivary OS biomarkers 8-hidroxy-desoxguanosine (8-HOdG), malondialdehyde (MDA), uric acid, total antioxidant capacity (TAC) and glutathione peroxidase (GPx) were evaluated. Bone loss markers such as C-terminal telopeptide of type I collagen (CTX I), matrix metalloproteinases-8 (MMP-8), osteocalcin and 25-hydroxy vitamin D3 (25- OH D) were detected in this study. The methods included general biochemical tests and ELISA. Results. Salivary 8-OHdG, MDA levels were significantly higher in the chronic periodontitis group compared with controls (p < 0.05). Salivary activities for uric acid, TAC and GPx were significantly decreased in patients with chronic periodontitis vs controls (p < 0.05). Salivary levels for CTX I, MMP-8, 25-OH D and Osteocalcin were significantly higher in the chronic periodontitis group compared to the controls (p < 0.05). A significant positive correlation was observed between salivary levels of MDA and CTX I. Significant negative correlations between uric acid and CTX I and between MMP-8 and uric acid have been found. Significant positive correlations were observed between CTX I, MMP-8, 25-OH D, osteocalcin and clinical parameters of periodontal disease. Conclusions. Important oxidative stress associated with alveolar bone loss biomarkers can be detected in saliva of patients with periodontal disease.

Expression of Multiple Stem Cell Markers in Dental Pulp Cells Cultured in Serum-free Media

INTRODUCTION Stem cell lines are usually grown in medium containing animal products. Fetal bovine serum (FBS) is an important additive for cell growth; however, the allergenic potential and the possibility of contamination when we use a medium containing serum would be a barrier to transplantation and consequently to the introduction of cell therapy methods into clinical applications. METHODS Dental mesenchymal cells were isolated and expanded in vitro and maintained in 4 different serum-free media (SFMs): SFM#1 (ITS-X, embryotrophic factor [ETF]); SFM#2 (ITS-X); SFM#3 (ETF); and SFM#4 (ETF, sodium pyruvate, ascorbic acid, fibroblast growth factor [FGF-a], acidic). Viability, proliferative, and immunocytochemical tests for the cells were performed by using 4 stem cell markers (CD44H, CK19, nestin, and P63) for ectoderm, mesoderm, and endoderm. RESULTS Viability tests showed a significant difference between the control and SFMs in both deciduous tooth pulp cells (DTPCs) and wisdom tooth pulp cells (WTPCs). However, all SFMs demonstrated 84%-90% viability, whereas the control showed 90%-93%. In both DTPCs and WTPCs, SFM#1 had the highest proliferation rate among the 4 SFMs. Immunocytochemistry stained positive stem cell markers most intensely in cells cultured with SFM#1. Furthermore, all stem cell markers for ectoderm, mesoderm, and endoderm were expressed in the cells cultured with SFM#1. CONCLUSIONS SFM#1 showed an acceptable survival rate, the highest proliferation rate, and the strongest expression of all the stem cell markers. SFM#1 proved to be a suitable medium for the culture of human dental pulp stem cells and to preserve pluripotency in differentiation.

Oral malodorous compound triggers mitochondrial-dependent apoptosis and causes genomic DNA damage in human gingival epithelial cells

BACKGROUND AND OBJECTIVE Volatile sulfur compounds are the main compounds causing halitosis. One of these compounds, hydrogen sulfide (H(2)S), which is responsible for physiological halitosis, is reported also to have periodontal pathogenic activities. Hydrogen sulfide has been shown to activate the apoptotic process in different tissues. Apoptosis plays an important role in the development of periodontitis. The aim of this study was to determine whether H(2)S causes apoptosis in human gingival epithelial cells and to examine the cellular signaling pathway initiating the process. MATERIAL AND METHODS Human gingival epithelial cells were incubated with 50 ng/mL H(2)S in air contining 5% CO(2) for 24, 48 or 72 h. To detect apoptosis, the cells were stained with annexin V and 7-amino actinomycin D, and analyzed using flow cytometry. Reactive oxygen species, mitochondrial membrane depolarization and release of cytochrome C into the cytosol were assessed using flow cytometry and enzyme-linked immunosorbent assay. Activity levels for the key apoptotic enzymes caspase-9, -8 and -3 were also determined. Genomic DNA damage was detected using single-cell gel electrophoresis. RESULTS Apoptosis was significantly increased to 24.5 +/- 5.7 at 24 h and 41.5 +/- 8.9% at 48 h (p < 0.01). Reactive oxygen species were enhanced and mitochondrial membrane depolarization was collapsed. Cytochrome C release was dramatically increased (0.12 +/- 0.02 vs. 0.02 +/- 0.01 at 24 h and 0.21 +/- 0.02 vs. 0.02 +/- 0.01 ng/mL at 48 h; p < 0.05). Caspase-9 and -3 were strongly activated, while caspase-8 activity remained low. The percentage of DNA strand breaks increased, especially at 48 h. CONCLUSION Hydrogen sulfide induces apoptosis in human gingival epithelial cells by activating the mitochondrial pathway.

Oral Malodorous Compound Causes Oxidative Stress and p53-Mediated Programmed Cell Death in Keratinocyte Stem Cells

BACKGROUND It is well known that hydrogen sulfide (H(2)S), the main substance causing physiologic halitosis, is also involved in the etiology of periodontitis. Gingival crevicular epithelium is the first barrier against periodontal pathogens and their products; keratinocyte stem cells play key roles in maintaining this barrier. An increased apoptotic process can affect keratinocyte stem cells, having a direct impact on oral epithelial tissue architecture. Our objective is to determine whether H(2)S induces apoptosis in human keratinocyte stem cells. METHODS Apoptosis levels; p53 activity; reactive oxygen species; mitochondrial membrane depolarization; cytochrome C release; and caspase-9, -8, and -3 were assessed using enzyme-linked immunosorbent assay and flow cytometry. Genomic DNA damage was examined using single-cell gel electrophoresis. Real-time polymerase chain reaction was used for Bax detection. RESULTS The percentage of apoptotic cells was significantly increased (20.5% +/- 1.6% versus 4.5% +/- 1.1% at 24 hours and 37.8% +/- 5.4% versus 4.8% +/- 0.9% at 48 hours; P <0.05, respectively; n = 5). Mitochondrial membrane potential was collapsed and reactive oxygen species levels were significantly increased compared to their control groups. At each time point the amount of released cytochrome C into the cytosol was significantly increased. Caspase-9 and -3 activities were significantly increased (P <0.05), whereas caspase-8 remained inactive. After both 24 and 48 hours, total and phosphorylated p53 levels were significantly increased. CONCLUSION We conclude that H(2)S can induce apoptosis in human keratinocyte stem cells, a key component of the epithelial barrier, following DNA damage and p53 activation.

Hydrogen sulfide induces apoptosis in epithelial cells derived from human gingiva.

Hydrogen sulfide (H(2)S) is not only one of the main causes of halitosis but is also an agent of toxicity against periodontal cells and tissues in biofilm-related periodontal diseases. Also, apoptosis of gingival epithelial cells may play an important role in the onset and progress of periodontitis. We examined the effect of H(2)S on the induction of apoptosis, using human gingival fibroblasts (HGF) and keratinocyte-like Ca9-22 cells derived from human gingiva. The cells were incubated with H(2)S (100 ng ml(-1)) for 24, 48 or 72 h by adding H(2)S to air containing 5% CO(2), supplied constantly to the culture environment during incubation. The incidence of apoptosis caused by H(2)S was determined with Annexin V staining by flow cytometry. The proportion of apoptotic cells was significantly increased by exposure to H(2)S for 48 h in comparison with the control in both Ca9-22 cells and HGF. A concentration of 100 ng ml(-1) H(2)S in air is possible in the gingival sulcus. This study indicates that apoptosis in gingival epithelial cells and HGF by H(2)S may occur in the oral cavity, which may cause a periodontal condition.

Magnetic separation and characterization of keratinocyte stem cells from human gingiva

BACKGROUND AND OBJECTIVE Although keratinocyte stem cells play a key role in tissue homeostasis, wound healing and neoplasia, they remain difficult to identify and characterize. The purpose of this study was to isolate and characterize an oral keratinocyte stem-cell population. MATERIAL AND METHODS Oral human keratinocytes obtained from keratinized oral mucosa were magnetically separated using α(6) β(4) integrin and a proliferation-related marker, CD71. The isolated cell fractions were analyzed for cell size, cell cycle stage (using flow cytometry) and colony-forming ability. The expression of stem cell markers p63 and cytokeratin 19 and of differentiation markers cytokeratin 10 and involucrin was checked using immunocytochemical analysis. RESULTS The stem cell CD71(neg) fraction had the smallest cell size compared with CD71(pos) and fractions [780.7 ± 141.5 (pixels), 1422.9 ± 264.6 (pixels) and 3844.4 ± 220.1 (pixels) respectively, p < 0.01; analysis of variance (ANOVA)]. Also, the CD71(neg) subpopulation consistently had the highest colony-forming ability among the three cell fractions (126.2 ± 21.7 vs. 32.8 ± 4.5 vs. 12.4 ± 2.1 compared with CD71(pos) and subpopulations, respectively, p < 0.01; ANOVA). Moreover, the CD71(neg) fraction contained more quiescent cells and fewer actively cycling cells than the CD71(pos) cell fraction. The candidate stem cells were positive for cytokeratin 19 and p63 keratinocyte stem cell markers, while differentiation markers such as cytokeratin 10 or involucrin were absent. CONCLUSION The human gingival CD71(neg) cell fraction, separated by a magnetic system, demonstrated several characteristics of gingival keratinocyte stem cells. It is also suggested that a magnetic system may be an important tool in acquiring oral keratinocyte stem cells for research.

Cancer stem cells: Involvement in pancreatic cancer pathogenesis and perspectives on cancer therapeutics

Pancreatic cancer is one of the most aggressive and lethal malignancies. Despite remarkable progress in understanding pancreatic carcinogenesis at the molecular level, as well as progress in new therapeutic approaches, pancreatic cancer remains a disease with a dismal prognosis. Among the mechanisms responsible for drug resistance, the most relevant are changes in individual genes or signaling pathways and the presence of highly resistant cancer stem cells (CSCs). In pancreatic cancer, CSCs represent 0.2%-0.8% of pancreatic cancer cells and are considered to be responsible for tumor growth, invasion, metastasis and recurrence. CSCs have been extensively studied as of late to identify specific surface markers to ensure reliable sorting and for signaling pathways identified to play a pivotal role in CSC self-renewal. Involvement of CSCs in pancreatic cancer pathogenesis has also highlighted these cells as the preferential targets for therapy. The present review is an update of the results in two main fields of research in pancreatic cancer, pathogenesis and therapy, focused on the narrow perspective of CSCs.

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.

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 Dulbeccos modified Eagles 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 Iscoves modified Dulbeccos 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.