Alkaline Phosphatase Activity In MG-63 Cells Research Articles

Decarboxylated osteocalcin, a possible drug for type 2 diabetes, triggers glucose uptake in MG63 cells.

BACKGROUNDUncarboxylated osteocalcin (GluOC) has been reported to improve glucose metabolism, prevent type 2 diabetes, and decrease the severity of obesity in mice with type 2 diabetes. GluOC can increase glucose uptake in a variety of cells. Glucose metabolism is the main source of energy for osteoblast proliferation and differentiation. We hypothesized that decarboxylated osteocalcin (dcOC), a kind of GluOC, can increase glucose uptake in MG63 cells (osteoblast-like osteosarcoma cells) and influence their proliferation and differentiation.AIMTo investigate the effects of dcOC on glucose uptake in human osteoblast-like osteosarcoma cells and the possible signaling pathways involved.METHODSMG63 cells (human osteoblast-like osteosarcoma cells) were treated with dcOC (0, 0.3, 3, 10, or 30 ng/mL) for 1 and 72 h, and glucose uptake was measured by flow cytometry. The effect of dcOC on cell proliferation was measured with a CCK-8 assay, and alkaline phosphatase (ALP) enzyme activity was measured. PI3K was inhibited with LY294002, and hypoxia-inducible factor 1 alpha (HIF-1α) was silenced with siRNA. Then, GPRC6A (G protein-coupled receptor family C group 6 subtype A), total Akt, phosphorylated Akt, HIF-1α, and glucose transporter 1 (GLUT1) levels were measured by Western blot to elucidate the possible pathways by which dcOC modulates glucose uptake.RESULTSThe glucose uptake of MG63 cells was significantly increased compared with that of the paired control cells after short-term (1 h) treatment with dcOC at different concentrations (0.3, 3, and 10 ng/mL groups, P < 0.01; 30 ng/mL group, P < 0.05). Glucose uptake of MG63 cells was significantly increased compared with that of the paired control cells after long-term (72 h) treatment with dcOC at different concentrations (0.3, 3, and 10 ng/mL groups, P < 0.01; 30 ng/mL group, P < 0.05). DcOC triggered Akt phosphorylation in a dose-dependent manner, and the most effective stimulatory concentration of dcOC for short-term (1 h) was 3 ng/mL (P < 0.01). LY294002 abolished the dcOC-mediated (1 h) promotion of Akt phosphorylation and glucose uptake without affecting GLUT1 protein expression. Long-term dcOC stimulation triggered Akt phosphorylation and increased the protein levels of HIF-1α, GLUT1, and Runx2 in a dose-dependent manner. Inhibition of HIF-1α with siRNA abolished the dcOC-mediated glucose uptake and substantially decreased GLUT1 protein expression. DcOC intervention promoted cell proliferation in a time- and dose-dependent manner as determined by the CCK-8 assay. Treatment with both 3 ng/mL and 10 ng/mL dcOC affected the ALP activity in MG63 cells after 72 h (P < 0.01).CONCLUSIONShort- and long-term dcOC treatment can increase glucose uptake and affect proliferation and ALP activity in MG63 cells. This effect may occur through the PI3K/Akt, HIF-1α, and GLUT1 signaling factors.

In vitro biological properties of 45S5 bioglass fabricated by spark plasma sintering (SPS)

Event Abstract Back to Event In vitro biological properties of 45S5 bioglass fabricated by spark plasma sintering (SPS) Zhong Li1, Brianna C. Thompson1, Huanlong Hu1 and Khiam Aik Khor1 1 Nanyang Technological University, School of Mechanical & Aerospace Engineering, Singapore Introduction: In our previous study, we reported the first use of Spark Plasma Sintering (SPS) to densify 45S5 Bioglass (45S5) and found that SPS could lead to better early-stage cytocompatibility compared with conventional sintering processes[1]. Although SPS has been utilized to fabricate bulk 45S5 and various 45S5-based composites, there is a lack of comprehensive investigation of the in vitro biological properties of such materials [2]-[4]. In this research, cell culture was carried out for 45S5 sintered by SPS at three different temperatures using two established cell lines: L929 and MG63 Materials and Methods: 45S5 powder (XL Sci-Tech Inc., US) was sintered for 3 min by an SPS system (Dr. Sinter 1050, SCM, Japan) at three different temperatures: 500, 550 and 600 °C. X-ray Diffraction (XRD) was utilized to identify the phase composition of the samples. Growth of L929 and MG63 was assessed with a CyQUANT Cell Proliferation Assay Kit (C7026) after culturing them for 3 days in the extractant media of sintered pellets. Alkaline phosphatase (ALP) activity was quantified using the Abcam ALP Assay Kit (ab83369) for MG63 cells cultured on the pellets’ surface for 4 days. In both experiments, the “new” samples were freshly polished and had not been soaked in any medium, while “old” samples had undergone prolonged soaking (~21 days) for other tests before the current study. Results and Discussion: It can be seen in Fig.1 that 45S5 sintered at 500 °C is amorphous, while pellets sintered at higher temperatures are crystalline with Na2CaSi3O8 as the major phase. Fig. 2 compares the cell number for different pellets. Statistical difference is represented by * (p≤0.05), ** (p≤0.01) and *** (p≤0.001) in Fig. 2 and Fig. 3, with asterisks in parentheses indicating differences between the old samples and their new counterparts. It is explicitly observed that 45S5 pellets all resulted in slower cell growth than the control (tissue culture plastic, TCP). Furthermore, both a smaller sintering temperature (and hence lower crystallinity) and long-term soaking prior to this research (and thus a smaller amount of ions released into the extractant media) could generally promote cell growth. Both old and new pellets compacted at 600 °C led to the slowest cell growth. It is interesting to find in Fig. 3 that 45S5 pellets all brought about remarkably higher ALP activity in MG63 than TCP, which is an important feature of the osteoblast phenotype. In addition, new samples could all result in significantly higher ALP activity than their old counterparts, indicating the presence of more bioactive substances which could facilitate cell differentiation and bone formation. Interestingly, 45S5 sintered at 550 °C led to much higher ALP activity in MG63 than those fabricated at higher or lower temperatures. This phenomenon suggests that neither fully amorphous nor highly crystalline 45S5 favors the differentiation of MG63 and bone formation [1]. Conclusions: This study investigated the in vitro biological properties of 45S5 pellets fabricated by SPS and reported for the first time the difference in ALP activity of MG63 cells cultured on 45S5 caused by different SPS temperatures. It was found that relatively low SPS temperatures (<600 °C) could bring about faster cell proliferation, and a moderate temperature of 550 °C could result in highest ALP activity. Therefore, it can be concluded that 550 °C should be used for sintering 45S5 used as orthopaedic implants. Z. LI thanks NTU for the University Research Scholarship

Cell response of nanographene platelets to human osteoblast‐like MG63 cells

The biologic/cytotoxic effects of dispersed nanographene platelets (NGPs) on human osteosarcoma cells (MG63 cell line) were first studied by examining cell viability, cycle, apoptosis, change in morphology, lactate dehydrogenase (LDH) release, alkaline phosphatase (ALP) activity, and inflammation. The results shown that the cell cytotoxicity of the dispersed NGPs exhibited dose-dependent characters, which had no obvious cytotoxic effects to MG63 cells at the concentration less than 10 μg mL(-1), whereas could postpone cell cycle, promote cell apoptosis, damage cell microstructure, induce serious tumor necrosis factor-α expression and greatly reduce ALP activity of MG63 cells at higher concentration of NGPs (>10 µg mL(-1)). Besides, NGPs had little influence on the LDH leakage. The cytotoxic mechanism of NGPs to MG63 cells was speculated to be intracellular activity with no physical damage of plasma membrane.

An effective approach by a chelate reaction in optimizing the setting process of strontium‐incorporated calcium phosphate bone cement

Strontium (Sr) plays a special role in enhancing the biological osteo-stimulation of calcium phosphate cement (CPC), not only increasing osteoblast-related gene expression and the alkaline phosphatase (ALP) activity of mesenchymal stem cells (MSCs), but also inhibiting the differentiation of osteoclasts. However, the incorporation of Sr unfortunately delays the setting of CPC and weakens its mechanical properties. The purpose of this study was to overcome the aforementioned problems by introducing a chelate reaction between Ca/Sr cations from the original solid phases and carboxyl groups from the liquid phases. As expected, the setting process of Sr-incorporated CPC was optimized and the cement body after rapid hardening was mostly consisting of unreacted original solid phases. After soaking in simulated body fluid for 14 and 28 days, the composition of the cement body gradually converted to the most thermodynamic stable phase, hydroxyapatite, indicating an in vitro bioactivity. The compressive strength was not impaired in the Sr-incorporated groups, but rather, further increased over time. Higher cell proliferation rate and better ALP activity of MG-63 cells cultured on the cement surface were obtained with the presence of Sr content, demonstrating potential abilities to favor new bone formation.

마가목 및 현지초 추출물의 골손실 및 연골손상 억제효과

본 연구에서는 마가목 (SC), 현지초 추출물 (GT) 및 이들의 1:1 혼합물 시료 (MIX)가 골손실 및 연골손상 억제에 효과가 있는지 알아보기 위해, 각각의 시료를 조골세포주인 MG-63 세포, 파골세포로의 분화를 유도한 Raw264.7 세포와 연골세포로의 분화를 유도한 ATDC5 세포에 처리하여 세포분화 조절 정도를 확인하였다. 각 세포의 분화 정도는 alkaline phosphatase (ALP) 활성 측정, tartrate-resistant acid phosphatase (TRAP) 염색법 및 alcian-blue 염색법으로 확인하였다. 이들 시료는 MG-63 세포에서 ALP 활성에는 영향을 미치지 않았으나, 마가목 추출물 (SC) 및 마가목과 현지초 추출물의 혼합시료 (MIX)는 농도 의존적으로 파골세포의 분화를 억제하고 연골세포의 분화를 촉진하는 것으로 나타났다. 이상의 결과를 종합하여 볼 때, 마가목과 현지초는 골손실과 연골 손상으로부터 보호할 수 있는 중요한 천연물 소재임을 확인할 수 있었다. 나아가 이들 추출물의 작용기전 및 활성물질 구명에 대한 연구는 추후 더 진행되어야 할 것이다. 【This study was carried out to investigate the effect of Sorbus commixta (SC), Geranium thunbergii (GT) and their mixture (SC:GT=1:1, MIX) on inhibition of bone loss and chondral defect. To examine their activities, we measured the alkaline phosphatase (ALP) activity in human osteoblast-like MG-63 cells and performed tartrate-resistant acid phosphate (TRAP) staining in osteoclast differentiated from Raw264.7 cells. To investigate the influence on chondrocyte differentiation, we performed alcian-blue staining in chondrocyte differentiated from ATDC5 cells. All of SC, GT and MIX did not increase ALP activity in MG-63 cells. However, SC and mixture (SC:GT=1:1, MIX) significantly inhibited osteoclastic differentiation. And they also induced chondrocyte differentiation. These results suggest that SC and GT may have a potential for the treatment of bone loss and chondral defect by suppression of osteoclast differentiation and stimulation of chondrocyte differentiation. Therefore, clarification of their mechanisms and active components will be needed.】

Development of magnesium calcium phosphate biocement for bone regeneration

Magnesium calcium phosphate biocement (MCPB) with rapid-setting characteristics was fabricated by using the mixed powders of magnesium oxide (MgO) and calcium dihydrogen phosphate (Ca(H(2)PO(4))(2).H(2)O). The results revealed that the MCPB hardened after mixing the powders with water for about 7 min, and the compressive strength reached 43 MPa after setting for 1 h, indicating that the MCPB had a short setting time and high initial mechanical strength. After the acid-base reaction of MCPB containing MgO and Ca(H(2)PO(4))(2).H(2)O in a molar ratio of 2 : 1, the final hydrated products were Mg(3)(PO(4))(2) and Ca(3)(PO(4))(2). The MCPB was degradable in Tris-HCl solution and the degradation ratio was obviously higher than calcium phosphate biocement (CPB) because of its fast dissolution. The attachment and proliferation of the MG(63) cells on the MCPB were significantly enhanced in comparison with CPB, and the alkaline phosphatase activity of MG(63) cells on the MCPB was significantly higher than on the CPB at 7 and 14 days. The MG(63) cells with normal phenotype spread well on the MCPB surfaces, and were attached in close proximity to the substrate, as seen by scanning electron microscopy (SEM). The results demonstrated that the MCPB had a good ability to support cell attachment, proliferation and differentiation, and exhibited good cytocompatibility.

Low molecular weight silk fibroin increases alkaline phosphatase and type I collagen expression in MG63 cells

Silk fibroin, produced by the silkworm Bombyx mori, has been widely studied as a scaffold in tissue engineering. Although it has been shown to be slowly biodegradable, cellular responses to degraded silk fibroin fragments are largely unknown. In this study, silk fibroin was added to MG-63 cell cultures, and changes in gene expression in the MG-63 cells were screened by DNA microarray analysis. Genes showing a significant (2-fold) change were selected and their expression changes confirmed by quantitative RT-PCR and western blotting. DNA microarray results showed that alkaline phosphatase (ALP), collagen type-I alpha-1, fibronectin, and transforming growth factor-beta1 expressions significantly increased. The effect of degraded silk fibroin on osteoblastogenic gene expression was confirmed by observing up-regulation of ALP activity in MG-63 cells. The finding that small fragments of silk fibroin are able to increase the expression of osteoblastogenic genes suggests that controlled degradation of silk fibroin might accelerate new bone formation. [BMB reports 2010; 43(1): 52-56].

Physiological strains induce differentiation in human osteoblasts cultured on orthopaedic biomaterial

We have developed an in vitro mechanical stretching model of osteoblastic cells cultured on metallic biomaterials in order to study the effects of mechanical strain on osteointegration of orthopaedic implants. Titanium alloy discs coated with alumina or hydroxyapatite were used as substrates. Three Dynacell ® devices were especially designed to apply cyclic strains on rigid biomaterials. The regimen (600 με strains, 0.25 Hz) was defined on the basis of physiological data and estimated deformation on hip stem prostheses. The performances of these apparatus were reproducible and provided controlled deformations. Human osteosarcoma cell line MG-63, human osteoblasts obtained from primary cultures and ROS 17/2.8 rat osteosarcoma cells were used as cell models. Cell behaviour was assessed in terms of growth and alkaline phosphatase (ALP) activity by in situ assays for two regimens: 15-min deformations repeated three times a day to mimic rehabilitation exercises and 24-h continuous deformations. We demonstrated that continuous deformation did not affect the growth and ALP activity of MG-63 cells, in contrast with sequential deformations which had no effect on cell number, but which stimulated ALP activity after 5 days of stretching. This sequential regimen can also modify the behaviour of human bone-derived cells resulting in increased proliferation after 5 days and stimulation of ALP activity after 15 days. ROS 17/2.8 rat osteosarcoma cells submitted to sequential deformations responded faster than other cell lines by increasing their ALP activity only after 1 day of stretching. Like MG-63 cells, proliferation of the ROS 17/2.8 rat osteosarcoma cell line was not affected by sequential deformations. This study suggests that short, repeated deformations defined to mimic rehabilitation exercises recommended after prostheses implantation are more likely to exert beneficial effects on implanted bone than continuous strains.