p53-Pathway activity and apoptosis in hydrogen sulfide-exposed stem cells separated from human gingival epithelium.

BACKGROUND AND OBJECTIVE: Hydrogen sulfide ( H2S ) is a volatile sulfur compound responsible for physiological halitosis. H2S was also reported as having periodontal pathologic activities. Gingival crevicular epithelium is the first barrier against periodontal pathogens and their products; oral keratinocyte stem cells OKSCs play key roles in maintaining this barrier. The p53 pathway is responsible for regulating key biological events. Increased apoptosis and cell-cycle arrest of DNA repair can affect keratinocyte stem cells, having a direct impact on the architecture of the oral epithelial tissue. However, the link between H2S , p53 activity and OKSCs has not yet been fully explored. The main objective of the present study was to explore the implications of the p53 pathway in OKSCs following exposure to H2S. MATERIAL AND METHODS: OKSCs were isolated from human gingival epithelium and incubated with physiological levels of H2S for 24 and 48 h. Apoptosis and the mitochondrial membrane potential were detected using flow cytometry. Cytochrome c, total p53, phosphorylated p53 and caspase activity were assessed using specific ELISAs. p53 Pathway gene activity was assayed using quantitative RT-PCR. RESULTS: The levels of apoptosis were significantly increased following incubation in the presence of H2S, especially after 48 h (36.95 ± 1.91% vs. 4.77 ± 0.74%). Caspases 9 and 3 were activated, whereas caspase-8 activity remained low. Total p53 activity and particularly phosphorylated p53 at serine 46, were significantly enhanced compared with controls (47.11 ± 9.84 units/mL vs. 1.5 ± 0 units/mL and 32.22 ± 10.23 units/mL vs. 0.15 ± 0 units/mL, respectively, at 48 h). Among p53 pathway genes, apoptosis-related genes [i.e. phosphatase and tensin homolog ( PTEN ), B-cell CLL/lymphoma 2 ( BCL2), sirtuin 3 ( SIRT3) and caspases]) were dramatically increased when compared with controls. Moreover, cell-cycle progression genes [i.e. E2F transcription factor (E2F) family and histone deacetylase ( HDAC )] and DNA-repair genes [i.e. growth arrest and DNA-damage-inducible, gamma ( GADD45G ) family and serine/threonine-protein kinase Chk2 ( CHEK2)] were also increased. CONCLUSION: Following incubation with H2 S , OKSCs express multiple p53-associated genes, including programmed cell death, cell-cycle control and DNA-repair genes.

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) ß(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.

[A study of linearity and reciprocity during shock applied with a hammer to human dry skull].

The authors used a human dry skull on which the cranial bone mandible had been joined with an artificial articulator disk to form a single unit. Impact acceleration corresponding to weak and strong tapping was considered a dynamic load in examining the vibration transfer characteristics of the facial cranial bone when impact was applied from the mentum section in a situation designed to be closer to reality. Flexion injection type (resonance frequency f0 = 100 to 150 Hz, produced by GC Corp.) was applied to the human dry skull as an artificial periodontal membrane at thickness of 0.3 mm. In addition, Exaflex heavy body type (f0 = 400 Hz, produced by GC Corp.) was applied as an artificial disk. This was then placed on a damper produced by spreading a rubber dam sheet with a thickness of 35 microns on a tire tube with a diameter of 35 cm and an air pressure of 35 kg/cm2. Investigations were then made concerning linearity and reciprocity to determine whether an experimental system could be achieved or not. This was then followed by modal analysis. As a result, the following matters were ascertained: (1) The resonating area differed according to the extent of the force. (2) An increase in the viscoelastic elements of the silicon was accompanied by attenuation of force. (3) Directionality of force attenuation was caused by the complexity of bone structure. (4) A tapping force of 0.3G or 1G was sufficiently attenuated by the facial cranial bone. (5) The transfer function at the bone seams and thinner areas of the bones was insufficient for modal analysis of the facial region and total cranial bone of the human dry skull.