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

Materials ◽  
2019 ◽  
Vol 12 (17) ◽  
pp. 2693
Author(s):  
Hyunjin Lee ◽  
Jihwan Son ◽  
Sae Kyung Min ◽  
Chae-Bin Na ◽  
Gawon Yi ◽  
...  
Keyword(s):  
Alkaline Phosphatase ◽  
Stem Cell ◽  
Phosphatase Activity ◽  
Alkaline Phosphatase Activity ◽  
Cationic Liposomes ◽  
Alizarin Red S ◽  
Cellular Viability ◽  
Alizarin Red ◽  
Cell Spheroids ◽  
Calcium Deposits

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.

scholarly journals The Effects of Morinda citrifolia (Noni) on the Cellular Viability and Osteogenesis of Stem Cell Spheroids

Medicina ◽  
2020 ◽  
Vol 56 (8) ◽  
pp. 389
Author(s):  
Sae Kyung Min ◽  
Jaekwen Oh ◽  
Jun-Beom Park
Keyword(s):  
Alkaline Phosphatase ◽  
Stem Cell ◽  
Phosphatase Activity ◽  
Alkaline Phosphatase Activity ◽  
Morinda Citrifolia ◽  
Herbal Remedies ◽  
Cellular Viability ◽  
Alizarin Red ◽  
Anthraquinone Dye ◽  
Cell Spheroids

Background and objectives: Morinda citrifolia (Noni) has been widely used in herbal remedies to treat and prevent various kinds of diseases. We conducted this study to evaluate the effects of Noni extract on the maintenance of morphology, the improvement of cellular viability, and the enhancement of osteogenesis of stem cell spheroids. Materials and Methods: We cultured stem cell spheroids made with gingiva-derived stem cells in the presence of Noni extract at concentrations of 10, 100 and 200 ng/mL. We performed analysis of the cell morphology and changes in the cellular viability. We conducted alkaline phosphatase activity assays using a kit, and mineralization assays using an anthraquinone dye to evaluate the osteogenesis of stem cell spheroids with the addition of Noni extract. Results: The applied cells formed spheroids well, and the addition of Noni at 10, 100 and 200 ng/mL concentrations did not produce significant morphological changes. The quantitative values for cellular viability on Day 3 showed that the absorbance values at 450 nm were 0.314 ± 0.013, 0.318 ± 0.008, 0.304 ± 0.000 and 0.300 ± 0.011 for Noni at 0, 10, 100 and 200 ng/mL concentrations, respectively. The results of alkaline phosphatase activity with absorbance values at 405 nm were 0.189 ± 0.019, 0.174 ± 0.023, 0.192 ± 0.014 and 0.210 ± 0.062 for Noni at 0, 10, 100 and 200 ng/mL concentrations, respectively, on Day 4. There were significantly higher values of Alizarin Red S staining for Noni in the 10, 100 and 200 ng/mL groups, with the highest value at 100 ng/mL when compared with the unloaded control on Day 14. Conclusions: Based on these findings, we concluded that Noni extract might be applied for the enhanced osteogenic differentiation of stem cell spheroids.

scholarly journals Evaluation of the Effects of Cuminum cyminum on Cellular Viability, Osteogenic Differentiation and Mineralization of Human Bone Marrow-Derived Stem Cells

Medicina ◽  
2021 ◽  
Vol 57 (1) ◽  
pp. 38
Author(s):  
Hyunjin Lee ◽  
Youngmin Song ◽  
Yoon-Hee Park ◽  
Md. Salah Uddin ◽  
Jun-Beom Park
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Alkaline Phosphatase ◽  
Osteogenic Differentiation ◽  
Phosphatase Activity ◽  
Alkaline Phosphatase Activity ◽  
Alizarin Red S ◽  
Control Group ◽  
Cellular Viability ◽  
Alizarin Red

Background and Objectives: Cuminum cyminum L. has long been used in the treatment of various diseases in multiple geographical regions. This study was performed to determine the effects of C. cyminum methanolic extract (CCT) on the cellular viability, alkaline phosphatase activity and mineralization of human mesenchymal stem cells. Materials and Methods: Bone marrow-derived stem cells were cultured in the presence of CCT at concentrations of 0, 0.001, 0.01, 0.1 and 1 μg/mL. Evaluations of cell morphology were performed on days 1, 3, 7 and 14. Cellular viability was evaluated on days 1, 3, 5 and 7. On the 7th and 14th day, alkaline phosphatase activity measurements and Alizarin red S staining were conducted to assess the osteogenic differentiation of stem cells. A real-time polymerase chain reaction was used to determine the expression levels of RUNX2, BSP, OCN, COL2A1 and β-catenin mRNAs. Results: Stem cells in the control group showed fibroblast-like morphology and the addition of CCT at 0.001, 0.01, 0.1 and 1 μg/mL did not generate noticeable changes in morphology compared with the untreated control group. The application of CCT did not produce significant changes in cellular viability or alkaline phosphatase activity compared with controls. Alizarin Red S staining was significantly increased with the application of CCT. Treatment with CCT increased the expressions of RUNX2, BSP and OCN. Conclusions: These results indicate that CCT enhanced the osteogenic differentiation of stem cells derived from bone marrow by regulating the expressions of RUNX2, BSP and OCN. Thus, the use of CCT may be applied to achieve beneficial effects on the mineralization of stem cells.

The inhibitory effects of vancomycin on rat bone marrow–derived mesenchymal stem cell differentiation

2021 ◽  
pp. 1-5
Author(s):  
Kari Hanson ◽  
Carly Isder ◽  
Kristen Shogren ◽  
Anthony L. Mikula ◽  
Lichun Lu ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Alkaline Phosphatase ◽  
Osteogenic Differentiation ◽  
Phosphatase Activity ◽  
Alkaline Phosphatase Activity ◽  
High Dose ◽  
Inhibitory Effects ◽  
Alizarin Red ◽  
Osteogenic Factors

OBJECTIVE The use of intrawound vancomycin powder in spine surgery has been shown to decrease the rate of surgical site infections; however, the optimal dose is unknown. High-dose vancomycin inhibits osteoblast proliferation in vitro and may decrease the rate of solid arthrodesis. Bone marrow–derived mesenchymal stem cells (BMSCs) are multipotent cells that are a source of osteogenesis in spine fusions. The purpose of this study was to determine the effects of vancomycin on rat BMSC viability and differentiation in vitro. METHODS BMSCs were isolated from the femurs of immature female rats, cultured, and then split into two equal groups; half were treated to stimulate osteoblastic differentiation and half were not. Osteogenesis was stimulated by the addition of 50 µg/mL l-ascorbic acid, 10 mM β-glycerol phosphate, and 0.1 µM dexamethasone. Vancomycin was added to cell culture medium at concentrations of 0, 0.04, 0.4, or 4 mg/mL. Early differentiation was determined by alkaline phosphatase activity (4 days posttreatment) and late differentiation by alizarin red staining for mineralization (9 days posttreatment). Cell viability was determined at both the early and late time points by measurement of formazan colorimetric product. RESULTS Viability within the first 4 days decreased with high-dose vancomycin treatment, with cells receiving 4 mg/mL vancomycin having 40%–60% viability compared to the control. A gradual decrease in alizarin red staining and nodule formation was observed with increasing vancomycin doses. In the presence of the osteogenic factors, vancomycin did not have deleterious effects on alkaline phosphatase activity, whereas a trend toward reduced activity was seen in the absence of osteogenic factors when compared to osteogenically treated cells. CONCLUSIONS Vancomycin reduced BMSC viability and impaired late osteogenic differentiation with high-dose treatment. Therefore, the inhibitory effects of high-dose vancomycin on spinal fusion may result from both reduced BMSC viability and some impairment of osteogenic differentiation.

scholarly journals Alkaline Phosphatase Activity of Exudative Leukocytes in Acute Leukemia

Blood ◽  
1961 ◽  
Vol 18 (5) ◽  
pp. 572-580 ◽  
Author(s):  
PASQUALE E. PERILLIE ◽  
STUART C. FINCH
Keyword(s):  
Alkaline Phosphatase ◽  
Stem Cell ◽  
Acute Leukemia ◽  
Phosphatase Activity ◽  
Alkaline Phosphatase Activity ◽  
Acute Monocytic Leukemia ◽  
Monocytic Leukemia ◽  
Leukocyte Alkaline Phosphatase

Abstract A method for studying the leukocyte alkaline phosphatase activity in patients with acute leukemia is described. The technic overcomes the problem of obtaining sufficient numbers of mature granulocytes for study in such patients. Seven of 11 patients with acute monocytic leukemia were shown to have elevated levels of alkaline phosphatase activity. It is suggested that this method may be of assistance in classifying the stem cell forms of leukemia.

299 A FEATURE OF SELF-RENEWAL PORCINE EMBRYONIC STEM CELL-LIKE CELL LINES ESTABLISHED BY INHIBITORS

2013 ◽  
Vol 25 (1) ◽  
pp. 297
Author(s):  
S. Haraguchi ◽  
T. Tokunaga ◽  
T. Furusawa ◽  
K. Ohkoshi ◽  
M. Nakai ◽  
...  
Keyword(s):  
Alkaline Phosphatase ◽  
Stem Cell ◽  
Embryonic Stem Cell ◽  
Phosphatase Activity ◽  
Cell Lines ◽  
Alkaline Phosphatase Activity ◽  
Embryonic Stem ◽  
Self Renewal ◽  
Established Cell Lines ◽  
Established Cell

Despite meticulous attempts for more than two decades, establishment of authentic porcine embryonic stem cell (ESC) from pig has never been successful. Although putative porcine ESC-like cells have been reported, such cell lines easily lose the ability of self-renewal, becoming extinct or differentiating after only a limited number of passages in culture. Porcine ESC-like cells exhibiting the property of self-renewal rather than pluripotency are considered a valuable resource in applications such as drug screening and toxicology testing in humans and livestock, and in veterinary medicine. In the present study, we evaluated the effect of glycogen synthase kinase 3β (GSK3β) inhibitor CHIR99021 and Erk signalling inhibitor PD184352 for use in establishing ESC-like cell lines derived from the inner cell mass (ICM) of porcine blastocysts produced in vitro. These ICM-derived cell lines were initially cultured and passaged in conventional human ES medium. They displayed so-called ESC-like morphology; for example, the isolated colonies began to grow as a monolayer with coarse cell–cell boundaries, in which the cells exhibited polygonal boundaries, high nuclear/cytoplasmic ratios, abundant lipid-like inclusions, alkaline phosphatase activity, and expression of markers of undifferentiated cells such as OCT4 and NANOG. After transfer to culture in ES medium containing the inhibitors, the morphology of the colony was dramatically changed, displaying a closely packed and smooth-edged colony with tight cell–cell boundaries. Remarkably, growth of the established cell lines is leukemia inhibitory factor (LIF)-dependent. The inclusion of inhibitors supports self-renewal, thus enabling continuous culture for over 100 passages while maintaining an undifferentiated state. High-passage-number cells continued to express undifferentiated marker genes and showed alkaline phosphatase activity and telomerase activity with an X chromosome status of XaXi. We further investigated the potential for differentiation of the established cell lines. The cells could easily form embryoid body-like spheres in suspension culture. When either the spheres or ESC-like cells were inoculated under the kidney or testis capsules of nude mice, classical teratoma formation was not observed after 2 to 3 months. However, histological analyses revealed apparent invasive proliferation derived from porcine cells. Although further analyses are required to characterise the property of the porcine ESC-like cells, we have recently succeeded in establishment of green fluorescent protein (GFP)-expressing stable cells lines, which will be useful for further investigation.

Mouse Fibroblasts Are Reprogrammed to Oct-4 and Rex-1 Gene Expression and Alkaline Phosphatase Activity by Embryonic Stem Cell Extracts

2007 ◽  
Vol 9 (3) ◽  
pp. 394-406 ◽  
Author(s):  
Tui Neri ◽  
Manuela Monti ◽  
Paola Rebuzzini ◽  
Valeria Merico ◽  
Silvia Garagna ◽  
...  
Keyword(s):  
Gene Expression ◽  
Alkaline Phosphatase ◽  
Stem Cell ◽  
Embryonic Stem Cell ◽  
Phosphatase Activity ◽  
Alkaline Phosphatase Activity ◽  
Embryonic Stem ◽  
Mouse Fibroblasts ◽  
Cell Extracts

Induction of Calcification in MC3T3-E1 Cells by Inorganic Polyphosphate

2004 ◽  
Vol 83 (8) ◽  
pp. 613-618 ◽  
Author(s):  
Y. Kawazoe ◽  
T. Shiba ◽  
R. Nakamura ◽  
A. Mizuno ◽  
K. Tsutsumi ◽  
...  
Keyword(s):  
Alkaline Phosphatase ◽  
Ascorbic Acid ◽  
Phosphatase Activity ◽  
Chain Length ◽  
Alkaline Phosphatase Activity ◽  
Average Chain Length ◽  
Inorganic Polyphosphate ◽  
Alizarin Red ◽  
Phosphate Source ◽  
In Cells

Relatively large amounts of inorganic polyphosphate [poly(P)] (400 μM) have been found in normal osteoblasts. The effect of poly(P) with an average chain length of 65 phosphate residues on cell calcification was therefore investigated with the use of MC3T3-E1 cells. Expression of both osteopontin and osteocalcin was induced by poly(P) (0.1 ~ 1 mM), and cells treated with poly(P) were strongly stained by alizarin red. In addition, the level of alkaline phosphatase activity induced in poly(P)-treated cells was two-fold higher than that in either orthophosphate-treated or control cells but not higher than that in cells treated with β-glycerophosphate and ascorbic acid. In contrast, however, polyphosphatase activities were activated by poly(P) treatment to levels up to six-fold greater than that in controls. MC3T3-E1 cells may utilize poly(P) as a phosphate source for calcification rather than phosphate sources that are mainly produced by ALPase. Poly(P)-dependent induction of polyphosphatase activities may therefore promote calcification in MC3T3-E1 cells.

scholarly journals The Effects of Bone Morphogenetic Protein-4 on Cellular Viability, Osteogenic Potential, and Global Gene Expression on Gingiva-Derived Stem Cell Spheroids

Coatings ◽  
2020 ◽  
Vol 10 (11) ◽  
pp. 1055
Author(s):  
Jae-Yong Tae ◽  
Yoon-Hee Park ◽  
Youngkyung Ko ◽  
Jun-Beom Park
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Alkaline Phosphatase ◽  
Mesenchymal Stem Cells ◽  
Stem Cell ◽  
Global Gene Expression ◽  
Bone Morphogenetic Protein 4 ◽  
Cellular Viability ◽  
Cell Spheroids ◽  
Morphogenetic Protein

Bone morphogenetic protein-4 (BMP-4) is engaged in the migration ability of mesenchymal stem cells and the transition of mesenchymal stem cells into osteogenic and adipocytic lines. The aim of this study was to evaluate the effects of BMP-4 on the cellular viability, osteogenic differentiation, and genome-wide mRNA levels using three-dimensional cell spheroids composed of stem cells. Stem cell spheroids were formed using concave microwells in the presence of BMP-4 with final concentrations of 0, 2, 6, and 10 ng/mL. Cellular viability was measured qualitatively using a microscope and quantitatively using an assay kit based on water-soluble tetrazolium salt. Osteogenic differentiation was assessed by measuring the level of alkaline phosphatase activity. Global gene expression was assessed using next-generation mRNA sequencing and performing gene ontology and pathway analyses. Spheroids were well-maintained with the addition of BMP-4 up to Day 7. No significant differences were observed in cell viability between each group. There were significantly higher alkaline phosphatase values in the 2 ng/mL BMP-4 groups when compared with the control (p < 0.05). A total of 25,737 mRNAs were differentially expressed. Expression of β-catenin (CTNNB1) was increased with higher dosages of BMP-4. The expression of runt-related transcription factor 2 (RUNX2) was increased up to 6 ng/mL. The phosphoinositide-3-kinase–protein kinase B/Akt signaling pathway was associated with the target genes. This study demonstrates that the application of BMP-4 enhanced alkaline phosphatase activity and the expression of CTNNB1 and RUNX2 without affecting cellular viability.

scholarly journals Application of Biodegradable PLGA-PEG-PLGA/CPC Composite Bone Cement in the Treatment of Osteoporosis

Coatings ◽  
2021 ◽  
Vol 11 (7) ◽  
pp. 827
Author(s):  
Chao Guo ◽  
Dongyang Niu ◽  
Jia Liu ◽  
Xiaogang Bao ◽  
Guohua Xu
Keyword(s):  
Computed Tomography ◽  
Alkaline Phosphatase ◽  
Bone Cement ◽  
Phosphatase Activity ◽  
Alkaline Phosphatase Activity ◽  
Micro Computed Tomography ◽  
Alizarin Red ◽  
Treatment Of Osteoporosis ◽  
Composite Bone

The aim of this study was to evaluate the biological activity, safety, and effectiveness of poly(lactic acid)–poly(glycolic acid)–poly(ethylene glycol)–calcium phosphate cement (PLGA-PEG-PLGA/CPC). Methods: The PLGA-PEG-PLGA/CPC composite bone cement was used for interaction with MC3T3-E1 mouse osteoblasts in vitro and its compatibility was tested using Cell Counting Kit-8 (CCK-8). Alizarin Red staining and alkaline phosphatase activity were used to detect the osteogenic properties. Twenty healthy female New Zealand rabbits were selected to establish osteoporosis models, which were randomly divided into two groups. The experimental group was treated with 30 wt.% PLGA-PEG-PLGA/CPC, while the control group was treated with polymethyl methacrylate (PMMA) bone cement. Imaging and histomorphology of the vertebral body were analyzed after 12 weeks. The distribution and degradation of bone cement were assessed using micro-computed tomography examination and hematoxylin–eosin (HE) staining. Results: In vitro, CCK-8 revealed significant proliferation of osteoblasts in the PLGA-PEG-PLGA/CPC composite bone cement. Alizarin Red staining showed that the degree of staining increased with time. Quantitative results showed that absorbance was significantly higher in this group than in the CPC group on days 7 and 14. The alkaline phosphatase activity levels on days 7 and 14 were significantly higher in the 30 wt.% PLGA-PEG-PLGA/CPC group than in the CPC group. In vivo, postoperative micro-computed tomography and histomorphology showed that the material was evenly distributed in the vertebral body and a small amount penetrated into the trabecular bone. After 12 weeks, CPC degradation and absorption and the formation of new bone matrix were observed and the formation of a callus was obvious. Conclusion: PLGA-PEG-PLGA/CPC composite bone cement has a positive effect on the treatment of osteoporosis.

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