The effect of blasting and anodizing-combined treatment of implant surface on response of osteoblast-like cell / 대한치과보철학회지

PURPOSE: The purpose of this study is to examine characteristics of implant surface with RBM and anodizing treatments, and to evaluate the responses of osteoblast-like cell (MG-63 cell). MATERIALS AND METHODS: Grade IV titanium disks were fabricated (Diameter 10 mm, thickness 3 mm). Anodizing treatment (ASD) group, RBM and anodizing treatment (RBM/ASD) group, control (machined surface) group were divided. In this study, osteoblast-like cell was used for experiments. The experiments consist of surface characteristics evaluation by FE-SEM images, energy dispersive spectroscopy and stereo-SEM. In order to evaluate cell adhesion evaluation by crystal violet assay and observe cells form by confocal laser microscopy. To assess cell proliferation by XTT assay, cell differentiation by RT-PCR and mineralization by Alizarin red S stain assay. ELISA analyzer was used for Quantitative evaluation. Comparative analysis was run by one-way ANOVA (SPSS version 18.0). Differences were considered statistically significant at P<.05. RESULTS: In ASD group and RBM/ASD group, the surface shape of the crater was observed and components of oxygen and phosphate ions in comparison with the control group were detected. The surface average roughness was obtained 0.08 +/- 0.04 microm in the control group, 0.52 +/- 0.14 microm in ASD group and 1.45 +/- 0.25 microm in RBM/ASD group. In cell response experiments, ASD group and RBM/ASD group were significantly higher values than control group in cell adhesion and mineralization phase, control group was the highest values in the proliferative phase. In RT-PCR experiments, RBM/ASD group was showed higher ALP activity than other groups. RBM/ASD group in comparison with ASD group was significantly higher value for cell adhesion and proliferation phase. CONCLUSION: In the limitation of this study, we are concluded that the surface treatment with RBM/ASD seems more effective than ASD alone or machined surface on cellular response.

Oscillatory fluid flow elicits changes in morphology, cytoskeleton and integrin-associated molecules in MLO-Y4 cells, but not in MC3T3-E1 cells

Interstitial fluid flow stress is one of the most important mechanical stimulations of bone cells under physiological conditions. Osteocytes and osteoblasts act as primary mechanosensors within bones, and in vitro are able to respond to fluid shear stress, both morphologically and functionally. However, there is little information about the response of integrin-associated molecules using both osteoblasts and osteocytes. In this study, we investigated the changes in response to 2 hours of oscillatory fluid flow stress in the MLO-Y4 osteocyte-like cell line and the MC3T3-E1 osteoblast-like cell line. MLO-Y4 cells exhibited a significant increase in the expression of integrin-associated molecules, including OPN, CD44, vinculin and integrin avp3. However, there was no or limited increase observed in MC3T3-E1 osteoblast-like cells. Cell area and fiber stress formation were also markedly promoted by fluid flow only in MLO-Y4 cells. But the numbers of processes per cell remain unaffected in both cell lines.

The effect of immobilization of heparin and bone morphogenic protein-2 to bovine bone substitute on osteoblast-like cell's function

PURPOSE: This study was performed to investigate the ability of recombinant human-bone morphogenic protein-2 immobilized on a heparin-grafted bone substrate to enhance the osteoblastic functions. MATERIALS AND METHODS: The Bio-Oss(R), not coated with any material, was used as a control group. In rhBMP-2-Bio-Oss(R) group, rhBMP-2 was coated with Bio-Oss(R) using only deep and dry methods (50 ng/mL, 24 h). In heparinized rhBMP-2-Bio-Oss(R) group, dopamine was anchored to the surface of Bio-Oss(R), and coated with heparin. rhBMP-2 was immobilized onto the heparinized- Bio-Oss(R) surface. The release kinetics of the rhBMP-2-Bio-Oss(R) and heparinized rhBMP-2-Bio-Oss(R) were analyzed using an enzyme-linked immunosorbent assay. The biological activities of the MG63 cells on the three groups were investigated via cytotoxicity assay, cell proliferation assay, alkaline phosphatase (ALP) measurement, and calcium deposition determination. Statistical comparisons were carried out by one-way ANOVA test. Differences were considered statistically significant at *P<.05 and **P<.001. RESULTS: The heparinized rhBMP-2-Bio-Oss(R) showed more sustained release compared to the rhBMP-2-Bio-Oss(R) over an extended time. In the measurement of the ALP activity, the heparinized group showed a significantly higher ALP activity when compared with the non-heparinized groups (P<.05). The MG63 cells cultivated in the group with rhBMP-2 showed increased calcium deposition, and the MG63 cells from the heparinized group increased more than those that were cultivated in the non-heparinized groups. CONCLUSION: Heparin increased the rhBMP-2 release amount and made sustained release possible, and heparinized Bio-Oss(R) with rhBMP-2 successfully improved the osteoblastic functions.

Effect of thyroid hormones on the expression of thyroid hormone receptor isoforms in human osteogenic sarcoma cell line MG-63 / 中华内分泌代谢杂志

Objective To examine the expression of thyroid hormone receptor isoforms (TR α1, α2, β1, and β2) in human osteoblast-like cell line MG-63 at the mRNA level and the effect of thyroid hormone (T3 or T4 ) on the expression. Methods Realtime quantitative PCR was performed. Results The expression of TRα1 mRNA was the highest, that was 10. 70± 0.45, TRβ1 was 5.75 ± 0. 10, TRβ2 was 3.34 ± 0. 08, and TRα2 was very low, only (3.66 ±0. 59) × 10-2. Only the expression of TRαl and TRα2 mRNA was down regulated significantly by the treatment of 10-10 ～ 10-6 mol/L T3, and there was a negative correlation between the expression of TRα1 or TRα2 mRNA and the concentration of T3. Conclusion TRα1 plays a primary role in mediating the effects of thyroid hormones in skeletal development.

The thyroid hornone receptor in human osteogenic sarcoma cell line MG-63 cell-T3 binding studies with nuclear extracts / 中华内分泌代谢杂志

To establish a method for radioligand binding assay of thyroid hormone receptors(TR)in human osteoblast-like osteosarcoma cell line MG-63 and to estimate the kinetic parameters of putative receptors. The MG-63cell was cultured in Ham's F12, the soluble TR was prepared from the intact nuclear extracts. The binding properties between TR and T3 were performed by using the traditional Scatchard analysis. The apparent Ka of TR in MG-63 is 7.68× 109 L/mol, and MBC(111. 25+ 10.77)fmol/mg protein. The study indicated that MG-63 cells possessed high affinity and limited-capacity of TR in its nuclear extracts. This may serve as the starting and basic work about TR in bone cell.

The effects of beta-TCP/rhBMP-2 on bone formation in osteoblast-like cells induced from bone marrow-derived mesenchymal stem cells

The present study aimed to investigate the osteogenic potentials of differentiated osteoblast-like cells (DOCs) induced from bone marrow-derived mesenchymal stem cells (MSCs) on beta-tricalcium phosphate (beta-TCP) with recombinant human bone morphogenetic protein (rhBMP-2) in vitro. Osteoblast differentiation was induced in confluent cultures by adding 100 nM dexamethasone, 10 mM beta-glycerophosphate, 50 mM L-ascorbic acid. The Alizarin red S staining and reverse transcriptase-polymerase chain reaction (RT-PCR) were perfomed to examine the mRNA expression of alkaline phosphatase (ALP), bone sialoprotein (BSP), osteocalcin (OCN), receptor activator for nuclear factor kappaB ligand (RANKL), runt-related transcription factor 2 (RUNX2), collagen-I (COL-I). There were no significant differences in the osteogenic potentials of DOCs induced from MSCs on beta-TCP(+/-). According to the incubation period, there were significant increasing of Alizadin red S staining in the induction 3 weeks. The mRNA expression of ALP, RUNX2, and RANKL were higher in DOCs/beta-TCP(-) than DOCs/beta-TCP(+). According to rhBMP-2 concentrations, the mRNA expression of BSP was significantly increased in DOCs/beta-TCP(+) compared to that of DOCs/beta-TCP(-) on rhBMP 10 ng/ml. Our study presented the beta-TCP will have the possibility that calcium phosphate directly affect the osteoblastic differentiation of the bone marrowderived MSCs.

The effect of permanent magnet connecting with dental implant on distribution and attachment of osteoblast-like cell around the dental implant / 대한치과보철학회지

PURPOSE: The purpose of this study is to find the effect of rare earth magnet's magnetic field of to the osteoblast around the implant by the means of observation number, and distribution around the implant which is connected to the permanent magnet but not, counted and compared by the number of cells attached to the surface of the implant. MATERIAL AND METHOD: The permanent magnets, made in the healing cap form, were connected to the implant fixture, and placed on the culture plate, The osteoblast-like cell: MC3T3-E1 were used for cell culture. As the control group, the implant were connected to normal healing cap, and cultured in the same conditions. 48 hours later, using inverted microscope, the number and distribution of osteoblast around the implant were observed, and 72 hours later, the number of the cells attached to the implant were counted. RESULTS: As a result, the implant connected to the permanent magnet had proved to have a more concentrated cell distribution rate than the control group. The implant connected to the permanent magnet, neck area: which has about 10 gauss magnetic force, had more cells than apex area. The implant connected to the permanent magnet had proven to attach to the osteoblast more productively than control group's implant. CONCLUSIONS: This research showed that the magnetic field of the permanent magnet affected the distribution and growth rate of the osteoblast around the implant. In order to support this study, it also had need to monitor the progress of the permanent magnet specifically shown on the neck area, which has10 gauss magnetic force. So after additional research on the distribution and attachment of the cells, and further more, on bone formation, it will be concluded that the clinical applications, such as immediate loading of implant treatment are possible.

Neoplastic transformation of human osteoblast-like cell line HOS TE85 / 第三军医大学学报

Objective To establish human osteoblast-like cell lines TE85 model of neoplastic transformation for exploring molecular mechanism in canceration process of osteosarcoma.Methods HOS TE85 cells were treated by MNNG(initiated factor) and TPA(promotor).The malignancy of transformed cells was identified by observing the cell form,colony forming frequency on soft agar and tumorigenesis in nude mice.Results Continuous passage after induction of neoplastic transformation led to the formation of a few paramorph foci that exhibited an extensively random orientation.The agglomeration in experiment group was more than that in control group.As compared with that of negative control cells,colony formation efficiency of transformed cells in semisolid agar showed a significant increase and the transformed cells could form tumor subcutaneously in the nude mice.The tumors were a poorly differentiated osteosarcoma confirmed by histopathological examination.Conclusion Simulating the process of malignant transformation of human cells,we establish neoplastic transformation of human osteoblast-like cell lines TE85 model.

Properties of stretch-activated K+ channels in an G292 osteoblast-like cell / 대한치과교정학회지

K+ -selective ion channels were studied in excised inside-out membrane patches from human osteoblast-like cells(G292). There classes of K+ channels were present and could be distinguished on the basis of conductance. Conductances were 270+/-27 pS, 113+/-12 pS, 48+/-8 pS according to their approximate conductances in symmetrical 140 mM KCI saline at holding potential of -80 mV. It was found that the small conductance (48 pS) K+ channel activation was dependent on membrane voltage. In current-voltage relationship, small conductance K+ channel showed outward rectification, and it was activated by the positive potential inside the membrane. In recordings, single channel currents were activayed by a negative pressure outside the membrane. The membrane pressure increased P(open) of the K+ channel in a pressure-dependent manner. In the excised-patch clamp recordings, G292 osteoblast-like cells have been shown to contain three types of K+ channels. Only the small conductance (48 pS) K+ channel is sensitive to the membrane stretch. These findings suggest that a hyperpolarzing current, mediated in part by this channel, may be associated with early events during the mechanical loading of the osteoblast. In G292 osteoblast-like cells, K+ channel is sensitive to membrane tension, and may represent a unique adaptation of the bone cell membrane to mechanical stress.

Bone Cell Viability after Exposure to Air / 대한정형외과학회잡지

Dryness has harmful effect on the harvested autogenous bone. But there was no report how the bone cells response to the drying condition and recuperate after exposure. Cytotoxic effect of dryness on the osteoblast-like cells were examined from 18- day chick calvaria. The cells were rinsed with lactated Ringer solution or lactated Ringer solution with 10% fetal bovine serum to see whether the added serum could help the cells live longer. Cultured cells were exposed to room air for period of time ranging from 0 minute to 35 minutes and viability was determined using the MTT test. Cultured cell death increased with the increase of exposure time to air between 25 and 30 minutes. No live cells existed after 35 minutes exposure. Lactated Ringers solution containing 10% fetal bovine serum did not prolong the survival time of osteoblast-like cells compared with lactated Ringers solution alone. These results suggest that the drying of bone cells that occurs in the operating room, especially after a prolonged reconstructive procedure, may severely damage the cell viability of exposed bone or grafted bone and thereby compromise the fusion rate.

The effects of changes in intracellular Ca2+ activity of osteoblast-like cell on fracture healing / 대한정형외과학회잡지

Bone formation by osteoblast may be closely related to the increase in intracellular Ca2+ activity of osteoblast. In order to study the effects of changes in Ca2+ activity of osteoblast-like cell on fracture healing, we changed intracellular Ca2+ activity of osteoblast-like cells by vanadate and verapamil. And the process of fracture healing was observed after injection of the treatment osteoblast-like cells into the fracture site by hematoxylin-eosin (H-E) stain and bromodeoxyuridine (BrdU) stain. The results were as follow: 1) The most effective range of concentration which could facilitate bone formation was 10-6 to 10-5 M. 2) H-E stain showed more abundant osteoblast and osteoid tissues, more active mitotic division of osteoblast, and earlier appearance of chondroblast and chondroid tissue, making the maturation of woven bone faster in the vanadate-treated group than in the control group. The opposite was true in the verapamil-treated group compared with the control group. 3) BrdU labeling index showed more active osteoblastic proliferation in the vanadate-treated than in the control group. The opposite was observed in the verapamil-treated group compared with the control group. From these results, the fracture healing appears to be facilitated and decelerated by vanadate which apparently increase intracellular Ca2+ activity of osteoblast and verapamil which decreases it, repectively. Therefore the change of intracellular Ca2+ activity of osteoblast can be considered to be one of fracture healing mechanisms and expected to be applied for clinical purpose.

The effect of stretch on the production of PGE_2 from osteoblast-like cells / 实用口腔医学杂志

0.05),319.97?11.83,337.20?13.13,424.65?15.53,450.53?14.23 and 508.38?9.26 respectively(each group vs control or vs each of the another stretched group,P

Effects of several cytokines on the functions of fetal rat osteoblast-like cells in vitro / 대한치과교정학회지

Effects of several cytokines(IL -1beta, TNFalpha, and IFNgamma) have been examined on fetal rat osteoblast-like cells. To investigate whether cytokines play direct causal roles in production of lysosomal enzyme, fetal rat osteoblast-like cells were treated with IL-1beta, TNFalpha, and IFNgamma, respectively or combined. And acid phosphatase was determined by biochemical method. Alkaline phosphatase was assayed to determine the effects of IL-1beta, TNFalpha, and IFNgamma on the expression of this enzyme. And also experiment of calcified nodule formation was performed to assess the effects of cytokines on the bone-forming activity of osteoblast-like cells in vitro. Acid phosphatase activity was significantly increased by the addition of IL-1beta and TNFalpha, whereas decreased by IFNgamma, However, no significant changes in alkaline phosphatase activity was observed when the osteoblast-like cells were treated with IL-1beta and TNFalpha. Interestingly, IFNalpha showed stimulatory effect on alkaline phosphatase activity. The number of calcified nodules was decreased by treatment of cultures with 1ng/ml IL -lbeta, 20ng/ml TNFalpha, and 500 u/ml IFNgamma continuously for 21 days, while considerable number of calcified nodules were formed in control group of osteoblast-like cell in culture for 21 days. These results seem to suggest that cytokines may play crucial roles in bone remodeling through the direct action on the osteoblast-like cell.