Yun Mi Song

Novel Effect of Biphasic Electric Current on In Vitro Osteogenesis and Cytokine Production in Human Mesenchymal Stromal Cells

Electrical stimulation (ES) can activate diverse biostimulatory responses in a range of tissues. Of various forms of ES, the application of biphasic electric current (BEC) is a new approach to bone formation. This study is to investigate the effects and mechanism of action of BEC in osteoblast differentiation and cytokine production in human mesenchymal stromal cells (hMSCs). Using an in vitro culture system with a modified version of the BEC stimulator chip used in our previous study, we exposed hMSCs to a 100 Hz ES with a magnitude of 1.5/15 muA/cm(2) for 250/25 mus. hMSCs showed increased proliferation during static BEC stimulation for 5 days. However, alkaline phosphatase activity and calcium deposition were enhanced in hMSCs 7 days after the stimulation, rather than during the period of ES. BEC induced vascular endothelial growth factor (VEGF) and BMP-2 production; the former can enhance the proliferation of human umbilical vein endothelial cells in culture using conditioned media from BEC cultures. Treatment with selective inhibitors of p38 MAPK (SB203580) or Erk (PD98059), as well as calcium channel blockers (verapamil and nifedipine), reduced the BEC-mediated increase of VEGF expression and cell proliferation. These findings reveal that BEC is involved in the osteoblast differentiation of hMSCs through enhancement of cell proliferation and modulation of the local endocrine environment through VEGF and BMP-2 induction through the activation of MAPK (Erk and p38) and the calcium channel. Thus, local stimulation using BEC might be most beneficial in promoting osteogenic differentiation of hMSCs, resulting in enhanced bone formation for bone tissue engineering.

Human Mesenchymal Stromal Cells are Mechanosensitive to Vibration Stimuli

Low-magnitude high-frequency (LMHF) vibrations have the ability to stimulate bone formation and reduce bone loss. However, the anabolic mechanisms that are mediated by vibration in human bone cells at the cellular level remain unclear. We hypothesized that human mesenchymal stromal cells (hMSCs) display direct osteoblastic responses to LMHF vibration signals. Daily exposure to vibrations increased the proliferation of hMSCs, with the highest efficiency occurring at a peak acceleration of 0.3 g and vibrations at 30 to 40 Hz. Specifically, these conditions promoted osteoblast differentiation through an increase in alkaline phosphatase activity and in vitro matrix mineralization. The effect of vibration on the expression of osteogenesis-related factors differed depending on culture method. hMSCs that underwent vibration in a monolayer culture did not exhibit any changes in the expressions of these genes, while cells in three-dimensional culture showed increased expression of type I collagen, osteoprotegerin, or VEGF, and VEGF induction appeared in 2 different hMSC lines. These results are among the first to demonstrate a dose-response effect upon LMHF stimulation, thereby demonstrating that hMSCs are mechanosensitive to LMHF vibration signals such that they could facilitate the osteogenic process.

Osteogenic Responses of Human Mesenchymal Stromal Cells to Static Stretch

Molecular signals driving the regenerative process in distraction osteogenesis (DO) involve a complex system of cellular behavior triggered by mechanical strain. However, it remains unclear how mesenchymal stromal cells (MSCs) adapt to osteogenic demands during DO. We hypothesized that human MSCs (hMSCs) modulate early osteogenic metabolism during exposure to static stretch. The proliferation of hMSCs was increased by static stretch, which, in turn, suppressed TGF-β1-mediated decreases in cell proliferation. The amount of stretching force applied had little effect on osteoblast differentiation of hMSCs induced by dexamethasone treatment. However, this strain induced sustained production of nitric oxide and vascular endothelial growth factor (VEGF), which are critical factors in angiogenesis, from differentiated hMSCs. Mechanical stretch involved ERK and p38 mitogen-activated protein kinase pathways, the selective inhibitors of which decreased static-stretch-induced VEGF production. These findings provide evidence that hMSCs act to facilitate early osteogenic metabolism during exposure to static stretch.

Synergistic action of static stretching and BMP-2 stimulation in the osteoblast differentiation of C2C12 myoblasts

Static stretching is a major type of mechanical stimuli utilized during distraction osteogenesis (DO), a general surgical method for the lengthening of bone. The molecular signals that drive the regenerative process in DO include a variety of cytokines. Among these, bone morphogenic protein (BMP, -2 and -4) has been reported to exhibit strongly enhanced expression following the application of mechanical strain during the distraction phase. We hypothesize that mechanical stretching enhances osteoblast differentiation in DO by means of interaction with BMP-2 induced cytokine stimulation. C2C12 pluripotential myoblasts were exposed to stretching load and the resulting cell proliferation and osteoblast differentiation were then examined. The application of static stretching force resulted in significant cell proliferation at day 3, although with variable intensity according to the magnitude of stretching. A combined treatment of stretching load with BMP-2 stimulation significantly increased alkaline phosphatase (ALP) activity and up-regulated the gene expression of osteogenic markers (ALP, type I collagen, osteopontin, osteocalcin, cbfa1, osterix and dlx5). Results obtained with the combined treatment yielded more activity than just the BMP-2 treatment or stretching alone. These results reveal that specific levels of static stretching force increase cell proliferation and effectively stimulate the osteoblast differentiation of C2C12 cells in conjunction with BMP-2 stimulation, thus indicating a synergistic interaction between mechanical strain and cytokine signaling.

Estrogen modulates BMP-induced sclerostin expression via the Wnt signaling pathway

Clinical data show that estrogen levels are inversely associated with the production of sclerostin, a Wnt antagonist that recently attracted great attention over the use of its antibody in the anabolic treatment of osteoporotic conditions. However, the molecular link between sclerostin expression and estrogen signaling is not yet known. We investigated the mechanisms by which estrogen modulates sclerostin (SOST) gene expression at the cellular level in human osteoblast cells in association with bone morphogenetic protein (BMP)2 signaling given that BMP2 is a potential inducer of SOST in human mesenchymal stromal cells (hMSCs). 17β-Estradiol (E2) alone had no effect on SOST expression, which was significantly induced by treatment with BMP2 in hMSCs and osteoblasts derived from the mandibles of female donors. However, E2 suppressed the induction of SOST and other BMP2 target genes by BMP2 in hMSCs. E2 signaling was independent of the Smad pathway, which plays a critical role in SOST induction mediated by BMP2. Instead, E2 increased the transcriptional expression of β-catenin and levels of its activated form. Silencing of the gene encoding estrogen receptor (ER)α decreased E2 activity in β-catenin activation and the suppression of SOST induction by BMP2, but had no influence on BMP2-mediated SOST induction in the same conditions. Similar results were obtained after treatment with ERα antagonist as a Wnt inhibitor. In human osteoblasts, the effect of E2 on SOST expression was either suppressive or absent, depending on the cell donor. Interestingly, the SOST expression pattern after treatment with BMP2 or BMP2/E2 in human osteoblasts showing a pattern of E2 suppression on SOST induction by BMP2 correlated with the ratio of receptor activator of nuclear factor kappa-B ligand (RANKL) to osteoprotegerin (OPG) expression. These results demonstrate that estrogen signaling in osteoblasts negatively regulates SOST expression in an indirect manner through interaction with BMP2 signaling and that this regulation involves the Wnt/ERα and β-catenin pathways. This study highlights several interactions between estrogen and BMP cascades in osteoblasts that may provide a basis for therapeutic intervention for the modification of bone mass density.

An implantable electrical bioreactor for enhancement of cell viability

Low survival of injected cells which are prepared by ex-vivo culture is main obstacle in cell-based tissue regeneration. To elevate cell adaptation, we designed an implantable electrical bioreactor where human mesenchymal stromal cells (hMSCs) can be cultured and stimulated electrically. Bioreactor was composed of biocompatible cylindrical Teflon body containing a flexible polyimide electrode and implantable stimulator. The Teflon body has about 300 holes with a diameter of 300um for effective nutrients supply inside the bioreactor and has a length of 17mm and a diameter of 8mm for implantation. After hMSCs seeded on the collagen sponge that serves as scaffold to form a bone tissue graft, they are cultured in the bioreactor with biphasic electric current (BEC) stimulation. BEC stimulation with amplitude of 20/40uA, duration of 100us and a frequency of 100Hz was applied for one week in the early stage of cultivation. Subsequently, after hMSCS were cultured for another week without electrical stimulation, cell response such as cell proliferation, cell attachment and gene expression are evaluated. In vitro and In vivo culture of hMSCs showed 19% and 22% increase in cell proliferation at stimulated groups, compared to unstimulated control. The expression of type I collagen increased significantly at stimulated group. These results suggest that the usage of implantable electrical bioreactor can be a good strategy to enhance the efficiency of stem cell-based tissue engineering.

Preliminary Approach of Real-Time Monitoring In Vitro Matrix Mineralization Based on Surface Plasmon Resonance Detection

Matrix mineralization is a terminal process in osteoblast differentiation, and several approaches have been introduced to characterize the process in tissues or cultured cells. However, an analytical technique that quantitates in vitro matrix mineralization of live cells without any labeling or complex treatments is still lacking. In this study, we investigate a simple and enhanced optical method based on surface plasmon resonance (SPR) detection that can monitor the surface-limited refractive index change in real-time. During monitoring MC3T3-E1 cells in vitro culture every 2 days for over 4 weeks, the SPR angle is shifted with a greater resonance change in cells cultured with osteogenic reagents than those without the reagents. In addition, the SPR results obtained have a close relevance with the tendency of conventional mineralization staining and an inductively coupled plasma-based calcium content measure. These results suggest a new approach of a real-time SPR monitoring in vitro matrix mineralization of cultured cells.

A surface plasmon resonance sensor for quantitative analysis of mineralization of osteoblast cells

Surface plasmon resonance (SPR) is a well-known optical technique for detection of protein absorption or dielectric concentration on a metal surface [1]. In this study, we developed an angle-scanning SPR biosensor that accurately measures the amount of mineralization using MC3T3-e1 cells without any damage to the cells. Since there have been few accurate methods to quantitatively measure mineralization of osteogenesis, our results are compared with the traditional method of Alizarin red S staining. The result showed that the SPR sensor is much more accurate and easier than the staining method without cell destruction. It is also proven that the SPR sensor is a useful method to analyze the mineralization of live cells.

Enhanced bone formation around dental implant using electrical stimulation

It has been known that electrical stimulation promotes osteogenesis. Electrical stimulation using direct current can be applied to dental implant without invasive technique. The purpose of study was to evaluate the effect of biphasic direct current (BDC) on osteoblast in vitro and on early bone formation around implant. Human mesenchymal stem cells were exposed to BDC (1.5 A/cm2, 1,000 Hz), which was compared to control without stimulation regarding to cell proliferation and cytokine production in vitro. For animal test, a micro-current stimulator chip was developed (120ldrs, 100 Hz 20ldrA/cm2). BDC was applied to the implant 7 days in beagle dog (n=10). No stimulation was applied to control group (n=10). The animals were sacrificed at 2 and 4 weeks after stimulation. Histological specimens was histomorphometrically analyzed. The number of osteoblasts was increased (31%, p<0.05) and VEGF was increased in the stimulated group (p<0.01). The bone volume (BV) and bone implant contact (BIC) in 2 weeks and BV in 4 weeks were significantly (p<0.05) increased in the test group compared to the control. BCS can contribute to enhanced osteogenesis, it could be applied clinically to the early bone formation around implants.