Effect of stimulation frequency on osteogenic capability of electrical ...

Bulletin of the JSME

Vol.9, No.3, 2014

Journal of Biomechanical Science and Engineering Effect of stimulation frequency on osteogenic capability of electrical muscle stimulation Shigeo M. TANAKA * * Institute of Nature and Environmental Technology, Kanazawa University Kakuma-machi, Kanazawa, Ishikawa, Japan E-mail: [email protected]

Received 5 March 2014 Abstract Exercise is effective as a preventive modality of osteoporosis because bone formation is accelerated by mechanical loading. However, elderly patients with osteoporosis are often difficult to exercise, which brings some risks of fractures during exercise with a fall. We focused on electrically-elicited muscle contraction to mechanically stimulate bone formation without any voluntary physical activities. The osteogenic effect of this method has been reported previously, however, it is unknown yet what the stimulation regimen is desirable to maximize the osteogenic effect. To provide basic knowledge to determine effective stimulation patterns in this method, this study aimed to investigate the effect of stimulation frequency on the osteogenic capability of electrical muscle stimulation in rats. The left quadriceps of rats were stimulated electrically for 30 min per day for 3 consecutive days under anesthesia. The stimulation waveform was a series of pulse trains composed of pulses with amplitude of 2 mA, 552 μs duration, and 50% duty ratio. The cyclic muscle contractions were generated by reversing the polarity of the electrical stimulation at 2, 10, 20, 40, or 80Hz. The stimulation effects were evaluated at the levels of gene expression and bone formation using RT-PCR and bone histomorphometry, respectively. Muscle stimulation at 20 Hz elicited a significantly higher expression level of osteocalcin mRNAs than those at the other frequencies. The 20-Hz stimulation also yielded the highest bone formation rate. However, these results did not correspond to the results of mechanical stimulation factors, including total number of contractions and average and cumulative muscle contraction forces during the stimulation period, suggesting a mechanism other than a dose-dependent effect of mechanical stimulation on osteogenesis. It was concluded that 20 Hz was the most effective frequency for eliciting the osteogenic effect of electric muscle stimulation in the rat. This finding provides useful information for establishing a stimulation regimen in clinical application of this method. Key words : Electrical stimulation, Muscle contraction, Mechanical stimulation, Osteoporosis, Osteogenesis

1. Introduction With its osteogenic effect, physical exercise such as walking (Shibata, et al., 2003) is clinically recommended for the elderly as an option effective for preventing osteoporosis with no pharmacological side effects (Gourlay, et al., 2003). Exercise also increases muscle power, thereby assisting in preventing accidental falls and reducing the risk of bone fracture (Gregg, et al., 1998). Osteoporotic fractures reduce quality of life (Magaziner, et al., 2000) as well as life expectancy (Dennison and Cooper, 2000). However, elderly people, especially those under bed rest conditions, often have difficulty exercising enough, owing to the decline in physical function (Metter, et al., 1997). Osteoporosis is also a major health problem in space flight. Astronauts suffer from bone loss as a result of mechano-adaptation of bone to zero gravity in a space flight (Droppert, 1990). Exercise is a feasible countermeasure against bone loss and was reported to be effective (Shibata, et al., 2003). However, space and time for exercise are limited in space flight, requiring ways other than physical exercise to mechanically stimulate bone. As an option for solving bone loss problems without voluntary physical activity, we investigated electrically-induced muscle contraction to stimulate bone formation. This is a promising, safe mechanical intervention in osteoporotic subjects in diverse situations for Paper No.14-00114 J-STAGE Advance Publication date : 2 October, 2014 [DOI: 10.1299/jbse.14-00114]

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© 2014 The Japan Society of Mechanical Engineers

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Tanaka, Journal of Biomechanical Science and Engineering, Vol.9, No.3 (2014)

preventing bone loss without physical exercise. Initially, electrical neuromuscular stimulation was researched and clinically used as an effective way to restore function in spinal cord injury (SCI) (Hooker, et al., 1990, Yarkony, et al., 1992). To date, electrical muscle stimulation is also widely recognized and commercialized as a non-pharmacological way to increase muscle mass and power (Porcari, et al., 2005). But osteogenesis is another benefit of electrical muscle stimulation and has been investigated using animal models (Burr, et al., 1984, Lee, et al., 2001, Zerath, et al., 1995). Using rabbits, Burr et al. showed that electrical stimulation to quadriceps prevented bone loss in cast-immobilized limbs (Burr, et al., 1984). Electrical muscle stimulation has comprehensive beneficial effects in the restoration of the neuromuscular skeletal system and is expected to be clinically applied to prevention, treatment, and rehabilitation in orthopedics. However, to date, an optimal pattern to elicit maximum osteogenesis has not been established. Stimulation frequency and resting time, in particular, are key factors in sustaining muscle contraction force over the stimulation time while avoiding muscle fatigue (Fitts, 2008). To elicit a beneficial osteogenic effect from electrical muscle stimulation, various combinations of frequencies and resting times(Lam and Qin, 2008, Lee, et al., 2001, Midura, et al., 2005, Wei, et al., 1998) have been experimentally tested in animal models. In our previous study (Tanaka and Kondo, 2009), electrically induced muscle contraction in the range up to 20 Hz showed a larger magnitude of muscle contraction at lower frequencies, a larger number of muscle contractions at higher frequencies, and the highest time accumulation at approximately 10 Hz. Inserting resting times into the stimulation period did not contribute to an increase in cumulative muscle force in repetitive electrical stimulation of rat quadriceps. It is not known to what degree osteogenic responses are caused by the muscle responses to oscillatory electrical stimulations. Higher frequencies of stimulation should be investigated, given that the frequency of muscle vibration signals is as high as 80 Hz (McAuley, et al., 1997) with three peaks at 10, 20, and 40 Hz, although the magnitude of the muscle vibration diminishes greatly at higher frequencies. The optimal electrical stimulation pattern for muscle is still controversial and awaits investigation of muscle contraction responses caused by electrical stimulation in a broader frequency range. This study aimed to investigate, at the levels of gene expression and bone formation in rats, the frequency dependency of osteogenic capability by electrical muscle stimulation at up to 80 Hz.

2. Materials and Methods 2.1 Electrical stimulation to rat quadriceps Eight-week-old female Sprague–Dawley rats were used. All experimental procedures were approved by the committee on animal experimentation of Kanazawa University (approval number: AP-132845). The method of electrical stimulation has been previously described (Tanaka and Kondo, 2009). Briefly, to elicit tetanic contractions from a rat quadriceps, the left quadriceps of rat was stimulated electrically using stainless steel needle electrodes under anesthesia (Fig. 1a). Stimulation waveforms were generated by a Windows -based personal computer using a Visual Basic program and sent to the quadriceps of rat through a 16-bit AD/DA board (Fig. 1b). A waveform was a series of pulse trains comprising pulses with amplitude of 2 mA, 552 μs duration, and 50% duty ratio (Fig. 2a). A pulse train stimulates the muscle at 906 Hz of pulse-repetition frequency, causing a single tetanic contraction that shows its maximum force at 50 ms after the start of the pulse train (Tanaka and Kondo, 2009). The repetitive muscle contraction was elicited by reversing the polarity of the electrical stimulation at 2, 10, 20, 40, or 80 Hz (Fig. 2b–f).

2.2 Muscle contraction force measurements Measurement of electrically induced muscle contraction was as follows: electrical stimulations were given to a rat quadriceps for 30 min, and the change in contraction force of the muscle with stimulation time was monitored with a force transducer as previously described (Tanaka and Kondo, 2009). Briefly, the quadriceps was disconnected from the patella by cutting the patellar tendon at the patella and tied with a nylon string at its distal end. The other end of the nylon string was connected to the cantilever beam of the force transducer. From the recorded muscle contraction data, the number of contractions, average contraction peak-to-peak force, and cumulative peak-to-peak force during 30 min of stimulation were calculated. In this study, a change exceeding one standard deviation of the variation under non-stimulation conditions was recognized as a muscle contraction.

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2.3 Semi-quantitative RT-PCR Right and left femurs were removed from the rats 3 h after the mechanical treatment of 30 min. Both the ends of the femurs were cut off, and the bone marrow was flushed out by syringe injection of saline. The femurs were frozen in liquid nitrogen and then pulverized with a sterilized stainless steel hammer that had previously been autoclaved-sterilized and cooled in liquid nitrogen. Total RNA was extracted from the bone fragments using a kit (#74104, Qiagen Inc., CA, USA), and the isolated RNA was reverse-transcribed as described previously (Tanaka, et al., 2003). Semi-quantitative RT-PCR was performed using a thermal cycler (ASTEC, PC-320, Fukuoka, Japan). The reverse-transcribed cDNA was amplified for 25 cycles as follows: 94°C for 1 min, 60°C for 30 s, and 72°C for 30 s, with primer pairs specific to rat osteocalcin or -actin as internal control (Table 1). Table 1

Sequences and sizes of primers designed for rat -actin and ostecalcin.

target mRNA

rat β-actin

rat osteocalcin

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primer sequence

product size

Forward: 5’-aagtaccccattgaacacgg-3’ Reverse: 5’-atcacaatgccagtggtacg-3’

Forward: 5’-ccaaccacagcatccttt-3’

257 bp

136 bp

Reverse: 5’-ggacttgtctgttctgca-3’


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The total cycle of PCR was adjusted to allow for linear amplification. PCR products were electrophoresed on 1.2% agarose gel and visualized with ethidium bromide. The intensity of electrophoretic bands was quantified using Photoshop (Adobe Systems Inc., CA, USA). RT-PCR analysis was conducted on four samples of each group for four independent experiments.

2.4 Bone histomorphometric analysis The left quadriceps of rat was stimulated electrically for 3 consecutive days. Mineralization fronts were fluorescence-labeled by intraperitoneal injection of calcein (1%, 0.5 ml) 2 and 9 days after stimulation, and the rats were sacrificed 13 days after the stimulation. Right and left femurs were removed, fixed in 10% formaldehyde, dehydrated in a graded ethanol series, and embedded in PMMA. Thin (50 m) sections were made perpendicular to the bone axis at the midshaft by sectioning and grinding the plastic-embedded femurs using a diamond blade (Maruto Instrument, ME, Tokyo, Japan) and 800- and 1000-grit sandpapers. Bone histomorphometric analyses were performed on the cross sections using a fluorescence microscope (KEYENCE, BZ-9000, Osaka, Japan). Bone histomorphometric parameters: mineralized surface/bone surface (MS/BS, %), mineral apposition rate (MAR, m/day), and bone formation rate/bone surface (BFR/BS, m3/m2/year) were measured, and corresponding values for the non-stimulated right femur (rMS/BS, rMAR, and rBFR/BS) were determined and compared among the groups (Tanaka, et al., 2003).

2.5 Data analysis To assess statistical significance of effects, one-way analyses of variance (ANOVA) followed by Tukey’s HDS post-hoc tests were performed among the groups using KaleidaGraph  software system (Version 3.6, Synergy Software, PA, USA). P-values of ≤0.05 were considered statistically significant.

3. Results 3.1 Muscle contraction force The time-course profile of the contraction force of rat quadriceps changed in response to the frequency of repetition of electrical stimulation. Fig. 3 shows the typical waveforms of muscle contraction force during the initial 2 s of a 30-min stimulation period. In synchrony with the repetitive stimulation, cyclic contractions in force were observed in all the groups. The highest contraction force appeared at the first contraction and decreased with increasing stimulation time at all the frequencies. The peak-to-peak force of each contraction decreased with increasing frequency. Fig. 4 shows the evaluation parameters analyzed from the 30-min data of muscle contraction force, representing the amounts of mechanical stimulation. Numbers of muscle contractions were highest at 40 Hz, 13.3-fold (p < 0.01), 2.7-fold (p < 0.05), 1.5-fold, and 6.3-fold (p < 0.01) higher than those at 2, 10, 20, and 80 Hz, respectively (Fig. 4a). The 2-Hz stimulation showed the fewest contractions but the largest average peak-to-peak force among the groups, 4.8-fold (p < 0.01), 10.4-fold (p < 0.01), 26.4-fold (p < 0.01), and 114-fold (p < 0.01) higher than those at 10, 20, 40, and 80 Hz, respectively (Fig. 4b). Cumulative peak-to-peak force showed similar values at 2, 10, and 20 Hz and decreased with increasing frequency over 20 Hz (Fig. 4c). However, there was no significant difference among the groups. 60

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Fig. 4 Comparison of the amount of mechanical stimulation among the frequencies. Stimulation amounts were evaluated in terms of (a) cycle number, (b) average peak-to-peak force, or (c) cumulative peak-to-peak force of muscle contraction during a stimulation of 30 min.

3.2 Messenger-RNA expression Muscle stimulation at 20 Hz elicited the highest level of OC mRNAs from femur (Fig. 5). This upregulation of OC gene was significantly different from that at 2 Hz (p < 0.05) and 80 Hz (p < 0.05). The levels of OC mRNA at 20 Hz were 1.24-, 1.05-, 1.05-, and 1.45-fold higher than those at 2, 10, 40, and 80 Hz, respectively. The 80-Hz stimulation showed the lowest expression of OC mRNA, which was significantly different from those at 10 (p < 0.05), 20 (p < 0.01), and 40 Hz (p < 0.05). p < 0.05 p < 0.01

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Fig. 5 Comparison of expression levels of osteocalcin mRNA among frequencies. Stimulation at 20 Hz showed a significantly higher expression level than that at 2 and 80 Hz. The expression levels were normalized by levels in -action and were expressed as the ratio to the non-stimulated control (right femurs).

3.3 Bone histmorphometric analysis Figs. 6a and b show typical mid-shaft cross section of rat femurs, which are stimulated at 20 Hz and not stimulated (control), respectively. In both samples, fluorescent labels were observed on either the periosteal or endosteal surface. However, the 20-Hz sample showed a larger distance between fluorescent labels on the lateral periosteal surface. Fig. 7 shows comparisons of rMS/BS, rMAR, or rBFR/BS over all the frequencies. The 2-, 10-, 20-, and 80-Hz stimulations showed positive rMS/BS, in contrast, a negative value was exhibited at 40 Hz (Fig. 7a). rMAR was not affected in the

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control and the 2-Hz groups. This parameter showed positive values at 10 and 20 Hz and negative values at 40 and 80 Hz (Fig. 7b). The 2-, 10-, and 20-Hz stimulations yielded positive values of rBFR/BS, with the largest value at 20 Hz, although there were no statistically significant differences among these groups (Fig. 7c). The 40- and 80-Hz stimulations did not increase rBFR/BS.

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Fig. 6 Bone morphometric observations on cross-sections of rat femur at mid-shaft for (a) a sample stimulated at 20 Hz and (b) non-stimulated control. The 20-Hz sample showed a larger interval between two fluorescent labels on the lateral periosteal surface. 20%

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Fig. 7 Comparison of bone morphometric parameters among the frequencies: (a) rMS/BS; (b) rMAR; and (c) rBFR/BS. The 20-Hz stimulation yielded the highest value for all three parameters. Both femurs were not stimulated in the control.

4. Discussion The RT-PCR data showed that the osteogenic effect of electrical muscle stimulation increased with stimulation frequency up to 20 Hz, but decreased beyond 20 Hz, with the frequencies applied in this study. This result at the mRNA level was supported at the tissue level by the histomorphometric result. The muscle contraction data did not qualitatively correspond to the data at mRNA and tissue levels. The average peak-to-peak force and the cyclic number of electrically induced oscillatory muscle contraction showed their highest values at 2 and 40 Hz, respectively. The magnitude of peak-to-peak force decreased depending on the degree of muscle fatigue, which is much greater under cyclic contractions at a higher frequency. Owing to perfect tetanic contraction at 80 Hz, a small number of contractions was recorded. However, the cumulative peak-to-peak forces showed no obvious differences among the frequencies selected. Our results suggested the possibility of an optimal stimulation frequency to elicit maximum osteogenesis from

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electrical muscle stimulation. Various frequencies of mechanical loading have been investigated for the osteogenic effect using animal models. Lanyon et al. found that dynamic mechanical loads applied directly to bone in vivo have higher osteogenic capability than static loads (Lanyon and Rubin, 1984), especially at higher frequencies. Rubin et al. reported the largest anabolic effect of whole-body vibration with a small magnitude at 30 Hz in an experiment using turkeys (Rubin, et al., 2001). Zhang et al. showed that mechanical loading on a mouse knee joint stimulated osteogenesis in the tibia at 5 Hz and the femur at 15 Hz (Zhang, et al., 2007). In mechanical loading induced by electrical muscle stimulation, in contrast, Lam and Qin et al. showed that electrical muscle stimulation elicited maximum osteogenesis in a hindlimb-suspended rat at 50 Hz (Lam and Qin, 2008, Qin, et al., 2010). In contrast, our data indicated 20 Hz-muscle stimulation to result in greatest osteogenesis. Although the loading frequency resulting in the maximum osteogenic effect is still in dispute, it appears to range from 5 to 50 Hz depending on the experimental model and conditions. Our data also suggest that the mechanical stimulation parameters of average peak-to-peak force, cyclic number, and cumulative peak-to-peak force are not determinant factors of osteogenesis after mechanical stimulation. Magnitude of bone strain over a threshold value is conventionally recognized to be the mechanical stimulant factor determining bone formation (Frost, 1992, Rubin and Lanyon, 1985, Turner, et al., 1994). However, bone strain alone cannot well explain all of the osteogenesis induced by mechanical stimuli, for example, the osteogenic effect of low-magnitude, high-frequency loading, which has been reported by Rubin et al. (Rubin, et al., 2001). In the daily stress stimulus (DSS) theory originally proposed by Carter et al. (Carter, 1987, Whalen, et al., 1988), mechanically induced osteogenesis is determined by summation of the stresses of daily loads given to a bone with a compensation factor “m”. This theory can well explain Rubin’s findings. However, in our results, osteogenesis after electrical muscle stimulation did not fully reflect the magnitude of cumulative peak-to-peak forces, suggesting another mechanism behind the result. Mechanisms underlying osteogenesis following electrically induced muscle contraction have been previously proposed and discussed. Qin et al. found that intramedullary pressure is elevated by electrically induced muscle contraction and showed its highest value at 20 Hz (Qin, et al., 2002). The increase in intramedullary pressure would be brought on by the pumping effect of muscle contraction through blood and suggests that interstitial fluid flow in the bone matrix stimulates the osteogenic response of the osteocyte network in the matrix. The strong tetanic contraction with small amplitude at 40 and 60 Hz would act as static pressure, and impede the fluid circulation, therefore, result in less stimulation to the cells. This might be a reason of the reduction of OC mRNA level at these higher frequencies. Interstitial fluid flow has been visually confirmed to be caused by direct mechanical loading to a long bone in vivo using nanomolecule tracers (Knothe Tate, et al., 1998), suggesting the possibility of the same event in a bone mechanically stimulated by muscle contraction. Fluid flow can stimulate more bone cells rather than matrix strain in vitro (Klein-Nulend, et al., 1995, Owan, et al., 1997, You, et al., 2000). The relationship between external loading pattern and the magnitude of fluid flow in the bone matrix is complex and demands further study. Another possible mechanism for the highest osteogenic response at 20 Hz in our results is related to cellular mechano-sensitivity, which changes depending on the frequency of mechanical loading. The frequency of loading to which bone cells are most sensitive has been previously investigated (Bacabac, et al., 2006, Han, et al., 2004, Jacobs, et al., 1998) and is still controversial. Complicating the issue is that the mechano-sensitivity of bone cells may change depending on their cellular maturation. Bone cells have a range of differentiation stages from mesenchymal stem cells to matured osteocytes, reflected in their locations in the bone matrix. Rosenberg et al. reported that the proliferation of osteoblastic cells is stimulated by vibration at 20 Hz, whereas alkaline phosphatase activity, which is a biomarker for osteoblastic differentiation, is elevated at 60 Hz (Rosenberg, et al., 2002). Given that the mechano-sensitivity of bone cells should be regulated by the status of mechano-sensors in the cell, which are Ca2+ channel, integrin (Duncan and Turner, 1995), and primary cilium (Praetorius and Spring, 2005, Whitfield, 2003), deeper investigations of the frequency and cellular maturation dependence of their behavior are needed to explain the mechanism underlying osteogenic responses after mechanical stimulation. The bone histomorphometric data did not clearly show differences in bone formation parameters among the frequencies selected in this study, although it had a tendency similar to that of osteocalcin mRNA expression. To elicit statistical differences among the experiment groups, longer stimulation periods and/or later observation points would be more appropriate. Given that this study was performed in rats, the data cannot be directly applied to humans, where a different stimulation frequency might exert the maximum osteogenenic effect owing to the larger size and mass of muscle and bone tissues.

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5. Conclusions This study investigated, at the gene expression and bone formation levels in rats, the frequency dependency of osteogenic capability of electrical muscle stimulation at up to 80 Hz. Stimulation at 20 Hz was the most effective frequency for the promotion of bone formation among those tested. This finding provides useful information for establishing a stimulation frequency in clinical application of electrical muscle stimulation for osteogenesis.

Acknowledgements The authors acknowledge the help of Mr. Shunsuke Kikuchi and Dr. Makiko Kakikawa in performing the gene expression experiments and Mr. Eigo Takahashi for performing the bone histomorphometric analysis in Kanazawa University.

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[DOI: 10.1299/jbse.14-00114]


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