High-acceleration whole body vibration stimulates cortical bone accrual and increases bone mineral content in growing mice

Journal of Biomechanics ∎ (∎∎∎∎) ∎∎∎–∎∎∎

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High-acceleration whole body vibration stimulates cortical bone accrual and increases bone mineral content in growing mice Vasily Gnyubkin a,b, Alain Guignandon a,b, Norbert Laroche a,b, Arnaud Vanden-Bossche a,b, Luc Malaval a,b, Laurence Vico a,b,n a b

INSERM U1059, 42023 Saint-Etienne, France Université de Lyon, 42023 Saint-Etienne, France

art ic l e i nf o

a b s t r a c t

Article history: Accepted 27 April 2016

Whole body vibration (WBV) is a promising tool for counteracting bone loss. Most WBV studies on animals have been performed at acceleration o1g and frequency between 30 and 90 Hz. Such WBV conditions trigger bone growth in osteopenia models, but not in healthy animals. In order to test the ability of WBV to promote osteogenesis in young animals, we exposed seven-week-old male mice to vibration at 90 Hz and 2g peak acceleration for 15 min/day, 5 days/week. We examined the effects on skeletal tissues with micro-computed tomography and histology. We also quantified bone vascularization and mechanosensitive osteocyte proteins, sclerostin and DMP1. Three weeks of WBV resulted in an increase of femur cortical thickness ( þ 5%) and area ( þ6%), associated with a 25% decrease of sclerostin expression, and 35% increase of DMP1 expression in cortical osteocytes. Mass-structural parameters of trabecular bone were unaltered in femur or vertebra, while osteoclastic parameters and bone formation rate were increased at both sites. Three weeks of WBV resulted in higher blood vessel numbers (þ 23%) in the distal femoral metaphysis. After 9-week WBV, we have not observed the difference in structural cortical or trabecular parameters. However, the tissue mineral density of cortical bone was increased by 2.5%. Three or nine weeks of 2g/90 Hz WBV treatment did not affect longitudinal growth rate or body weight increase under our experimental conditions, indicating that these are safe to use. These results validate a potential of 2g/90 Hz WBV to stimulate trabecular bone cellular activity, accelerate cortical bone growth, and increase bone mineral density. & 2016 Elsevier Ltd. All rights reserved.

Keywords: Osteocytes Sclerostin DMP1 mCT Immunohistochemistry Bone vascularization

1. Introduction An increase of bone mass during the bone acquisition stage is an important osteoporosis prevention factor (Babatunde and Forsyth, 2014). This is why various physical exercises are widely used to stimulate bone formation during growth period (Parfitt, 1994). At the same time bone adaptation to physical activity depends on the intensity of loading and the number of loading cycles (Qin et al., 1998). Therefore, whole body vibration (WBV) can be an addition or even an alternative to physical exercise, as it produces thousands of low-impact strain events in a relatively short period of time, with no significant efforts from a recipient. A pioneering study performed by Rubin et al. (2001) showed that one year of 30 Hz vibration at 0.3g leads to a 30% increase of n Correspondence to: INSERM U1059, UFR Médecine, Campus Santé Innovation, 10 Chemin de la Marandière, St-Priest en Jarez, 42270, France. Tel.: þ 33 477421857. E-mail address: [email protected] (L. Vico).

the trabecular bone volume and density, as well as bone strength and stiffness in sheep (Rubin et al., 2001). Even though in recent years there have been numerous WBV studies on both animals and humans, it is still difficult, due to high variability of experimental procedures, to decide which WBV protocol is the most beneficial for bones (Prisby et al., 2008). Among published WBV animal studies, acceleration ranges between 0.3g and 3g (1g ¼9.81 m/s2) (Rubinacci et al., 2008), and frequencies between 8 Hz and 90 Hz (Judex et al., 2007; Pasqualini et al., 2013). The studies also vary in duration of exposure and resting times (Xie et al., 2006; Zhang et al., 2014), choice of studied species (rat, mouse, sheep), and age of the animals (Lynch et al., 2010; Rubinacci et al., 2008; Rubin et al., 2002). Even though various combinations were possible, most WBV experiments have been performed with magnitudes below 1g, and frequencies from 30 Hz to 50 Hz on physiologically challenged (ovariectomized, unloaded, or immobilized) animals. This has been done in order to test WBV ability to prevent an induced bone loss. Notably, effects of low-magnitude WBV were mostly

http://dx.doi.org/10.1016/j.jbiomech.2016.04.031 0021-9290/& 2016 Elsevier Ltd. All rights reserved.

Please cite this article as: Gnyubkin, V., et al., High-acceleration whole body vibration stimulates cortical bone accrual and increases bone mineral content in growing mice. Journal of Biomechanics (2016), http://dx.doi.org/10.1016/j.jbiomech.2016.04.031i

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V. Gnyubkin et al. / Journal of Biomechanics ∎ (∎∎∎∎) ∎∎∎–∎∎∎

beneficial on osteopenic models, but did not promote bone accrual in control animals. The effects of high-acceleration WBV (above 1g) have not been much investigated yet. It could be expected that higher acceleration would result in higher strain, and therefore bone formation (Ozcivici et al., 2010), even in healthy animals. However, despite the high g-level, WBV at 2g/50 Hz and 3g/30 Hz appeared osteogenic for ovariectomized animals but not for controls (Flieger et al., 1998; Rubinacci et al., 2008). Such results were presumably informed by suboptimal frequencies. Indeed, strain rate appeared to be an important component of the skeleton response to mechanical stimulation (Turner et al., 1995). Additionally, a study performed in our laboratory comparing effects of WBV frequencies (8 Hz, 52 Hz and 90 Hz) at the same acceleration (0.7g) revealed that only a frequency of 90 Hz stimulated bone formation in healthy rats (Pasqualini et al., 2013). In the present study, our goal was to establish a WBV regimen, which promotes bone gain in healthy young animals. We hypothesized that, due to an interrelationship between loading cycles and bone adaptation (Ozcivici et al., 2010), a combination of 90 Hz frequency and high acceleration (2g) would efficiently stimulate bone growth in healthy mice. Young animals were chosen to verify that (a) WBV at a high acceleration does not impair bone growth or increase in body weight and (b) WBV applied during the period of active skeletal growth accelerates bone growth and/or bone mineralization leading to a higher peak bone mass. Blood vessels

are known to play an essential role (delivery of osteoclast and osteoblast precursors and endocrine factors) in basic multicellular units where bone remodeling takes place (Sims and Martin, 2014). Also, bone angiogenesis is indispensable for bone gain induced by physical exercises (Zao et al., 2004). Thus, we assessed vascularization parameters using a previously validated quantification technique (Roche et al., 2012). Osteocyte proteins sclerostin and Dentin Matrix acidic Phosphoprotein 1 (DMP1) were selected for analysis because they are known for bone formation control (Lin et al., 2009, 2005) and mineralization (Feng et al., 2006; Ling et al., 2005; Maciejewska et al., 2009) respectively, and their expression is affected by mechanical signals (Gluhak-Heinrich et al., 2003; Harris et al., 2007; Moustafa et al., 2012; Robling et al., 2008). We showed that in a short run (three weeks) a high acceleration WBV regimen stimulated mouse bone vascularization and bone cellular activity, accelerated cortical bone size acquisition, and, in the long run (nine weeks), resulted in a net increase in bone matrix mineral content. 2. Materials and methods 2.1. Animal care Seven-week-old C57BL/6J male mice (Charles River Laboratories, l’Arbresle, France) were housed in the PLEXAN facility (Platform for Experiments and Analysis,

Fig. 1. Vibration device and a scheme of the experimental design. a – descriptive photo of the vibration device during a vibration session; b – diagram of the experimental design, number of animals and performed analyses are listed for each group/experiment. Ctrl¼control, WBV ¼whole body vibration.

Please cite this article as: Gnyubkin, V., et al., High-acceleration whole body vibration stimulates cortical bone accrual and increases bone mineral content in growing mice. Journal of Biomechanics (2016), http://dx.doi.org/10.1016/j.jbiomech.2016.04.031i

V. Gnyubkin et al. / Journal of Biomechanics ∎ (∎∎∎∎) ∎∎∎–∎∎∎ Faculty of Medicine, University of Saint-Etienne, France). The procedures for the animal care were done in accordance with the European Community Standards (Ministère de l’Agriculture, France, Authorization 04827). All animal experiments were approved by the local ethical committee (Comité d’Ethique en Expérimentation Animale de la Loire CEEAL-UJM, agreement n° 98) and the Animal Welfare Committee of the PLEXAN. Mice were housed by four in standard cages (36  20  14 cm) with bedding material, in a quiet room with constant temperature (23 72 °C) with 50% relative humidity, in a light controlled environment (12 h light/12 h dark cycle). Food and water were provided ad libitum (Safe diets A04, Augy, France). The light/dark cycle was inverted so that WBV sessions were performed during the active day phase in a dark quiet room.

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2.3. Experimental design The experimental design is summarized in Fig. 1b. A total of 58 animals were distributed as follows: 10 animals in basal control group euthanized at the beginning, 32 mice in the three weeks experiment randomly divided in control and experimental groups (n ¼16/group). At the end-point, 8 animals from each group were processed for microcomputed tomography (mCT), histomorphometry, and immunohistochemistry (IHC) analysis. The other 8 were perfused with barium sulfate for bone vascular imaging. For the nine-week experiment, 16 mice were randomly divided in control and experimental groups (n¼ 8/group) and processed at the end-point for mCT and histomorphometry.

2.2. Material set-up

2.4. Microtomography

Shakers (TIRA TV 52120) with attached aluminum table (30 cm diameter, 4 mm thickness) were used to generate vibrations (Fig. 1a). The g-level was controlled by an accelerometer (PBB Piezotronics, 100 mV/g, ref 352C33) glued under the table at midradius distance. During each WBV session, four mice from the same cage were put on the table and left free to move. WBV was applied 15 min/day, 5 days/week, at 90 Hz frequency (sinusoidal signal) for three or nine weeks. To avoid potential stress, the peak acceleration level was gradually increased from 0.5g to 2g within the first week of the experiment. Mice in the control group were put on an inactive shaker for a sham-WBV for the same duration/day.

Formalin-fixed and ethanol-dehydrated femur and L2 vertebral body were scanned with a high-resolution mCT (Viva CT 40; Scanco Medical, Switzerland). Data were acquired at 55 keV energy, and 145 mA current for 10 mm cubic resolution. The distal right femur was scanned through 4.1 mm starting from the distal growth plate (Fig. 2a) in order to include regions of interest (ROIs) for both trabecular and cortical compartments. The L2 vertebra was scanned entirely (Fig. 2e). Threedimensional reconstructions were generated with the following parameters: Sigma, 1.2; Support, 2; Threshold, 160 (spongiosa) or 280 (cortex). In the femur distal metaphysis, ROI was restricted to the trabecular area (Fig. 2b and c). The

Fig. 2. Locations of the regions of interest for mCT analysis of trabecular and cortical bones. Mice femur, scanned area of 4.1 mm height (a), manual selection of region of interest for analysis on distal metaphysis (0.6 mm, 60 successive sections) starting below the primary spongiosa (b), up to the middle of the secondary spongiosa (c), and in the diaphysis (0.6 mm, 60 successive sections) (d); L2 vertebral body, scanned area (e), manual selection of region of interest (1.8 mm, 180 sections) for analysis from primary spongiosa (f), and including entire secondary spongiosa (g).

Please cite this article as: Gnyubkin, V., et al., High-acceleration whole body vibration stimulates cortical bone accrual and increases bone mineral content in growing mice. Journal of Biomechanics (2016), http://dx.doi.org/10.1016/j.jbiomech.2016.04.031i

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cortical area was assessed at femur diaphysis (Fig. 2d). 3D structural parameters were obtained from 60 sections (0.6 mm) for both trabecular (secondary spongiosa) and cortical bone in femur. In the L2 vertebral body, trabecular bone (180 Section, 1.8 mm) was assessed between two growth plates (Fig. 2f and g). These ROI were manually defined by the same operator on transversal 2D images (Fig. 2b, c, f, g). Tissue Mineral Density (TMD, mg/cm3), cortical thickness (Ct.Th, mm), and cortical area (Ct.Ar, mm2) were measured in the cortical bone. In the trabecular bone, we measured bone volume/trabecular volume (BV/TV, %), trabecular separation (Tb.Sp, mm), trabecular thickness (Tb.Th, mm), trabecular number (Tb.N, mm  1), connectivity density (Conn.D, mm  3), structure model index (SMI), and degree of anisotropy (DA). 2.5. Histomorphometry Six and two days before euthanasia, the mice were injected intraperitoneally with 200 ml of a 0.37% tetracycline saline solution. After the mCT analysis, undecalcified femur metaphysis and L2 vertebra were embedded in methyl methacrylate and processed for histomorphometry, as previously described (David et al., 2006). Bone cellular parameters were measured in the secondary spongiosa with a digitizing tablet (Summasketch; Summagraphics, Paris, France) and software designed in our laboratory (David et al., 2006). In brief, we measured osteoid surfaces (OS/BS, %), surfaces double labeled with tetracycline (dLS/BS, %) and mineral apposition rate (MAR, mm/day), from which the bone formation rate (BFR/ BS, μm3/μm2/day) was calculated. Osteoclast surfaces (Oc.S/BS, %) were assessed through tartrate-resistant acid phosphatase (TRAP) labeling of osteoclast. In the primary spongiosa of the femur, longitudinal growth rate (LGR, mm/day) was determined through tetracycline labeling by measuring the distance in mm/day between two fluorochrome labelings (parallel to the growth plate) in the primary spongiosa.

In the cortical bone we assessed endosteal MAR in the same region that was used for mCT measurements (between 4.1 and 3.5 mm from the distal growth plate, Fig. 2a). Due to bone surface scraping during dissection it was not possible to reliably measure periosteal bone formation. 2.6. Bone vessel imaging and quantification Vascular imaging was done as previously described in our laboratory (Roche et al., 2012). In brief, euthanized mice received an intracardiac perfusion of BaSO4 solution (Micropaque Guerbet, Paris, France). Femurs were embedded in Neg 50 (Thermo Scientific, Ref. 6502) by snap-freezing in liquid nitrogen. Nine mm-thick longitudinal sections (Fig. 3) of a whole bone were cut with 27 mm increments with a cryotome (Microm HM 525, Thermo Scientific) and counterstained with Goldner's stain. ROI for quantitative analysis was located in the secondary spongiosa of the femur diaphysis. It consisted of four fields (0.51  0.51 mm, with total area of 1.02 mm) covering most of the secondary spongiosa. Marrow area was derived from total tissue area (i.e., ROI) minus bone area. Bone area was assessed from Goldner's staining (David et al., 2006). Measurements of vascular parameters were performed with a 100-point eyepiece square grid. Results are expressed as Vessel Number/mm2 of Marrow Area (Ves.N/Mar.Ar) and Vessel Volume/Marrow Volume (Ves.V/Mar.V), as described in Roche et al. (2012). 2.7. Quantitative immunohistochemistry We used fluorescent IHC as in Gerdes et al. (2013) to perform semi-automatic quantitative analysis of protein expression level, i.e., the amount of osteocyte proteins, by measuring IHC staining intensity (Gnyubkin et al., 2015). Femurs were fixed in 10% neutral buffered formalin and then in 70% ethanol. Three mm long

Fig. 3. Effects of 3-week whole body vibration on distal femur metaphysis vascularization. Cryosection (9 mm-thick) of a mice femur perfused with barium sulfate at x16 (a) and x200 (b) magnifications, t ¼trabecula, v ¼vessel; vessels number over bone marrow area (c); vessel volume over bone marrow volume (d). Control¼ age-matched control, WBV ¼ whole body vibration. Boxes represent 50% of values (25% to 75%), whiskers represent minimum and maximum values, middle point represents median. Nonparametric Mann–Whitney U-test, *p o 0.05.

Please cite this article as: Gnyubkin, V., et al., High-acceleration whole body vibration stimulates cortical bone accrual and increases bone mineral content in growing mice. Journal of Biomechanics (2016), http://dx.doi.org/10.1016/j.jbiomech.2016.04.031i

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entirely match the osteocyte lacuna. Thus, pixels of the surrounding bone matrix with zero gray level were sometimes included in the ROI. Therefore, we did not calculate the Mean Gray Level and used the Raw Integrated Density (RawIntDens) parameter of ImageJ instead, which sums up gray levels of all pixels within the ROI. The gray levels of collagen and osteocyte background autofluorescence were obtained from a negative control bone sections (tissue, which received the same treatment but no primary antibody was applied) and then removed with the Subtract function of ImageJ, prior to sclerostin or DMP1 analysis. Negative control bone sections were treated within the same sclerostin or DMP1 IHC run. RawIntDens values were obtained for individual osteocytes labeled for sclerostin or DMP1, and the percentage of cells positive for these proteins was calculated. To ensure representativeness of osteocyte populations, we analyzed three to four cortical transversal sections from 5 mice per group; the average number of osteocytes analyzed per group was 7500. Intra- and inter-observer coefficients of variation (measuring three times with the same operator and three times with a different operator on five sections) were less than 3%. 2.8. Circulating sclerostin immunoassay Serum from control (n¼ 8) and three-week WBV (n¼ 8) mice was collected, aliquoted and stored at  80 °C. The Quantikine mouse/rat sclerostin ELISA kit (R&D systems, MSST00) was used in accordance with the manufacturer's protocol. 2.9. Statistical analysis s

Data analysis was performed with the STATISTICA software (version 8.2; StataCorp, College Station, TX). When age-matched control and experimental groups were compared, data were assessed with the non-parametric Mann– Whitney U-test. When basal controls, age-matched controls and experimental groups were compared altogether (to access growth effects), data were assessed with the non-parametric Kruskal–Wallis ANOVA test; if significant differences were detected, it was followed by the non-parametric post-hoc Mann–Whitney U-tests with Bonferroni correction. A p-value r 0.05 was considered statistically significant.

3. Results 3.1. A 2g/90 Hz WBV regimen does not affect mouse growth

Fig. 4. Effects of whole body vibration on growth processes. Animals body weight (a); longitudinal growth rate (b). BC ¼ basal control, Ctrl¼ age-matched control, WBV ¼whole body vibration, w¼weeks. Boxes represent 50% of values (25% to 75%), whiskers represent minimum and maximum values, middle point represents median. Non-parametric Kruskal–Wallis ANOVA test followed by post-hoc Mann– Whitney U-tests with Bonferroni correction, † indicates difference (p o 0.01) between BC, and Ctrl 3w or Ctrl 9w.

To assess the growth-related differences between seven weekold (basal control), ten week-old (three-week WBV experiment) and 16 week-old (nine-week WBV experiment) mice, we compared the basal control group with respective age-matched controls. While, as expected, we found an increase in body weight (p o0.05), which usually comes with age, and a decrease in LGR (p o0.002), WBV did not affect these parameters, neither after three weeks, nor after nine weeks (Fig. 4). 3.2. Three weeks of WBV accelerate cortical bone growth

sections of the mid-diaphysis (5 and 8 mm from the distal epiphysis) were cut out with a precision diamond wire saw (ESCIL Well, France), decalcified for 24 h (Immunocal, Decal Chemical Corp., Ref 1440), and embedded in paraffin. 5 mm thick transversal sections of the diaphysis, separated by 250 mm, were cut from the distal part of the sample with a Leica RM2245 microtome and immunolabeled for sclerostin (Primary antibody: polyclonal, goat, R&D Systems, AF1589, 1:400), and DMP1 (dentin matrix acidic phosphoprotein 1, Primary antibody: polyclonal, rabbit, Takara, M176, 1:500). Sclerostin was detected using a biotinylated secondary polyclonal rabbit anti-goat antibody (Dako, E0466) and Streptavidin-Alexa Fluor (Life technologies, S 11225, 1:500). DMP1 was detected using a biotin-free readyto-use PowerFluor immunofluorescence detection kit (MaxVision Biosciences, PF 21-M). Cell nuclei were visualized with DAPI (Santa Cruz, 28718-90-3). To avoid batch-to-batch variations, all slides used for the analysis of a specific protein were stained within the same run. Black and white images of sclerostin, DMP1 and DAPI labeling in the entire cortical area were obtained with an AxioObserver Z1 microscope (Zeiss, Darmstad, Germany), at a magnification of 400 (oil immersed objective), and automatically merged using the Mosaic plugin of Axiovision 4.7 software (Zeiss). Mosaic images were directly analyzed with the ImageJ software (http://imagej.nih.gov/ij/), without any brightness or contrast correction. ROIs were restricted to the cortical bone. In order to provide fluorescence intensity data for individual osteocytes, a mask of DAPI stained cells was merged with the sclerostin or DMP1 staining. As the lacunar size exceeds the area of DAPI staining, we applied two successive dilatations of the DAPI mask to fit with lacunar areas. Nonetheless, the “dilated DAPI” area did not

After three-week WBV dynamic bone formation parameters were increased in the trabecular bone of distal femur and L2 vertebral body, as compared to age-matched controls (Fig. 5). In addition, OS/BS only detectable in femur was also increased by WBV (Fig. 5). Osteoclast surfaces (Oc.S/BS) were higher after threeweek WBV (Fig. 5e and i). Three-week WBV led to 23% increase of vessels number in the femur metaphysis vs. age-matched controls, with no change of vessel volume (Fig. 3c and d). However, the stimulation of bone cell activity did not translate into a net increase of any trabecular parameters, as measured by mCT (Table 1). In the cortical bone, three-week WBV resulted in 5% increase of Ct.Th and 6% increase of Ct.Ar, as compared to age-matched controls (Fig. 6). Also, cortical MAR in the endosteum was 60% higher (p o0.05) in the vibrated group (2.17 0.4 mm/day), as compared to age-matched controls (1.370.3 mm/day, Supplementary Fig. 1). While the percentage of sclerostin positive osteocytes was 11.5% lower in the WBV group than in controls, the percentage of DMP1 positive osteocytes remained unchanged (Table 3). Analysis of the fluorescence intensity of individual osteocytes showed that

Please cite this article as: Gnyubkin, V., et al., High-acceleration whole body vibration stimulates cortical bone accrual and increases bone mineral content in growing mice. Journal of Biomechanics (2016), http://dx.doi.org/10.1016/j.jbiomech.2016.04.031i

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Fig. 5. Effects of 3-week whole body vibration on trabecular bone cellular activities. dLS¼ double labeled surface, MAR¼ mineral apposition rate, BFR ¼ bone formation rate, OS ¼ osteoid surface, Oc.S ¼osteoclast surface, BS ¼bone surface. Analysis was performed in distal femoral metaphysis (a–e), and L2 vertebral body (f–i). BC ¼ basal control, Control¼ age-matched control, WBV ¼whole body vibration. Boxes represent 50% of values (25% to 75%), whiskers represent minimum and maximum values, middle point represents median. Non-parametric Kruskal–Wallis ANOVA test followed by post-hoc Mann–Whitney U-tests with Bonferroni correction. † indicates difference (p o 0.01) between BC, and Control, * indicates difference (*p o 0.05, **p o 0.01, ***p o0.001) between Control and WBV.

Please cite this article as: Gnyubkin, V., et al., High-acceleration whole body vibration stimulates cortical bone accrual and increases bone mineral content in growing mice. Journal of Biomechanics (2016), http://dx.doi.org/10.1016/j.jbiomech.2016.04.031i

V. Gnyubkin et al. / Journal of Biomechanics ∎ (∎∎∎∎) ∎∎∎–∎∎∎ Table 1 mCT analysis of femoral distal metaphysis and L2 vertebral body in basal control, age-matched controls and whole body vibration (WBV) groups. Groups

BC

Ctrl 3 w

WBV 3 w

Ctrl 9 w

WBV 9 w

Femur, Trabecular bone BV/TV, % 17.47 4.7 Tb.Th, mm 50.5 7 5 Tb.N, mm  1 5.67 0.5 Tb.SP, mm 1707 20 3 Conn.D, mm 161.37 48.3 SMI 2.23 7 0.3 DA 1.77 0.04

8.67 0.9* 357 2* 2.47 0.2* 3777 27* 45.0 7 11.2* 3.17 0.1* 1.337 0.07*

8.47 1.0 357 2 2.47 0.4 390 7 55 46.8 7 17.8 3.17 0.1 1.427 0.12

12.2 74* 41 76* 2.9 70.6* 322 793* 60.6 724* 2.6 70.5*,** 1.4 70.2*

12.9 7 3 41 7 4 3.1 7 0.5 287 7 60 74 7 23 2.5 7 0.4 1.5 7 1.2

Vertebra, Trabecular bone BV/TV, % 17.17 1.9 Tb.Th, mm 43 7 2 1 Tb.N, mm 5.47 0.1 Tb.SP, mm 179 7 4 Conn.D, mm  3 191.27 19.1 SMI 1.87 0.2 DA 1.827 0.07

21.77 2* 39 7 2* 5.67 0.3 1417 11* 240.47 13.5* 1.27 0.2* 1.597 0.04*

21.57 2 39 7 1 5.57 0.3 143 7 12 231.77 30.7 1.27 0.1 1.67 0.05

23.6 73* 43 74 5.5 7 0.2 140 711* 199 715** 0.9 70.3*,** 1.8 70.1**

26.3 7 4 45 7 4 5.9 7 0.2 125 7 9 200 7 19 0.8 7 0.3 1.9 7 0.1

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In relation to bone cell activity, it has been shown that WBV stimulates bone formation and decreases osteoclastic parameters (Prisby et al., 2008; Xie et al., 2006). We also found that three

Data are shown as mean7SD. BC¼ basal control group, Ctrl¼age-matched control, WBV¼ whole body vibration, w¼weeks. BC compared to Ctrl 3w and Ctrl 9w; Ctrl compared to relevant WBV (non-parametric Kruskal–Wallis ANOVA test followed by post-hoc Mann–Whitney U-tests with Bonferroni correction). Marked with (*) when there is significant po0.05 difference between BC and Ctrl 3w or Ctrl 9w; marked with (**) when there is significant po0.05 difference between Ctrl 3w and Ctrl 9w.

sclerostin protein expression was 25% lower (Fig. 7d) and DMP1 expression 35% higher (Fig. 7b) in the WBV group than in agematched controls (Table 3, Fig. 7a and c). No difference in the osteocyte density was detected. Additionally, no difference in the levels of circulating sclerostin was observed between control (1947 36 pg/ml, n ¼8) and WBV mice (184 737 pg/ml, n ¼8). 3.3. Nine weeks of WBV result in a net increase in bone matrix mineral content To test whether bone cell activity changes would finally result in bone mass alteration, we performed a longer WBV experiment. After nine-week 2 g/90 Hz WBV the femoral cortical TMD increased by 2.5%, as compared to age-matched controls (p o0.001, Fig. 6). Bone structural or cellular parameters in both femur and vertebra did not show any difference between the nineweek WBV and age-matched control groups (Table 2).

4. Discussion Despite the high g-level of the vibration regimen used in the study, we did not observe any difference in animal body weight between WBV and control groups. Also, there were no detectable negative effects on bone growth or mass-structural parameters, establishing that WBV at 2g/90 Hz was safe for the skeleton, at least within the limits of our protocol. The main conclusion that we have made is that animals subjected to WBV during the phase of active growth accelerated skeletal growth processes, thus reaching higher peak cortical mineral density at the age of skeletal maturity, as compared to age-matched controls. This suggested that vibrated animals had stronger bones (Jiang et al., 2000), which would have to be assessed in the future. Several studies reported a stimulatory effect of WBV on cortical bone in ovariectomized animals (Rubinacci et al., 2008; Sehmisch et al., 2009; Stuermer et al., 2010). However, to the best of our knowledge, only one study released by our research group reported an increase of Ct.Th in healthy rodents (Pasqualini et al., 2013).

Fig. 6. Effects of whole body vibration on cortical bone mass-structural parameters. Cortical thickness (a); cortical area (b); tissue mineral density (c). BC¼basal control, Ctrl¼age-matched control, WBV¼ whole body vibration, w¼ weeks. Boxes represent 50% of values (25% to 75%), whiskers represent minimum and maximum values, middle point represents median. Non-parametric Kruskal–Wallis ANOVA test followed by post-hoc Mann–Whitney U-tests with Bonferroni correction. † indicates difference (po0.01) between BC, and Ctrl 3 w or Ctrl 9 w. * indicates difference (*po0.05, **po0.01, ***po0.001) between Ctrl and relevant WBV groups.

Please cite this article as: Gnyubkin, V., et al., High-acceleration whole body vibration stimulates cortical bone accrual and increases bone mineral content in growing mice. Journal of Biomechanics (2016), http://dx.doi.org/10.1016/j.jbiomech.2016.04.031i

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Fig. 7. Representative pictures of immunohistochemistry on cortical bone. DAPI (blue) and DMP1 staining (green) of control (a) and 3-week WBV experimental (b) groups; DAPI (blue) and sclerostin (red) staining of control (c) and 3-week WBV experimental (d) groups. Blood vessels, bone marrow and collagen are visible due to their natural autofluorescence. Pictures are part of total cortical bone mosaic images, which were used for fluorescence quantitative analysis (chapter materials and methods). Images were equally adjusted for brightness and contrast for demonstrative purposes. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Table 2 Histomorphometry analysis of femoral diaphysis and L2 vertebral body in basal control, age-matched control and 9-week Whole Body Vibration (WBV) groups. Groups

BC

Control

WBV

Femur Oc.S/BS, % OS/BS, % dLS/BS, % MAR, mm/day BFR/BS, μm3/μm2/day

36.9 7 5.6 45.9 7 9.5 26.5 7 0.9 1.9 7 0.3 0.5 7 0.08

17.9 71.9* 6 72.1* 8.5 72.3* 0.6 70.08* 0.05 70.01*

16.3 7 3.9 12.2 7 7.9 8.7 7 1.9 0.7 7 0.1 0.067 0.02

Vertebra Oc.S/BS, % dLS/BS, % MAR, mm/day BFR/BS, μm3/μm2/day

Table 3 Effects of three-week WBV on osteocyte quantitative parameters in femur cortical bone compared to control: osteocyte density (DAPI staining); immunohistochemistry: percentage of osteocyte positive for sclerostin or DMP1 and fluorescence levels (RawIntDens, arbitrary unit). Groups Osteocyte density, 1/1000 mm2

20.1 72.9* 8.7 71.7* 0.7 70.1* 0.06 70.01*

19.7 7 3 8.6 7 4.3 0.7 7 0.1 0.067 0.05

Data are shown as mean 7SD. BC ¼basal control group, Control¼ age-matched control, WBV ¼ whole body vibration. BC compared to Control, Control compared to WBV (non-parametric Kruskal–Wallis ANOVA test followed by post-hoc Mann– Whitney U-tests with Bonferroni correction). Marked with (*) when there is significant p o0.05 difference between BC and Control.

weeks of 2g/90 Hz WBV resulted in augmented osteoid and mineralized surfaces, presumably due to an increase of osteoblast recruitment and/or lifespan. Along with a higher bone formation rate, we detected an increase of osteoclastic surfaces in cancellous tissues of both the femur metaphysis and vertebra. It is possible that in growing healthy animals, a 2g/90 Hz WBV regimen would

WBV

0.82 7 0.08

0.84 7 0.04

Sclerostin Positive osteocytes, % RawIntDens per group

14.6 7 2.6 17.6 7 3.7 1.2 7 0.1 0.2 7 0.06

Control

77% 3656

DMP1 71.5% 2430

Sclerostin *

68.1% 2751*

DMP1 70.8% 3300*

Data are shown as mean for osteocytes' density and percentage of positive; as medians for level of fluorescence. Non-parametric Mann–Whitney U-test, WBV compared to age-matched controls. *

p r 0.002.

stimulate both formation and resorption in a balanced manner, thus maintaining trabecular microarchitecture. Bone cell activity and vascularization are closely linked (Barou et al., 2002). A relationship between bone remodeling and vascularization has been previously demonstrated in unloaded rats (Fei et al., 2010) or those subjected to physical exercises (Yao et al., 2004). Further, high frequency WBV has been shown to stimulate angiogenesis in normal and osteoporotic rat bones (Cheung et al., 2012). The increase of vessel number coupled with high bone

Please cite this article as: Gnyubkin, V., et al., High-acceleration whole body vibration stimulates cortical bone accrual and increases bone mineral content in growing mice. Journal of Biomechanics (2016), http://dx.doi.org/10.1016/j.jbiomech.2016.04.031i

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remodeling observed in our study is in line with the previous findings. Our results further emphasize the importance of vascularization in bone adaptation processes. In experimental models of high mechanical loading, osteocyte expression of sclerostin as well as the percentage of sclerostinpositive osteocytes has been reported to be reduced, while that of DMP1 was increased (Gluhak-Heinrich et al., 2003; Harris et al., 2007; Robling et al., 2008). Our quantitative IHC analysis allowed us to detect reduction of the percentage of osteocytes positive for sclerostin along with shifts in sclerostin and DMP1 expression, which were congruent with the changes in structural and cellular parameters measured in the whole bone (i.e., stimulated bone formation and increased TMD, respectively). We did not find any change in circulating sclerostin levels, suggesting that serum levels do not necessarily reflect alterations of local production in osteocytes. Additionally, in mice after ovariectomy, sclerostin level changed in a site-specific manner in bones, whereas sclerostin serum level remained unchanged (Jastrzebski et al., 2013). It further demonstrates that there is no direct correlation between tissue and serum sclerostin levels. Because after three-week WBV we observed altered cell activity in trabecular bone, a thicker cortex and higher DMP1 levels, we assumed that a longer exposure to WBV may further stimulate adaptation processes and result in trabecular bone gain or cortex mineralization. However, after nine weeks of 2g/90 Hz WBV, we still observed no changes of trabecular bone microarchitecture, and, in contrast to three-week WBV, no difference in bone cell activity either in femur or vertebra. The latter may indicate that a steady state has been achieved in the adaptation of the skeleton to this mechanical environment. While after three-week WBV cortical size was increased, after nine-week the cortex of the controls reached the same thickness as in WBV mice. However, after nine-week WBV we detected higher cortical TMD in the vibrated group. Importantly, at the end of the experiment the four month-old mice had reached their skeletal maturity (Amblard et al., 2003; Somerville et al., 2004). This indicated that an absolute gain in peak TMD has been achieved through WBV, which may translate in higher mechanical performance (Jiang et al., 2000). In relation to the processes, our findings suggest that young mice adapted to WBV by accelerating their natural cortical bone growth that would leave more time for subsequent secondary mineralization. During this process the higher expression of DMP1 after three-week WBV might have contributed to the higher TMD, as observed in DMP1 overexpressing mice (Bhatia et al., 2012). In conclusion, 2g/90 Hz WBV affected healthy growing mice: it accelerated cortical bone growth after three weeks, and increased cortical TMD in older animals after nine weeks. Our results suggest that WBV could intensify the mineralization process during skeletal growth, leading to higher cortical peak mineral density in mature skeleton. Because the risk of osteoporosis is negatively correlated with the bone mineral density achieved at skeletal maturity (Hernandez et al., 2003; Tatò et al., 1996), we believe that a proper WBV regimen might be a solution permitting to defer the onset of osteoporosis.

Ethical approval All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. Conflict of interest The authors declare that they have no conflict of interest.

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Acknowledgments We acknowledge financial support from the French National Space Agency (CNES) (Program ‘Microgravity and Development’), the ‘Agence Nationale de la Recherche’ ANR-09-BLAN-0148 (AdapHyG), and INSERM (Institut National de la Santé et de la Recherche Médicale). V. Gnyubkin had a scholarship from French Ministry of Higher Education and Research. We thank the staff of the PLEXAN animal facility of the University of Saint-Etienne. We are also grateful to E. Berger, V. Mucci, F. Louis and W. Bouleftour for technical assistance.

Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.jbiomech.2016.04.031.

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