http://repub.eur.nl/res/aut/18279/ List of Publications en http://repub.eur.nl/static-eur/img/logo.png http://repub.eur.nl http://repub.eur.nl/res/pub/20930/ 2010-08-26T00:00:00Z Background: Although 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. Methods: BMSCs 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. Results: Biochemical 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-B ligand expression was primarily stimulated on day 14. ERK1/2 phosphorylation was not affected by PEMF stimulation. Conclusions: PEMF 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. © 2010 Jansen et al; licensee BioMed Central Ltd. http://repub.eur.nl/res/pub/20409/ 2010-05-12T00:00:00Z Bone is a surprisingly versatile tissue. It provides body shape, protection of vital organs, and enables locomotion. Additionally, it forms a reservoir for calcium, phosphate and bone marrow cells, and is the endocrine organ involved in phosphate homeostasis. It is also a dynamic tissue, while it has the capacity to regenerate, and to change in shape, mass, and quality. Bone is remodeled at such a high speed that approximately 5-10% of the total bone content is replaced per year in adult humans1. Furthermore, bone is a mechanosensitive tissue. Mechanical loading plays a crucial role in maintenance of bone: disuse or unloading may cause osteoporosis, which results in increased fracture risk. Both socially and economically, osteoporosis and its complications have a severe impact on the individual and society. One way to prevent osteoporosis is physical activity. Moreover, physical stimuli have proven to be beneficial in fracture healing and remodeling of malunions. Prerequisite for the maintenance of skeletal homeostasis are cells that degrade old bone (osteoclasts), and those that deposit new bone in its place (osteoblasts). In order to respond to mechanical stress, the physical stimulus needs to be converted into a biochemical signal, eventually resulting in a biological response. This process is called mechanotransduction. Osteoblasts and osteocytes are the main effectors to facilitate this process, equipped with mechanoreceptors on the cell membrane, signal transduction pathways in the cytoplasm, and dedicated ‘mechanosensitive’ genes in the nucleus. Although this concept is generally accepted, little is known on the exact molecular mechanisms that play a role in mechanotransduction in bone. While osteoblasts and osteocytes are both part of the same differentiating lineage, these cells have entirely different characteristics. This thesis focuses on the effects of mechanical loading and human osteoblast differentiation in vitro. This first chapter will provide background information and the rationale for this work. http://repub.eur.nl/res/pub/17603/ 2010-03-01T00:00:00Z Wnt signaling is important for bone formation and osteoblastic differentiation. Recent findings indicate a stimulating role of Wnt signaling in bone mechanotransduction. However, negative effects of Wnt signaling on osteoblast differentiation and mineralization have been described as well. We conducted in vitro stretch experiments using human pre-osteoblasts to study short- and long-term effects of mechanical loading on Wnt/beta-catenin signaling. As the extracellular regulated kinase (ERK) pathway is known to be involved in mechanotransduction in osteoblasts, we also evaluated its role in Wnt/beta-catenin signaling. Stretch experiments up to 21 days (using stretch episodes of 15 min, alternated with 90 min rest) resulted in higher mineralization compared to static control cultures. We found that 15 min of stretch initially increased nuclear beta-catenin, but ultimately resulted in significant decrease at 12 and 40 h after stretch. Downregulation of Wnt-responsive element activity 16 h after stretch, using a luciferase construct, further supported these findings. The presence of the ERK inhibitor U0126 did not alter the stretch-induced decrease of beta-catenin levels. Our data indicate a biphasic effect of mechanical loading on beta-catenin in mineralizing human differentiating osteoblasts, which is independent of the ERK pathway. The osteogenic potential of our loading regime was confirmed by an increase in osteogenic differentiation markers such as alkaline phosphatase activity and calcium deposition after 3 weeks of culture. We conjecture that the biphasic aspect of Wnt/beta-catenin signaling with a strong decrease up to 40 h after the stretch induction, is important for the anabolic effects of mechanical stretch on bone. http://repub.eur.nl/res/pub/15422/ 2006-07-01T00:00:00Z Matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) produced by osteoblasts play an essential role in bone remodeling. Hence, these proteins could provide an interesting means by which mechanical loading leads to adaptation of bone. Here, we examined the effect of stretch on MMP-1, -2, -3, -8, -9, -13, and -14, as well as TIMP-1 and -2 gene expression in differentiating, mineralizing, and nonmineralizing human SV-40 immortalized preosteoblast cells. In the mineralizing osteoblast culture, but not in the nonmineralizing cultures, cyclic stretch for only 15 min resulted in an increase of MMP-1 (fourfold) and -3 (depending on differentiation stage up to 25-fold) transcript abundance. No clear effect was observed for other MMPs, TIMP-1 or -2. The increase of MMP-1 and -3 was confirmed on the protein level. Stretching experiments performed in the presence of a specific inhibitor of extracellular signal-regulated kinase (ERK) showed a strong suppression of the stretch-induced increase in MMP-1 and -3. In conclusion, we show that MMP-1 and MMP-3 are mechanosensitive genes in mineralizing the human osteoblast, and that the mechano-induction of these genes is mediated via the ERK pathway. Our findings implicate that these MMPs are important factors in the mechanoregulation of bone turnover. With the ability to generate MMPs at highly stretched sites, osteoblasts can potantially direct osteoclasts to specific bone surface areas prepared for resorption. http://repub.eur.nl/res/pub/15374/ 2004-10-01T00:00:00Z The goal of this study was to investigate the effect of mechanical loading on osteoblasts and extracellular signal-regulated kinase (ERK1/2) signaling in relation to osteoblast differentiation and mineralization. A human osteoblast cell line (SV-HFO) was triggered to differentiate to the advanced state of mineralization by addition of the osteogenic factors dexamethasone and beta-glycerophosphate. Osteoblasts were subjected to cyclic, equibiaxial stretch for 5, 15, or 60 min at different stages of differentiation (days 7, 14, and 21). Baseline (static) phosphorylated ERK1/2 and total ERK1/2 levels gradually increased during osteoblast differentiation. Cyclic stretch induced a rapid increase in ERK1/2 phosphorylation with a maximum between 5 and 15 min. Prolonged stretching for 60 min resulted in a decrease of phosphorylated ERK1/2 towards baseline level, suggesting a desensitization mechanism. The effect of stretch on ERK1/2 phosphorylation was strongest at later stages of differentiation (days 14 and 21). At day 21, the increase of ERK1/2 phosphorylation in response to stretch was significantly lower in non-differentiating than in differentiating osteoblasts. This could not be explained by differences in cell density, but did correlate with the formation of extracellular matrix, collagen fibrils. Mineralization of the extracellular matrix did not lead to a further increase of ERK1/2 phosphorylation. In conclusion, the current study demonstrates that the extent of activation of the ERK1/2 pathway is dependent on the differentiation or functional stage of the osteoblast. The presence of an extracellular matrix, but not per se mineralization, seems to be the predominant determinant of osteoblastic response to strain.