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Muscle and bone, a common world?

Juffer, P.

2013

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citation for published version (APA)Juffer, P. (2013). Muscle and bone, a common world? Biological pathways in muscle and bone adaptation tomechanical loading.

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Download date: 25. Apr. 2021

CHAPTER 4

Mechanically loaded myotubes affect

osteoclast formation

Petra Juffer1

Richard T. Jaspers2 Jenneke Klein-Nulend1

Astrid D. Bakker1

1 Department of Oral Cell Biology, Academic Centre for Dentistry Amsterdam (ACTA), University of Amsterdam and VU University Amsterdam, MOVE Research Institute Amsterdam,

Amsterdam, The Netherlands

2 MOVE Research Institute Amsterdam, Faculty of Human Movement Sciences, VU University Amsterdam, Amsterdam, The Netherlands

Submitted for publication

62

ABSTRACT

In response to mechanical loading skeletal muscle produces numerous growth factors and cytokines that enter the circulation. We hypothesized that myotubes produce soluble factors that affect osteoclast formation, and aimed to identify which osteoclastogenesis-modulating factors are differentially produced by mechanically-stimulated myotubes. C2C12 myotubes were subjected to mechanical loading by cyclic strain for 1h, and either or not post-incubated without cyclic strain for 24h. The effect of cyclic strain on gene expression in myotubes was determined by PCR array. Conditioned medium (CM) was collected from cultures of unloaded and loaded myotubes and from MLO-Y4 osteocytes. CM was added to mouse bone marrow cells containing osteoclast precursors, and after 6 days osteoclasts were counted. Compared to unconditioned medium, CM from unloaded osteocytes increased osteoclast formation, while CM from unloaded myotubes decreased osteoclast formation. Cyclic strain strongly enhanced IL-6 expression in myotubes. CM from cyclically strained myotubes increased osteoclast formation compared to CM from unloaded myotubes, but this effect did not occur in the presence of an IL-6 antibody. In conclusion, mechanically loaded myotubes secrete soluble factors, amongst others IL-6, which affect osteoclast formation. These results suggest that muscle could potentially affect bone homeostasis in vivo via production of growth factors and/or cytokines.

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INTRODUCTION

Musculoskeletal diseases are a major cause of disability (40, 50). In order to improve therapies for musculoskeletal diseases such as osteoporosis and sarcopenia, many studies are performed that focus on identification of the molecular pathways involved in the anabolic response of skeletal muscle or bone to exercise (1, 21, 22, 39, 49). The adaptation of these two tissues to mechanical loading is mostly studied separately, even though a close relationship exists between skeletal muscle and bone. Mechanical forces generated by muscle contractions are essential to acquire bone strength and structure during skeletal growth (33). Since skeletal muscles produce numerous growth factors and cytokines, it has been suggested that the interaction between muscle and bone is not solely mechanical but also biochemical (16, 33). Anabolic and metabolic factors produced by muscle, such as insulin-like growth factor 1 (IGF-I), hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF), and myostatin are known to affect osteoblast differentiation (9, 10, 16, 20, 30). Muscle could thus potentially affect osteoblast formation and activity in a paracrine/endocrine fashion. Muscle-derived factors could also potentially affect bone resorption by osteoclasts (22, 24). Osteoclasts are derived from hematopoietic precursor cells in the bone marrow. Differentiation of pre-osteoclasts into mature osteoclasts requires the presence of receptor activator of nuclear factor kappa-B ligand (RANKL) and macrophage colony-stimulating factor (M-CSF), expressed by osteocytes and osteoblasts (29). Many growth factors and cytokines produced by osteoblasts and osteocytes, such as tumor necrosis factor-alpha (TNFα), IGF-I, and IL-6, affect osteoclast formation, often by modulating RANKL expression by osteoblasts or osteocytes (2, 18, 23, 26, 38). In bone, mechanical loading modulates expression of these growth factors and cytokines by osteoblasts and osteocytes (28, 45). Several of the osteoclastogenesis-modulating factors that are expressed by osteoblasts and osteoclasts are also expressed within skeletal muscle, and their expression levels may also change as a result of mechanical loading (14, 44). If muscle-derived factors reach the cells within bone, this muscle loading may affect osteoclastogenesis similar to bone related growth factors and cytokines. In this study, we hypothesized that C2C12 myotubes secrete factors that inhibit osteoclast formation, and that mechanical loading modulates the secretion of these factors. We aimed to identify soluble factors produced by C2C12 myotubes in response to mechanical loading that affect osteoclast formation.

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MATERIALS AND METHODS

Myotube cultureThe C2C12 myoblast cell line, obtained through serial passage of myoblasts cultured from the thigh muscle of C3H mice, was used (51). C2C12 myoblasts were cultured in Dulbecco’s modified Eagle medium (D-MEM; Gibco, Paisly, UK) supplemented with 10 µg/ml penicillin (Sigma-Aldrich, St. Louis, MO, USA), 10 µg/ml streptomycin (Sigma-Aldrich), 50 µg/ml fungizone (Gibco) (hereafter called “antibiotics”), and 10% fetal bovine serum (FBS; Gibco). Upon 70% confluence, cells were harvested using 0.25% trypsin and 0.1% EDTA, and seeded at 2x104 cells/well of laminin-coated 6-well Bioflex® plates and cultured in 2 mL medium. (Dunn Labortechnik GmbH, Asbach, Germany). After 3 days, medium (2 mL) was replaced by medium (2 mL) consisting of D-MEM supplemented with 2% horse serum (Gibco) and antibiotics. This “differentiation medium” was refreshed daily for another 3 days. Differentiated myotubes were treated with or without mechanical loading by cyclic strain, mimicking in vivo dynamic muscle fiber strain deformations during different types of exercise (5, 46, 48), and resembling a physiological regime resulting in a biologically relevant response (17, 43).

Cyclic uni-axial strainOne hour before mechanical loading of C2C12 myotubes by cyclic strain, “differentiation medium” of C2C12 myotube cultures was replaced by D-MEM containing 2% FBS and antibiotics. Uni-axial cyclic strain was applied using a FlexCell® FX4000™ Tension system (FlexCell® Int Corp, Hillsborough, NC) in a sinusoidal pattern with a frequency of 1 Hz, and a maximum elongation of 15% for 1 h. C2C12 myotube-conditioned medium (CM) was collected immediately after 1 h cyclic strain or static culture, and 24 h later, i.e. after 24 h post-incubation without cyclic strain. Myotube-CM was stored at −20°C.

Fibroblast culturedetermineIt is well known that in the absence of mechanical stimuli osteocytes and osteoblasts secrete soluble factors that stimulate the formation of osteoclasts (27). To test whether this stimulatory effect is unique for bone cells, we determined whether conditioned medium from muscle cells and other non-skeletal cells, were different from those of bone cells. Five pieces of skin (5 mm2) of 18 day-old C57BL6 mouse embryos were cultured in D-MEM containing 10% FBS and antibiotics, in 6-well plates (Greiner Bio-One GmbH). After 7 days, skin fibroblasts were harvested using 0.25% trypsin and 0.1% EDTA, and seeded at 2x104 cells per well in 2 mL medium in a 6-well plate. Upon confluence the medium was replaced by D-MEM containing 2% FBS and antibiotics. After 1 and 24 h incubation, 2 mL skin fibroblast-CM was collected.

Osteocyte culture We used MLO-Y4 osteocytes as a positive control, since it is well known that in the absence of mechanical stimuli osteocytes and osteoblasts secrete soluble factors that stimulate the formation of osteoclasts (27). MLO-Y4 osteocytes (kindly provided by Prof. L.F. Bonewald) were

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cultured in α-minimal essential medium (α-MEM; Gibco) supplemented with 5% FBS, 5% calf serum (Gibco) and antibiotics. Upon sub-confluence, cells were harvested using 0.25% trypsin and 0.1% EDTA and seeded at 3x105 cells/well in 2 mL α-MEM containing 1% FBS and 1% calf serum and antibiotics in 6-well plates (Greiner Bio-One GmbH, Frickenhausen, Germany). After 1 h and 24 h of incubation, 2 mL MLO-Y4 osteocyte-CM was collected per well and stored at −20°C.

Osteoclast formationOsteoclast formation was determined using bone marrow cells obtained from mouse tibiae and femora as described earlier (8). Bone marrow cells (100k cells) were cultured in a 96-wells plate in a 1:1 (vol/vol) ratio of 75 µL fresh α-MEM supplemented with 5% FBS, antibiotics, 30 ng/mL rmM-CSF (R&D Systems, Minneapolis, MN), and 20 ng/mL rmRANKL (R&D Systems), and 75 µL of either myotube-CM, fibroblast-CM, osteocyte-CM, fresh DMEM with 2% FBS and antibiotics, or fresh α-MEM with 1% FBS and 1% calf serum and antibiotics. In some cultures with myotube-CM, 5 ng/mL IL-6 antibody (clone # MP5-20F3; R&D systems) was added to the medium. After 6 days of culture, cells were fixed in 4% formaldehyde, and stained for tartrate-resistant acid phosphatase (TRACP) according to the manufacturer’s instructions (Sigma). Nuclei were visualized with 4’,6-diamidino-2-phenylindole (DAPI) staining. Osteoclast formation was assessed by counting the number of TRACP-positive osteoclasts, containing 3 or more nuclei per cell.

RNA isolation and real-time PCR Total RNA was isolated using a RiboPure™ Kit (Applied Biosystems, Foster City, CA). RT2 mouse Cytokines and Chemokines PCR Array (Qiagen) was performed according to the manufacturer’s protocol on pooled RNA samples from 3 separate independent experiments of myotubes treated with or without 1h cyclic strain. Expression levels of genes showing higher mRNA expression levels than the median level, as well as expression levels of genes showing a cyclic strain effect >200% compared to basal levels, were verified using qPCR. PCR analysis of chemokine (c-motif) ligand (Ccl)-7, Ccl-19, chemokine (C-X-C-motif) ligand (Cxcl)-1, leukemia inhibitory factor (Lif), and IL-6 was repeated with Taqman® qPCR using inventoried Taqman® gene expression assays (Applied Biosystems). GAPDH was used as housekeeping gene.

Statistical analysisData were obtained from 6 separate independent experiments. Data per experiment were average values of duplicates. Groups were compared using one way ANOVA with Bonferroni-adjusted t-tests as post-hoc tests. Differences were considered significant if p<0.05. All data are expressed as mean ± SEM.

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RESULTS

CM from statically cultured C2C12 myotubes affected osteoclast formationIn the absence of a mechanical stimulus, myotube-CM and fibroblast-CM decreased the number of TRACP+ osteoclasts compared to un-conditioned control medium or osteocyte- CM (fig. 1A-C). CM harvested from C2C12 myotubes cultured for 1 h decreased the number of osteoclasts by 3.5-fold (fig. 1B), and CM from C2C12 myotubes cultured for 24 h decreased the number of osteoclasts by 7.0-fold (fig. 1C). CM from mouse skin fibroblasts also decreased the number of osteoclasts, by 3.5-fold (fig. 1B) to 7.5-fold (fig. 1C). In contrast, CM from MLO-Y4 osteocytes increased the number of osteoclasts in bone marrow cultures by 1.6-fold (fig. 1C).

Figure 1. (A-C) Effect of CM from 1 h or 24 h cultured myotubes, osteocytes, and skin fibroblasts on osteoclast formation. (A) Micrographs of TRACP-stained mouse bone marrow cells after 6 days of stimulation with CM from osteocytes (OCY-CM), fibroblasts (FB-CM), or myotubes (MYO-CM). Arrows indicate TRACP+ multinucleated osteoclasts. (B) CM from 1 h cultured myotubes and skin fibroblasts, but not from 1 h cultured MLO-Y4 osteocytes, decreased the number of TRACP+ multinucleated osteoclasts. (C) CM from 24 h cultured myotubes and skin fibroblasts decreased the number of TRACP+ multinucleated osteoclasts, whereas CM from 1 h cultured MLO-Y4 osteocytes increased the number of TRACP+ multinucleated osteoclasts. (D) CM harvested after 1 h cyclic strain of C2C12 myotubes increased the number of TRACP+ multinucleated osteoclasts compared to CM harvested after 1 h static culture. (E) CM harvested after 24 h post-cyclic strain of C2C12 myotubes did not change the number of TRACP+ multinucleated osteoclasts compared to CM harvested after 24 h post-static culture. Values are mean ± SEM (n=6). TRACP, tartrate-resistant acid phosphatase; CS, cyclic strain; PI, post-incubation. Bar, 100 µm. *Significant effect of CM, p<0.05.

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Mechanically loaded myotubes affect osteoclast formation

Cyclic strain modulated the production of soluble factors affecting osteoclast formation by C2C12 myotubesCM from 1 h cyclically strained C2C12 myotubes inhibited the number of osteoclasts to a lesser extend compared to CM from static C2C12 myotube cultures (fig. 1D). The differrence by 1.7-fold (p<0.05) between CM from cyclically-strained myotubes and CM from static cultured C2C12 myotubes was not seen in myotube-CM that was obtained 24 h after cyclic strain (fig. 1E).

Mechanical loading of C2C12 myotubes altered expression of factors involved in osteoclastogenesisWe questioned which factors differentially expressed by cyclically-strained C2C12 myotubes could be responsible for the changes in osteoclast formation. We found that cyclic strain of C2C12 myotubes increased the RANKL/OPG ratio at 1 h cyclic strain (p= 0.077) and significantly at 3 h post-incubation (fig. 2). Based on a PCR array of 84 genes, C2C12 myotubes subjected to cyclic strain seemed to increase mRNA levels of Lif (+376%), IL-6 (+292%), Ccl7 (+251%), and Cxcl1 (+205%) compared to unloaded myotubes. Cyclic strain seemed to decrease mRNA levels of Ccl19 (-770%; fig 3). Quantitative PCR confirmed that cyclic strain increased mRNA levels of Ccl7 by 2.6 fold (p<0.05), Cxcl1 by 2.5 fold (p<0.05), Lif by 2.2 fold (p<0.05), and IL-6 by 3.0 fold (p<0.05). mRNA levels of Ccl19 were not detectable (fig 4A).

Figure 2: Effect of cyclic strain on RANKL and OPG mRNA levels in C2C12 myotubes. Cells were subjected to cyclic uni-axial strain for 1 h (1h CS) and post-incubated without CS for 3, 6, and 24 h. (A, B) Cyclic strain decreased RANKL and OPG mRNA levels in C1C12 myotubes at 6 h post-incubation. (C) The RANKL/OPG ratio was increased at 3 h post-incubation. Values are mean ± SEM of CS-treated over control ratios (n=6). CS, cyclic strain; dashed line, no effect of CS. *Significant effect of CS; p<0.05.

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IL-6 expression by mechanically loaded C2C12 myotubes increased osteoclast formation Since cyclic strain significantly affected IL-6 mRNA levels in C2C12 myotubes, we questioned whether IL-6 is a key factor causing cyclic strain-induced stimulation of osteoclast formation. Increased osteoclast formation as a result of 1 h cyclic strain on myotubes was nullified by adding IL-6 antibody to the CM from these myotubes (fig. 4B).

Figure 3: Effects of cyclic strain on mRNA levels of cytokines and chemokines in C2C12 myotubes. Basal gene expression levels are shown on the y-axis. The relative change in gene expression level as a result of 1 h cyclic strain is shown on the x-axis. Mechanical loading by cyclic strain increased mRNA expression of Ccl7, Cxcl1, IL-6, and Lif. Cyclic strain decreased Ccl19 mRNA expression. Horizontal dashed line, median; vertical dashed line, no effect of cyclic strain; vertical solid lines, >200% change in gene expression as a result of cyclic strain; 1, Ccl19; 2, IL-16; 3, Csf2; 4, Ccl4; 5, Ccl24; 6, IL-9; 7, Cxcl3; 8, Spp1; 9, Mif; 10, Gpi1; 11, Cxcl5; 12, Ccl2; 13, VEGFa; 14, Csf1; 15, BMP4; 16, Tgfβ2; 17,Cxcl12; 18, Ccl11; 19, IL-18; 20, Cx3cl13; 21, IL-15; 22, Cxcl13; 23, IL-12a; 24, Ctf1; 25, IL-1β; 26, Cntf; 27, Cxcl16; 28, Ccl5; 29, IL-1rn; 30, BMP6; 31, Tnfrsf11b; 32, Ppbp; 33, Ccl17; 34, IL-11; 35, IL-7; 36, Lta; 37, Mstn; 38, Cxcl9; 39, Pf4; 40, Ltb; 41, IL-17f; 42, IL-10; 43, Ccl3; 44, Thpo; 45, IL-1α; 46, Tnf; 47, Csf3; 48, Hc; 49, IL-13; 50, BMP2; 51, IL-3; 52, IL-12β ;53, Cd70; 54, Ccl1; 55, IL-24; 56, Ifna2; 57, Ccl7; 58, Cxcl1; 59, IL-6; 60, Lif; 61, Cxcl10; 62, Xcl1; 63, IL-27; 64, Ccl20; 65, Ifng. (See supplementary material on pages 75 and 76 for details and explanation of abbreviations.)

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Mechanically loaded myotubes affect osteoclast formation

Figure 4. (A) Effects of cyclic strain on mRNA levels of IL-6, Lif, Ccl7, Ccl19, and Cxcl1 in C2C12 myotubes. Cyclic strain for 1 h increased mRNA levels of Ccl7, Cxcl1, Lif, and IL-6. (B) Addition of IL-6 antibody to myotube-CM nullified the modulatory effect of mechanical loading of myotubes on osteoclast formation. CM from cyclically-strained myotubes supplemented with IL-6 antibody did not affect the number of TRACP+ multinucleated osteoclasts compared to CM from unloaded myotubes. Values are mean ± SEM (n=3). TRACP, tartrate-resistant acid phosphatase. Dashed line, no effect of CS. *Significant effect of cyclic strain or IL-6 antibody, p<0.05.

70

DISCUSSION

In addition to its contractile function, skeletal muscle can be considered an endocrine organ that produces numerous growth factors and cytokines in response to mechanical loading (35). To the best of our knowledge, this is the first study on biochemical communication between mechanically-loaded myotubes and osteoclasts in vitro. The aim of this study was twofold, i.e. first to investigate whether soluble factors produced by mechanically-loaded C2C12 myotubes affect osteoclast formation, and second to determine which factors contribute to an effect of myotubes on osteoclastogenesis. Unloaded myotubes were shown to inhibit osteoclast formation, while cyclic stretching of myotubes diminished this inhibition of osteoclast formation, and enhanced the expression of IL-6. These observations indicate that cyclic strain stimulates the production of IL-6 by myotubes, which may explain in part why mechanically-loaded myotubes inhibit osteoclast formation to a lesser extent than unloaded myotubes. This suggests that exercise leads to more osteoclasts and may result in a higher bone remodeling rate, which agrees with earlier reported increased bone resorption as well as bone formation markers in plasma in response to exercise (3). Our results support the hypothesis that biochemical communication between bone and muscle is possible (15, 22, 24). This communication may occur via mechanical loading induced expression of muscle derived factors. Myotubes are not the only cells that produce factors affecting osteoclast formation, since soluble factors secreted by mouse skin fibroblasts also significantly inhibited osteoclast formation. It is unlikely that this inhibition of osteoclast formation by CM from myotubes or skin fibroblasts was caused by medium depletion, since CM harvested from myotubes or skin fibroblasts cultured for only 1 h already inhibited osteoclast formation. In addition, CM from unloaded MLO-Y4 osteocytes increased the number of osteoclasts, which is in agreement with observations by others (6, 45). Our findings suggest that osteoclast formation in vivo is particularly encouraged in bone tissue, but not in muscle and skin, as cells from these tissues produced factors which limit osteoclast formation. Possible candidate soluble factors produced by myotubes causing inhibition of osteoclast formation are osteoprotegerin (OPG), IL-10, IL-18, IL-27, and/or transforming growth factor-β2 (TGF-β2), that are known to inhibit osteoclast formation, and which are expressed by myotubes as shown by our PCR array (7, 12, 29, 34, 47). Mechanically-loaded myotubes inhibit osteoclast formation to a lesser extent than unloaded myotubes. Cyclic strain may 1) have reduced the productionof osteoclastogenesis-inhibitory factors, or may 2) have increased the production of osteoclastogenesis-enhancing factors by myotubes. With regard to the first hypothesis, cyclic strain only marginally decreased mRNA levels of OPG and IL-10. In addition, IL-18 and TGFβ2 mRNA levels seemed not affected by cyclic strain. The significant increase in IL-27 mRNA levels after cyclic strain would theoretically result in a stronger inhibition of osteoclastogenesis by mechanically-stimulated myotubes, whereas the opposite effect was observed. In favor of the second hypothesis, cyclic strain increased RANKL/OPG mRNA ratio in myotubes. RANKL and OPG are crucial regulators of osteoclast formation, and an increased RANKL/OPG ratio is indicative for increased osteoclast formation (4, 29). Here, only a mild change in RANKL/

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Mechanically loaded myotubes affect osteoclast formation

OPG ratio was observed as a result of mechanical stimulation, but cyclic strain also increased mRNA levels of Ccl-7, Cxcl-1, IL-6, and Lif. Ccl-7 and Cxcl-1 are chemokines involved in muscle homeostasis (19, 36). So far it is unknown whether Ccl-7 and/or Cxcl-1 stimulate osteoclast formation and/or activity. No such effect is known for Lif either; the Lif-receptor is not expressed by osteoclasts (42) and osteoclast formation is unaffected in bones of Lif-/- mice undergoing bone remodeling (37). In contrast, IL-6 is a well known stimulator of osteoclast formation in vitro (32), and the effect of cyclic strain on IL-6 expression by myotubes was more pronounced than the effect on Lif expression. Therefore we explored the role of IL-6 in biochemical signaling between myotubes and osteoclasts. We found that CM from mechanically-loaded myotubes and unloaded myotubes inhibited osteoclast formation to the same extends after blocking IL-6 in the CM from the mechanically-loaded myotubes. Our data are in agreement with observations in dystrophic mice, where osteoclast formation is mediated by muscle-derived IL-6 (41). This suggests that muscle-derived IL-6 may affect osteoclast formation in vivo in response to exercise. IL-6-/- mice exhibit a low bone mass phenotype, suggesting that IL-6 is not just a catabolic agent (52). Since IL-6 affects both bone resorption and bone formation, increased IL-6 serum levels in humans as a result of endurance or peak power exercise (44), may increase the rate of bone turnover, and enhance the rate of mechanical adaptation of bone (11, 31). In summary, our data indicate that C2C12 myotubes secrete soluble factors that inhibit osteoclast formation, and that mechanical loading of C2C12 myotubes by cyclic strain stimulates osteoclast formation, possibly via IL-6 produced by myotubes. Since IL-6 plasma concentrations increase substantially during muscular activity (13), our data suggest that IL-6 produced by mechanically loaded muscle cells in vivo might affect osteoclast formation. Our current data strengthen the hypothesis that biochemical communication between muscle and bone exists, as has been suggested by others as well (15, 22, 24, 25). Additional research on the biological cross-talk between bone cells and muscle cells in vivo may reveal new targets for the prevention and/or treatment of sarcopenia and/or osteoporosis.

ACKNOWLEDGEMENTS

The authors thank D.C. Jansen and J. Vermeer for the isolation of mouse bone marrow cells, and K. Hermes and G. Sowidjojo for their technical assistance.

GRANTS

This work was supported by a grant from the MOVE Research Institute Amsterdam of the VU University Amsterdam, The Netherlands.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

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Chapter 4

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Mechanically loaded myotubes affect osteoclast formation

SUPPLEMENTARY MATERIAL I

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SUPPLEMENTARY MATERIAL II

Chapter 4