Enhancement of Osteogenic Differentiation by Combination Treatment with 5-azacytidine and Thyroid-Stimulating Hormone in Human Osteoblast Cells

BACKGROUND AND OBJECTIVES: The role of thyroid-stimulating hormone (TSH) signaling on osteoblastic differentiation is still undetermined. The aim of this study was to investigate the effects of 5-aza-2′-deoxycytidine (5-azacytidine) on TSH-mediated regulations of osteoblasts. MATERIALS AND METHODS: MG63, a human osteoblastic cell-line, was treated with 5-azacytidine before inducing osteogenic differentiation using osteogenic medium (OM) containing L-ascorbic acid and β-glyceophosphate. Bovine TSH or monoclonal TSH receptor stimulating antibody (TSAb) was treated. Quantitative real-time PCR analyses or measurement of alkaline phosphatase activities were performed for evaluating osteoblastic differentiation. RESULTS: Studies for osteogenic-related genes or alkaline phosphatase activity demonstrated that treatment of TSH or TSAb alone had no effects on osteoblastic differentiation in MG63 cells. However, treatment of 5-azacytidine, per se, significantly increased osteoblastic differentiation and combination treatment of 5-azacytidine and TSH or TSAb in the condition of OM showed further significant increase of osteoblastic differentiation. CONCLUSION: Stimulating TSH signaling has little effects on osteoblastic differentiation in vitro. However, in the condition of epigenetic modification using inhibitor of DNA methylation, TSH signaling positively affects osteoblastic differentiation in human osteoblasts.

Dikkopf-2, -3 and -4 Enhance Early Osteoblastic Differentiation in Mouse Mesenchymal Progenitor Cells and Stimulate Apoptosis in Osteoblastic Cells

OBJECTIVES: The inhibitory effect of Dickkopf (Dkk)-1 on osteoblastic differentiation through blocking Wnt signaling has been well studied. However, the role of other members of the subfamily of Dkks remains unclear. We have examined the role of different Dkks on osteoblastic differentiation of mesenchymal progenitor cells and apoptosis of osteoblasts. METHODS: Osteoblastic differentiation was induced by treatment of Wnt-3a with Dkks or vehicle in C3H10T1/2 cells and alkaline phosphatase (ALP) activity was measured. Serum deprivation induced apoptosis was performed with pre-treatment of Dkks or vehicle in MC3T3-E1 cells and methyl thiazolyl tetrazolium (MTT) assay was done. RESULTS: Dkk-2 at low concentrations (5 and 20 nM) and Dkk-3, -4 at any concentrations (5 to 100 nM) significantly increased Wnt-3a-induced ALP activity, whereas Dkk-2 at high concentration (100 nM) significantly reduced. Treatment of Dkk-2, -3 and -4 at high concentration (100 nM) showed significant decreases of Wnt/beta-catenin transcriptional activity, whereas no effects were seen at low concentration (20 nM). In parallel experiments, treatment of Dkk-1 showed robust dose dependent inhibition not only in ALP activity but also in Wnt/beta-catenin transcriptional activity. Dkk-2, -3 and -4 increased serum deprivation-induced apoptosis in MC3T3-E1 mouse osteoblasts, while Dkk-1 had no effect. CONCLUSIONS: We found that unlike Dkk-1, Dkk-3 and -4 stimulated early osteoblastic differentiation at various concentrations regardless of their inhibitory effects on Wnt/beta-catenin transcriptional activity at high concentration. Dkk-2 had a biphasic effect where the lower doses significantly increased ALP activity while the high dose was inhibitory. Dkk-2, -3 and -4 stimulated osteoblast apoptosis whereas Dkk-1 had no effect.

Osteogenic Potential of Human Mesenchymal Stem Cells During Serial Subculture / 대한정형외과연구학회지

PURPOSE: The purpose of this study was to evaluate the osteogenic differentiation potential of human mesenchymal stem cells during serial subculture. MATERIALS AND METHODS: Human bone marrow-derived MSCs were serially subcultured and then maintained in basal or osteogenic medium for 14 days. Then we performed FAC analysis, RT-PCR, alkaline phosphatase activity and stains. RESULTS: Human MSCs had different morphologies, immunophenotypes, and growth rates that were correlated with the length of serial subculture. The phenotype changed from small spindle-shaped cells at passage 1 into large cuboidal or flattened cells at passage 7. The osteogenic capacity of human MSCs decreased during serial subculture. Using RT-PCR, the mRNA levels of bone-specific genes, such as cbfa1/runx2 and osteocal-cin, decreased with increasing passage number. Strong positive staining was observed for ALP and Alizarin reds in osteogenic medium on day 14, but declined significantly with increasing passage number. CONCLUSION: We have shown that osteogenic potential of human MSCs decreased during serial subculture. This result can provide the helpful information to decide the timing of human MSC transplantation during in vitro culture expansion for treatment of bone defects and so on.

ERK1/2 Activation Modulates Matrix Metalloproteinase 2 Activity and Ezrin Expression in Osteosarcoma Cell Lines / 대한정형외과연구학회지

PURPOSE: In this study, we aimed to investigate the influence of mitogen-activated protein kinase (MAPK) signal pathway and related factors of metastatic property on the osteosarcoma cell lines. MATERIALS AND METHODS: Osteosarcoma cell lines, MG-63, HOS, U2OS, SAOS-2, and osteoblast-like cell line, MC3T3-E1 were used for evaluating the activation of MAPK signal pathway and the effect of ERK1/2 inhibiton by 15 micrometer U0126 for 6 days. Then we measured the activity of matrix metalloproteinases (MMP) by gelatin zymography analysis. RESULTS: ERK was strongly activated in three osteosarcoma cell lines, MG-63, HOS, U2OS, and was weakly activated in SAOS-2 cell line. MMP2 was strongly activated in MG-63, HOS, U2OS cell lines. However, SAOS-2 cell line, in which ERK1/2 was weakly activated, showed weak activation of MMP2. When the ERK1/2 was inhibited by U0126 for 6 days, the ezrin expression was remarkably suppressed in all osteosarcoma cell lines. These data showed that the suppression of ezrin expression might be dependent on the ERK1/2 activation in osteosarcoma cell lines. CONCLUSION: Our results suggested that the ERK1/2 activation might be related to the activity of MMP2 and the suppression of ezrin expression in osteosarcoma cell lines. We suggest that ERK1/2 activation might be closely related to the ezrin expression and the ezrin expression might play an important role in the activation of MMP2 which is known to involve in the activated MMP2 involves in early metastatic properties in osteosarcoma.

Comparison of Phenotypic Characterization between "Alginate Bead" and "Pellet" Culture Systems as Chondrogenic Differentiation Models for Human Mesenchymal Stem Cells

Chondrogenesis involves the recruitment of mesenchymal cells to differentiate into chondroblasts, and also the cells must synthesize a cartilage-specific extracellular matrix. There were two representative culture systems that promoted the chondrogenic differentiation of human mesenchymal stem cells. These systems were adaptations of the "pellet" culture system, which was originally described as a method for preventing the phenotypic modulation of chondrocytes, and the "alginate bead" culture system, which was used to maintain encapsulated cells at their differentiated phenotype over time, and also it was used to maintain the cells' proteoglycan synthesis at a rate similar to that of primary chondrocytes. We performed test on the differences of phenotypic characterization with the two methods of differentiating human mesenchymal stem cells into chondrocytes. The typical gene for articular cartilage, collagen type II, was more strongly expressed in the "alginate bead" system than in the "pellet" culture system, in addition, specific gene for hypertrophic cartilage, collagen type X, was more rapidly expressed in the "pellet" system than in "alginate bead" culture system. Therefore, the "alginate bead" culture system is a more phenotypical, practical and appropriate system to differentiate human mesenchymal stem cells into articular chondrocytes than the "pellet" culture system.