• Archives of Pharmacal Research
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• v.26 no.12
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• pp.1029-1035
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• 2003

Osteoporosis is recognized as one of the major hormonal deficiency diseases, especially in menopausal women and the elderly. When estrogen is reduced in the body, local factors such as IL-1 $\beta$ and IL-6, which are known to be related with bone resorption, are increased and promote osteoclastogenesis, which is responsible for bone resorption. In the present study, we investigated whether glucosidic isoflavones (Isocal, PIII) extracted from Sophorae fructus affect the proliferation of osteoblasts and prevent osteoclastogenesis in vitro by attenuating upstream cytokines such as IL-1$\beta$ and IL-6 in a human osteoblastic cell line (MG-63) and in a primary osteoblastic culture from SD rat femurs. Interestingly, IL-1$\beta$ and IL-6 mRNA were significantly suppressed in osteoblast-like cells treated with 17$\beta$-estradiol (E2) and PIII when compared to positive control (SDB), and this suppression was more effective at $10^{-8}$% than at the highest concentration of $10^{-4}$%. In addition, these were confirmed in protein levels using ELISA assay. In the cell line, the cells showed that E2 was the most effective in osteoblastic proliferation over the whole range of concentration ($10^{-4}%-10^{-12}$%), even though PIII also showed the second greatest effectiveness at $10^{-8}$%. Nitric oxide (NO) was significantly (p<0.05) upregulated in PIII and E2 over the concentration range $10^{-6}% to 10^{-8}$% when compared to SDB, without showing any dose dependency. In bone marrow primary culture, we found by TRAP assay that PIII effectively suppressed osteoclastogenesis next to E2 in comparison with SDB and culture media (control). In conclusion, these results suggest that local bone-resorbing cytokines can be regulated by PIII at lower concentrations and that, therefore, PIII may preferentially induce anti-osteoporosis response by attenuating osteoclastic differentiation and by upregulating NO.

• Journal of the Korean institute of surface engineering
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• v.41 no.2
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• pp.69-75
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• 2008

Electrochemical behaviors of surface modified and MC3T3-E1 cell cultured Ti-30Ta alloys have been investigated using various electrochemical methods. The Ti alloys containing Ta were melted by using a vacuum furnace and then homogenized for 6 hrs at $1000^{\circ}C$. MC3T3-E1 cell culture was performed with MC3T3-E1 mouse osteoblasts for 2 days. The microstructures and corrosion resistance were measured using FE-SEM, XRD, EIS and potentiodynamic test in artificial saliva solution at $36.5{\pm}1^{\circ}C$. Ti-Ta alloy showed the martensite structure of ${\alpha}+{\beta}$ phase and micro-structure was changed from lamellar structure to needle-like structure as Ta content increased. Corrosion resistance increased as Ta content increased. Corrosion resistance of cell cultured Ti-Ta alloy increased predominantly in compared with non cell cultured Ti- Ta alloy due to inhibition of the dissolution of metal ion by covered cell. $R_p$ value of MC3T3-E1 cell cultured Ti-40 Ta alloy showed $1.60{\times}10^6{\Omega}cm^2$ which was higher than those of other Ti alloy. Polarization resistance of cell-cultured Ti-Ta alloy increased in compared with non-cell cultured Ti alloy.

• Bulletin of the Korean Chemical Society
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• v.34 no.6
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• pp.1857-1863
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• 2013

Most of biomaterials come in direct contact with the body, making standardized methods of evaluation and validation of biocompatibility an important aspect to biomaterial development. However, biomaterial validation guidelines have not been fully established, until now. This study was to compare the in vitro behavior of osteoblasts cultured on nanomaterial $TiO_2$ surfaces to osteoblast behavior on culture plates. Comparisons were also made to cells grown in conditioned media (CM) that creates an environment similar to the in vivo environment. Comparisons were made between the different growth conditions for osteoblast adhesion, proliferation, differentiation, and functionality. We found that the in vivo-like system of growing cells in concentrated CM provided a good validation method for biomaterial development and in vivo implant therapy. The $TiO_2$ materials were biocompatible, showing similar behavior to that observed in vivo. This study provided valuable information that would aid in the creation of guidelines into standardization and evaluation of biocompatibility in $TiO_2$ biomaterials.

• The Journal of Korean Academy of Prosthodontics
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• v.42 no.3
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• pp.307-326
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• 2004

Statement of problem. The long-term success of implants is the development of a stable direct connection between bone and implant surface, which must be structural and functional. To improve a direct implant fixation to the bone, various strategies have been developed focusing on the surface of materials. Among them, altering the surface properties can modify cellular responses such as cell adhesion, cell motility and bone deposition. Purpose. This study was to evaluate the cellular behaviors on the surface-modified titanium by morphological observation, cellular proliferation and differentiation. Material and methods. Specimens were divided into five groups, depending on their surface treatment: electropolishing(EP) anoclizing(AN), machining(MA), blasting with hydroxyapatite particle(RBM) and electrical discharge machining(EDM). Physicochemical properties and microstructures of the specimens were examined and the responses of osteoblast-like cells were investigated. The microtopography of specimens was observed by scanning electron microscopy(SEM). Surface roughness was measured by a three-dimensional roughness measuring system. The microstructure was analyzed by X-ray diffractometer(XRD) and scanning auger electron microscopy(AES). To evaluate cellular responses to modified titanium surfaces, osteoblasts isolated from neonatal rat were cultured. The cellular morphology and total protein amounts of osteoblast-like cell were taken as the marker for cellular proliferation, while the expression of alkaline phosphatase was used as the early differentiation marker for osteoblast. In addition, the type I collagen production was determined to be a reliable indicator of bone matrix synthesis. Results. 1. Each prepared specimen showed specific microtopography at SEM examination. The RBM group had a rough and irregular pattern with reticulated appearance. The EDM-treated surface had evident cracks and was heterogeneous consisting of broad sheet or plate with smooth edges and clusters of small grains, deep pores or craters. 2. Surface roughness values were, from the lowest to the highest, electropolished group, anodized group, machined group, RBM group and EDM group. 3. All groups showed amorphous structures. Especially anodized group was found to have increased surface oxide thickness and EDM group had titaniumcarbide(TiC) structure. 4. Cells on electropolished, anodized and machined surfaces developed flattened cell shape and cells on RBM appeared spherical and EDM showed both. After 14 days, the cells cultured from all groups were formed to be confluent and exhibited multilayer proliferation, often overlapped or stratified. 5. Total protein amounts were formed to be quite similar among all the group at 48 hours. At 14 days, the electropolished group and the anodized group induced more total protein amount than the RBM group(P<.05). 6. There was no significant difference among five groups for alkaline phosphatase(ALP) activity at 48 hours. The AN group showed significantly higher ALP activity than any other groups at 14 days(P<.05). 7. All the groups showed similar collagen synthesis except the EDM group. The amount of collagen on the electropolished and anodized surfaces were higher than that on the EDM surface(P<.05).

• The Journal of Korean Academy of Prosthodontics
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• v.41 no.6
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• pp.699-713
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• 2003

Statement of problem. Titanium is the most important material for biomedical and dental implants because of their high corrosion resistance and good biocompatibility. These beneficial properties are due to a protective passive oxide film that spontaneously forms on the surface. Purpose. The purpose of this study was to evaluate the responses of osteoblast-like cells on different surface treatments on Ti discs. Material and Methods. Group 1 represented the machined surface with no treatment. Group 2 surfaces were sandblasted with $50{\mu}m\;Al_2O_3$ under $5kgf/cm^2$ of pressure. Groups 3 and 4 were sandblasted under the same conditions. The samples were treated on a titanium oxide surface with reactive sputter depositioning and thermal oxidation at $600^{\circ}C$ (Group 3) and $800^{\circ}C$ (Group 4) for one hour in an oxygen environment. The chemical composition and microtopography were analyzed by XRD, XPS, SEM and optical interferometer. The stability of $TiO_2$ layer was studied by petentiodynamic curve. To evaluate cell response, osteoblast extracted from femoral bone marrow of young adult rat were cultured for cell attachment, proliferation and morphology on each titanium discs. Results and Conclusion. The results were as follows : 1. Surface roughness values were, from the lowest to the highest, machined group, $800^{\circ}C$ thermal oxidation group, $600^{\circ}C$ thermal oxidation group and blasted group. The Ra value of blasted group was significantly higher than that of $800^{\circ}C$ thermal oxidation group (P=0.003), which was not different from that of $600^{\circ}C$ thermal oxidation group (P<0.05). 2. The degree of cell attachment was highest in the $600^{\circ}C$ thermal oxidation group after four and eight hours (P<0.05), but after 24 hours, there was no difference among the groups (P>0.05). 3. The level of cell proliferation showed no difference among the groups after one day, three days, and seven days (P>0.05). 4. The morphology and arrangement of the cells varied with surface roughness of the discs.

• The Korean Journal of Physiology and Pharmacology
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• v.18 no.4
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• pp.347-352
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• 2014

Most known osteoporosis medicines are effective for bone resorption, and so there is an increasing demand for medicines that stimulate bone formation. Watercress (N. officinale R. Br.) is widely used as a salad green and herbal remedy. This study analyzed a watercress extract using ultra-performance liquid chromatography/mass spectrometry, and identified a rutin as one of its major constituents. Osteogenic-related assays were used to compare the effects of watercress containing rutin (WCR) and rutin alone on the proliferation and differentiation of human osteoblast-like MG-63 cells. The reported data are expressed as percentages relative to the control value (medium alone; assigned as 100%). WCR increased cell proliferation to $125.0{\pm}4.0%$ ($mean{\pm}SD$), as assessed using a cell viability assay, and increased the activity of alkaline phosphatase, an early differentiation marker, to $222.3{\pm}33.8%$. In addition, WCR increased the expression of collagen type I, another early differentiation marker, to $149.2{\pm}2.8%$, and increased the degree of mineralization, a marker of the late process of differentiation, to $122.9{\pm}3.9%$. Rutin alone also increased the activity of ALP (to $154.4{\pm}12.2%$), the expression of collagen type I (to $126.6{\pm}6.2%$), and the degree of mineralization (to $112.3{\pm}5.0%$). Daidzein, which is reported to stimulate bone formation, was used as a positive control; the effects of WCR on proliferation and differentiation were significantly greater than those of daidzein. These results indicate that WCR and rutin can both induce bone formation via the differentiation of MG-63 cells. This is the first study demonstrating the effectiveness of either WCR or rutin as an osteoblast stimulant.

• Journal of Periodontal and Implant Science
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• v.37 no.sup2
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• pp.427-445
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• 2007

To improve osseointegration at the boneto-implant interface, several studies have been carried out to modify titanium surface. Variations in surface texture or microtopography may affect the cellular response to an implant. Osteoblast-like cells attach more readily to a rougher titanium surface, and synthesis of extracellular matrix and subsequent mineralization were found to be enhanced on rough or porous coated titanium. However, regarding the effect of roughened surface by physical and mechanical methods, most studies carried out on the reactions of cells to micrometric topography, little work has been performed on the reaction of cells to nanotopography. The purpose of this study was to examme the response of osteoblast-like cell cultured on blasted surfaces and alkali treated surfaces, and to evaluate the influence of surface texture or submicro-scaled surface topography on the cell attachment, cell proliferation and the gene expression of osteoblastic phenotype using ROS 17/2.8 cell lines. In scanning electron micrographs, the blasted, alkali treated and machined surfaces demonstrated microscopic differences in the surface topography. The specimens of alkali treatment had a submicro-scaled porous sur-face with pore size about 200 nm. The blasted surfaces showed irregularities in morphology with small(<10 ${\mu}m$) depression and indentation among flatter-appearing areas of various sizes. Based on profilometry, the blasted surfaces was significantly rougher than the machined and the alkali treated surfaces (p$TiO_2$) were observed on alkali treated surfaces, whereas not observed on machined and blasted surfaces. The attachment morphology of cells according to time was observed by the scanning electron microscope. After 1 hour incubation, the cells were in the process of adhesion and spreading on the prepared surfaces. After 3 hours, the cells on all prepared surfaces were further spreaded and flattened, however on the blasted and alkali treated surfaces, the cells exhibited slightly irregular shapes and some gaps or spaces were seen. After 24 hours incubation, most cells of the all groups had a flattened and polygonal shape, but the cells were more spreaded on the machined surfaces than the blasted and alkali treated surfaces. The MTT assay indicated the increase on machined, alkali treated and blasted surfaces according to time, and the alkali treated and blasted surfaces showed significantly increased in optical density comparing with machined surfaces at 1 day (p<0.01). Gene expression study showed that mRNA expression level of ${\alpha}\;1(I)$ collagen, alkaline phosphatase and osteopontin of the osteoblast-like cells showed a tendency to be higher on blasted and alkali treated surfaces than on the machined surfaces, although no siginificant difference in the mRNA expression level of ${\alpha}\;1(I)$ collagen, alkaline phosphatase and osteopontin was observed among all groups. In conclusion, we suggest that submicroscaled surfaces on osteoblast-like cell response do not over-ride the one of the surface with micro-scaled topography produced by blasting method, although the microscaled and submicro-scaled surfaces can accelerate osteogenic cell attachment and function compared with the machined surfaces.

• The Journal of Korean Academy of Prosthodontics
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• v.43 no.6
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• pp.751-763
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• 2005

Statement of problem. To improve a direct implant fixation to the bone, various strategies have been developed focusing on the surface of materials. The surface quality of the implant depends on the chemical, physical, mechanical and topographical properties of the surface. The different properties will interact with each other and a change in thickness of the oxide layer may also result in a change in surface energy, the surface topography and surface, chemical composition. However, there is limited the comprehensive study with regard to changed surface and biologic behavior of osteoblast by anodization. Purpose of study. The aim of this study was to analyze the characteristics of an oxide layer formed and to evaluate the cellular biologic behaviors on titanium by anodic oxidation (anodization) by cellular proliferation, differentiation, ECM formation and gene expression. And the phospholipase activity was measured on the anodized surface as preliminary study to understand how surface properties of Ti implant are transduced into downstream cellular events. Methods and Materials. The surface of a commercially pure titanium(Grade 2) was modified by anodic oxidation. The group 1 samples had a machined surface and other three experimental specimens were anodized under a constant voltage of 270 V(Group 2), 350 V(Group 3), and 450 V(Group 4). The specimen characteristics were inspected using the following five categories; the surface morphology, the surface roughness, the thickness of oxide layer, the crystallinity, and the chemical composition of the oxide layer. Cell numbers were taken as a marker for cell proliferation. While the expression of alkaline phosphatase and Runx2 (Cbfa1) was used as early differentiation marker for osteoblast. The type I collagen production was determined, which constitutes the main structural protein of the extracellular matrix. Phospholipase $A_2$ and D activity were detected. Results. (1) The anodized titanium had a porous oxide layer, and there was increase in both the size and number of pores with increasing anodizing voltage. (2) With increasing voltage, the surface roughness and thickness of the oxide film increased significantly (p<0.01), the $TiO_2$phase changed from anatase to rutile. During the anodic oxidization, Ca and P ions were more incorporated into the oxide layer. (3) The in vitro cell responses of the specimen were also dependant on the oxidation conditions. With increasing voltage, the ALP activity, type I collagen production, and Cbfa 1 gene expression increased significantly (p<0.01), while the cell proliferation decreased. (4) In preliminary study on the relation of surface property and phospholipase, PLD activity was increased but $PLA_2$ activity did not changed according to applied voltage. Conclusion. The anodized titanium shows improved surface characteristics than the machined titanium. The surface properties acquired by anodization appear to give rise more mature osteoblast characteristics and might result in increased bone growth, and contribute to the achievement of a tight fixation. The precise mechanism of surface property signaling is not known, may be related to phospholipase D.

• Journal of the Korean Society of Food Science and Nutrition
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• v.39 no.2
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• pp.203-209
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• 2010

In this study, the effects of Petasites japonicus and Momordica charantia L. extracts on MC3T3-ET1 osteoblastic cells were investigated. Since the activity of osteoblastic cell is one of the important factors for bone formation, the cellular proliferation of osteoblast was evaluated by MTT and alkaline phosphatase (ALP) activity. Compared to control, the cell proliferation was elevated to 114% and 112% by the treatment of Petasites japonicus and Momordica charantia L. extracts, respectively at the concentration of $10\;{\mu}g/mL$. The cell differentiation was also measured by alkaline phosphatase (ALP) activity at 3, 7, 14, and 27 days treatments with one of the extracts, respectively. As results, the ALP activity was significantly increased at 3 days, compared to control (p<0.05). To evaluate the effect of Petasites japonicus and Momordica charantia L. extracts on bone nodule formation, MC3T3-E1 cells were cultured in $\alpha$-MEM for 3, 14, and 21 days and then stained by alizarin red. To determine the expression patterns of bone-related proteins during the MC3T3-E1 osteoblast-like cell differentiation, osteoblast cells were cultured in $\alpha$-MEM for 24 hr. RNA was extracted and RT-PCR analysis was performed to examine the expression of OPG, RANKL and osteocalcin. Petasites japonicus extract exhibited the significant increment of osteocalcin compared with the positive control, which suggests that Petasites japonicus may have beneficial effects on bone health through the proliferation of osteoblast cells.

• Proceedings of the Korean Society of Precision Engineering Conference
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• 2002.05a
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• pp.154-157
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• 2002

A cyto-indentation technique was used to obtain the biomechanical compressive compliance property of an chondrocyte cell attached to glass surface, which was tried to generate joint cartilage by tissue engineering. Piezo-transducer system and dual photo-diode system were used to conduct mechanical indentation through displacement-controlled testing and the measurement of corresponding cell reaction force. The Poisson's ratio of 0.37 was quoted from other report. The compressive compliance of chondrocyte, that was determined by elastic contact theory, was 1.38${\pm}$0.057 kPa. This value is 30% higher than that of MG63 osteoblast-like cell. The cyto-indentation technique employed in this study is so precise that it can quantify the biomechanical property of single cell.