Hyung-Keun You

Dynamic changes of gangliosides expression during the differentiation of embryonic and mesenchymal stem cells into neural cells

Stem cells are used for the investigation of developmental processes at both cellular and organism levels and offer tremendous potentials for clinical applications as an unlimited source for transplantation. Gangliosides, sialic acid-conjugated glycosphingolipids, play important regulatory roles in cell proliferation and differentiation. However, their expression patterns in stem cells and during neuronal differentiation are not known. Here, we investigated expression of gangliosides during the growth of mouse embryonic stem cells (mESCs), mesenchymal stem cells (MSCs) and differentiated neuronal cells by using high-performance thin-layer chromatography (HPTLC). Monosialoganglioside 1 (GM1) was expressed in mESCs and MSCs, while GM3 and GD3 were expressed in embryonic bodies. In the 9-day old differentiated neuronal cells from mESCs cells and MSCs, GM1 and GT1b were expressed. Results from immunostaining were consistent with those observed by HPTLC assay. These suggest that gangliosides are specifically expressed according to differentiation of mESCs and MSCs into neuronal cells and expressional difference of gangliosides may be a useful marker to identify differentiation of mESCs and MSCs into neuronal cells.

Growth and osteogenic differentiation of alveolar human bone marrow‐derived mesenchymal stem cells on chitosan/hydroxyapatite composite fabric

Scaffolds can be used for tissue engineering because they can serve as templates for cell adhesion and proliferation for tissue repair. In this study, chitosan/hydroxyapatite (CS/HAp) composites were prepared by coprecipitation synthesis. Then, CS and CS/HAp fabrics were prepared by wet spinning. CS fibers with a diameter of 15 ± 1.3 μm and CS/HAp fibers with a diameter of 22 ± 1.2 μm were successfully produced; incorporation of HAp into the CS/HAp fibers was confirmed by X-ray diffraction analysis. Biological in vitro evaluations showed that human mesenchymal stem cells (hMSCs) cultured on CS/HAp fabric showed increased proliferation compared to those cultured on pure CS fabric, which was observed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, DNA content assay, and [(3) H] thymidine incorporation assay. Neither the CS nor CS/HAp scaffold exhibited any cytotoxicity to hMSCs, as shown by viability staining and cytotoxicity fluorescence image assays. After 10 days of culturing, the attachment of cells onto the scaffold was observed by scanning electron microscopy. Furthermore, under osteogenic differentiation conditions, alkaline phosphatase (ALP) activity and calcium accumulation was higher in cells cultured on the CS/HAp scaffold than in cells cultured on the CS scaffold. The mRNA expression of osteoblast markers, including ALP, osteocalcin, Co1Ia1, and runt-related transcription factor 2, was higher in cells cultured on CS/HAp than in cells cultured on the CS fabric. The results of this study indicate that the CS/HAp composite fabric may serve as a good scaffold for bone tissue engineering applications.

Effects of nicotine on proliferation and osteoblast differentiation in human alveolar bone marrow-derived mesenchymal stem cells

AIMS Nicotine is a risk factor for various diseases, including osteoporosis, oral cancer, and periodontal disease. Numerous studies have elucidated the effects of nicotine on cell proliferation and differentiation. The purpose of this study was to determine the effects of nicotine on the proliferation and osteoblast differentiation of human alveolar bone marrow-derived mesenchymal stem cells (hABMMSCs). MAIN METHODS In this study, we treated hABMMSCs with different doses (1 μM to 5 mM) of nicotine. The survival and proliferation of hABMMSCs were evaluated by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium (MTT) assay and crystal violet assay. TUNEL and propidium iodide (PI) double staining assay were also performed. The effect of nicotine on osteoblast differentiation of hABMMSCs was determined by measuring calcium accumulation using alizarin red-sulfate (AR-S) staining, measurement of alkaline phosphatase (ALP) activity, and semi-quantitative PCR of osteoblast markers. KEY FINDINGS The survival and proliferation of hABMMSCs did not differ when they were exposed to nicotine at concentrations ranging from 1 μM to 100 μM; however, cell proliferation increased when the cells were exposed to nicotine at concentrations of 1-2 mM and decreased significantly when exposed to 5mM of nicotine. A number of cells were stained by PI but not by TUNEL, and membrane vacuolization was observed in hABMMSCs treated with 5mM nicotine. Calcium accumulation; ALP activity; and mRNA levels of ALP, bone sialoprotein (BSP), collagen type I α 1 (Col1αI), and runt-related transcription factor 2 (Runx2) were significantly decreased by treatment with 2mM of nicotine, while osteocalcin transcripts decreased by treatment with 1 to 2 mM of nicotine. SIGNIFICANCE These results suggest that nicotine has a bimodal effect on the proliferation and osteoblast differentiation in hABMMSCs.

Cellular attachment and osteoblast differentiation of mesenchymal stem cells on natural cuttlefish bone

The purpose of this study was to describe an approach that aims to provide fundamental information for the application of natural cuttlefish bone. Before applying cuttlefish bone as a bone defect filling material, we evaluated proliferation, adhesion, and cell viability of human mesenchymal stem cells (hMSCs) cultured on cuttlefish bone. Cuttlefish bone was separated into two parts (dorsal shield and lamellar region) and each part was used. Cell proliferation and viability were assessed using the MTS assay and live/dead fluorescence staining method. The morphology was observed using scanning electron microscopy (SEM). hMSCs were stimulated with osteogenic medium and osteoblast differentiation was evaluated. The fluorescence images showed that the seeded cells grew well and that cell distribution was in accordance with the surface morphology of the cuttlefish bone. Compared with the dorsal shield, cells penetrated deeper into the three-dimensional inner space of the lamellar part. Furthermore, under osteogenic differentiation conditions, alkaline phosphatase activity increased and the mRNA expression of ALP, runt-related transcription factor 2, and collagen type I α1 was increased in hMSCs cultured on both the dorsal shield and lamellar block. These results indicate the potential of cuttlefish bone as an ideal scaffold for bone regenerative materials.

Effects of gangliosides on the differentiation of human mesenchymal stem cells into osteoblasts by modulating epidermal growth factor receptors

Gangliosides are sialic acid-conjugated glycosphingolipids that are believed to regulate cell differentiation as well as the signals of several signal molecules, including epidermal growth factor receptors (EGFR). These compounds are localized in a glycosphingolipid-enriched microdomain on the cell surface and regulated by the glycosphingolipid composition. However, the role that gangliosides play in osteoblastogenesis is not yet clearly understood, therefore, in this study, the relationship between gangliosides and EGFR activation was investigated during osteoblast differentiation in human mesenchymal stem cells (hMSCs). The results of high-performance thin-layer chromatography (HPTLC) showed that ganglioside GM3 expression was decreased, whereas ganglioside GD1a expression was increased during the differentiation of hMSCs into osteoblasts. In addition, an increase in the activation of alkaline phosphatase (ALP) was observed in response to treatment with EGF (5 ng/ml) and GD1a (1 microM) (p<0.05). The activation of ALP was significantly elevated in response to treatment of ganglioside GD1a with EGF when compared to control cells (p<0.01). However, treatment with GM3 (1muM) resulted in decreased ALP activation (p<0.01), and treatment of hMSCs with a chemical inhibitor of EGFR, AG1478, removed the differential effect of the two gangliosides. Moreover, incubation of the differentiating cells with GD1a enhanced the phosphorylation of EGFR, whereas treatment with GM3 reduced the EGFR phosphorylation. However, AG1478 treatment inhibited the effect of ganglioside GD1a elicitation on EGFR phosphorylation. Taken together, these results indicate that GD1a promotes osteoblast differentiation through the enhancement of EGFR phosphorylation, but that GM3 inhibits osteoblast differentiation through reduced EGFR phosphorylation, suggesting that GM3 and GD1a are essential molecules for regulating osteoblast differentiation in hMSCs.

Effects of the Dichloromethane Fraction of Dipsaci Radix on the Osteoblastic Differentiation of Human Alveolar Bone Marrow-Derived Mesenchymal Stem Cells

Dipsaci Radix is the dried root of Dipsacus asper Wall. It has been used in Korean herbal medicine to treat bone fractures. In this study, we examined the effect of the dichloromethane fraction of Dipsaci Radix (DRDM) on the osteoblastic differentiation of human alveolar bone marrow-derived MSCs (ABM-MSCs). The ABM-MSCs were isolated from healthy subjects and cultured in vitro, followed by phenotypic characterization. They showed a fibroblast-like morphology and expressed CD29, CD44, CD73, and CD105, but not CD34. Calcified nodules were generated in response to both dexamethasone (DEX) and DRDM. There was a significant increase in the alkaline phosphatase (ALP) activity and protein expression of bone sialoprotein (BSP) and osteocalcin (OC) in response to DEX and DRDM as compared to control. These results provide evidence for the osteogenic potential of cultured ABM-MSCs in response to DRDM. Also, an active single compound was additionally isolated from DRDM. The single compound (hederagenin 3-O-(2-O-acetyl)-α-L-arabinopyranoside) also significantly increased ALP activity and the level of protein expression of BSP and OC. These results highlight the possible clinical applications of DRDM and hederagenin 3-O-(2-O-acetyl)-α-L-arabinopyranoside in bone regeneration.

Healing pattern of the mucous membrane after tooth extraction in the maxillary sinus

Purpose To investigate the healing pattern of the mucous membrane after tooth extraction necessitated by periodontal disease in the maxillary sinus. Methods One hundred and three patients with 119 maxillary sinuses were investigated. Before implant placement, cone-beam computed tomography (CT) scanning was performed. The causes of extraction, the time elapsed since extraction, smoking, periodontal disease in adjacent teeth, and gender were recorded. In addition, the thickness of the mucous membrane of the maxillary sinus and the height of residual alveolar bone at the extracted area were calculated from CT images. Results The thickness of the mucous membrane in the periodontal disease group (3.05±2.71 mm) was greater than that of the pulp disease group (1.92±1.78 mm) and the tooth fracture group (1.35±0.55 mm; P<0.05). The causes of extraction, the time elapsed since extraction, and gender had relationships with a thickening of the mucous membrane of the maxillary sinus (P<0.05). In contrast, the height of the residual alveolar bone at the extracted area, periodontal disease in adjacent teeth, and smoking did not show any relation to the thickening of the mucous membrane of the maxillary sinus. Conclusions The present study revealed distinct differences in healing patterns according to the causes of extraction in the maxillary sinus, especially periodontal disease, which resulted in more severe thickening of the mucous membrane.

Inhibitory effect of Aralia continentalis on the cariogenic properties of Streptococcus mutans.

ETHNOPHARMACOLOGICAL RELEVANCE Aralia continentalis has been used in traditional Korean medicine for dental diseases such as toothache, dental caries, periodontal disease and gingivitis, and also has been used for neuralgia, analgesia, sweating, and as an antirheumatic. AIM OF THE STUDY The present study was designed to investigate the inhibitory effect of Aralia continentalis extract on cariogenic properties of Streptococcus mutans, which is one of the most important bacteria in the formation of dental caries and dental plaque. MATERIALS AND METHODS The inhibitory effects of Aralia continentalis extract on the growth, acid production, water-insoluble glucan synthesis, and adhesion were investigated in Streptococcus mutans. The biofilm formation of Streptococcus mutans was determined by scanning electron microscopy (SEM) and safranin staining. RESULTS The ethanol extract of Aralia continentalis showed concentration dependent inhibitory activity on the growth of Streptococcus mutans and significant inhibition of acid production at the concentrations of 0.25, 0.5, 1, 2 and 4 mg/ml compared to the control group. The synthesis of water-insoluble glucan by glucosyltransferase (GTFase) was decreased in the presence of 0.5-4 mg/ml of the extract of Aralia continentalis. The extract markedly inhibited Streptococcus mutans adherence to saliva-coated hydroxyapatite beads (S-HAs). The extract of Aralia continentalis has an inhibitory effect on the formation of Streptococcus mutans biofilms at the concentrations higher than 2mg/ml. CONCLUSIONS These results suggest that Aralia continentalis may inhibit cariogenic properties of Streptococcus mutans, and also may support the scientific rationale that native inhabitants used the extract for the treatment of dental diseases.

Fabrication of 3D alginate scaffold with interconnected pores using wire-network molding technique

In this study, we fabricated 3D porous scaffold by ‘Wire-Network Molding’ technique with alginate gel which has been used for cartilage regeneration because of the chemical similarity. Firstly, prepared ETPCS-S wires with size of rectangular cross section 600 μm by 600 μm, 400 μm by 400 μm, respectively, and the wires are inserted in designed mold. Secondly, sterilized 2 wt% alginate gel within hMSC (human Mesenchymal stem cell) was injected into the assembled mold. The concentration of hMSC in the used alginate gel is about 5000 cells per scaffold. For the gelation of alginate gel, the mold was soaked in 5 wt% CaCl2 solution for 5 min. Subsequently, wires are separated from the mold and the mold is removed from alginate gel. Consequently, the remained alginate scaffold has interconnected pores with a configuration of wire-network. Additionally, to analyze the cell-culturing characteristics, 1-day, 3-day, and 7-day cultured scaffolds which encapsulate hMSC are assessed using MTS assay. Consequently, the optical density of 400 μm-WNM scaffolds and 600 μm-WNM scaffolds are clearly more increased than control scaffolds without pores.

TGF-β Signaling Regulates Cementum Formation through Osterix Expression

TGF-β/BMPs have widely recognized roles in mammalian development, including in bone and tooth formation. To define the functional relevance of the autonomous requirement for TGF-β signaling in mouse tooth development, we analyzed osteocalcin-Cre mediated Tgfbr2 (OCCreTgfbr2fl/fl) conditional knockout mice, which lacks functional TGF-β receptor II (TβRII) in differentiating cementoblasts and cementocytes. Strikingly, OCCreTgfbr2fl/fl mutant mice exhibited a sharp reduction in cellular cementum mass with reduced matrix secretion and mineral apposition rates. To explore the molecular mechanisms underlying the roles of TGF-β signaling through TβRII in cementogenesis, we established a mouse cementoblast model with decreased TβRII expression using OCCM-30 cells. Interestingly, the expression of osterix (Osx), one of the major regulators of cellular cementum formation, was largely decreased in OCCM-30 cells lacking TβRII. Consequently, in those cells, functional ALP activity and the expression of genes associated with cementogenesis were reduced and the cells were partially rescued by Osx transduction. We also found that TGF-β signaling directly regulates Osx expression through a Smad-dependent pathway. These findings strongly suggest that TGF-β signaling plays a major role as one of the upstream regulators of Osx in cementoblast differentiation and cementum formation.