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Vibrational stimulation induces osteoblast differentiation and the upregulation of osteogenic gene expression in vitro.

ABSTRACT: Vibrational stimulation is an accepted non-invasive method used to improve bone remodeling. However, the underlying mechanisms of this phenomenon remain unclear. In this study, we developed a new vibration-loading system to apply vibrational stimulation to cells based on a previously reported in vivo study. We hypothesized that osteoblasts respond to vibrational strain by expressing osteogenic marker genes, such as alkaline-phosphatase (ALP), Runx2, and Osterix. To test our hypothesis, we developed a vibration-loading system to apply a precise vibrational force to an osteoblast culture on a silicone membrane. The system regulated frequency and acceleration of the vibration, and strain on the silicone membrane culture surface was measured using the strain gauge method. After vibrational stimulation, cellular gene expression was analyzed using real-time polymerase chain reaction. We obtained clear strain signals from the culture surface at vibrational ranges of 1.0-10 m/s2 acceleration and frequencies of 30, 60, and 90 Hz, respectively. The strain increased in a linear fashion, depending on the acceleration magnitude. Vibrational stimulation also significantly upregulated expression of the osteogenic marker genes Runx2, Osterix, type I collagen, and ALP. In conclusion, we developed a new vibration-loading system that can precisely regulate frequency and acceleration, and we established the presence of dynamic cellular strain on a culture surface. Our findings suggest that vibrational stimulation may directly induce osteoblast differentiation.

SUBMITTER: Ota T

PROVIDER: S-EPMC5101299 | biostudies-literature | 2016 Dec

REPOSITORIES: biostudies-literature

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Vibrational stimulation induces osteoblast differentiation and the upregulation of osteogenic gene expression in vitro.

Ota Takeru T Chiba Mirei M Hayashi Haruhide H

Cytotechnology 20160917 6

Vibrational stimulation is an accepted non-invasive method used to improve bone remodeling. However, the underlying mechanisms of this phenomenon remain unclear. In this study, we developed a new vibration-loading system to apply vibrational stimulation to cells based on a previously reported in vivo study. We hypothesized that osteoblasts respond to vibrational strain by expressing osteogenic marker genes, such as alkaline-phosphatase (ALP), Runx2, and Osterix. To test our hypothesis, we develo ...[more]

PMID: 27639712

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Project description:BackgroundOsteomyelitis is a severe and often debilitating disease characterized by inflammatory destruction of bone. Despite treatment, chronic infection often develops which is associated with increased rates of treatment failure, delayed osseous-union, and extremity amputation. Within affected bone, bacteria exist as biofilms, however the impact of biofilms on osteoblasts during disease are unknown. Herein, we evaluated the effect of S. aureus biofilms on osteoblast viability, osteogenic potential, and the expression of the pro-osteoclast factor, receptor activator of NF-kB ligand (RANK-L).MethodsOsteoblasts were exposed to biofilm conditioned media (BCM) from clinical wound isolates of Staphylococcus aureus under normal growth and osteogenic conditions to assess cellular viability and osteoblast differentiation, respectively. Cell viability was evaluated using a live/dead assay and by quantifying total cellular DNA at days 0, 1, 3, 5, and 7. Apoptosis following treatment with BCM was measured by flow-cytometry using the annexin V-FITC/PI apoptosis kit. Osteogenic differentiation was assessed by measuring alkaline phosphatase activity and intracellular accumulation of calcium and osteocalcin for up to 21 days following exposure to BCM. Expression of genes involved in osteogenic differentiation and osteoclast regulation, were also evaluated by quantitative real-time PCR.ResultsBCM from clinical strains of S. aureus reduced osteoblast viability which was accompanied by an increase in apoptosis. Osteogenic differentiation was significantly inhibited following treatment with BCM as indicated by decreased alkaline phosphatase activity, decreased intracellular accumulation of calcium and inorganic phosphate, as well as reduced expression of transcription factors and genes involved in bone mineralization in viable cells. Importantly, exposure of osteoblasts to BCM resulted in up-regulated expression of RANK-L and increase in the RANK-L/OPG ratio compared to the untreated controls.ConclusionsTogether these studies suggest that soluble factors produced by S. aureus biofilms may contribute to bone loss during chronic osteomyelitis simultaneously by: (1) reducing osteoblast viability and osteogenic potential thereby limiting new bone growth and (2) promoting bone resorption through increased expression of RANK-L by osteoblasts. To our knowledge these are the first studies to demonstrate the impact of staphylococcal biofilms on osteoblast function, and provide an enhanced understanding of the pathogenic role of staphylococcal biofilms during osteomyelitis.

Project description:BackgroundAlthough pulsed electromagnetic field (PEMF) stimulation may be clinically beneficial during fracture healing and for a wide range of bone disorders, there is still debate on its working mechanism. Mesenchymal stem cells are likely mediators facilitating the observed clinical effects of PEMF. Here, we performed in vitro experiments to investigate the effect of PEMF stimulation on human bone marrow-derived stromal cell (BMSC) metabolism and, specifically, whether PEMF can stimulate their osteogenic differentiation.MethodsBMSCs derived from four different donors were cultured in osteogenic medium, with the PEMF treated group being continuously exposed to a 15 Hz, 1 Gauss EM field, consisting of 5-millisecond bursts with 5-microsecond pulses. On culture day 1, 5, 9, and 14, cells were collected for biochemical analysis (DNA amount, alkaline phosphatase activity, calcium deposition), expression of various osteoblast-relevant genes and activation of extracellular signal-regulated kinase (ERK) signaling. Differences between treated and control groups were analyzed using the Wilcoxon signed rank test, and considered significant when p < 0.05.ResultsBiochemical analysis revealed significant, differentiation stage-dependent, PEMF-induced differences: PEMF increased mineralization at day 9 and 14, without altering alkaline phosphatase activity. Cell proliferation, as measured by DNA amounts, was not affected by PEMF until day 14. Here, DNA content stagnated in PEMF treated group, resulting in less DNA compared to control.Quantitative RT-PCR revealed that during early culture, up to day 9, PEMF treatment increased mRNA levels of bone morphogenetic protein 2, transforming growth factor-beta 1, osteoprotegerin, matrix metalloproteinase-1 and -3, osteocalcin, and bone sialoprotein. In contrast, receptor activator of NF-κB ligand expression was primarily stimulated on day 14. ERK1/2 phosphorylation was not affected by PEMF stimulation.ConclusionsPEMF exposure of differentiating human BMSCs enhanced mineralization and seemed to induce differentiation at the expense of proliferation. The osteogenic stimulus of PEMF was confirmed by the up-regulation of several osteogenic marker genes in the PEMF treated group, which preceded the deposition of mineral itself. These findings indicate that PEMF can directly stimulate osteoprogenitor cells towards osteogenic differentiation. This supports the theory that PEMF treatment may recruit these cells to facilitate an osteogenic response in vivo.

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Vibrational stimulation induces osteoblast differentiation and the upregulation of osteogenic gene expression in vitro.

Publications

Vibrational stimulation induces osteoblast differentiation and the upregulation of osteogenic gene expression in vitro.

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