**3. Comprehensive results of animal studies: BMD and BMC**

In biomedical science, animal experiments are performed to establish models that mimic human physiological phenomena. Either estimation methods or experimental designs, which may not be feasible in humans, can then be performed to further investigate possible mechanisms. Rodent models of treadmill activity are commonly used to investigate the effects and mechanisms of exercise on bone metabolism. This section reviews the findings of

The Effects of Endurance Running Training on

age

Wistar, 11 wk

Wistar, 10wk

Wistar, 8wk

5wk

7 wk

7wk

4 wk

4 wk

S.D., 4 wk

Wistar, 4 wk

Author strain,

Nordsletten et al.53

Brourrin et al.50

Horcajada-Molteni et al.51

Huang et al.54 Wistar,

Huang et al.52 Wistar,

Huang et al.55 Wistar,

Joo et al.43 Wistar,

Kiuchi, et al.44 Wistar,

Notomi et al.46

Sakamoto & Grunewald47

Young Adult Bone: Densitometry vs. Biomaterial Properties 335







CON\*

CON\*

+ NS Femurs BMD

+ NS BMC:

BW or BW gain (g)

BMD or BMC Biomechanical

N/A No data

Femoral BMD: NS

Tibiae BMD(g/cm2 ):NS

Tibia and femur BMD: NS

EXE < CON\* in total femur (p=0.04)

(g/cm2) EXE > CON

EXE > CON\*

+ NS NS Vertebra and

+ NS N/A Tibia breaking

N/A Ultimate bending

testing

moment(N‧m×10- <sup>2</sup>) EXE > CON

Femoral failure load (N): EXE > CON\*

No data

Three-point bending load (N), energy (mJ), stress (MPa), toughness (mJ/mm3): EXE > CON\*

Femoral midshaft bending energy and toughness (mJ & mJ/mm3): EXE > CON

Femoral middiaphysis Bending stress (N/mm2): NS Maximum load (*N*): EXE > CON

No data

femoral maximal load (N): NS

strength (kg): NS

control

Protocol BW

27m/min, 60min/d, 10% inclination, 5d/wk, 4 wk

30m/min, 1.5h/d, 5d/wk, 5wk

20m/min to 30m/min, 60min/d, 6d/wk, 90d

24m/min, 60min/d, 5 d/wk, 10wk

22m/min, 60min/d, 5d/wk, 8wk

Two groups: 22m/min, 60min/day vs. 30m/min, 5d/wk, 8wk

30 m/min, 60 min/d, 5 d/wk, 10wk

35m/min, 5 inclination, 60min/d, 5d/wk, 10 wks

24m/min, 60min/d, every other d, 4 wk

24m/min, 75min/d, 5d/wk, 8 wk

previous animal studies. Briefly, the results of animal studies using rodents as subjects showed gender differences, which might affect their further applicability in human subjects. As mentioned above, intensity-trained runners usually have a lower body mass and often have equal or even lower bone mass than non-athletes. Therefore, an animal model of ERT would be expected to reveal the same phenomena. Animal studies reviewed in the present article were selected according to training type (typically endurance treadmill training) and the age of animals (growing or young adult rats).

#### **3.1 Rodents adapted to endurance exercise showed gender differences 3.1.1 Male rodent studies**

Table 2 summarizes the outcomes of studies using male rats as subjects. The studies were reviewed and classified into two categories. The first category includes those using diet control or adjustment to achieve equivalent BW gains between exercise and control groups.42-47 These studies demonstrated that trained animals have a higher BMD.43-45 Tissue mechanical properties were not available in every study, and only one of them shows a higher load-withstanding capacity in the femoral diaphysis.43 However, it must be mentioned that diet prohibition for the purpose of equalizing body weights among groups might cause an additional negative effect on tissue mechanical properties. Diet prohibition impairs the tissue levels (intrinsic) and mechanical properties of bone, suggesting that dietary manipulation of a control group might not be appropriate.48, 49

The second category of studies included animals fed *ad libitum*. In these studies, the exercise groups revealed significantly less BW gain after a programmed ERT.50-55 With lower BW, exercise trained animals showed no difference or lower BMD values as compared to the sedentary control group. As in human subjects, male rats undergoing intense ERT exhibit lower BW gain and no benefits to bone health when considering BMD or BMC as a predictor. However, the higher load-withstanding and energy-absorption capacity of the bones in training rats introduced new research into how endurance exercise benefits bone quality (see section 4).

### **3.1.2 Female rodent studies**

Compared with the treadmill training results for male rats, those for female rats are inconsistent with human subjects, and the data are somewhat controversial. Table 3 summarizes the results of ERT in growing or young adult female rats. Most studies indicate that female growing or young adult rats exhibit no change in BW after a period of ERT.56-64 One study even reported increased BW in female rats after training.58 Of the studies performing BMD analysis in female rats, many report positive effects from endurance running not only in site-specific increments but also in whole bone. Although densitometric measurements demonstrate this advantage, female rats rarely show improved biomechanical properties and may even reveal adverse effects after an intense training program (see Table 3). Therefore, female rats acclimate to ERT differently than do male rats. In human beings, distance runners are also expected to exhibit gender differences in physiological response to similar ERT. However, it seems inappropriate to use the gender difference found in rats to explain the one found in humans, since a period of programmed ERT would commonly reduce BW either in women or men. Thus, the phenomena observed in female rats may not be applicable to female humans. Based on the idea that animal models should mimic the phenomena shown in human subjects, studies using female rats may not be applicable to female humans, since female rats and women have been shown to respond differently to ERT. Possible reasons are discussed in the next section.

previous animal studies. Briefly, the results of animal studies using rodents as subjects showed gender differences, which might affect their further applicability in human subjects. As mentioned above, intensity-trained runners usually have a lower body mass and often have equal or even lower bone mass than non-athletes. Therefore, an animal model of ERT would be expected to reveal the same phenomena. Animal studies reviewed in the present article were selected according to training type (typically endurance treadmill training) and

Table 2 summarizes the outcomes of studies using male rats as subjects. The studies were reviewed and classified into two categories. The first category includes those using diet control or adjustment to achieve equivalent BW gains between exercise and control groups.42-47 These studies demonstrated that trained animals have a higher BMD.43-45 Tissue mechanical properties were not available in every study, and only one of them shows a higher load-withstanding capacity in the femoral diaphysis.43 However, it must be mentioned that diet prohibition for the purpose of equalizing body weights among groups might cause an additional negative effect on tissue mechanical properties. Diet prohibition impairs the tissue levels (intrinsic) and mechanical properties of bone, suggesting that

The second category of studies included animals fed *ad libitum*. In these studies, the exercise groups revealed significantly less BW gain after a programmed ERT.50-55 With lower BW, exercise trained animals showed no difference or lower BMD values as compared to the sedentary control group. As in human subjects, male rats undergoing intense ERT exhibit lower BW gain and no benefits to bone health when considering BMD or BMC as a predictor. However, the higher load-withstanding and energy-absorption capacity of the bones in training rats introduced new research into how endurance exercise benefits bone

Compared with the treadmill training results for male rats, those for female rats are inconsistent with human subjects, and the data are somewhat controversial. Table 3 summarizes the results of ERT in growing or young adult female rats. Most studies indicate that female growing or young adult rats exhibit no change in BW after a period of ERT.56-64 One study even reported increased BW in female rats after training.58 Of the studies performing BMD analysis in female rats, many report positive effects from endurance running not only in site-specific increments but also in whole bone. Although densitometric measurements demonstrate this advantage, female rats rarely show improved biomechanical properties and may even reveal adverse effects after an intense training program (see Table 3). Therefore, female rats acclimate to ERT differently than do male rats. In human beings, distance runners are also expected to exhibit gender differences in physiological response to similar ERT. However, it seems inappropriate to use the gender difference found in rats to explain the one found in humans, since a period of programmed ERT would commonly reduce BW either in women or men. Thus, the phenomena observed in female rats may not be applicable to female humans. Based on the idea that animal models should mimic the phenomena shown in human subjects, studies using female rats may not be applicable to female humans, since female rats and women have been shown to

respond differently to ERT. Possible reasons are discussed in the next section.

**3.1 Rodents adapted to endurance exercise showed gender differences** 

dietary manipulation of a control group might not be appropriate.48, 49

the age of animals (growing or young adult rats).

**3.1.1 Male rodent studies** 

quality (see section 4).

**3.1.2 Female rodent studies** 


The Effects of Endurance Running Training on

36m/min, grade15%, 60min/day, 5days/wk for 10wk

49cm/s, 12% grade, 60min/d, 5d/wk, 10 wk (~75-80% of maximum oxygen capacity)

25m/min, 60min/d, 5d/wk, for 7 or 11 wk

20m/min,30 mim/d, 5° inclination, 5d/wk, 17wk

45 cm/s, 5% grade, 60 min/d, 3d/wk, 10wk

none available.3.1.3Gender differences revealed by animal studies

Raab et al.60 Fischer

Hou et al.61 S.D., 8

Shimamura et al.62

van der Wiel et al.63

Salem et al.64 S.D., 8

344, 2.5 month

wk

Wistar, 6 wk

Wistar, 5 month

wk

Young Adult Bone: Densitometry vs. Biomaterial Properties 337

withstand torsion test.

force (kg/mm): NS

maximum load (N): NS Energy to maximum load (mJ): NS

N/A

Femoral neck maximal load (N): NS Femoral shaft maximal load (N): NS

maximal load (N):NS

BW: NS N/A Femur ultimate


tibia (mg): EXE > CON\*

BMC (g): NS CON:9.3±1. 1 EXE:9.9±1.0




Note: Protocol, training protocols were presented serially by the final training intensity (m or cm per minute), training time per day (minute or hour per day), training frequency (times per week), and training periods (day or week); d, day; wk, week; min, minute; NS, none significant difference; N/A,

As mentioned above, male and female rats adapt differently to endurance treadmill training, especially in densitometric measurements. The reasons for this gender difference in rodents have been comprehensively investigated elsewhere. According to the theory of Frost,10 this difference may partially contribute to different adaptations in BW gain. Female rats usually exhibit a similar or sometimes higher body mass after training; they therefore may acquire a greater advantage from local mechanical loading than male rats with lower BW gain after forced endurance treadmill training or voluntary running.65-68 The mechanisms of this gender difference in BW gain associated with ERT are unknown. A possible explanation is the involvement of gonadal hormones in BW regulation. Endurance exercise reportedly lowers plasma testosterone levels in male rats.69 The down regulation of this anabolic

Table 3. Studies of endurance running training vs. growing or young adult female rats


Note: Protocol, training protocols were presented serially by the final training intensity (m or cm per minute), training time per day (minute or hour per day), training frequency (times per week), and training periods (day or week); d, day; wk, week; min, minute; NS, none significant difference; N/A, none available.

Table 2. Studies of endurance running training vs. growing or young adult male rats


+ EXE >

Note: Protocol, training protocols were presented serially by the final training intensity (m or cm per minute), training time per day (minute or hour per day), training frequency (times per week), and training periods (day or week); d, day; wk, week; min, minute; NS, none significant difference; N/A,

Table 2. Studies of endurance running training vs. growing or young adult male rats

Protocol BW control BW or BW

CON

gain and tissue measurement

Muscle weight & Femoral length (12wk): EXE > CON\*

Femoral length: EXE > CON\*

B > A\*





Femur BMD & BMC: EXE > CON\*

+ NS NS N/A

N/A

BMD or BMC Biomechanical

Femoral and L6 vertebral BMD (mg/L): NS Femoral and L6 vertebral bone volume (L), wet weight (mg): EXE > CON\*

Tibial BMC (g): EXE > CON\* Tibial BMD (g/cm2): NS

Tibial trabecular BMD (mg/cm3): B, C, D, E > A group\*. But, NS in tibial cortical BMD.

(g/cm2): EXE > CON

testing

N/A

N/A

N/A

Group trained at 75% V‧O2max and 90min/d showed higher stiffness but lower energy to

Newhall et al.45

Ferreira et al.42

none available.

Iwamoto et al.56

Iwamoto et al.57

Hagihara et al.58

Wheeler et al.59

Author Strain

and age

S.D., 4wk

Wistar, 4wk

Wistar rats, 8wk

S.D., 120 d

S.D., 47 d10.2km/d, for

Wistar, 10 wk

6 wks (voluntary running)

12m/min, 1h/d, 10 wks.

24m/min, 60min/d, 5 d/wk, 8wk or 12wk

24m/min, 60min/d, 5 d/wk, 7wk or 11wk

A group: control group B-E group: 4~7d/wk, running at 15m/min, 30min/d, 8wk

55%, 65%, 75% V‧O2max, 30min/d, 60min/d, 90min/d, 4d/wk, 10wk


Note: Protocol, training protocols were presented serially by the final training intensity (m or cm per minute), training time per day (minute or hour per day), training frequency (times per week), and training periods (day or week); d, day; wk, week; min, minute; NS, none significant difference; N/A, none available.3.1.3Gender differences revealed by animal studies

Table 3. Studies of endurance running training vs. growing or young adult female rats

As mentioned above, male and female rats adapt differently to endurance treadmill training, especially in densitometric measurements. The reasons for this gender difference in rodents have been comprehensively investigated elsewhere. According to the theory of Frost,10 this difference may partially contribute to different adaptations in BW gain. Female rats usually exhibit a similar or sometimes higher body mass after training; they therefore may acquire a greater advantage from local mechanical loading than male rats with lower BW gain after forced endurance treadmill training or voluntary running.65-68 The mechanisms of this gender difference in BW gain associated with ERT are unknown. A possible explanation is the involvement of gonadal hormones in BW regulation. Endurance exercise reportedly lowers plasma testosterone levels in male rats.69 The down regulation of this anabolic

The Effects of Endurance Running Training on

animals were fed ad libitum and data were not adjusted

correlated to tissue-level changes (e.g. collagen fiber orientation).

**5. Effects on tissue-level (material) properties** 

Young Adult Bone: Densitometry vs. Biomaterial Properties 339

However, in considering the applicability of exercise, a training program with moderate exercise intensity would be expected to show a higher compliance and therefore be more appropriate for the general population. As shown in Table 2, animals trained at a relatively moderate intensity (20-24m/min), which corresponds to 70% V‧O2 max,76 also had lower body masses and slightly lower (~5% lower) total BMD (p = 0.04), but were not found to have enhanced structural bone strength.55 The authors' previous study used body mass as a covariate to equalize raw data, which then revealed a comprehensively stronger bone tissue either in structural or tissue-level biomaterial properties.52 With lower body mass, the data of the exercise group would be adjusted to a higher level, and the effects of ERT seemed to become "good" for animal bones. However, it would be a more relevant and natural study if

In aspects of biological efficiency, an athlete at her/his optimal physiological status will not necessarily be absolutely higher in every physiological parameter. Therefore, a smaller muscle mass or skeleton size seems to be a benefit, rather than a weakness, for a distance runner or an endurance athlete. With such a smaller bone size, moderate ERT rats did not show absolutely enhanced structural bending load values but, interestingly, they showed better energy absorption capacity in long bone tissue that ERT rats were found to have a four-fold increase in energy absorption after long bone tissue reached the yield-point (postyield energy).55 As mentioned in previous studies,41 post-yield behaviors are highly

In our previous studies, we used mathematical methods to estimate tissue-level biomaterial properties. Through calculating long bone's cross-sectional moment of inertia, we normalized load-displacement data to stress and strain. Under such conditions, ERT rats' worse structural material properties disappeared. Additionally, exercise and control groups showed no differences in yield stress, yield toughness or elastic modulus (Young's modulus),55 suggesting that endurance training is not harmful for bone material properties. ERT's benefits on the post-yield biomaterial behaviors seemed to be more size-independent and associated with tissue-level (e.g. bone matrix, collagen) changes. Because measuring post-yield mechanical properties using beam bending theory is only valid in the pre-yield regime,75 reporting post-yield stress, strain or toughness is inappropriate. Therefore, we discussed this tissue-level adaptation base on the post-yield parameters measured from load-displacement data. As shown in our two recent studies, either moderate ERT or endurance swimming training benefits bone tissue more in terms of energy absorption capacity,55,77 especially in post-yield energy. Similar results of enhanced post-yield behavior were provided by another ERT animal study, which showed a short-term treadmill running (21 days) enhanced tibia post-yield deformation in mice.78 Moreover, such effects on postyield behavior changes seem to apply not only to endurance training. After a short-term (5 days) freefall landing exercise, Wistar rats revealed an increased post-yield energy absorption in ulnae.79 Such an enhanced absorption capability is more likely due to tissuelevel (e.g. bone matrix, collagen orientation etc) changes rather than structural adaptation. As mentioned in previous studies, tissue-level properties can be divided into the inorganic mineral phase (e.g., hydroxyapatite), which determines tissue stiffness and strength, 80, 81 and the organic bone matrix, which plays a key role in energy absorption, 82, 83 It has been suggested that the networks of collagen, one of the major components of bone matrix, could affect the energy dissipation between the yield point and fracture point in bone tissue.84-86

hormone in growing male rats may account for the significantly lower protein mass gain and BW gain. In female rats, however, estrogens would suppress body mass, food consumption and fat deposition.70, 71 Progesterone, on the other hand, has been verified to increase body fat and body mass.72, 73 Moreover, a previous study suggests that regular treadmill training results in extended periods of progesterone secretion, which was associated with significant weight gain.74 Women may respond to ERT with similar regulation in progesterone. However, the up-regulation of progesterone may be more pronounced in rats than in women, since female rats reveal no decrease in BW even under vigorous ERT.60

### **3.2 Studies of male rats mimic human practice**

An analysis of gender differences observed in the above animal studies reveals that ERT increases BMD and BMC in female rats but not in male rats. However, the physiological response (*e.g.* BW gain) of female rats to ERT differs from that in female humans. Given that animal studies are intended to clarify the mechanisms of biological phenomena in humans, female rats may not be a suitable model for investigating the effects of ERT on developing or young adult bone. On the other hand, the changes in BW and densitometric parameters associated with ERT in male rats were similar to those in humans, indicating that male rats are a suitable model for investigating the effects of endurance running.
