Objective. To investigate the effects of the hypoxia inducible factor-1 (HIF-1) activation–mimicking agent cobalt chloride (CoCl2) on the osteogenic differentiation of human mesenchy-mal stem cells (hMSCs) and elucidate the underlying mole-cular mechanisms. Study design. The dose and exposure periods for CoCl2 in hMSCs were optimized by cell viability assays. After confirmation of CoCl2-induced HIF-1α and vas-cular endothelial growth factor expression in these cells by RT-PCR, the effects of temporary preconditioning with CoCl2 on hMSC osteogenic differentiation were evaluated by RT- PCR analysis of osteogenic gene expression, an alkaline phos-phatase (ALP) activity assay and by alizarin red S staining. Results. Variable CoCl2 dosages (up to 500 µM) and exposure times (up to 7 days) on hMSC had little effect on hMSC survival. After CoCl2 treatment of hMSCs at 100 µM for 24 or 48 hours, followed by culture in osteogenic differentiating media, several osteogenic markers such as Runx-2, osteocal-cin and osteopontin, bone sialoprotein mRNA expression level were found to be up-regulated. Moreover, ALP acti-vity was increased in these treated cells in which an accele-rated osteogenic capacity was also verified by alizarin red S staining. Conclusions. The osteogenic differentiation poten-tial of hMSCs could be preserved and even enhanced by CoCl2 treatment.

MicroRNAs (miRNAs, miRs) are about 21-25 nucleotides in length and regulate mRNA translation by base pairing to partially complementary sites, predominantly in the 3’-untranslated region (3’-UTR) of the target mRNA. In this study, the expression profile of miRNAs was compared and analyzed for the establishment of miRNA-related odontoblast differentiation using MDPC-23 cells derived from mouse dental papilla cells. To determine the expression profile of miRNAs during the differentiation of MDPC-23 cells, we employed miRNA microarray analysis, quantitative real-time PCR (qRT-PCR) and Alizaline red-S staining. In the miRNA microarray analysis, 11 miRNAs were found to be up- or down-regulated more than 3-fold between day 0 (control) and day 5 of MDPC-23 cell differentiation among the 1,769 miRNAs examined. In qRT-PCR analysis, the expression levels of two of these molecules, miR-194 and miR-126, were increased and decreased in the control MDPC-23 cells compared with the MDPC-23 cells at day 5 of differentiation, respectively. Importantly, the overexpression of miR-194 significantly accelerated mineralization compared with the control cultures during the differentiation of MDPC-23 cells. These results suggest that the miR-194 augments MDPC-23 cell differentiation, and potently accelerates the mineralization process. Moreover, these in vitro results show that different miRNAs are deregulated during the differentiation of MDPC-23 cells, suggesting the involvement of these genes in the differentiation and mineralization of odontoblasts.

Hertwig's epithelial root sheath (HERS) consists of bilayered cells derived from the inner and outer dental epithelia and plays important roles in tooth root formation as well as in the maintenance and regeneration of periodontal tissues. With regards to the fate of HERS, and although previous reports have suggested that this entails the formation of epithelial rests of Malassez, apoptosis or an epithelialmesenchymal transformation (EMT), it is unclear what changes occur in the epithelial cells in this structure. This study examined whether HERS cells undergo EMT using a keratin-14 (K14) cre:ROSA 26 transgenic reporter mouse. The K14 transgene is expressed by many epithelial tissues, including the oral epithelium and the enamel organ. A distinct K14 expression pattern was found in the continuous HERS bi-layer and the epithelial diaphragm were visualized by detecting the β-galactosidase (lacZ) activity in 1 week postnatal mice. The 2 and 4 week old mice showed a fragmented HERS with cell aggregation along the root surface. However, some of the lacZ-positive dissociated cells along the root surface were not positive for pan-cytokeratin. These results suggest that the K14 transgene is a valuable marker of HERS. In addition, the current data suggest that some of the HERS cells may lose their epithelial properties after fragmentation and subsequently undergo EMT.

Teeth develop via a reciprocal induction between the ectomesenchyme originating from the neural crest and the ectodermal epithelium. During complete formation of the tooth morphology and structure, many cells proliferate, differentiate, and can be replaced with other structures. Apoptosis is a type of genetically-controlled cell death and a biological process arising at the cellular level during development. To determine if apoptosis is an effective mechanism for eliminating cells during tooth development, this process was examined in the rat mandible including the developing molar teeth using the transferase-mediated dUTP-biotin nick labeling (TUNEL) method. The tooth germ of the mandibular first molar in the postnatal rat showed a variety of morphological appearances from the bell stage to the crown stage. Strong TUNEL-positive reactivity was observed in the ameloblasts and cells of the stellate reticulum. Odontoblasts near the prospective cusp area also showed a TUNEL positive reaction and several cells in the dental papilla, which are the forming pulp, were also stained intensively in this assay. Our results thus show that apoptosis may take place not only in epithelial-derived dental organs but also in the mesenchyme-derived dental papilla. Hence, apoptosis may be an essential biological process in tooth development.

Recently, new antibacteri al strategy has been demanded because of the incr eased occurrence 0 1' drug-resistant bact ena Accordingly , phot odynarnic therapy has been attempted for clinical appli cation against drug-resistant bact eri a, Antimi crobial photodyna rni c t herapy combines a nontoxic photosensiti zer with harmJ ess vi sible light to generate singlet oxygen and free radicals that kill lni croor ganism. In thi s study , we investigated bactericida l effect of photodynarnic ther apy by using phot osensiti zer chl orin e6 to pathogenic bacteria including a gram- positive Stapbylococuus a ureus and gram- negative strains including Pseudomonas aeruginosa, EscbeJicbia coù; and SaJmoneJla en terica sero v，없' 7γpbimurium， To exa때 n e antimicrobial ef fec t 0 1' photodynamic t herapy, we measured inhibition zone‘ colony forrning units (CFU) , and in situ viability of bacteri al cell s after illumination with an energy density (Diode pumped laser driver LD203이 01 20J/cm2 in the presence 0 1' lOuM chlorin e6, We found the incr ease 0 1' inhibition zone on agar plat es‘ the reduction 0 1' colony forming unit , and the rapid decrease 0 1' viable cell number 0 1' all bacterial species exarnined while those 0 1' control bacteria treated so ley wi th ei t her light 0 1' photosensiti zer were unchanged. The susceptibility of 8. aureush and P. aeru - ginosa was much higher t han that 01' the other strains These resu lts show tha t photodynamic t hera py using photosensi ti zer chlorin e6 is very effective to inhibit bacterial s urviva l, suggesti ng t hat t his system can be clin ically appli cable as an alternative antibacterial strategy to treat mul t iple drug-resistant bacteria

Photodynamic therapy (PDT) is a clinically approved and ra pidly developing cancer treatment regimen, It is a minimally invasive procedure that requires the administration of a photosens iti zer foll owed by the illumination of the tumor with Iigh t of an appropriate wavelength, In the presence of molecular oxygen, cytotoxic intermedi a ries a re produced‘ thus damaging cellular structures containing the photosensitizer , In the present study. we exa mined the effectiveness of newly d evelped chlorin e6- induced PDT on malignant animal tumor model of 3prague-Da wley (3D) ra t Three-week-old male 3D rats we re inocula ted s ,c, on the right f1 ank with our previously esta blished k- ras-trans formed RK3E cell line (RK3E- ras. tota l, 5xl07 cell s) , The experiments were carried out 1 week after inoculation of tumor cell s , by which time the tumors had r eached about 0,7 mm to 1.0 cm in diameter, L3-chlorin e6 (L8 Pharm Co" Gwa ngju, Korea) was admin istrated intravenous ly by the tail vein of 3D rat at a dosage of 10 mg/kg after inhalation a nesthesia of ether, Twenty- four hours a fter L8-chlorin e6 ad ministration, PDT was pe rfol‘med using a laser diode (Geumgwang Co ‘ Ltd‘’ Daejeon, Korea) a t a light dose of 100 J /cm2 and wavelength of 664 nm, A..nimals were monitered daily and tumor volume was measured by calipel The tumor t reated with PDT using Ce6 had significant reduction in tumor s ize examined by gross tumor volume , softex x- ray image, molecular imaging a nalysis, respectively, PCNA immunostaining and TUNEL assay revealed that the treat ed tu mor caused signifi cant inlübition of tumor formati on with decreased tumor cell proliferation a nd increased a poptosis , Our dat a showed Ce6-induced PDT effecti vely arrested the tumor growth by inhibi t ing cell proliferation a nd inducing a poptosis , These findings provide the potential value of Ce6- induced PDT as an a lternative candidate for a nt i- tumor therapy, Furthel bi ochemical and cellular studies will reveal the precise molecul ar mecha ni sm of cell death induced by PDT