Osteoarthritis improvement effect of Chrysanthemum zawadskii var. latilobum extract in relation to genotype.

Objectives: To determine whether SNPs of osteoarthritis (OA)-related genes predict the effect of Chrysanthemum zawadskii var. latilobum (CZ) extract in OA patients with OA. Subjects/methods: To analyze correlations between CZ extract effects in humans and their genotypes, 121 Korean patients with OA were recruited. Patients ingested 600 mg/day of the CZ extract GCWB106 (one tablet daily), including 250-mg CZ, or placebo (one tablet daily) for 12 weeks. Twenty SNPs were genotyped in 11 genes associated with OA pathogenesis, including tumor necrosis factor-alpha (TNF-α) and matrix metalloproteinases (MMPs), and 9 genes involved in OA-related dietary intervention. The Visual Analogue Scale (VAS) and Korean Western Ontario and McMaster Universities (K-WOMAC) were measured as indicators of GCWB106 effect. Statistical comparisons were performed using Kruskal-Wallis tests to identify associations between these scales and genotyped loci in patients with OA. Results: Three SNPs (PPARG rs3856806, MMP13 rs2252070, and ZIP2 rs2234632) were significantly associated with the degree of change in VAS pain score. Homozygous CC genotype carriers of rs3856806, G allele carriers (GA or GG) of rs2252070, and T allele carriers (GT or TT) of rs2234632 showed lower VAS score (i.e., less severe symptoms) in the GCWB106 group (n=53) than the placebo group (n=57) (p=0.026, p=0.009, and p=0.025, respectively). Gene-gene interaction effects on GCWB106-mediated pain relief were then examined, and it was found that the addition of each genotype resulted in a greater decrease in VAS pain score in the GCWB106 group (p=0.0024) but not the placebo group (p=0.7734). Conclusions: These novel predictive markers for the pain-relieving effects of GCWB106 may be used in the personalized treatment of patients with OA.

Evaluation of the Age- and Sex-Related Changes of the Osteogenic Differentiation Potentials of Healthy Bone Marrow-Derived Mesenchymal Stem Cells.

Background andObjectives: Human bone marrow-derived mesenchymal stem cells (BMSCs) are promising sources for cell-based regenerative therapy. The purpose of the present study was to elucidate the roles of age and sex on the cellular viability and osteogenic potential of BMSCs cultured in osteogenic media. Materials and Methods: Human BMSCs were isolated and expanded from 3 age groups-20s, 30s, and 50s-from both sexes. The total number of aspirates was ten, and each subgroup had five for 20s (two females and three males), three for 30s (one female and two male), and two for 50s (one female and one male). Analyses of the cell morphology, the cell viability, the expression of the stem cell marker SSEA-4, the secretion of human vascular endothelial growth factor (VEGF), the expression of Runx2 and collagen I, the metabolic activity, and the formation of mineralization nodules were performed. Results: No significant differences were found in the cell viability of human BMSCs cultured in osteogenic media among the different age groups. There were no significant differences in the expression of SSEA among the age groups or between males and females. There were no significant differences in the secretion of human VEGF between males and females. No significant differences in Runx2 or collagen I expression were noted by age or gender. Moreover, no significant differences were shown in osteogenesis by alizarin red staining. Conclusions: The human BMSCs showed no age-related decreases in cellular viability or osteogenic differentiation potential.

PARsylated transcription factor EB (TFEB) regulates the expression of a subset of Wnt target genes by forming a complex with ß-catenin-TCF/LEF1.

Wnt signaling is mainly transduced by ß-catenin via regulation of the ß-catenin destruction complex containing Axin, APC, and GSK3ß. Transcription factor EB (TFEB) is a well-known master regulator of autophagy and lysosomal biogenesis processes. TFEB's nuclear localization and transcriptional activity are also regulated by various upstream signals. In this study, we found that Wnt signaling induces the nuclear localization of TFEB and the expression of Wnt target genes is regulated by TFEB-ß-catenin-TCF/LEF1 as well as ß-catenin-TCF/LEF1 complexes. Our biochemical data revealed that TFEB is a part of the ß-catenin destruction complex, and destabilization of the destruction complex by knockdown of either Axin or APC causes nuclear localization of TFEB. Interestingly, RNA-sequencing analysis revealed that about 27% of Wnt3a-induced genes were TFEB dependent. However, these "TFEB mediated Wnt target genes" were different from TFEB target genes involved in autophagy and lysosomal biogenesis processes. Mechanistically, we found that Tankyrase (TNKS) PARsylates TFEB with Wnt ON signaling, and the nuclear localized PARsylated TFEB forms a complex with ß-catenin-TCF/LEF1 to induce the "TFEB mediated Wnt target genes". Finally, we found that in various types of cancer, the levels of TFEB mediated Wnt target genes exhibit strong correlations with the level of Axin2, which represents the activity of Wnt signaling. Overall, our data suggest that Wnt signaling induces the expression of a subset of genes that are distinct from previously known genes regulated by the ß-catenin-TCF/LEF1 complex or TFEB, by forming a transcription factor complex consisting of PARsylated TFEB and ß-catenin-TCF/LEF1.

A Study of the Effects of Doxorubicin-Containing Liposomes on Osteogenesis of 3D Stem Cell Spheroids Derived from Gingiva.

The objective of the present investigation is to determine the effects of neutral, anionic, and cationic liposomes loaded with doxorubicin with thin-lipid-film-hydration method on the cellular viability and osteogenesis of stem cell spheroids. Spheroid formation and morphology of the three-dimensional spheroid were noted with an inverted microscope. Quantitative cellular viability was assessed using a commercially available kit. Osteogenic potential was evaluated by applying alkaline phosphatase activity and anthraquinone dye of Alizarin Red S. Western blot analysis was performed using collagen I expression. Spheroids were formed in each silicon elastomer-based concave microwell on Day 1. Noticeable changes of the spheroid were seen with a higher concentration of doxorubicin, especially in the cationic liposome group at Days 5 and 7. We found that the application of doxorubicin for 5 days significantly reduced the cellular viability. A higher concentration of doxorubicin produced a significant decrease in alkaline phosphatase activity. Alizarin Red S staining showed that extracellular calcium deposits were evenly noted in each group. An increase of calcium deposits was noted on Day 14 when compared to Day 7. The morphology of the groups with higher concentrations of doxorubicin showed to be more dispersed. We noticed that doxorubicin-loaded cationic liposomes resulted in the highest uptake of the examined cell spheroids and that doxorubicin-loaded liposomes affected the osteogenic differentiation. The implication of this study is that the type of liposome should be selected based on the purpose of the application.

The effects of simvastatin on cellular viability, stemness and osteogenic differentiation using 3-dimensional cultures of stem cells and osteoblast-like cells.

BACKGROUND: Simvastatin has been reported to increase the therapeutic effects of many kinds of stem cells by increasing the number of those cells. However, the effects of simvastatin on the differentiation potential of stem cells have not been clearly determined. OBJECTIVES: The aim of the study was to evaluate the effects simvastatin has on cellular viability, stemness and osteogenic differentiation using 3-dimensional cell spheroids of stem cells and osteoblast-like cells. MATERIAL AND METHODS: Three-dimensional cell spheroids were fabricated using concave silicon elastomerbased microwells in the presence of simvastatin at concentrations of 1 µM and 10 µM. Qualitative cellular viability was determined with a confocal microscope, and quantitative cellular viability was evaluated using a cell-counting assay kit. The expression of stem cell surface markers was tested. A quantitative real-time polymerase chain reaction (qRT-PCR) was performed to evaluate the expression of collagen I and RUNx2. Alkaline phosphatase activity and alizarin red S staining were used to assess osteogenic differentiation. RESULTS: The spheroids formed well in the concave silicon elastomer-based microwells, and the application of simvastatin caused no significant morphological changes. No significant changes in cellular viability were noted with the addition of simvastatin on days 1, 3 and 5. Secretion of the vascular endothelial growth factor (VEGF) was observed on day 1 and remained stable throughout the culture period. Expression of the CD90 surface marker was seen on day 7. The addition of simvastatin caused a statistically significant increase in the expression of collagen I and RUNX2. It also caused decreases in alkaline phosphatase activity and alizarin red S staining. CONCLUSIONS: The study clearly showed that the application of simvastatin enhanced collagen I and RUNX2 expression; however, this did not lead to increases in alkaline phosphatase activity or alizarin red S staining.

The effects of doxorubicin-loaded liposomes on viability, stem cell surface marker expression and secretion of vascular endothelial growth factor of three-dimensional stem cell spheroids.

The aim of the present study was to evaluate the effects of anionic, cationic and neutral liposomes containing doxorubicin on the cellular viability and osteogenic differentiation of three-dimensional stem cell spheroids. Doxorubicin-loaded liposomes were prepared using the traditional thin-lipid-film-hydration method and were characterized using transmission electron microscopy and a zeta potential analyzer. The doxorubicin release profile from these liposomes was also analyzed in vitro. Three-dimensional cell spheroids were fabricated using silicon elastomer-based concave microwells. Qualitative results of cellular viability were observed using a confocal microscope and quantitative cellular viability was evaluated using a Cell-Counting Kit-8 (CCK-8) assay. Furthermore, the secretion of vascular endothelial growth factor was evaluated. Western blot analysis was performed to assess the expression of collagen I and glyceraldehyde 3-phosphate. Results indicated that the spheroids were well formed in silicon elastomer-based concave microwells on day 1. In general, the shapes of the cells in the in the doxorubicin-loaded anionic, cationic and neutral liposome groups were similar to the control group except for the 10 µg/ml groups on days 3, 5, and 7. No significant changes in cellular viability were noted with the addition of doxorubicin at day 1 but significant decreases in cellular viability were noted with application of doxorubicin at day 5. Notably, higher concentrations of doxorubicin reduced the secretion of vascular endothelial growth factor and stem cell marker expression. To conclude, the present study indicated that doxorubicin-loaded anionic liposomes produced the most sustained release profile and cationic liposomes produced the highest uptake of the stem cell spheroids. These findings suggested that higher concentrations of doxorubicin-loaded liposomes affected cellular viability, the secretion of vascular endothelial growth factor and stem cell marker expression.

Effects of Simvastatin on the Viability and Secretion of Vascular Endothelial Growth Factor of Cell Spheroids Cultured in Growth Media.

PURPOSE: This study evaluates the effects of simvastatin on the morphology, viability, secretion of vascular endothelial growth factor (VEGF) and expression of stemness markers and messenger RNA of cell spheroids cultured in growth media. MATERIALS AND METHODS: Three-dimensional cell spheroids with stem cells and osteoblast-like cells were fabricated using concave, silicon, elastomer-based microwells in the presence of simvastatin at concentrations of 1 and 10 µM. Qualitative cellular viability was determined with a confocal microscope, and quantitative cellular viability was evaluated using a Cell Counting Kit-8 assay. The expression of stem cell surface markers was tested, and a quantitative real-time polymerase chain reaction was performed to evaluate the expression of collagen I and Runx2. RESULTS: The cell spheroids were well formed in the microwells, but the addition of simvastatin produced significant changes in the morphology of spheroids. No significant changes in cellular viability were noted with the addition of simvastatin on day 1, but the addition of simvastatin significantly decreased cellular viability on day 5. The addition of simvastatin significantly increased the secretion of VEGF. The expression of the CD90 surface marker was seen regardless of whether simvastatin was added. The addition of simvastatin significantly decreased the expression of collagen I. CONCLUSIONS: Based on these findings, the application of simvastatin clearly decreased the cellular viability of the cell spheroids made with stem cells and osteoblast-like cells but increased the secretion of VEGF by the cell spheroids.