MICROSCOPY RESEARCH AND TECHNIQUE 78:784–791 (2015)
The Osteogenic Effects of Swimming on Bone Mass, Strength, and Microarchitecture in Rats With Unloading-Induced Bone Loss JOSE BATISTA VOLPON,1 ADRIANA VALADARES SILVA,1 MAURICIO JOSE FALCAI,1 MARIO JEFFERSON QUIRINO LOUZADA,2 ARIANE ZAMARIOLI,1* ~ PAULO MARDEGAN ISSA3 BRUNA GABRIELA DOS SANTOS KOTAKE,1 AND JOAO 1 2 3
Department of Biomechanics, Medicine and Rehabilitation, School of Medicine of Ribeir~ ao Preto, University of S~ ao Paulo, Brazil Department of Production and Health Animal, University of S~ ao Paulo State, Brazil Department of Morphology, Physiology and Basic Pathology, University of S~ ao Paulo, Brazil
KEY WORDS
bone tissue; osteoporosis; nonweight-bearing exercise; hindlimb suspension
ABSTRACT The effect of nonweight-bearing exercise on osteoporotic bones remains controversial and inconclusive. The purpose of this study was to evaluate the effects of swimming on osteoporotic tibias of rats submitted to hindlimb suspension. Initially, 20 Wistar rats were used to confirm a significant bone loss following 21 days of unloading. Thirty rats were then divided into 3 groups and followed during 51 days: CON (nonsuspended rats), S 1 WB (suspended rats for 21 days and then released for regular weight-bearing) and, S 1 Swim (suspended rats for 21 days and then released from suspension and submitted to swimming exercise). We observed that swimming exercise was effective at fully recovering the bone deterioration caused by suspension, with significant increments in BMD, bone strength and bone volume. On the other hand, regular weight-bearing failed at fully restoring the bone loss induced by unloading. These results indicate that swimming exercise may be a potential tool to improve bone density, strength, and trabecular volume in tibias with bone loss induced by mechanical unloading in suspended rats. We conclude that this modality of activity could be beneficial in improving bone mass, strength, and architecture in osteoporotic individuals induced by disuse, such as bed rest or those exposed to microgravity, who may not be able to perform weight-bearing exercises. Microsc. Res. Tech. 78:784–791, 2015. V 2015 Wiley Periodicals, Inc. C
INTRODUCTION Bone interacts with internal and external environments and adapts its microstructure to meet mechanical demands via a mechanosensory mechanism. Therefore, the skeleton normally reaches the best mass and resistance required for proper functioning (Cowin, 1998). The decrease or absence of mechanical loading results in bone loss, with architectural disarray, tissue deterioration, and osteopenia (Ohira et al., 2006), leading to increased bone fragility. This correlation is commonly observed in clinical conditions associated with reduced motor function, such as spinal cord injury (Kiratli et al., 2000; McCarthy et al., 2012), stroke sequela, cerebral palsy (Stark et al., 2010), and in patients exposed to prolonged bed rest (Leblanc et al., 1990) or temporary immobilized in plaster casts (Uhthoff et al., 1985). Additionally, microgravity conditions such as those experienced by astronauts induce significant bone mass reduction, which may not be completely recovered even after six months back on Earth (Vico et al., 2000). Osteoporosis can be treated by pharmacological agents, which favorably affect bone tissue metabolism (Migliore et al., 2013), combined with a healthy lifestyle, which includes a balanced diet, avoidance of harmful substances and habits (e.g., smoking) and regC V
2015 WILEY PERIODICALS, INC.
ular physical activities. Increases on mechanical loading demand lead to augment in bone formation, where bone becomes stronger (Baxter-Jones et al., 2008; Karlsson et al., 2008). Compared with nonimpact exercises such as swimming and cycling, high-impact exercise seems to be more osteogenic due to increased mechanical load, both in physically immature individuals and in the elderly population (Gomez-Bruton et al., 2013; Guadalupe-Grau et al., 2009). In agreement, previous studies have shown that gymnasts, tract runners, soccer, volleyball, and basketball players have higher bone mass than swimmers, which confirms the more effectiveness of weight-bearing activity in increasing bone mass (Bassey and Ramsdale, 1995; Duncan et al., 2002; Lee et al., 1995; Taaffe et al., 1995). However, the effects of swimming at improving bone quality *Correspondence to: Ariane Zamarioli, Ph.D., Department of Biomechanics, Medicine and Rehabilitation, School of Medicine of Ribeir~ ao Preto, University of Sao Paulo, Brazil. E-mail: [email protected] Received 12 February 2015; accepted in revised form 16 June 2015 Conflict of interest: The authors declare that they have no conflict of interest. REVIEW EDITOR: Prof. George Perry Contract grant sponsor: Foundation for Research Support of the State of S~ ao Paulo (FAPESP), Brazil. DOI 10.1002/jemt.22541 Published online 15 July 2015 in Wiley Online Library (wileyonlinelibrary.com).
EFFECT OF SWIMMING ON OSTEOPOROTIC BONE TISSUE
remain controversial (Gomez-Bruton et al., 2013; Huang et al., 2003). A study evaluating the effects of a water-based exercise program for osteopenic women concluded that the intervention successfully improved functional fitness, but had no effect on the skeletal system (Bravo et al., 1997). Furthermore, a recent systematic review identified 55 published articles involving studies of the effects of swimming on the bone tissue. The authors demonstrated that nearly 30% of these studies showed a positive effect of swimming on bone quality, whereas 47% observed no effect of swimming on bone tissue (Gomez-Bruton et al., 2013). Several animal studies have been performed to clarify the controversial association between swimming and osteogenic stimulation. Huang et al. (2003) compared the effects of swimming and running on the bone quality of maturing rats and revealed that both methods increased bone strength in tibias and femurs, as well as bone density. Although the bone mineral density (BMD) increment was greater in the running group, swimming activity exerted a beneficial effect on bone structure. Furthermore, swimming activities are shown to play a positive effect at reverting prednisolone-induced osteoporosis in aging rats (Swissa-Sivan et al., 1992), ovariectomized rats (Melton et al., 2004), and aging female mice (Hoshi et al., 1998), likely due to the bone stimuli caused by muscle contraction. However, the effect of nonweight-bearing exercise on osteoporotic bones remains inconclusive. The purpose of this study was to evaluate the effects of swimming on osteoporotic tibias of rats submitted to hindlimb suspension. In spite of the scientific evidence of swimming as a beneficial agent against bone deterioration, its effect on bone tissue remain inconclusive due to the difficulty to compare the experimental results existent in the literature. Several and different methods were used to induce bone deterioration at varying levels in animals of different ages and gender, as well as different exercise protocols and different methods for bone evaluation. Tail suspension is a well-established model of disuse osteoporosis in rats, which leads to unloading and hypoactivity thus inducing bone loss in the hindlimb bones (Morey-Holton and Globus, 2002). Furthermore, this method may be reversed with reloading, allowing investigations regarding the effects of physical exercises on the treatment of bone loss induced by suspension. Thus, this study aimed to assess the effectiveness of swimming in reverting the osteoporosis induced by tail suspension in rats. METHODS Animals In this study, we used 50 female young adult Wistar rats, with body mass of 252 g (64.24). Our experiments were conducted in accordance with the recommendations of the Guide for the Care and Use of Laboratory Animals, which were approved by the Institutional Ethics Committee for Animal Experimentation. The rats were allocated in cages (four rats per cage) and allowed free access to water and food. All rats were kept in a quiet environment with a 12-h lightdark cycle, at a temperature of 22 6 28C and air humidity of 55 6 10%. Rats were randomly divided into five groups (n 5 10/group): (1): (CON1); nonsusMicroscopy Research and Technique
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pended animals allocated in regular cages for 21 days; (2): (S); rats submitted to suspension for 21 consecutive days; (3): (CON2); nonsuspended animals allocated in regular cages for 51 days; (4): (S1WB); suspended animals for 21 consecutive days and released into regular cages for the remaining 30 days and; (5): (S 1 Swim); suspended rats for 21 consecutive days and then submitted to swimming exercise in the remaining 30 days. Procedures Suspension Technique. Rat tail suspension was carried out using the Morey-Holton and Globus method (Morey-Holton and Globus, 2002). Briefly, tail was cleansed under gentle manual restraint of the animal and without anesthetic use. A 20% tincture of benzoin (Styrax spp; Rioquımica, Rio de Janeiro, RJ, Brazil) was applied for skin protection. Subsequently, a self-adhering foam strip (Reston 1560M, 3M, Sumare, SP, Brazil) was applied from the base to the proximal two-thirds of the tail, so as form a distal loop, leaving the end of the tail free. A bandage wrapping was applied to the foam. The suspension was performed in a special cage (width 35.0 cm and height 21.5 cm) with an adjustable transversal bar fixed in its upper part, from which to hang the rat. The loop formed by the foam strip was connected to a metal swivel that was attached to the transversal bar. The rod height was adjusted to keep the animal suspended with the trunk tilted 308. This position allowed forelimb floor locomotion, but hindlimbs could not touch the floor or cage walls. The swivel allowed the animal to rotate and move enough to reach water and food. Throughout the suspension period, animals were examined on a daily basis to identify injuries, painful, or stressing conditions, for example, grooming disturbance, mobility, eating, drinking, hair loss, and porphyrin pigments on face. Swimming Protocol. Swimming activity was performed in a rectangular tank with a base measuring 100 cm 3 60 cm and a height of 60 cm (filled with water to a height of 40 cm). Water temperature was approximately 328C and animals were continuously monitored. Animals were simply placed in water, after which they spontaneously started swimming to avoid drowning. No weights were attached to the rats. There was no surface for resting, no resting period, and no need to stimulate the animals. After swimming, rats were dried with warm air jet. Protocol training consisted of 20 swimming sessions of 60 min each, five days/week for four weeks (Renno et al., 2007). Experimental Analysis. At the end of each experimental period, rats were euthanized with intraperitoneal lethal dose of sodium thiopental. Afterwards, animals were weighed, right and left tibias were removed, and cleaned of soft tissues, whence fibulas were excised. Left tibias were stored in a freezer for subsequent densitometry and mechanical testing, while the right tibias were fixed in neutral formalin for 24 h and then reserved for histological processing. BMD was measured in left tibias using the dualenergy absorptiometry technique (DXA, Lunar – X-ray Bone Densitometer, DPX-alpha with SmartScan, version 4.7, Madison, WI), which provided high-resolution images. As the BMD apparatus is designed for human patients, a special software was adapted for small
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Day 0
Day 21
252 282.5 SD 6 3.59 P < 0.005 112.1%
TABLE 1. Mean body mass (g) among the different groups CON2 S 1 WB
S Day 0
Day 21
249.2 220.8 SD 6 1.40 P < 0.005 211.39%
Day 0
Day 21
252.5 267.9 SD 6 1.4 P < 0.005 16.09%
S 1 Swim
Day 51
Day 0
Day 21
Day 51
Day 0
Day 21
Day 51
312.9 SD 6 8.35 P < 0.005 116.79%
255.2 SD 6 9.8
219.5
272.7
252.3 SD 6 10.2
SD, 6 7.7 P < 0.005 213.79%
SD, 67.6 P < 0.005 124.23%
213.3 SD 6 4.2 P < 0.005 215.4%
289.1 SD 6 6.4 P < 0.005 135.5%
specimens (Lunar), which provided a scanning reproducibility assessed using root mean square coefficient of variation (RMS-CV) of 4%. First, the whole bone was scanned. Then BMD was measured at the proximal part of the tibia, from the articular surface to the metaphysis, spanning a distance of 7.0 mm. Finally, BMD was measured at the center of the diaphysis over a 2.5-cm-long segment. Following BMD measurement, tibias were stored overnight in a refrigerator dried in order to avoid microdamages to bone structure due to water molecules dilatation. The next day, bones were kept moistened in saline and were allowed to reach a thermal balance with the environment (22 6 28C). All mechanical tests were performed under similar conditions, using universal testing machine EMIC DL10000 (S~ ao Jose dos Pinhais, PR, Brazil) with a 500 N load cell. Load rate was set at 1 mm/min with a 5.0-N preloading force and a 30-second accommodation time. Mechanical test consisted of three-point bending, during which the left tibia extremities rested on two metal supports with a 2.0 cm span, to ensure that diaphysis received the majority of bending force. A vertical force was applied to the center of the posterior face of bone until it fractured. Load and deformation curves were R software (EMIC, recorded in real time and, the TescV S~ ao Jose dos Pinhais, PR, Brazil) was used to obtain ultimate load and stiffness. During the test, specimens were sprayed with saline solution to prevent dehydration. Special attention was paid to the generated curve to identify any change in specimen position or bizarre curves that could indicate lack of reproducibility. For histological studies, right tibias were decalcified in a 0.5 M EDTA solution for 30 days, followed by a quick wash under running water. Dehydration was manually performed with successive passages of alcohol and xylol solutions. Specimens were then embedded in paraffin, cut longitudinally in frontal plane into serial 5-mm slices, and stained with hematoxylin-eosin. For quantification of trabecular bone in proximal region of tibias, 12 histological fields were randomly chosen. The use of 12 photomicrographies of each animal resulted in a total of 120 images per group. Trabecular volume was measured stereologically using the Axiovision program (Zeiss, Germany) and expressed in percentage of total volume (BV/TV), according to Parfitt et al. (Martins-Pinge et al., 2005). Statistical Analyses The software programs GraphPad and Prism version 5.00 (San Diego, CA) were used for the statistical analyses. The D’Agostino-Pearson test was applied for normality, and the homogeneity of variance was examined with Bartlett’s test. Student’s t-test for paramet-
ric data was applied to compare between the S and CON1 groups. For the other comparisons, Fisher’s analysis of variance was used as a rating criterion, along with Tukey’s post-hoc test. All values were considered to be significantly different at P 0.05) on day zero, showing baseline homogeneity of specimens at the beginning of experiment. However, on day 51, mean body mass was significantly different among groups (P < 0.05), whence all rats gained weight along the experiment, but suspended rats did not gain weight at the same degree as control rats. The body mass of CON2 rats (123.9%) was significantly higher than both S 1 WB (16.86%) (P < 0.0001) and S 1 Swim rats (114.59%; P 5 0.0017). No significant difference was shown with regards to the body mass between S1WB and S 1 Swim groups (P 5 0.0660). Deleterious Effects of Suspension on Bone Quality Unloading significantly affected bone quality of rats exposed to 21 days of hindlimb suspension, where significant decreases in bone mass, strength, and volume were observed in the suspended rats when compared with the weight-matched control rats. BMD in the metaphyseal proximal region of tibia was reduced in 17.62% in the S rats (0.145 6 0.0119 g/cm2), when compared with CON1 rats (0.176 6 0.020 g/cm2; P 5 0.019; Fig. 1). Unloading also induced bone weakness; ultimate load was significantly lower in the S group (214.03%, P 5 0.0003; 36.08 6 4.42 N) than in the CON1 group (41.97 6 4.15 N; Fig. 2), as well as stiffness, which was significantly lower (221.68%, P < 0.005) in the S group (45.93 6 11.84 N/mm) than in the CON1 group (58.64 6 7.84 N/mm; Fig. 3). Bone microarchitecture was also negatively affected by 21 days of unloading, where BV/TV ratio was significantly lower (257.2%, P < 0.0001) in the S group (19.81 6 1.3%) than in the CON1 group (46.28 6 7.71%; Figs. 4 and 5). Microscopy Research and Technique
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Fig. 1. BMD comparison among the different groups CON1 5 control rats 1 (21 days); S= rats submitted to tail suspension for 21 days; CON2 5 control 2 (51 days); S 1 WB 5 suspended rats for 21 days and
then allowed regular weight-bearing for 30 days; and S 1 Swim 5 suspended rats for 21 days and then trained for 30 days (swimming). (The vertical line interval expresses the standard deviation).
Fig. 2. Ultimate load comparison among the different groups CON1 5 control rats 1 (21 days); S 5 rats submitted to tail suspension for 21 days; CON2 5 control 2 (51 days); S 1 WB 5 suspended rats for 21
days and then allowed regular weight-bearing for 30 days; and S 1 Swim 5 suspended rats for 21 days and then trained for 30 days (swimming). (The vertical line interval expresses the standard deviation).
Swimming as a Therapeutic Approach to Increase Bone Quality Swimming exercise was shown to have an important effect at improving bone quality, with improvements in bone mass, strength, and volume.
We found that swimming increased by 9.46% (P < 0.05) the BMD in tibias of S 1 Swim rats (0.201 6 0.018 g/cm2) when compared to the S 1 WB rats (0.182 6 0.127 g/cm2). Swimming not only improved bone density, but also completely reverted
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Fig. 3. Stiffness comparison among the different groups CON1 5 control rats 1 (21 days); S 5 rats submitted to tail suspension for 21 days; CON2 5 control 2 (51 days); S 1 WB 5 suspended rats for 21 days and then allowed regular weight-bearing for 30 days; and
S 1 Swim 5 suspended rats for 21 days and then trained for 30 days (swimming). (The vertical line interval expresses the standard deviation).
Fig. 4. Comparison of metaphyseal trabecular bone content (BV/ TV) among the different groups CON1 5 control rats 1 (21 days); S 5 rats submitted to tail suspension for 21 days; CON2 5 control 2
(51 days); S 1 WB 5 suspended rats for 21 days and then allowed regular weight-bearing for 30 days; and S 1 Swim 5 suspended rats for 21 days and then trained for 30 days (swimming).
bone density to values considered normal [no significant difference was found between S 1 Swim (0.201 6 0.018 g/cm2) and CON2 (0.210 6 0.12 g/cm2) groups]. However, BMD in the S 1 WB group (0.182 6 0.127 g/cm2) was significantly lower (213.34%, P < 0.05) than in the CON2 group (0.210 6 0.12 g/cm2;
Fig. 1), which demonstrates the higher efficacy of swimming over weight-bearing activity only. Swimming also showed positive effects in bone strength. The ultimate load in the S 1 Swim group (46.12 6 3.08 N) was significantly higher (110.23, P < 0.05) than in the S 1 WB group (41.40 6 3.37 N) Microscopy Research and Technique
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Fig. 5. Proximal tibia sections stained with HE qualitatively showing loss in trabecular bone after 21 days of suspension (B 5 suspended rats), when compared with age-matched control rats (A 5 Con1). The deleterious effects of suspension on the trabecula
bone were mitigated by swimming exercise (D 5 S 1 Swim), but not by regular activity in cage (C 5 S 1 WB), when compared with agematched control rats (E 5 Con2). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
and statistically similar (P > 0.05) to the CON2 group (48.36 6 4.57 N). Conversely, the ultimate load in S 1 WB group was significantly lower than in the CON2 group (214.4%, P < 0.05; Fig. 2). Similarly to the ultimate load, stiffness in the S 1 Swim group (69.85 6 10.37 N/mm) was also higher (121.91%, P < 0.001) than in the S 1 WB group (54.55 6 10.88 N/ mm). Furthermore, no significant difference was found between S 1 Swim (69.85 6 10.37 N/mm) and CON2 (72.93 6 8.99 N/mm) groups (P > 0.05). Conversely, stiffness in the S 1 WB group (54.55 6 10.88 N/mm) was lower (225.21%, P < 0.005) than in the CON2 group (72.93 6 8.99 N/mm; Fig. 3). The osteogenic effects of swimming exercise were also seen in bone microarchitecture, whence rats that swam showed an increase of 48.82% (P < 0.001) in BV/ TV ratio when compared with rats submitted to regular weight-bearing loading (S 1 Swim: 44.37 6 2.70% vs. S 1 WB: 22.71 6 8.07%). Additionally, no significant difference was found between S 1 Swim and CON2 groups (P > 0.05), whereas BV/TV ratio was significantly lower (252.06%, P < 0.001) in the S 1 WB group than in the CON2 group (Figs. 4 and 5).
Rats, in special, are suitable models to study several aspects of bone quality and repair (Martins-Pinge et al., 2005; Morey-Holton et al., 2002). The rat tail suspension method was originally used to simulate the effects of microgravity on several organs and systems, as experienced by astronauts in space (Morey-Holton et al., 2002); however, it was later accepted as a standard model for the study of bone loss induced by hypoactivity or disuse (Morey-Holton and Globus, 1998; Turner et al., 1979). In this study, hypoactivity secondary to three weeks of tail suspension effectively impaired bone quality, as shown by reduced bone mass, mechanical weakness, and deterioration of trabecular microarchitecture in suspended rats, when compared to the weightmatched controls. These findings are in agreement with previous reports in literature, which state that hypoactivity due to hindlimb suspension quickly leads to osteopenia (Falcai et al., 2012; Shimano and Volpon, 2009). Our histological and BMD data showed severe trabecular bone loss caused by a relatively short duration of suspension. By means of mechanical test, we also demonstrated the impairment of unloading on bone strength, where both ultimate load and stiffness were significantly decreased in animals suspended for three weeks. As the mechanical force was applied to the diaphysis of bones, mainly composed of compact
DISCUSSION Animal models can be used to investigate the main biological mechanisms involved in bone properties. Microscopy Research and Technique
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bone, it is reasonable to accept that compact bone was also affected by hypoactivity. It is well known that increased physical activity triggers structural, metabolic, hormonal, neural, and molecular adaptations, which positively affect muscle force and power (den Hoed et al., 2008). Likewise, the skeleton requires mechanical stimuli to maintain its trophism. Several modalities of bone mechanical stimulation, including jumping and vibration are shown to exert positive effects on the bone tissue. Although exercises requiring greater mechanical loading (e.g., running and jumping) are known to play an important role at increasing bone formation, these modalities of exercise (high-impact exercises) may not be recommended to specific populations, such as elderly or individuals with certain disabilities or systemic disease. To these populations, aquatic exercises are more adequate and beneficial. In a systemic review, Gomez-Bruton et al. (Gomez-Bruton et al., 2013) concluded that swimming, besides playing an osteogenic role, also has the benefit of being a low-impact activity, which is not hazardous to joints and other structures (Bravo et al., 1997). In our study, swimming improved bone quality to a larger level than regular weight-bearing activity. Furthermore, swimming exercise completely reversed the osteopenic condition induced by unloading, whereas spontaneous cage activity (regular weight-bearing) only ameliorated bone loss. Thus, we found that swimming, in spite of being a low-impact exercise, exerted a positive effect at stimulating bone formation and increasing bone quality both on cancellous and lamellar bones, whence trabecular bone was more affected by unloading and reloading, since it is metabolically more active than cortical bone (Smith, 1993). According to DiVasta and Gordon (DiVasta and Gordon, 2013), the mechanisms involved in skeletal changes in response to physical exercise are still unclear. Gains in bone mass due to exercise are related to increased mechanical strain as well as other factors, such as endocrine responses (DiVasta and Gordon, 2013). As swimming involves no impact and minimizes the effects of gravity, it seems reasonable to assume that the positive effects of swimming on bone are due to muscle activity. Biomechanically, muscle transmits efforts to the bone, which causes microdeformations thus stimulating bone turnover by mechanotransduction mechanisms. Through these mechanisms, mechanical stimulus is converted into biological responses that stimulates bone formation and/or inhibits bone resorption. In bone, this process is initiated by mechanical stimuli caused by gravity, physical impact, vibration or muscular action. The pathways involved in this process have not yet been completely elucidated. Initially, microdeformations create a pressure gradient in the canalicular system of bone, which leads to variation in the flow of pericellular space fluids. These flow variations are transferred to the osteocyte, which seems to be the main mechanosensor of bone (Duncan and Turner 1995) as they release biochemical mediators (e.g., prostaglandins) and regulate the activity of osteoblasts and osteoclasts (Klein-Nulend et al., 1995). Transmission to the effector cells may be achieved through many substances (Robling, 2009), such as nitrous acid, prostaglandins, sclerostin, IGF
(Hamrick, 2012), TGF-beta, RANK, and OPG (Bergmann et al., 2010). The Wnt signaling pathway has recently been identified as a mediator related to bone homeostasis (Gaudio et al., 2010; Morse et al., 2013), which is essential for osteoblastic differentiation. Wnt binds to a coreceptor complex that includes the Frizzled receptor and low-density lipoprotein receptorrelated protein (LRP)25 or 26, which are both present on osteoblasts. This binding stabilizes cytoplasmic bcatenin and causes its translocation to the nucleus, activating the transcription of genes that promote osteoblast proliferation, differentiation, and function, and thus resulting in bone formation (Morse et al., 2013). Conversely, sclerostin, which is upregulated in response to mechanical unloading, inhibits the Wnt pathway through competitive binding to LRP5/6 (Armstrong et al., 2007; Kim et al., 2013; Li et al., 2005; Miyagawa et al., 2011; Robling et al., 2008). Thus, mechanical unloading causes decreased Wnt/beta-catenin signaling activity as well as sclerostin upregulation, which are associated with lower bone mineral content and density (Morse et al., 2013). Conversely, sclerostin upregulation may be reversed by reloading (Morse et al., 2013), whence mechanical bone stimulation reduces the number of SOST, particularly in highstrain regions of the bone. Previous authors have shown a 73% reduction of sclerostin mRNA levels in the ulnar diaphysis of mice submitted to mechanical loading (Robling et al., 2008). During exercise, tissue deformation occurs and stretches resident bone cells (e.g., osteocyte), which results in the expression of several genes involved in mechanotransduction and osteoblast/osteoclast differentiation, thus increasing bone formation (i.e., IGF, FGF, MMP, BMP, osteocalcin) and decreasing bone resorption (i.e., osteoprogeterin, IL, sclerostin, and TRAP). We concluded that swimming exercise completely revert the deleterious effects of three weeks of unloading in tibias of rats, whence bone mass, strength and microarchitecture structure were fully restored to normal levels. Furthermore, we believe the osteogenic effect of swimming exercise was triggered by muscular action, as aquatic activities have no mechanical impact, thus confirming the important role of muscle at stimulating bone formation. ACKNOWLEDGMENTS The authors declare that they have no conflict of interest, no professional relationships with companies or manufacturers who will benefit from the results of this study. REFERENCES Armstrong VJ, Muzylak M, Sunters A, Zaman G, Saxon LK, Price JS, Lanyon LE. 2007. Wnt/beta-catenin signaling is a component of osteoblastic bone cell early responses to load-bearing and requires estrogen receptor alpha. J Biol Chem 282:20715–20727. Bassey EJ, Ramsdale SJ. 1995. Weight-bearing exercise and ground reaction forces: a 12-month randomized controlled trial of effects on bone mineral density in healthy postmenopausal women. Bone 16:469–476. Baxter-Jones AD, Kontulainen SA, Faulkner RA, Bailey DA. 2008. A longitudinal study of the relationship of physical activity to bone mineral accrual from adolescence to young adulthood. Bone 43: 1101–1107. Bergmann P, Body JJ, Boonen S, Boutsen Y, Devogelaer JP, Goemaere S, Kaufman J, Reginster JY, Rozenberg S. 2010. Loading
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McCarthy ID, Bloomer Z, Gall A, Keen R, Ferguson-Pell M. 2012. Changes in the structural and material properties of the tibia in patients with spinal cord injury. Spinal Cord 50:333–337. Melton SA, Hegsted M, Keenan MJ, Morris GS, O’Neil CE, ZablahPimentel EM. 2004. Water exercise prevents femur density loss associated with ovariectomy in the retired breeder rat. J Strength Cond Res 18:508–512. Migliore A, Broccoli S, Massafra U, Cassol M, Frediani B. 2013. Ranking antireabsorptive agents to prevent vertebral fractures in postmenopausal osteoporosis by mixed treatment comparison meta-analysis. Eur Rev Med Pharmacol Sci 17:658–667. Miyagawa K, Kozai Y, Ito Y, Furuhama T, Naruse K, Nonaka K, Nagai Y, Yamato H, Kashima I, Ohya K, Aoki K, Mikuni-Takagaki Y. 2011. A novel underuse model shows that inactivity but not ovariectomy determines the deteriorated material properties and geometry of cortical bone in the tibia of adult rats. J Bone Miner Metab 29:422–436. Morey-Holton E, Globus RK, Kaplansky A, Durnova G. 2005. The hindlimb unloading rat model: literature overview, technique update and comparison with space flight data. Adv Space Biol Med 10:7–40. Morey-Holton ER, Globus RK. 1998. Hindlimb unloading of growing rats: a model for predicting skeletal changes during space flight. Bone 22:83S–88S. Morey-Holton ER, Globus RK. 2002. Hindlimb unloading rodent model: technical aspects. J Appl Physiol (1985) 92:1367–1377. Morse LR, Sudhakar S, Lazzari AA, Tun C, Garshick E, Zafonte R, Battaglino RA. 2013. Sclerostin: a candidate biomarker of SCIinduced osteoporosis. Osteoporos Int 24:961–968. Ohira Y, Kawano F, Wang XD, Sudoh M, Iwashita Y, Majima HJ, Nonaka I. 2006. Irreversible morphological changes in leg bone following chronic gravitational unloading of growing rats. Life Sci 79: 686–694. Renno AC, Silveira Gomes AR, Nascimento RB, Salvini T, Parizoto N. 2007. Effects of a progressive loading exercise program on the bone and skeletal muscle properties of female osteopenic rats. Exp Gerontol 42:517–522. Robling AG. 2009. Is bone’s response to mechanical signals dominated by muscle forces? Med Sci Sports Exerc 41:2044–2049. Robling AG, Niziolek PJ, Baldridge LA, Condon KW, Allen MR, Alam I, Mantila SM, Gluhak-Heinrich J, Bellido TM, Harris SE, Turner CH. 2008. Mechanical stimulation of bone in vivo reduces osteocyte expression of Sost/sclerostin. J Biol Chem 283:5866– 5875. Shimano MM, Volpon JB. 2009. Biomechanics and structural adaptations of the rat femur after hindlimb suspension and treadmill running. Braz J Med Biol Res 42:330–338. Smith R. 1993. Bone physiology and the osteoporotic process. Respir Med 87:3–7. Stark C, Nikopoulou-Smyrni P, Stabrey A, Semler O, Schoenau E. 2010. Effect of a new physiotherapy concept on bone mineral density, muscle force and gross motor function in children with bilateral cerebral palsy. J Musculoskelet Neuronal Interact 10:151– 158. Swissa-Sivan A, Statter M, Brooks GA, Azevedo J, Viguie C, Azoury R, Greenfield C, Oman S, Leichter I, Zinker BA and et al. 1992. Effect of swimming on prednisolone-induced osteoporosis in elderly rats. J Bone Miner Res 7:161–169. Taaffe DR, Snow-Harter C, Connolly DA, Robinson TL, Brown MD, Marcus R. 1995. Differential effects of swimming versus weightbearing activity on bone mineral status of eumenorrheic athletes. J Bone Miner Res 10:586–593. Turner RT, Morey ER, Liu C, Baylink DJ. 1979. Altered bone turnover during spaceflight. Physiologist 22:S73274. Uhthoff HK, Sekaly G, Jaworski ZF. 1985. Effect of long-term nontraumatic immobilization on metaphyseal spongiosa in young adult and old beagle dogs. Clin Orthop Relat Res 278–283. Vico L, Collet P, Guignandon A, Lafage-Proust MH, Thomas T, Rehaillia M, Alexandre C. 2000. Effects of long-term microgravity exposure on cancellous and cortical weight-bearing bones of cosmonauts. Lancet 355:1607–1611.
The Osteogenic Effects of Swimming on Bone Mass, Strength, and Microarchitecture in Rats With Unloading-Induced Bone Loss JOSE BATISTA VOLPON,1 ADRIANA VALADARES SILVA,1 MAURICIO JOSE FALCAI,1 MARIO JEFFERSON QUIRINO LOUZADA,2 ARIANE ZAMARIOLI,1* ~ PAULO MARDEGAN ISSA3 BRUNA GABRIELA DOS SANTOS KOTAKE,1 AND JOAO 1 2 3
Department of Biomechanics, Medicine and Rehabilitation, School of Medicine of Ribeir~ ao Preto, University of S~ ao Paulo, Brazil Department of Production and Health Animal, University of S~ ao Paulo State, Brazil Department of Morphology, Physiology and Basic Pathology, University of S~ ao Paulo, Brazil
KEY WORDS
bone tissue; osteoporosis; nonweight-bearing exercise; hindlimb suspension
ABSTRACT The effect of nonweight-bearing exercise on osteoporotic bones remains controversial and inconclusive. The purpose of this study was to evaluate the effects of swimming on osteoporotic tibias of rats submitted to hindlimb suspension. Initially, 20 Wistar rats were used to confirm a significant bone loss following 21 days of unloading. Thirty rats were then divided into 3 groups and followed during 51 days: CON (nonsuspended rats), S 1 WB (suspended rats for 21 days and then released for regular weight-bearing) and, S 1 Swim (suspended rats for 21 days and then released from suspension and submitted to swimming exercise). We observed that swimming exercise was effective at fully recovering the bone deterioration caused by suspension, with significant increments in BMD, bone strength and bone volume. On the other hand, regular weight-bearing failed at fully restoring the bone loss induced by unloading. These results indicate that swimming exercise may be a potential tool to improve bone density, strength, and trabecular volume in tibias with bone loss induced by mechanical unloading in suspended rats. We conclude that this modality of activity could be beneficial in improving bone mass, strength, and architecture in osteoporotic individuals induced by disuse, such as bed rest or those exposed to microgravity, who may not be able to perform weight-bearing exercises. Microsc. Res. Tech. 78:784–791, 2015. V 2015 Wiley Periodicals, Inc. C
INTRODUCTION Bone interacts with internal and external environments and adapts its microstructure to meet mechanical demands via a mechanosensory mechanism. Therefore, the skeleton normally reaches the best mass and resistance required for proper functioning (Cowin, 1998). The decrease or absence of mechanical loading results in bone loss, with architectural disarray, tissue deterioration, and osteopenia (Ohira et al., 2006), leading to increased bone fragility. This correlation is commonly observed in clinical conditions associated with reduced motor function, such as spinal cord injury (Kiratli et al., 2000; McCarthy et al., 2012), stroke sequela, cerebral palsy (Stark et al., 2010), and in patients exposed to prolonged bed rest (Leblanc et al., 1990) or temporary immobilized in plaster casts (Uhthoff et al., 1985). Additionally, microgravity conditions such as those experienced by astronauts induce significant bone mass reduction, which may not be completely recovered even after six months back on Earth (Vico et al., 2000). Osteoporosis can be treated by pharmacological agents, which favorably affect bone tissue metabolism (Migliore et al., 2013), combined with a healthy lifestyle, which includes a balanced diet, avoidance of harmful substances and habits (e.g., smoking) and regC V
2015 WILEY PERIODICALS, INC.
ular physical activities. Increases on mechanical loading demand lead to augment in bone formation, where bone becomes stronger (Baxter-Jones et al., 2008; Karlsson et al., 2008). Compared with nonimpact exercises such as swimming and cycling, high-impact exercise seems to be more osteogenic due to increased mechanical load, both in physically immature individuals and in the elderly population (Gomez-Bruton et al., 2013; Guadalupe-Grau et al., 2009). In agreement, previous studies have shown that gymnasts, tract runners, soccer, volleyball, and basketball players have higher bone mass than swimmers, which confirms the more effectiveness of weight-bearing activity in increasing bone mass (Bassey and Ramsdale, 1995; Duncan et al., 2002; Lee et al., 1995; Taaffe et al., 1995). However, the effects of swimming at improving bone quality *Correspondence to: Ariane Zamarioli, Ph.D., Department of Biomechanics, Medicine and Rehabilitation, School of Medicine of Ribeir~ ao Preto, University of Sao Paulo, Brazil. E-mail: [email protected] Received 12 February 2015; accepted in revised form 16 June 2015 Conflict of interest: The authors declare that they have no conflict of interest. REVIEW EDITOR: Prof. George Perry Contract grant sponsor: Foundation for Research Support of the State of S~ ao Paulo (FAPESP), Brazil. DOI 10.1002/jemt.22541 Published online 15 July 2015 in Wiley Online Library (wileyonlinelibrary.com).
EFFECT OF SWIMMING ON OSTEOPOROTIC BONE TISSUE
remain controversial (Gomez-Bruton et al., 2013; Huang et al., 2003). A study evaluating the effects of a water-based exercise program for osteopenic women concluded that the intervention successfully improved functional fitness, but had no effect on the skeletal system (Bravo et al., 1997). Furthermore, a recent systematic review identified 55 published articles involving studies of the effects of swimming on the bone tissue. The authors demonstrated that nearly 30% of these studies showed a positive effect of swimming on bone quality, whereas 47% observed no effect of swimming on bone tissue (Gomez-Bruton et al., 2013). Several animal studies have been performed to clarify the controversial association between swimming and osteogenic stimulation. Huang et al. (2003) compared the effects of swimming and running on the bone quality of maturing rats and revealed that both methods increased bone strength in tibias and femurs, as well as bone density. Although the bone mineral density (BMD) increment was greater in the running group, swimming activity exerted a beneficial effect on bone structure. Furthermore, swimming activities are shown to play a positive effect at reverting prednisolone-induced osteoporosis in aging rats (Swissa-Sivan et al., 1992), ovariectomized rats (Melton et al., 2004), and aging female mice (Hoshi et al., 1998), likely due to the bone stimuli caused by muscle contraction. However, the effect of nonweight-bearing exercise on osteoporotic bones remains inconclusive. The purpose of this study was to evaluate the effects of swimming on osteoporotic tibias of rats submitted to hindlimb suspension. In spite of the scientific evidence of swimming as a beneficial agent against bone deterioration, its effect on bone tissue remain inconclusive due to the difficulty to compare the experimental results existent in the literature. Several and different methods were used to induce bone deterioration at varying levels in animals of different ages and gender, as well as different exercise protocols and different methods for bone evaluation. Tail suspension is a well-established model of disuse osteoporosis in rats, which leads to unloading and hypoactivity thus inducing bone loss in the hindlimb bones (Morey-Holton and Globus, 2002). Furthermore, this method may be reversed with reloading, allowing investigations regarding the effects of physical exercises on the treatment of bone loss induced by suspension. Thus, this study aimed to assess the effectiveness of swimming in reverting the osteoporosis induced by tail suspension in rats. METHODS Animals In this study, we used 50 female young adult Wistar rats, with body mass of 252 g (64.24). Our experiments were conducted in accordance with the recommendations of the Guide for the Care and Use of Laboratory Animals, which were approved by the Institutional Ethics Committee for Animal Experimentation. The rats were allocated in cages (four rats per cage) and allowed free access to water and food. All rats were kept in a quiet environment with a 12-h lightdark cycle, at a temperature of 22 6 28C and air humidity of 55 6 10%. Rats were randomly divided into five groups (n 5 10/group): (1): (CON1); nonsusMicroscopy Research and Technique
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pended animals allocated in regular cages for 21 days; (2): (S); rats submitted to suspension for 21 consecutive days; (3): (CON2); nonsuspended animals allocated in regular cages for 51 days; (4): (S1WB); suspended animals for 21 consecutive days and released into regular cages for the remaining 30 days and; (5): (S 1 Swim); suspended rats for 21 consecutive days and then submitted to swimming exercise in the remaining 30 days. Procedures Suspension Technique. Rat tail suspension was carried out using the Morey-Holton and Globus method (Morey-Holton and Globus, 2002). Briefly, tail was cleansed under gentle manual restraint of the animal and without anesthetic use. A 20% tincture of benzoin (Styrax spp; Rioquımica, Rio de Janeiro, RJ, Brazil) was applied for skin protection. Subsequently, a self-adhering foam strip (Reston 1560M, 3M, Sumare, SP, Brazil) was applied from the base to the proximal two-thirds of the tail, so as form a distal loop, leaving the end of the tail free. A bandage wrapping was applied to the foam. The suspension was performed in a special cage (width 35.0 cm and height 21.5 cm) with an adjustable transversal bar fixed in its upper part, from which to hang the rat. The loop formed by the foam strip was connected to a metal swivel that was attached to the transversal bar. The rod height was adjusted to keep the animal suspended with the trunk tilted 308. This position allowed forelimb floor locomotion, but hindlimbs could not touch the floor or cage walls. The swivel allowed the animal to rotate and move enough to reach water and food. Throughout the suspension period, animals were examined on a daily basis to identify injuries, painful, or stressing conditions, for example, grooming disturbance, mobility, eating, drinking, hair loss, and porphyrin pigments on face. Swimming Protocol. Swimming activity was performed in a rectangular tank with a base measuring 100 cm 3 60 cm and a height of 60 cm (filled with water to a height of 40 cm). Water temperature was approximately 328C and animals were continuously monitored. Animals were simply placed in water, after which they spontaneously started swimming to avoid drowning. No weights were attached to the rats. There was no surface for resting, no resting period, and no need to stimulate the animals. After swimming, rats were dried with warm air jet. Protocol training consisted of 20 swimming sessions of 60 min each, five days/week for four weeks (Renno et al., 2007). Experimental Analysis. At the end of each experimental period, rats were euthanized with intraperitoneal lethal dose of sodium thiopental. Afterwards, animals were weighed, right and left tibias were removed, and cleaned of soft tissues, whence fibulas were excised. Left tibias were stored in a freezer for subsequent densitometry and mechanical testing, while the right tibias were fixed in neutral formalin for 24 h and then reserved for histological processing. BMD was measured in left tibias using the dualenergy absorptiometry technique (DXA, Lunar – X-ray Bone Densitometer, DPX-alpha with SmartScan, version 4.7, Madison, WI), which provided high-resolution images. As the BMD apparatus is designed for human patients, a special software was adapted for small
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Day 0
Day 21
252 282.5 SD 6 3.59 P < 0.005 112.1%
TABLE 1. Mean body mass (g) among the different groups CON2 S 1 WB
S Day 0
Day 21
249.2 220.8 SD 6 1.40 P < 0.005 211.39%
Day 0
Day 21
252.5 267.9 SD 6 1.4 P < 0.005 16.09%
S 1 Swim
Day 51
Day 0
Day 21
Day 51
Day 0
Day 21
Day 51
312.9 SD 6 8.35 P < 0.005 116.79%
255.2 SD 6 9.8
219.5
272.7
252.3 SD 6 10.2
SD, 6 7.7 P < 0.005 213.79%
SD, 67.6 P < 0.005 124.23%
213.3 SD 6 4.2 P < 0.005 215.4%
289.1 SD 6 6.4 P < 0.005 135.5%
specimens (Lunar), which provided a scanning reproducibility assessed using root mean square coefficient of variation (RMS-CV) of 4%. First, the whole bone was scanned. Then BMD was measured at the proximal part of the tibia, from the articular surface to the metaphysis, spanning a distance of 7.0 mm. Finally, BMD was measured at the center of the diaphysis over a 2.5-cm-long segment. Following BMD measurement, tibias were stored overnight in a refrigerator dried in order to avoid microdamages to bone structure due to water molecules dilatation. The next day, bones were kept moistened in saline and were allowed to reach a thermal balance with the environment (22 6 28C). All mechanical tests were performed under similar conditions, using universal testing machine EMIC DL10000 (S~ ao Jose dos Pinhais, PR, Brazil) with a 500 N load cell. Load rate was set at 1 mm/min with a 5.0-N preloading force and a 30-second accommodation time. Mechanical test consisted of three-point bending, during which the left tibia extremities rested on two metal supports with a 2.0 cm span, to ensure that diaphysis received the majority of bending force. A vertical force was applied to the center of the posterior face of bone until it fractured. Load and deformation curves were R software (EMIC, recorded in real time and, the TescV S~ ao Jose dos Pinhais, PR, Brazil) was used to obtain ultimate load and stiffness. During the test, specimens were sprayed with saline solution to prevent dehydration. Special attention was paid to the generated curve to identify any change in specimen position or bizarre curves that could indicate lack of reproducibility. For histological studies, right tibias were decalcified in a 0.5 M EDTA solution for 30 days, followed by a quick wash under running water. Dehydration was manually performed with successive passages of alcohol and xylol solutions. Specimens were then embedded in paraffin, cut longitudinally in frontal plane into serial 5-mm slices, and stained with hematoxylin-eosin. For quantification of trabecular bone in proximal region of tibias, 12 histological fields were randomly chosen. The use of 12 photomicrographies of each animal resulted in a total of 120 images per group. Trabecular volume was measured stereologically using the Axiovision program (Zeiss, Germany) and expressed in percentage of total volume (BV/TV), according to Parfitt et al. (Martins-Pinge et al., 2005). Statistical Analyses The software programs GraphPad and Prism version 5.00 (San Diego, CA) were used for the statistical analyses. The D’Agostino-Pearson test was applied for normality, and the homogeneity of variance was examined with Bartlett’s test. Student’s t-test for paramet-
ric data was applied to compare between the S and CON1 groups. For the other comparisons, Fisher’s analysis of variance was used as a rating criterion, along with Tukey’s post-hoc test. All values were considered to be significantly different at P 0.05) on day zero, showing baseline homogeneity of specimens at the beginning of experiment. However, on day 51, mean body mass was significantly different among groups (P < 0.05), whence all rats gained weight along the experiment, but suspended rats did not gain weight at the same degree as control rats. The body mass of CON2 rats (123.9%) was significantly higher than both S 1 WB (16.86%) (P < 0.0001) and S 1 Swim rats (114.59%; P 5 0.0017). No significant difference was shown with regards to the body mass between S1WB and S 1 Swim groups (P 5 0.0660). Deleterious Effects of Suspension on Bone Quality Unloading significantly affected bone quality of rats exposed to 21 days of hindlimb suspension, where significant decreases in bone mass, strength, and volume were observed in the suspended rats when compared with the weight-matched control rats. BMD in the metaphyseal proximal region of tibia was reduced in 17.62% in the S rats (0.145 6 0.0119 g/cm2), when compared with CON1 rats (0.176 6 0.020 g/cm2; P 5 0.019; Fig. 1). Unloading also induced bone weakness; ultimate load was significantly lower in the S group (214.03%, P 5 0.0003; 36.08 6 4.42 N) than in the CON1 group (41.97 6 4.15 N; Fig. 2), as well as stiffness, which was significantly lower (221.68%, P < 0.005) in the S group (45.93 6 11.84 N/mm) than in the CON1 group (58.64 6 7.84 N/mm; Fig. 3). Bone microarchitecture was also negatively affected by 21 days of unloading, where BV/TV ratio was significantly lower (257.2%, P < 0.0001) in the S group (19.81 6 1.3%) than in the CON1 group (46.28 6 7.71%; Figs. 4 and 5). Microscopy Research and Technique
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Fig. 1. BMD comparison among the different groups CON1 5 control rats 1 (21 days); S= rats submitted to tail suspension for 21 days; CON2 5 control 2 (51 days); S 1 WB 5 suspended rats for 21 days and
then allowed regular weight-bearing for 30 days; and S 1 Swim 5 suspended rats for 21 days and then trained for 30 days (swimming). (The vertical line interval expresses the standard deviation).
Fig. 2. Ultimate load comparison among the different groups CON1 5 control rats 1 (21 days); S 5 rats submitted to tail suspension for 21 days; CON2 5 control 2 (51 days); S 1 WB 5 suspended rats for 21
days and then allowed regular weight-bearing for 30 days; and S 1 Swim 5 suspended rats for 21 days and then trained for 30 days (swimming). (The vertical line interval expresses the standard deviation).
Swimming as a Therapeutic Approach to Increase Bone Quality Swimming exercise was shown to have an important effect at improving bone quality, with improvements in bone mass, strength, and volume.
We found that swimming increased by 9.46% (P < 0.05) the BMD in tibias of S 1 Swim rats (0.201 6 0.018 g/cm2) when compared to the S 1 WB rats (0.182 6 0.127 g/cm2). Swimming not only improved bone density, but also completely reverted
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Fig. 3. Stiffness comparison among the different groups CON1 5 control rats 1 (21 days); S 5 rats submitted to tail suspension for 21 days; CON2 5 control 2 (51 days); S 1 WB 5 suspended rats for 21 days and then allowed regular weight-bearing for 30 days; and
S 1 Swim 5 suspended rats for 21 days and then trained for 30 days (swimming). (The vertical line interval expresses the standard deviation).
Fig. 4. Comparison of metaphyseal trabecular bone content (BV/ TV) among the different groups CON1 5 control rats 1 (21 days); S 5 rats submitted to tail suspension for 21 days; CON2 5 control 2
(51 days); S 1 WB 5 suspended rats for 21 days and then allowed regular weight-bearing for 30 days; and S 1 Swim 5 suspended rats for 21 days and then trained for 30 days (swimming).
bone density to values considered normal [no significant difference was found between S 1 Swim (0.201 6 0.018 g/cm2) and CON2 (0.210 6 0.12 g/cm2) groups]. However, BMD in the S 1 WB group (0.182 6 0.127 g/cm2) was significantly lower (213.34%, P < 0.05) than in the CON2 group (0.210 6 0.12 g/cm2;
Fig. 1), which demonstrates the higher efficacy of swimming over weight-bearing activity only. Swimming also showed positive effects in bone strength. The ultimate load in the S 1 Swim group (46.12 6 3.08 N) was significantly higher (110.23, P < 0.05) than in the S 1 WB group (41.40 6 3.37 N) Microscopy Research and Technique
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Fig. 5. Proximal tibia sections stained with HE qualitatively showing loss in trabecular bone after 21 days of suspension (B 5 suspended rats), when compared with age-matched control rats (A 5 Con1). The deleterious effects of suspension on the trabecula
bone were mitigated by swimming exercise (D 5 S 1 Swim), but not by regular activity in cage (C 5 S 1 WB), when compared with agematched control rats (E 5 Con2). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
and statistically similar (P > 0.05) to the CON2 group (48.36 6 4.57 N). Conversely, the ultimate load in S 1 WB group was significantly lower than in the CON2 group (214.4%, P < 0.05; Fig. 2). Similarly to the ultimate load, stiffness in the S 1 Swim group (69.85 6 10.37 N/mm) was also higher (121.91%, P < 0.001) than in the S 1 WB group (54.55 6 10.88 N/ mm). Furthermore, no significant difference was found between S 1 Swim (69.85 6 10.37 N/mm) and CON2 (72.93 6 8.99 N/mm) groups (P > 0.05). Conversely, stiffness in the S 1 WB group (54.55 6 10.88 N/mm) was lower (225.21%, P < 0.005) than in the CON2 group (72.93 6 8.99 N/mm; Fig. 3). The osteogenic effects of swimming exercise were also seen in bone microarchitecture, whence rats that swam showed an increase of 48.82% (P < 0.001) in BV/ TV ratio when compared with rats submitted to regular weight-bearing loading (S 1 Swim: 44.37 6 2.70% vs. S 1 WB: 22.71 6 8.07%). Additionally, no significant difference was found between S 1 Swim and CON2 groups (P > 0.05), whereas BV/TV ratio was significantly lower (252.06%, P < 0.001) in the S 1 WB group than in the CON2 group (Figs. 4 and 5).
Rats, in special, are suitable models to study several aspects of bone quality and repair (Martins-Pinge et al., 2005; Morey-Holton et al., 2002). The rat tail suspension method was originally used to simulate the effects of microgravity on several organs and systems, as experienced by astronauts in space (Morey-Holton et al., 2002); however, it was later accepted as a standard model for the study of bone loss induced by hypoactivity or disuse (Morey-Holton and Globus, 1998; Turner et al., 1979). In this study, hypoactivity secondary to three weeks of tail suspension effectively impaired bone quality, as shown by reduced bone mass, mechanical weakness, and deterioration of trabecular microarchitecture in suspended rats, when compared to the weightmatched controls. These findings are in agreement with previous reports in literature, which state that hypoactivity due to hindlimb suspension quickly leads to osteopenia (Falcai et al., 2012; Shimano and Volpon, 2009). Our histological and BMD data showed severe trabecular bone loss caused by a relatively short duration of suspension. By means of mechanical test, we also demonstrated the impairment of unloading on bone strength, where both ultimate load and stiffness were significantly decreased in animals suspended for three weeks. As the mechanical force was applied to the diaphysis of bones, mainly composed of compact
DISCUSSION Animal models can be used to investigate the main biological mechanisms involved in bone properties. Microscopy Research and Technique
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bone, it is reasonable to accept that compact bone was also affected by hypoactivity. It is well known that increased physical activity triggers structural, metabolic, hormonal, neural, and molecular adaptations, which positively affect muscle force and power (den Hoed et al., 2008). Likewise, the skeleton requires mechanical stimuli to maintain its trophism. Several modalities of bone mechanical stimulation, including jumping and vibration are shown to exert positive effects on the bone tissue. Although exercises requiring greater mechanical loading (e.g., running and jumping) are known to play an important role at increasing bone formation, these modalities of exercise (high-impact exercises) may not be recommended to specific populations, such as elderly or individuals with certain disabilities or systemic disease. To these populations, aquatic exercises are more adequate and beneficial. In a systemic review, Gomez-Bruton et al. (Gomez-Bruton et al., 2013) concluded that swimming, besides playing an osteogenic role, also has the benefit of being a low-impact activity, which is not hazardous to joints and other structures (Bravo et al., 1997). In our study, swimming improved bone quality to a larger level than regular weight-bearing activity. Furthermore, swimming exercise completely reversed the osteopenic condition induced by unloading, whereas spontaneous cage activity (regular weight-bearing) only ameliorated bone loss. Thus, we found that swimming, in spite of being a low-impact exercise, exerted a positive effect at stimulating bone formation and increasing bone quality both on cancellous and lamellar bones, whence trabecular bone was more affected by unloading and reloading, since it is metabolically more active than cortical bone (Smith, 1993). According to DiVasta and Gordon (DiVasta and Gordon, 2013), the mechanisms involved in skeletal changes in response to physical exercise are still unclear. Gains in bone mass due to exercise are related to increased mechanical strain as well as other factors, such as endocrine responses (DiVasta and Gordon, 2013). As swimming involves no impact and minimizes the effects of gravity, it seems reasonable to assume that the positive effects of swimming on bone are due to muscle activity. Biomechanically, muscle transmits efforts to the bone, which causes microdeformations thus stimulating bone turnover by mechanotransduction mechanisms. Through these mechanisms, mechanical stimulus is converted into biological responses that stimulates bone formation and/or inhibits bone resorption. In bone, this process is initiated by mechanical stimuli caused by gravity, physical impact, vibration or muscular action. The pathways involved in this process have not yet been completely elucidated. Initially, microdeformations create a pressure gradient in the canalicular system of bone, which leads to variation in the flow of pericellular space fluids. These flow variations are transferred to the osteocyte, which seems to be the main mechanosensor of bone (Duncan and Turner 1995) as they release biochemical mediators (e.g., prostaglandins) and regulate the activity of osteoblasts and osteoclasts (Klein-Nulend et al., 1995). Transmission to the effector cells may be achieved through many substances (Robling, 2009), such as nitrous acid, prostaglandins, sclerostin, IGF
(Hamrick, 2012), TGF-beta, RANK, and OPG (Bergmann et al., 2010). The Wnt signaling pathway has recently been identified as a mediator related to bone homeostasis (Gaudio et al., 2010; Morse et al., 2013), which is essential for osteoblastic differentiation. Wnt binds to a coreceptor complex that includes the Frizzled receptor and low-density lipoprotein receptorrelated protein (LRP)25 or 26, which are both present on osteoblasts. This binding stabilizes cytoplasmic bcatenin and causes its translocation to the nucleus, activating the transcription of genes that promote osteoblast proliferation, differentiation, and function, and thus resulting in bone formation (Morse et al., 2013). Conversely, sclerostin, which is upregulated in response to mechanical unloading, inhibits the Wnt pathway through competitive binding to LRP5/6 (Armstrong et al., 2007; Kim et al., 2013; Li et al., 2005; Miyagawa et al., 2011; Robling et al., 2008). Thus, mechanical unloading causes decreased Wnt/beta-catenin signaling activity as well as sclerostin upregulation, which are associated with lower bone mineral content and density (Morse et al., 2013). Conversely, sclerostin upregulation may be reversed by reloading (Morse et al., 2013), whence mechanical bone stimulation reduces the number of SOST, particularly in highstrain regions of the bone. Previous authors have shown a 73% reduction of sclerostin mRNA levels in the ulnar diaphysis of mice submitted to mechanical loading (Robling et al., 2008). During exercise, tissue deformation occurs and stretches resident bone cells (e.g., osteocyte), which results in the expression of several genes involved in mechanotransduction and osteoblast/osteoclast differentiation, thus increasing bone formation (i.e., IGF, FGF, MMP, BMP, osteocalcin) and decreasing bone resorption (i.e., osteoprogeterin, IL, sclerostin, and TRAP). We concluded that swimming exercise completely revert the deleterious effects of three weeks of unloading in tibias of rats, whence bone mass, strength and microarchitecture structure were fully restored to normal levels. Furthermore, we believe the osteogenic effect of swimming exercise was triggered by muscular action, as aquatic activities have no mechanical impact, thus confirming the important role of muscle at stimulating bone formation. ACKNOWLEDGMENTS The authors declare that they have no conflict of interest, no professional relationships with companies or manufacturers who will benefit from the results of this study. REFERENCES Armstrong VJ, Muzylak M, Sunters A, Zaman G, Saxon LK, Price JS, Lanyon LE. 2007. Wnt/beta-catenin signaling is a component of osteoblastic bone cell early responses to load-bearing and requires estrogen receptor alpha. J Biol Chem 282:20715–20727. Bassey EJ, Ramsdale SJ. 1995. Weight-bearing exercise and ground reaction forces: a 12-month randomized controlled trial of effects on bone mineral density in healthy postmenopausal women. Bone 16:469–476. Baxter-Jones AD, Kontulainen SA, Faulkner RA, Bailey DA. 2008. A longitudinal study of the relationship of physical activity to bone mineral accrual from adolescence to young adulthood. Bone 43: 1101–1107. Bergmann P, Body JJ, Boonen S, Boutsen Y, Devogelaer JP, Goemaere S, Kaufman J, Reginster JY, Rozenberg S. 2010. Loading
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