Effect of whole-body vibration on bone properties in aging mice

Bone 47 (2010) 746–755

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Effect of whole-body vibration on bone properties in aging mice Karl H. Wenger a,b,⁎, James D. Freeman c, Sadanand Fulzele a, David M. Immel d, Brian D. Powell c, Patrick Molitor c, Yuh J. Chao e, Hong-Sheng Gao e, Mohammed Elsalanty b,f, Mark W. Hamrick a,h, Carlos M. Isales a,b, Jack C. Yu b,g a

Medical College of Georgia, Department of Orthopaedic Surgery, USA Medical College of Georgia, Institute for Molecular Medicine and Genetics, USA Medical College of Georgia, USA d Savannah River National Laboratory, Non-Destructive Engineering Section of the Engineered Equipment & Systems Department, USA e University of South Carolina—Columbia, Department of Mechanical Engineering, USA f Medical College of Georgia, Department of Oral and Maxillofacial Surgery, USA g Medical College of Georgia, Department of Surgery, Section of Plastic Surgery, USA h Medical College of Georgia, Department of Cell Biology & Anatomy, USA b c

a r t i c l e

i n f o

Article history: Received 7 March 2009 Revised 29 June 2010 Accepted 13 July 2010 Available online 16 July 2010 Edited by: David Burr Keywords: Whole-body vibration Computed tomography Bone biomechanics Aging Femur

a b s t r a c t Recent studies suggest that whole-body vibration (WBV) can improve measures of bone health for certain clinical conditions and ages. In the elderly, there also is particular interest in assessing the ability of physical interventions such as WBV to improve coordination, strength, and movement speed, which help prevent falls and fractures and maintain ambulation for independent living. The current study evaluated the efficacy of WBV in an aging mouse model. Two levels of vibration — 0.5 and 1.5 g — were applied at 32 Hz to CB57BL/6 male mice (n = 9 each) beginning at age 18 months and continuing for 12 weeks, 30 min/day, in a novel pivoting vibration device. Previous reports indicate that bone parameters in these mice begin to decrease substantially at 18 months, equivalent to mid-fifties for humans. Micro-computed tomography (micro-CT) and biomechanical assessments were made in the femur, radius, and lumbar vertebra to determine the effect of these WBV magnitudes and durations in the aging model. Sera also were collected for analysis of bone formation and breakdown markers. Mineralizing surface and cell counts were determined histologically. Bone volume in four regions of the femur did not change significantly, but there was a consistent shift toward higher mean density in the bone density spectrum (BDS), with the two vibration levels producing similar results. This new parameter represents an integral of the conventional density histogram. The amount of high density bone statistically improved in the head, neck, and diaphysis. Biomechanically, there was a trend toward greater stiffness in the 1.5 g group (p = 0.139 vs. controls in the radius), and no change in strength. In the lumbar spine, no differences were seen due to vibration. Both vibration groups significantly reduced pyridinoline crosslinks, a collagen breakdown marker. They also significantly increased dynamic mineralization, MS/BS. Furthermore, osteoclasts were most numerous in the 1.5 g group (p ≤ 0.05). These findings suggest that some benefits of WBV found in previous studies of young and mature rodent models may extend to an aging population. Density parameters indicated 0.5 g was more effective than 1.5 g. Serological markers, by contrast, favored 1.5 g, while biomechanically and histologically the results were mixed. Although the purported anabolic effect of WBV on bone homeostasis may depend on location and the parameter of interest, this emerging therapy at a minimum does not appear to compromise bone health by the measures studied here. © 2010 Elsevier Inc. All rights reserved.

Introduction Therapeutic whole-body vibration (WBV) has been shown to mitigate bone loss and improve neuromuscular function in certain clinical studies. Bone quality issues, in particular, are a significant ⁎ Corresponding author. MCG Orthopaedic Surgery, CB-2509, 1459 Laney Walker Blvd., Augusta, GA 30912, USA. Fax: + 1 706 721 4850. E-mail address: [email protected] (K.H. Wenger). 8756-3282/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.bone.2010.07.014

concern for post-menopausal women and the aging population in general. In these groups there is increased risk of fracture primarily from repeated falls on the hip, and spontaneous vertebral body collapse as a sequela to osteoporosis. In one study, early postmenopausal women experiencing 10 min of low-level WBV (0.2 g, where 1 g = acceleration due to gravity, 9.81 m/s2) twice daily for one year showed retention of bone mineral density (BMD), whereas controls showed a loss of 2–3% [1]. Extrapolated over the full period of sharp decline for such a cohort, the finding suggests a potential for

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substantial improvement in the prognosis for aging bone health. This result was similar to the difference found in a study of related subjects comparing resistance training to light exercise on a WBV platform at high accelerations (2.3–5.1 g). Over six months there was a significant 1% increase in BMD in the WBV group, whereas both the control and resistance training groups were statistically unchanged, with slight mean decreases. [2]. In the elderly, WBV of 0.6–9.5 g added to a physical therapy regimen was shown in one study to improve balance as well as speed of rising from a seated position, whereas physical therapy alone did not improve these measures [3]. Similar intervention with WBV at a less elderly age also has demonstrated beneficial effects [4]. A more recent study in elderly women found that high-magnitude WBV up to 4.3 g, applied for 3 min/day for 3 months, improved movement speed, maximum excursion, and directional control [5]. At a lower magnitude of 0.3 g, however, a similar cohort recently showed no difference in excursion and control, although BMD was better retained than in controls [6]. Propensity to fall has been shown to be equally mitigated in elderly men due to either WBV for short-durations of 0.5–1 min at 6.2–16.1 g or a more rigorous exercise regimen [7]. Prevention of falls can be a significant prophylaxis against femoral neck fractures, in particular. Many study designs, notably in the field of sports science, have examined the relative effect of WBV either in direct comparison to or in combination with various exercise routines. One such study in elderly females, using a similar protocol to that of [7], found improvements in strength and counter-movement jump performance to be similar between WBV and resistance training groups [8]. This suggested that the retention of neuromuscular health in aging may not require the physical demands of a disciplined exercise regimen. Rodent models commonly have indicated a qualified anabolic response, in which WBV often induces increased bone formation rate and remodeling, but at restricted anatomical sites. The quality of the bone improves selectively, and the degree to which signaling is sustained is equivocally reported. For instance, a 5-week study in adult mice subjected to 1 g of WBV resulted in an increase in the ratio of bone volume to tissue volume (BV/TV) of 43% in the proximal tibia [9]. Yet there was no difference in BV/TV in the femoral condyles, distal femoral metaphysis, proximal femur, or L5 vertebral body. Mineralizing surface was found in a study of adult BALB mice to increase 75% in the proximal tibia trabecular metaphysis due to WBV of 0.3 g, but was effectively unchanged in the epiphysis [10]. Periosteal and bone marrow area also increased with WBV, along with type I and especially type II muscle fiber area. Recently, a study comparing adult to aged BALB/c mice showed that WBV increased bone mineral content (BMC) in the lower leg at a low-amplitude WBV of 0.3 g in both ages, but only in the adult mice at a high-amplitude WBV of 1.0 g [11]. This appears to be the first report on the effect of WBV in an aging model. Current literature suggests a provisional consensus is building toward low-amplitude, high-frequency vibration as the most effective and least invasive form for therapeutic stimulation. This combination is thought to approximate the subtle in vivo stress of nominal basal twitch in some muscles. The less-is-more movement has extended down to nano-intensity levels, in which bone strains as low as 10 micro-strain (1 part in 100,000) have been shown to effect an anabolic response [10]. Certainly, high-amplitude vibration has unwanted consequences when chronically applied, as noted by a generation of studies correlating low back pain and other ailments to occupational vibration [12–14]. Such high-amplitude vibration also is often found in efficacy studies from the sports and exercise science field. The recent application of WBV to the health care population, however, normally operates at a categorically lower level of stimulation, as this less invasive realm has proven to be effective in some areas, as well, while it enhances safety. The goal of the current study was to bridge the low- and mediumamplitude protocols of recent investigations in determining the

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degree to which WBV can influence the aging process of the skeleton, which normally is associated with significant bone loss. The two vibration amplitudes have been shown to be well tolerated clinically and in rodents. The model was C57BL/6 male mice, which are available at advanced age and provide an efficient means to stimulate and assay a statistically useful sample size. WBV was applied by a novel vibration device operating on a single pivot axis. This design produces a continuum of acceleration across the vibrating surface, allowing multiple acceleration magnitudes to be applied simultaneously for a given frequency through varying displacement amplitudes. The analysis of the study focused on both established and new measurement tools for characterizing morphological, structural, and biomechanical changes in the femur, radius, and spine of the experimental groups, along with serological markers and histological parameters. Methods Specimens C57BL/6 male mice were obtained from Harlan Sprague Dawley, Inc. (Indianapolis, IN) at 17 months of age, and allowed 1 month to acclimate to our facility until the starting age of the study at 18 months. Initial mean weight was 28.1 g. Three groups of 9 mice each were formed, with 3 mice housed in each 11 × 7-inch cage and fed ad libidum. One group received 0.5 g at 32 Hz for 30 min/day, 5 day/week, for 3 months (LOW). A second group received 1.5 g at 32 Hz for the same period (HIGH). A third group was not stimulated (CTRL), serving as age-matched controls. A fourth group of 9 mice representing time-zero specimens (TIME-0) at 18 months were sacrificed following the acclimation period. All procedures were approved by our campus IACUC. At sacrifice, whole blood was collected by cardiac puncture and dissections were made of the two radii for biomechanical testing, the L5 vertebra for biomechanical testing, and the left femur and L4 vertebra for micro-CT scanning. Pivoting vibration device (PVD) The strategy for the design of the vibration device was to provide a range of displacement amplitudes for a given frequency, such that more than one vibration magnitude could be applied at one time (Fig. 1). Using a single axis to pivot the vibration table allowed multiple 12 × 1.5-inch polycarbonate cages — divided into 3 individual mouse compartments each — to be placed at locations along the length of the table corresponding to the desired acceleration magnitudes. The vibration table was fabricated from 2-inch wide, 8-inch thick aluminum angle plates fixed flush on either side of a central 1 × 1 inch aluminum channel beam for stability, with threaded holes in the angle plates to allow variable attachment of the cages every half-inch along the overall 12-inch length. The motor (Mavilor BLT-055, Barcelona, Spain; through Automotion, Inc., Ann Arbor, MI) mounted to a baseplate underneath the vibration table and connected to the table through a free-angle bearing and 3-inch pistoning elbow. Eccentric bearings of various small offsets were fabricated to attach to the motor shaft, providing several ranges of acceleration. The circular motion of the motor was converted to vertically constrained motion through the pivot axis bearings in the back of the device and a pair of aluminum square columns in the front coated with teflon tape and separated to fit closely against the central channel beam. The speed of the motor was adjustable through a potentiometer on the ACS200 control card. Maximum acceleration for a given combination of eccentric bushing, motor speed, and distance from the pivot axis was estimated by the formula of radius times rotational-velocity-squared. True acceleration at standardized intervals along the central support beam, as well as at the far ends of the mounted cages, was determined at

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Fig. 1. Pivoting vibration device (PVD). Continuously increasing displacement amplitude ranged from negligible at the axis in the back to a maximum at the free end. The bearing between the motor shaft and right-angled arm that connects the motor to the plate (inset) is positioned eccentrically to generate the vertical motion. The acceleration at the two cage positions corresponded to 0.5 and 1.5 g for the 32 Hz vibration frequency. Each cage held 3 mice in separate compartments.

frequencies of 30 and 60 Hz, using 500-g accelerometers (model 352A10, PCB Peziotronics, Depew, NY) with a sensitivity of 9.8 mV/g and resolution of 0.006 g. True acceleration deviated from calculated acceleration by less than 2% along the central channel beam, while the difference reached values up to 10% for the higher frequency at the far ends of the mounted cages. The use of 32 Hz for the stimulation frequency minimized this effect. μCT Femurs and L4 vertebrae were stored frozen until the time of scanning, when they were immersed in a saline-filled tube and placed into a micro-computed tomography imaging system developed at the Savannah River National Laboratory (Aiken, SC). Scans consisted of 12-second exposures at 0.5° rotational increments, using 60 kV and 0.148 mA, with the source input filtered through 2 mm high density polyethylene and 0.5 mm aluminum. The hardware included a 160 kV micro-focus X-ray machine (Kevex Inc., model 16010), a four-axis positioning system (New England Affiliated Technologies series 300, Lawrence, MA) and an amorphous silicon imager (Varian Inc, Paxscan 4030, Palo Alto, CA). Voxel size was fixed at 15 μm per side. Electronic warming of the micro-CT system appeared to produce an artifact of 1– 2% increase in BMD of the bone standard over the course of a week of daily use. Datasets were re-calculated accordingly. To provide a bone density standard, a series of bone plugs, cored to 5 mm in diameter with a dentistry drill and cut to 1-mm-thick sections on a diamond wafer saw, were harvested from a fresh-frozen bovine femur. Five plugs were ashed to determine mean bone mineral density, while another plug was quartered for placement within the scanned specimen tube. Ash weights indicated a density of 1.34 ± 0.02 g/cm3 (mean ± standard deviation, SD) for the standards. Bone morphology and density parameters were measured at four locations in the femur (Fig. 2). Initially, the dataset was aligned with the long axis of the bone. From this orientation, transverse single slices were made at the condyle, corresponding to the level of the largest circumference (typically just below the growth plate), and of the diaphysis, located halfway along the length of the femur. For analysis of the proximal region, the dataset was re-oriented parallel to the neck axis. Transverse slices were made at the largest circumference of the femoral head and halfway between the smallest circumference of the neck and its blending with the trochanters. The neck and the diaphysis served as locations relevant to clinical

fracture conditions, the neck being of particular significance in riskmanagement of the elderly. The head and condyle regions provided areas of relatively high trabecular content, although the comparatively oversized trabecular thickness in rodents yielded less structure for analysis than in humans. Tissue volume (TV) was determined by thresholding the image at 0.50 g/cm3, then counting the enclosed voxels, with an additional dilation-erosion step to fill in occasional small voids in the cortical wall. Bone volume (BV) was the subset of tissue volume above threshold. The ratio of BV/TV represented the normalized bone volume. Polar moment of inertia (MMI) was determined on a Skyscan system (Kontich, Belgium), using the CTAn software package, version 1.7.0.5. MMI is a measure of the relative deformability of a structure for a given load. The CTAn software also calculated fractal dimension (FD), and trabecular thickness (Tb.Th; plate model) for the head and condyle slices. FD is a measure of the complexity of the morphological bone pattern. Bone mass and density measures were based on integrating the histogram plot of the number of voxels for each calibrated 8-bit value of density. After converting voxel number to total volume, the resulting curve indicated the mass of bone at each density, or the bone density spectrum (BDS). Total bone mineral mass (TB) was calculated by summing the area under the mass-density curve. To provide a quantitative measure of the BDS, the amount of bone above the peak, or mode, of the mass-density curve (Hi.D, or high density) was summed, using the mode of the longitudinal control group as baseline. Due to handling issues, it was necessary to obtain a second set of baseline controls for μCT analysis of the femur. Biomechanics The radius was freed from the ulna, wrist, and humerus, cleaned of soft tissues, stored frozen until testing, then placed in a 3-point bending fixture with a free-length span of 6.5 mm. Specimens were kept moist with periodic saline spraying; total exposure time was less than 1 h. The fixture was mounted in a dynamic mechanical analyzer (DMA, model RSA3, TA Instruments, New Castle, DE), provided with a pressurized air source to negate bearing losses due to friction. Displacement was applied at 1 mm/min transversely to the radius axis until failure. Load and displacement data were recorded at 10 Hz, with a force resolution of 0.002 g, and displacement resolution of 1 μm. Two biomechanical parameters were determined. Peak force

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Fig. 2. Micro-computed tomography of the femur. Single slices were isolated from the condyle at the widest circumference, and from the diaphysis at the midpoint of total femur length. A data subset of the proximal femur was transformed to be parallel to the neck axis. Slices were isolated from the head at the widest circumference, and from the neck just distal from where it blends with the trochanter.

(Fp) was the maximum load sustained in the test. Whole-bone stiffness (S) was the slope of the load-displacement curve between 100 and 250 g. This consistently bracketed a portion of the linear elastic range, which characterized virtually the whole curve. The 250 g upper limit approximated half of the typical failure load. Material properties were not estimated, as this would have required an elusively reliable measure of cross-sectional geometry, and, barring numerical modeling, an assumption that the free length of the tested bone be simplified as a tube of constant shape. Given the relatively small differences expected in our experimental groups, the unavoidable inaccuracies introduced by these issues excluded a meaningful characterization of material properties. For analysis of vertebral body stiffness, axial compression to 200 g was applied to L5 at 1 mm/min (model 1011, Instron, Cambridge, MA), following resection of the posterior elements, but leaving a portion of the disc cephalically and caudally to allow distribution of load across the full surfaces of the specimen ends. Stiffness was calculated between 100 and 200 g, beyond the broader influence of the remnant soft tissue. The tall aspect ratio of the mouse vertebral bodies discouraged failure analysis, as the use of adequate restraint to prevent tilting of the specimen under high loads introduced a stabilization artifact. Serological markers Blood obtained from the specimens at sacrifice was centrifuged to isolate the serum, stored at − 80 °C, then analyzed for osteocalcin (BT470, Biomedical Technologies, Inc., Stoughton, MA) pyridinoline (PyD) crosslinks (8019, Quidel Corp., San Diego, CA), and alkaline phosphatase (CBA-301, Cell Biolabs, Inc., San Diego, CA) following manufacturer instructions, as described previously [15]. Dynamic mineralization and cell counts Thirteen days prior to sacrifice, the mice in the three longitudinal groups were injected with 15 μg/g tetracycline. A second label was not used, as double labels normally are not detectable beyond 6 months of age in mice. At sacrifice, the right femur was fixed in 70% ETOH and the distal third was cut using a diamond wire saw. The

specimen was then dehydrated and embedded in methyl methacrylate and sectioned in the horizontal (transverse) plane at 6–8 μm using a Leitz Polycut S microtome. Sections were viewed using a Zeiss Axioplan2 fluorescent microscope and captured using a SPOT® digital camera to image labeled bone surfaces. Forming surface was calculated as the percentage of non-eroded, single-labeled surface / total surface × 100 (MS/BS). Cell counts were performed on 4–5 μm sections of the proximal tibia after the specimens were decalcified in 4% EDTA for 1 week, dehydrated, cleared in xylene, then embedded in paraffin. Osteoblasts were counted on sections with von Giessen staining, and osteoclasts on sections stained for tartrate-resistant acid phosphatase (TRAP) activity. Standardized peripheral locations from the metaphysis were measured in a fixed region of interest applied to 5 neighboring areas. Statistics Student's t-test was performed to compare the TIME-0, LOW and HIGH groups versus CTRL group for Fp of the radius and BV of the femoral neck, diaphysis, and vertebral body, and the three serological markers (α = 0.05). For these parameters, which previous work suggests to be the most informative among those in this study, an effect size in the range of 0.83–0.92 was expected based on reported data and preliminary tests. The design of the sample size provided for an anticipated power of 0.80 at a level of significance of α = 0.05. The large number of remaining parameters necessitated reliance on parastatistical comparisons between individual groups. Regarding the morphological micro-CT results, graphical presentation is made of the “effect index,” defined as one minus the pairwise p-value. The index is signed negative when the median value of the experimental group is lower than that of controls. Thus, a marker appearing toward the midline at zero represents little change from the control group, while a marker appearing toward the maximum of 1 or minimum of − 1 represents a relatively high degree of change, respectively greater or less than controls. While only those markers above 0.95 and below −0.95 indicate statistical significance, the relative positioning of the “effect index” value is suggestive of a greater or lesser effect of WBV in the LOW versus the HIGH groups due to probability differences.

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Results Monitoring Mean weight of the CTRL and HIGH groups changed by less than 0.1 g over the 3-month course of the study, while that of the LOW group was reduced by 0.3 g (not significant; NS). Food intake followed a similar pattern in all three groups, with slight fluctuation in the first few weeks, then stable consumption for the duration of the study. One mouse from the CTRL group had to be euthanized due to an infection surrounding the eye. No behavioral changes were readily evident either due to aging or as a result of vibration. Femur: μCT morphology In the femoral head, bone volume (BV) was highest in the LOW group, at 85.8 ± 12.3 mm3 (mean ± SD; Table 1). The HIGH and CTRL groups had a BV on average about 5 mm3 less than that of LOW. Comparisons of the TIME-0, LOW, and HIGH endpoint groups versus CTRL, however, were not significant, although the relative p-values tended toward a greater effect in the LOW group (Fig. 3). Among the endpoint groups, BV/TV also was highest in the LOW group, reaching 0.537 ± 0.057, with the HIGH group improved over its value in the non-normalized BV alone. Moment of inertia (MMI) also was highest in the LOW group at 3.41 ± 0.56 cm4, with a similar proportion of reduction to that of BV in the other two groups (not significant). Fractal dimension (FD), as well, was highest in the LOW group at 1.40 ± 0.05, with a slightly smaller proportional reduction in the HIGH and CTRL groups. In this case, LOW versus CTRL was significant. For Tb.Th, the four groups compared nearly equally, with the LOW group producing 0.383 ± 0.015 mm. In the femoral neck, BV again was highest in the LOW group at 41.0 ± 3.9 mm3, as was MMI at 0.66± 0.11 cm4. For both parameters, the mean values for the HIGH and CTRL groups were less than 10% lower than that of the LOW group, with no statistical significance in the comparisons but a stronger trend in the LOW group. TIME-0 ranked behind LOW. BV/TV values were higher in the neck than in the head, with the LOW group producing the highest ratio among the endpoint groups; TIME-0 was slightly higher than LOW. FD was trendwise elevated in the LOW group, at 1.24± 0.10, but unchanged from controls in the HIGH and TIME-0 groups. Tb.Th was not analyzed as it did not directly apply in the neck. In the diaphysis, BV was 76.0 ± 12.2 mm3 in the LOW group. In contrast to the proximal regions, however, this was trendwise lower than that of the CTRL group (77.9 ± 7.9 mm3). The HIGH group, at 71.4 ± 5.7 mm3, notably approached a statistically significant de-

crease versus controls, and TIME-0 ranked between HIGH and LOW. Normalization to BV/TV resulted in TIME-0 ranking highest. Likewise, the HIGH group showed the lowest MMI at 5.19 ± 0.66 cm4, differing trendwise from the LOW and CTRL groups by about 10%. FD increased significantly in the LOW group (1.24 ± 0.04) versus controls. Tb.Th did not apply in the diaphysis. In the condyle, BV produced a different ranking among the groups than was found in the diaphyseal and proximal regions, with the HIGH group trendwise highest at 163.6 ± 23.2 mm3 . BV/TV further extended the mean differences due to a reduced TV. MMI was trendwise highest in the HIGH group at 22.14 ± 3.58 cm4, while there was a significant aging effect between TIM ± E-0 and CTRL. Regarding FD, there likewise was a small but significant aging effect, with the same difference found in the HIGH (1.36 ± 0.04) versus CTRL (1.32 ± 0.02) groups. Tb.Th was similar among the groups, trendwise highest for controls. Femur: uCT density (BDS, Hi.D) In all four femoral regions, the plot of bone mass versus mineral density revealed a subtle but characteristic shift in the peak of the bone density spectrum toward higher density in both the LOW and HIGH groups compared to the CTRL group (Fig. 4). The peak represents the mode, or most commonly occurring density. The peak of the CTRL group was used as the threshold for defining high density bone (Hi.D) for all groups, which represents the proportion of bone mass beyond the peak. Hi.D was significantly greater for the LOW group than it was for the CTRL group in the head (0.425 ± 0.060 vs. 0.369 ± 0.043), neck (0.506 ± 0.095 vs. 0.399 ± 0.065), and diaphyseal regions (0.588 ± 0.057 vs. 0.470 ± 0.094; Table 1). Hi.D in the HIGH group also was significantly greater than that in the CTRL group in the head and neck regions, but the difference was not significant in the diaphyseal region (p = 0.062). In the condyles, neither level of WBV achieved a significant difference from controls. TIME-0 values indicated a significant aging effect in the neck and diaphysis, but not in the head and condyle. Total bone (TB) was not statistically different among the groups in any region. However, the trends in the proximal regions suggested the LOW group realized a somewhat stronger effect than did the HIGH group. In the region where the shift toward higher density due to vibration was most evident — in the diaphysis — TB nonetheless was about the same among the groups (e.g., p = 0.673: LOW vs. CTRL). Consequently, mean BV for the LOW and HIGH groups was at least trendwise lower than that in the CTRL group, indicating there may have been a tendency to concentrate the bone mineral in a reduced net volume in that region, with essentially no net effect on bone mass itself.

Table 1 Micro-CT morphology and density results. BV (mm3) Head

Neck

Diaphysis

Condyle

TIME-0 CTRL LOW HIGH TIME-0 CTRL LOW HIGH TIME-0 CTRL LOW HIGH TIME-0 CTRL LOW HIGH

85.0 79.9 85.8 80.1 39.4 38.6 41.0 37.3 74.0 77.9 76.0 71.4 177.7 157.2 156.0 163.6

BV/TV (ratio) 6.1 7.0 12.3 4.1 2.6 3.1 3.9 3.5 6.4 7.9 12.2 5.7 15.7 22.2 20.0 23.2

0.592 0.509 0.537 0.520 0.750 0.702 0.714 0.688 0.410 0.389 0.379 0.372 0.479 0.364 0.361 0.377

MMI (cm4) 0.045 0.041 0.057 0.032 0.061 0.054 0.045 0.030 0.034 0.033 0.047 0.029 0.021 0.049 0.045 0.040

3.53 3.22 3.41 3.20 0.65 0.61 0.66 0.59 5.45 5.78 5.75 5.19 24.79 21.57 21.34 22.14

Tb.Th (mm) 0.21 0.35 0.56 0.27 0.08 0.09 0.11 0.13 0.88 0.71 1.18 0.66 3.76 3.19 2.75 3.58

0.387 0.386 0.383 0.385 n/a n/a n/a n/a n/a n/a n/a n/a 0.367 0.363 0.356 0.359

Tb.Sp (mm) 0.012 0.018 0.015 0.012

0.011 0.016 0.014 0.010

0.464 0.505 0.537 0.496 n/a n/a n/a n/a n/a n/a n/a n/a 1.288 1.282 1.356 1.326

Tb.N (mm) 0.062 0.057 0.075 0.040

0.120 0.222 0.117 0.128

0.825 0.784 0.770 0.791 n/a n/a n/a n/a n/a n/a n/a n/a 0.502 0.504 0.484 0.491

FD (ratio) 0.020 0.030 0.043 0.020

0.024 0.066 0.024 0.035

1.37 1.35 1.40 1.36 1.19 1.19 1.24 1.18 1.12 1.17 1.24 1.21 1.36 1.32 1.34 1.36

Hi.D (ratio) 0.04 0.04 0.05 0.03 0.08 0.05 0.10 0.05 0.02 0.05 0.04 0.05 0.03 0.02 0.03 0.04

0.332 0.369 0.425 0.425 0.493 0.399 0.506 0.480 0.595 0.470 0.588 0.559 0.294 0.253 0.295 0.260

0.018 0.043 0.060 0.039 0.044 0.065 0.095 0.065 0.054 0.094 0.057 0.072 0.028 0.051 0.042 0.052

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Fig. 3. Effect index in the femur. The probability of statistical significance is shown as a continuum between a decrease (− 1) or increase (+1) over controls (CTRL) due to LOW or HIGH vibration. LOW vibration had a stronger effect than HIGH in all (A–C) but the condyle region (D), with a neutral effect for LOW in the diaphysis (C) and a decrease in BV for HIGH that approached significance.

Fig. 4. Bone density spectrum (BDS) was determined by integrating the curve of voxel number (from the conventional histogram) versus mineral density. In all regions (head: A; neck: B; diaphysis: C; condyles: D) there was a notable shift toward more mineral mass at higher densities due to vibration. Display focuses on LOW compared to CTRL for visual clarity; HIGH mostly overlapped LOW, and TIME-0 was somewhat higher. The strongest difference in BDS was in the diaphysis, although the bone morphology parameters were least distinct in this region.

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Radius: biomechanics Failure load (Fp) in the radius was effectively unchanged by aging or by either level of vibration, with a mean of 4.33 ± 0.63 N in the CTRL group, 4.34 ± 0.49 N in LOW, 4.45 ± 0.33 N in HIGH, and 4.30 ± 0.41 in TIME−0. Whole-bone stiffness (S) also did not produce a statistical difference among the groups (21.9 ± 2.2 N/mm in CTRL, 22.6 ± 1.6 N/ mm in LOW, 23.1 ± 2.3 N/mm in HIGH, 21.6 ± 1.7 N/mm in TIME-0), although the HIGH group approached significance (p = 0.139) with a mean increase of 4.9% over controls. Variance in the dataset for wholebone stiffness was about half of that for failure load, indicating that stiffness may be the more reliable measure in this context for assessing biomechanical differences between groups. Vertebra: μCT morphology, density, and biomechanics BV was not significantly affected by either vibration level in L4, with mean values slightly lower than that of controls at 73.3±19.6 mm3 (Table 1). Combined with a trendwise increase in TV, the BV/TV parameter approached a significant decrease for both vibration levels versus controls at 0.184±0.045. MMI, Tb.Th, and FD were all comparable between the groups, with nominal increases due to vibration except for FD of the HIGH group. TIME-0 values in the morphological parameters did not indicate an aging effect. BDS showed a similar distribution of bone density among the groups, but with somewhat reduced bone mass in the vibration groups at the lower ranges of density. The Hi.D portion was not affected by vibration, with controls averaging 0.285±0.105. Mechanically, there were no significant differences among the groups. The LOW group produced the highest mean value of structural stiffness at 45.0±11.0 N/mm. Serological markers PyD was significantly reduced by both levels of vibration. From a maximum of 1.75 ± 0.46 nmol/L in the CTRL group, the intervention resulted in 1.35 ± 0.34 nmol/L in the LOW group and 1.17± 0.39 nmol/ L in the HIGH group (Fig. 5). The aging effect was not significant but notable, increasing from 1.49 ± 0.45 nmol/L at TIME-0. OCN decreased significantly with aging (TIME-0: 13.2± 5.2 U/L; CTRL: 9.6 ± 3.4 U/L), but was not affected by vibration (LOW: 7.9 ± 3.2 U/L; HIGH: 11.3 ± 1.3 U/L). Likewise, ALP was unaffected by vibration (LOW: 7.9 ± 3.2 U/ L; HIGH: 11.3 ± 1.3 U/L) and did not show a significant aging effect (TIME-0: 13.2 ± 5.2 U/L; CTRL: 9.6 ± 3.4 U/L). Dynamic mineralization and cell counts MS/BS was highest in the HIGH group (21.8 ± 7.1%; Fig. 6a), still high in the LOW group (19.5 ± 7.9%), and significantly lower than both vibration groups in the CTRL group (10.0 ± 4.3%).

Osteoclasts were most numerous in the HIGH group (40.3 ± 9.4 mm− 2; Fig. 6b), which was statistically greater than the count in both the LOW group (26.7 ± 9.3 mm− 2) and CTRL group (20.3 ± 5.5 mm− 2). Osteoblasts were not different among the groups, reaching the highest mean value in the HIGH group (51.8 ± 16.8 mm− 2; Fig. 6c), next highest in the LOW group (43.1 ± 20.5 mm− 2), and lowest in the CTRL group (42.0 ± 18.3 mm− 2). Discussion This study aimed to further establish the degree to which wholebody vibration influences bone health, in this case in an aging population. Based on lifespan, the 18-month starting age of the mice was equivalent to the human age of mid-fifties. The ending age of 21 months was equivalent to mid-sixties in humans. The study sought, furthermore, to differentiate between conventionally low and medium acceleration amplitudes, as a considerable disparity still exists in the range of vibrational forces applied in various clinical, sports, animal, and cell studies. The primary finding of this study is that vibration can subtly affect aging bone characteristics, depending on the region and parameter examined. For example, an acceleration of 0.5 g in this model appears favorable to bone density through most of the femur, whereas 1.5 g is largely ineffective in the femur although it may help bone strength and stiffness in the radius. There also was a significant increase in mineralizing surface in the distal femur for both vibration magnitudes. This is generally consistent with previous studies of pre-aged animals [9,10,16]. The most statistically significant results in the current study relate to bone density spectrum (BDS), where the lower magnitude of 0.5 g produced the strongest increase in high density bone. For several of the morphological parameters in the femur, the higher level of 1.5 g actually resulted in trendwise reduced bone parameters, in particular in the diaphysis. In the vertebral body, there was little distinction between 0.5 and 1.5 g results, with both showing trendwise reduced BV/TV mean values versus controls. As in previous studies, the effect of vibration varied regionally. [9] In the femur, low vibration had its strongest effect on bone volume in the head and neck regions, although its highest impact on bone density was in the diaphysis, where it had essentially no effect on bone volume. The high magnitude of 1.5 g affected bone density trendwise in all regions, but generally had no positive impact on bone morphology, except for slight improvement at the condyles. In the context of an aging population, where femoral neck fractures are especially common, the conclusion is that a vibration level of 0.5 g is recommended over that of 1.5 g for maintaining healthy femora (to the extent these results in mice can be extrapolated to the clinic). Still, relatively high vibration cannot be summarily discounted, as 1.5 g had the highest trendwise impact on stiffness and strength in the radius.

Fig. 5. Serum markers indicated a significant reduction (p ≤ 0.05) in the collagen breakdown marker, PyD, in both the HIGH and LOW vibrated groups compared to longitudinal controls (CTRL; panel A). A significant aging effect was found for osteocalcin (panel B), which was not affected by either vibration level. For alkaline phosphatase, there were no significant differences detected (panel C).

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Fig. 6. MS/BS (single-labeled surface) was significantly higher in the HIGH and LOW vibration groups than it was in the longitudinal control group (CTRL; panel A). Osteoclasts were significantly more numerous in the HIGH group than in controls (CTRL; panel B), with similar trends but no pronounced difference for osteoblasts (panel C).

The effect of aging in the range tested in this study was not significant for most parameters, with the exception of micro-CT assessments in the condyle (MMI, FD), and high density bone (Hi.D) in the neck and diaphysis, where vibration also had its greatest effect. Interestingly, the neck and diaphysis were the two regions with predominantly cortical bone, indicating a heightened sensitivity in this bone type to both aging and vibration. Positive effects of vibration on cortical bone have been corroborated in another study, [16] although most reports find the largest changes in trabecular bone [17]. Regarding safety concerns, the amplitudes of 0.5 and 1.5 g used here are within reported therapeutic levels and considerably below the much higher magnitudes found in some exercise studies. Such magnitudes also may be encountered occupationally, where extended exposure results in a range of clinical manifestations. [18] Caution is warranted in therapeutic application, since the use of vibration training as an exercise tool has popularized a class of devices that can exceed safety standards established in ISO 2631-1. [13] At the lower levels used in this study, however, safety in a non-healing environment does not appear to be compromised. There was no lift-off effect with these magnitudes despite that one level exceeded the acceleration due to gravity. This was partly due to the energy storage and dissipation capacity of the soft tissues, and potentially to compensatory motion produced by the muscle response. It should be pointed out, however, that lower frequencies, especially in the 10–20 Hz subclinical range, can result in several-fold amplification of the input vibration, according to one accelerometer study in volunteers. [19] Consideration of the rodent anatomy in the distal appendicular skeleton would suggest that the vibration magnitudes used here were dampened progressively upward through the limbs. Indeed, potentially substantial dampening of the input at the level of the spine, as suggested by the cited accelerometer study, could explain the more negligible effect in that area. Others have shown significant decrease in transmissibility with increasing knee flexion angle in volunteers [20], further suggesting a weakening of the vibration signal proximally in this rodent model. The aging specimens evaluated in this study represent a specialized cohort, which, along with other under-studied groups, is beginning to gain attention in the therapeutic application of WBV for improved bone health. In another study, a group of disabled pediatric patients receiving a 6-month course of 0.3 g WBV increased BMD 6.3% over baseline, while controls lost 11.9% in the same period [21]. Smaller but still significant changes were noted in a study of young women with low BMD who were given 0.3 g WBV for 12 months [22]. In a post-menopausal study, BMD was found to be increased similarly between groups receiving alendronate, with or without 0.6–3.4 g WBV [23]. A secondary finding was that WBV showed a differentiated effect from that of alendronate alone in the improvement of low back

pain. The rationale for clinical trials of WBV has derived in part from a seminal study in sheep earlier in the decade, showing a significant increase in trabecular bone volume after one year of 0.3 g vibration on a single hindlimb [17]. An important corollary finding from that study was that the forelimb was unaffected, indicating the osteogenic stimulation was direct and not systemic. A further study by the group showed an increase in bone mineral content of 10% due to vibration of varying magnitudes, leading to a meaningful increase in strength of 25% [24]. In animal models, bone formation rate has been shown to be retained in a model of disuse osteopenia when treated with 0.25 g WBV [25]. Similarly, in an oophorectomized rat model, 3.0 g WBV best promoted both endosteal and periosteal bone formation, as well as retention of muscle strength otherwise lost to the hormonal deficiency [26]. Translation of WBV to surgical interventions has been reported only recently. In a model of tibial fracture fixation, young female rats were given 0.3 g WBV for 20 min/day over 4 weeks, and found to have an improved healing rate and faster induction of mRNA for type II collagen and bone morphogenetic protein-2 [27]. The stability of the gene expression pattern, however, was mixed. A recent report on rabbits instrumented for distraction osteogenesis found that 0.3 g WBV resulted in a recovery of nearly 100% of intact failure torque and stiffness of the contralateral limbs, whereas the unstimulated group showed a 30–50% loss in mechanical properties [28]. Localizing the vibration cephalically in a rat calvarial defect model, one new study reported that the increase in bone area in the 0.4 g vibration group versus controls continued several weeks beyond the 4-week stimulation period, suggesting the triggering of anabolic cellular processes proceeded autonomically with limited de-activating feedback of the interrupted mechanical signal [29]. A limitation of the osteogenic response was that the defect filled in by a surfacespreading pattern, and did not grow through the thickness of the healing region, with or without a carrier sponge. With this expanded scope of application for WBV, there are several pertinent surgical procedures for the elderly to consider, including treatment of femoral neck fracture. Studies elucidating the effect of WBV specifically on cells are now gaining interest. To this point, it has been demonstrated that 0.2 g WBV increases mineralization of adherent stromal cells [30], and a range of WBV counter-regulates nitric oxide positively and PGE2 negatively in MC3T3 cells [31]. That study proposed using the Stokes– Einstein relation to model the kinetics of the nucleus within the cytoplasm during vibration, but the validation process remains challenging. A recent report of irradiated mice re-infused with GFPlabeled bone marrow cells showed that WBV increased the number of labeled cells in the marrow, and that this group showed the fewest adipocytes, suggesting WBV acts also at the progenitor cell level [30].

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As with in vivo experiments, the in vitro cell studies can produce confounding results. In one study of growing mice, osteoclast number did not change with WBV [10], although in a similar study adding a rest-insertion period, there was a 30% decrease in osteoclast activity [16]. A related study reported an increase in RANKL gene expression in 0.3 g vibrated mouse limbs, indicative of increased osteoclasts [32]. The potential benefits of WBV are under investigation also for other clinical conditions. In cerebral palsy patients, for example, WBV at ultra-low frequencies (ULF) of 2–4.4 Hz was shown to improve sensory organization and timed up-and-go over controls [33]. This corroborates findings in elderly individuals in nursing homes. From at least one study, however, it is not clear that the benefits of ULF WBV exceed those also obtained with conventional exercise programs [34]. In Parkinson's patients, one study found an improvement in postural control due to ULF WBV [35], although the presumptive proprioceptive mechanism for modulating postural control was shown to be unaffected in another ULF study [36]. By contrast, in chronic stroke patients, WBV of 5.4 g was found to improve proprioceptive control [37], whereas another study in postactute patients using the same magnitude indicated no improvement over a range of kinematic control measures [38]. The broadest body of work on stimulatory vibration comes out of the exercise and sports science field. WBV of 7 g has been shown in one study to increase skin blood flow, with the effect sustained at least 10 min following exercise, whereas the effect of exercise alone was seen to be more readily dissipated [39]. Likewise, in a training study, muscle deoxygenation in exercising subjects was sustained longest in those exposed to 2.3 g WBV [40]. In a study of brief squatting, time to exhaustion was shortened and neuromuscular excitability was enhanced with a WBV of 16.3 g [41]. The effect of WBV on muscle strength, EMG activity, and flexibility has been equivocal, depending on the cohort and context, with some studies generally reporting an increase [13,41–45], and others no change or a decrease [46–49]. Those showing an increase used WBV magnitudes typically in the mid-teens, with a maximum of 40 g [44], whereas the others specified levels of 5 g or less. Interestingly, one of the common findings of WBV is that it improves jumping capacity, in some cases as a countermovement jump and in others as jump height [43,47,50–53]. One study, however, has contradicted these findings [54]. The body of exercise studies collectively shows that WBV directly affects a range of tissues in the musculoskeletal system as well as its neurovascular support network. The conclusions that can be drawn from the current study are limited by the extent of the vibratory stimulation, which was restricted to a 3-month period in an animal that lacks secondary bone remodeling. Also, the age of the mice likely had not quite reached the timepoint at which the normal loss of bone associated with aging is at a maximum. An earlier study identified a window between 18 and 24 months for C57BL/6 mice in which bone mineral density experiences a significant decrease [55]. However, it is not clear that the first half of that period — corresponding to that of the current study — represented the period of greatest change. Longerduration studies, beginning at a later age and designed with larger sample sizes, would help elucidate whether the trends seen in this study potentially reflect statistically meaningful changes that may be achieved with vibration. The primary findings of a shift toward higher bone density spectra in the femur due to vibration, plus an increase in mineralizing surface, with suggestive effects on bone strength in the radius and a reduction in the marker for bone collagen breakdown, encourage further study in an aging model. Acknowledgments This work was financially supported by the Medical College of Georgia School of Medicine, Department of Orthopaedic Surgery and Section of Plastic Surgery. The authors wish to thank: 1) Greg

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