The Clinicopathological and Prognostic Significance of Nrf2 and Keap1 Expression in Hepatocellular Carcinoma.

Nuclear factor E2-related factor2 (Nrf2) activation is associated with both cytoprotective effects and malignant behavior of cancer cells. This study aimed to evaluate the clinicopathological implications of the expression of Nrf2, pNrf2, and its regulator Keap1 in human hepatocellular carcinomas (HCCs). Tissue microarrays consisting of 285 surgically resected HCCs were immunohistochemically stained with pNrf2, Nrf2, Keap1, stemness-related markers (keratin 19 (K19), epithelial cell adhesion molecule (EpCAM)), carbonic anhydrase IX (CAIX), epithelial-mesenchymal transition (EMT)-related markers (ezrin, uPAR, E-cadherin), and p53, and the results were correlated with the clinicopathological features. pNrf2 expression was significantly associated with increased proliferative activity, as well as EpCAM, ezrin, p53, and CAIX expression and E-cadherin loss (p < 0.05, all). Strong cytoplasmic Nrf2 expression was associated with CAIX and ezrin expression (p < 0.05, both). Keap1 was associated with increased proliferative activity, portal vein invasion, EMT-related markers, and p53 expression in CAIX-negative HCCs (p < 0.05, all). Both pNrf2 and cytoplasmic Nrf2 expression were associated with decreased overall survival (p < 0.05, both), and cytoplasmic Nrf2 expression was an independent predictor of decreased overall survival on multivariate analysis (hazard ratio 4.15, p < 0.001). Both pNrf2 and cytoplasmic Nrf2 expression were associated with poor survival and aggressive behavior of HCC. In addition, Keap1 expression was also associated with aggressive HCC behavior in CAIX-negative HCCs, suggesting that Keap1 expression should be interpreted in the context of hypoxia status.

Evaluation of the Apical Complex and the Coronal Pulp as a Stem Cell Source for Dentin-pulp Regeneration.

INTRODUCTION: This study compared the stemness and differentiation potential of stem cells derived from the apical complex (apical complex cells [ACCs]) and coronal pulp (dental pulp stem cells [DPSCs]) of human immature permanent teeth with the aim of determining a more suitable source of stem cells for regeneration of the dentin-pulp complex. METHODS: ACC and DPSC cultures were established from 13 human immature permanent teeth using the outgrowth method. The proliferation capacity and colony-forming ability of ACCs and DPSCs were evaluated. ACCs and DPSCs were analyzed for mesenchymal stem cell markers using flow cytometry. The adipogenic and osteogenic differentiation potential of ACCs and DPSCs were evaluated using the quantitative real-time polymerase chain reaction and histochemical staining. ACCs and DPSCs were transplanted subcutaneously in immunocompromised mice using macroporous biphasic calcium phosphate as a carrier. The histomorphologic characteristics of the newly formed tissues were verified using hematoxylin-eosin staining and immunohistochemical staining. Quantitative alkaline phosphatase analysis and quantitative real-time polymerase chain reaction using BSP, DSPP, POSTN, and ColXII were performed. RESULTS: ACCs and DPSCs showed similar cell proliferation potential and colony-forming ability. The percentage of mesenchymal stem cell markers was similar between ACCs and DPSCs. In the in vitro study, ACCs and DPSCs showed adipogenic and osteogenic differentiation potential. In the in vivo study, ACCs and DPSCs formed amorphous hard tissue using macroporous biphasic calcium phosphate particles. The quantity and histomorphologic characteristics of the amorphous hard tissue were similar in the ACC and DPSC groups. Formation of periodontal ligament-like tissue, positive to Col XII, was observed in ACC transplants, which was absent in DPSC transplants. CONCLUSIONS: ACCs and DPSCs showed similar stemness, proliferation rate, and hard tissue-forming capacity. The notable difference was the periodontal ligament-like fiber-forming capacity of ACCs, which indicates the presence of various lineages of stem cells in the apical complex compared with the coronal pulp. Regarding regeneration of the dentin-pulp complex, the coronal pulp can be a suitable source of stem cells considering its homogenous lineages of cells and favorable osteo/odontogenic differentiation potential.

Relief of Upper Airway Obstruction Using a Cervical Splint for Young Patients with Cerebral Palsy.

13-year old boy with spastic quadriplegia cerebral palsy visited dental clinic with chief complaints of mouth breathing and malocclusion. His mouth was constantly open at the resting position, with his mandible and tongue displaced downward. He breathed through his mouth, making a constant gurgling sound, a sign of upper airway obstruction. To enhance his mandible position, vertical chin cap was first considered, but it was not sufficient to reduce the gurgling sound or ease breathing. Then, cervical splint was considered, which effectively decreased the gurgling sound by repositioning his mandible to the anterior-superior position. Oxygen saturation was increased when the cervical splint was used. Cervical splint can effectively assist breathing in patients with cerebral palsy, but it should be carefully applied as long-term use can result in unexpected complications. Under instruction by a physician regarding proper usage, a cervical splint can be applied to assist breathing in patients with cerebral palsy.

Cytokine Expression of Stem Cells Originating from the Apical Complex and Coronal Pulp of Immature Teeth.

INTRODUCTION: The aim of this study was to measure and compare the expression levels of cytokines from developing apical complex cells (DACCs) and dental pulp stem cells (DPSCs) of the immature tooth. METHODS: DPSC-conditioned medium (CM) and DACCs-CM were obtained from human young teeth, and 174 cytokines secreted from each CM were identified and compared. A cytokine membrane array and enzyme-linked immunosorbent assay were used to measure and compare the expression levels of the cytokines. Immunocytochemistry targeting insulin-like growth factor-1 and neurotrophin-3 was additionally performed. RESULTS: There were statistically significant differences in the expression levels of 25 cytokines: 22 and 3 were expressed more strongly in DPSCs-CM and DACCs-CM, respectively. Odontoblast differentiation-related cytokines were more strongly expressed in DPSCs-CM, while cell-proliferation-related cytokines were more strongly expressed in DACCs-CM. Proinflammatory and anti-inflammatory cytokines were predominantly expressed in DPSCs-CM and DACCs-CM, respectively. CONCLUSIONS: DPSCs may exert a stronger paracrine effect than DACCs on regeneration of the dentin-pulp complex, in terms of odontoblast differentiation.

Biological efficacy of two mineral trioxide aggregate (MTA)-based materials in a canine model of pulpotomy.

The aim of this study was to compare the biocompatibility of Endocem Zr® and ProRoot MTA® by histopathologic analysis in a canine model of pulpotomy. This study utilized 39 teeth of two beagle dogs. The exposed pulp tissues were treated by pulpotomy using ProRoot MTA (n=19) or Endocem Zr (n=20). After 8 weeks, the teeth were extracted and processed with hematoxylin-eosin staining for histologic evaluation. Most of the specimens in both groups developed a calcific barrier at the pulp amputation site and formed an odontoblast layer. However, some of the Endocem Zr specimens showed less calcific barrier formation with a greater inflammatory response and less odontoblast layer formation when compared with the ProRoot MTA specimens. ProRoot MTA and Endocem Zr specimens developed a calcific barrier; however, ProRoot MTA was more biocompatible than Endocem Zr.

Comparative Gene Expression Analysis of the Coronal Pulp and Apical Pulp Complex in Human Immature Teeth.

INTRODUCTION: This study determined the gene expression profiles of the human coronal pulp (CP) and apical pulp complex (APC) with the aim of explaining differences in their functions. METHODS: Total RNA was isolated from the CP and APC, and gene expression was analyzed using complementary DNA microarray technology. Gene ontology analysis was used to classify the biological function. Quantitative reverse-transcription polymerase chain reaction and immunohistochemical staining were performed to verify microarray data. RESULTS: In the microarray analyses, expression increases of at least 2-fold were present in 125 genes in the APC and 139 genes in the CP out of a total of 33,297 genes. Gene ontology class processes found more genes related to immune responses, cell growth and maintenance, and cell adhesion in the APC, whereas transport and neurogenesis genes predominated in the CP. Quantitative reverse-transcription polymerase chain reaction and immunohistochemical staining confirmed the microarray results, with DMP1, CALB1, and GABRB1 strongly expressed in the CP, whereas SMOC2, SHH, BARX1, CX3CR1, SPP1, COL XII, and LAMC2 were strongly expressed in the APC. CONCLUSIONS: The expression levels of genes related to dentin mineralization, neurogenesis, and neurotransmission are higher in the CP in human immature teeth, whereas those of immune-related and tooth development-related genes are higher in the APC.

Characteristics of stem cells from human exfoliated deciduous teeth (SHED) from intact cryopreserved deciduous teeth.

The aim of this study is to compare the characteristics of stem cells derived from human exfoliated deciduous teeth (SHED) from cryopreserved intact deciduous teeth with those of fresh SHED. In total, 20 exfoliated deciduous teeth were randomly divided into a fresh group (f-SHED; n = 11) and cryopreserved group (c-SHED; n = 9; stored for 1-8 months). Following thawing and separation of the pulp, the SHED cells were cultured, and the characteristics as mesenchymal stem cells were investigated using proliferation assays, cell-cycle analysis, colony-forming unit-fibroblast (CFU-F) assays, and flow cytometry analyses. Furthermore, differentiation into adipogenic and osteogenic lineages was investigated in vitro as well as in vivo via transplantation in mice. We found no significant differences between the two groups in the proliferation analyses, in the expression of mesenchymal stem cell markers, or in the adipogenic and osteogenic differentiation in vitro (p < 0.05). Furthermore, the in vivo transplantation results showed no significant differences in the quantity of bone tissue that formed or in histochemistry performance (p < 0.05). In conclusion, cryopreservation of intact exfoliated deciduous teeth appears to be a useful method for preserving SHED.

Effects of In Vitro Osteogenic Induction on In Vivo Tissue Regeneration by Dental Pulp and Periodontal Ligament Stem Cells.

INTRODUCTION: The aim of this study was to determine the effects of in vitro odontogenic/cementogenic differentiation on the in vivo tissue regeneration of dental pulp stem cells (DPSCs) and periodontal ligament stem cells (PDLSCs). METHODS: DPSCs and PDLSCs were predifferentiated for 0, 4, or 8 days with an odontogenic/cementogenic medium and then transplanted into subcutaneous pockets in immunocompromised mice. The transplants were harvested 9 weeks after transplantation, and the characteristics of the newly formed tissues in vivo were analyzed by histologic staining; examining alkaline phosphate activity; immunohistochemical staining for osteocalcin, dentin sialoprotein, and type XII collagen; and quantitative real-time polymerase chain reaction to analyze the expression patterns of the following genes: RUNX2, OC, DMP1, DSPP, POSTN, CP23, and Col XII. RESULTS: In DPSC transplants, the amount of new tissues was similar in all groups, whereas in predifferentiated transplants the OC and DSPP expression were higher than undifferentiated transplants. Predifferentiated PDLSC transplants generated more hard tissue and expressed higher alkaline phosphatase activity than undifferentiated transplants. In particular, 8-day predifferentiated PDLSC transplants formed tissue closer to the cementum/PDL complex in vivo as confirmed by the higher expression levels of POSTN, CP23, and Col XII. CONCLUSIONS: Although there was no significant increase in tissue-forming ability among DPSCs after predifferentiation, predifferentiated DPSCs generated hard tissue closer to dentin. Also, predifferentiated PDLSCs appeared to be able to generate higher-quality and greater amounts of tissue for dental regeneration than undifferentiated PDLSCs.

Ectopic Hard Tissue Formation by Odonto/Osteogenically In Vitro Differentiated Human Deciduous Teeth Pulp Stem Cells.

There have been many attempts to use the pulp tissue from human deciduous teeth for dentin or bone regeneration. The objective of this study was to determine the effects of odonto/osteogenic in vitro differentiation of deciduous teeth pulp stem cells (DTSCs) on their in vivo hard tissue-forming potential. DTSCs were isolated from extracted deciduous teeth using the outgrowth method. These cells were exposed to odonto/osteogenic stimuli for 4 and 8 days (Day 4 and Day 8 groups, respectively), while cells in the control group were cultured in normal medium. The in vitro differentiated DTSCs and the control DTSCs were transplanted subcutaneously into immunocompromised mice with macroporous biphasic calcium phosphate and sacrificed at 8 weeks post-implantation. The effect of odonto/osteogenic in vitro differentiation was evaluated using alkaline phosphatase (ALP) staining and quantitative reverse transcription polymerase chain reaction (RT-PCR). The in vivo effect was evaluated by qualitative RT-PCR, assessment of ALP activity, histologic analysis, and immunohistochemical staining. The amount of hard tissue was greater in Day 4 group than Day 8 group (p = 0.014). However, Day 8 group generated lamellar bone-like structure, which was immunonegative to anti-human dentin sialoprotein with significantly low expression level of DSPP compared with the control group (p = 0.008). This study demonstrates that odonto/osteogenic in vitro differentiation of DTSCs enhances the formation of bone-like tissue, instead of dentin-like tissue, when transplanted subcutaneously using MBCP as a carrier. The odonto/osteogenic in vitro differentiation of DTSCs may be an effective modification that enhances in vivo bone formation by DTSCs.

Continued root development of a surgically repositioned human incisor tooth germ.

Conventional orthodontic traction may not be the treatment of choice in cases of inverted impaction of a maxillary incisor, especially when located near the alveolar crest. Poor prognosis is associated with the limited space for proper root development, resulting in a root too short for normal function and/or a severely dilacerated root interrupting the force-induced positioning. The surgical repositioning of ectopic impacted toothgerm before the development of root could be a valuable alternative choice of treatment before the decision of extraction. In this case report, an impacted immature incisor toothgerm in complete inversion was surgically repositioned using a closed-flap technique in a boy who was 6 years 8 months old. Continued root formation and spontaneous eruption were observed after surgery over the 51-month follow-up period, without pulpal or periodontal complications.

In vitro and in vivo characteristics of stem cells derived from the periodontal ligament of human deciduous and permanent teeth.

In many studies, adult stem cells have been found in human periodontal ligament (PDL), but in most cases they were found in the permanent teeth. The aim of the present study was to characterize stem cells from the PDL of deciduous teeth (dPDLSCs) and compare them with those from the PDL of permanent teeth (pPDLSCs). Stem cell markers were examined by a flow cytometric analysis. The results of in vitro differentiation into adipogenic and osteogenic lineages were analyzed by histochemical staining and quantitative reverse transcription-polymerase chain reaction (RT-PCR). The results of in vivo transplantation were analyzed by histological staining, immunohistochemical staining, and quantitative RT-PCR. There were no significant differences in the proliferation rate, cell cycle distribution, expressions of stem cell markers such as Stro-1 and CD146, or in vitro differentiation. The pPDLSC transplants made more typical cementum/PDL-like tissues and expressed more cementum/PDL-related genes (CP23 and collagen XII) than did the dPDLSC transplants. Together, these results suggest that pPDLSCs are better candidates for use in reconstructing periodontium.

Pedobacter agri sp. nov., from soil.

A Gram-negative strain, PB92(T), which belongs to the family Sphingobacteriaceae, was isolated from soil (Daejeon, Korea). Phylogenetic analysis based on 16S rRNA gene sequences showed that strain PB92(T) was associated with the genus Pedobacter and was most closely related to the type strains Pedobacter sandarakinus DS-27(T) (97.7 %), Pedobacter roseus CL-GP80(T) (97.5 %) and Pedobacter suwonensis 15-52(T) (97.5 %). The major cellular fatty acid components of strain PB92(T) were C(16 : 1)omega7c (21.4 %), iso-C(15 : 0) (30.8 %), iso-C(17 : 0) 3-OH (9.3 %) and iso-C(15 : 0) 2-OH (11.2 %). The G+C content of the genomic DNA from strain PB92(T) was 41.4 mol%. Analysis of 16S rRNA gene sequences, as well as physiological and biochemical tests, indicated that strain PB92(T) could be differentiated genotypically and phenotypically from reference species of the genus Pedobacter. Strain PB92(T) (=KCTC 12511(T)=DSM 19486(T)) is proposed as the type strain of a novel species, Pedobacter agri sp. nov.