Adaptive Medicine 4(1): 27-32, 2012 DOI: 10.4247/AM.2012.

ABB016

27

Enhancements of Free-Fall Landing on the Trabecular Bone Structure in Tibiae of Growing Female Rats: A Time Course Study
Hsin-Shih Lin 1, 2, Tsang-Hai Huang 2, Ho-Seng Wang 1, and Rong-Sen Yang 3
1 2

Department of Physical Education, National Taiwan Normal University Institute of Physical Education, Health and Leisure Studies, National Cheng Kung University 3 Department of Orthopaedics, National Taiwan University & Hospital, Taipei, Taiwan, Republic of China

The purpose of this study is to investigate the effects of one- and four-week mechanical loading on the bones in young female rats. Seven-week-old female SD rats (n = 40) were randomly assigned into four groups, which were the 1WCON, 1WLAND, 4WCON, and 4WLAND groups (n = 10 for each). Animals of the 1WLAND and 4WLAND groups were respectively subjected to 1- and 4-week free-fall landing program, in which animals were free-fallen from a height of 40 cm for 10 times per day; 5 days per week. The 1WCON and 4WCON groups served as the time-matched control groups. Methods of histomorphometry, tissue geometry, and tissue weight were performed on the animals femora and tibiae. Two-way (landing time) ANOVA was used for statistical analysis ( = .05). In the histomorphometric analysis, the landing animals showed significantly higher bone volume ratio (BV/TV, %) and trabecular thickness (Tb. Th, m). The effect of time was also evident in the bone tissue wet weight (WW) and fat-free dry weight (FFDW), while the landing showed no effects on the WW and the FFDW. Our study preliminarily showed that 1- and 4week landing training was beneficial on the trabecular structure of the proximal tibia in female rats. Further study would be valuable to investigate related cellular response as well as tissue material properties of the spongy bone in free-fall landing rats. Key Words: trabecular bone, landing, mechanical load

Introduction
Mechanical loading has been well proven for its efficacies on local bone metabolism. As mentioned in previous investigations, local mechanical loadinginduced bone strain has been reported to be one of the important factors affecting local bone metabolism as well as local bone mineral properties (3, 27). However, the mechanisms of how local mechanical loading affects bone metabolism or bone development have not been demonstrated.

Various animal models (e.g. four-point bending, axial loading and jump/landing etc.) (9, 15, 22, 28, 31) were designed to investigate the relationship between mechanical load and bone metabolism. Among those experimental models, jumping as well as freefall landing model were more similar to human physical activity. In a previous study, low frequency (< 0.1 Hz) free-fall landing has been suggested to benefit bone formation (31). In addition, merely 10 repetitions per day of free-fall landing ( 30 cm) were suggested to favor bone formation activity (18, 31). In human studies, intervention of daily free-fall landing for 3-8 months was also reported to increase bone mineral density in elementary children (6, 17, 19, 20, 24). Up-to-date, all previous animal studies using free-fall landing models only investigated the landing effects by a single time point (8 weeks or 1 week) (18, 30, 31), which were more likely to be either longterm or a short-term studies. Therefore, it would be valuable to design a time-course study to observe the effects of free-fall landings on local bone metabolism. On the other hand, all the previous studies focused on BMD (31), bone strength (18, 30, 31) and dynamic histomorphometry (15, 18) of the cortical bone. None of them had been designed to investigate the landing effects on the histomorphometry of the trabecular bone. Although one of those previous studies investigated the histomorphometry of the trabecular on ulna after a landing training used micro CT (30), but no data of bone tissue of the hind limbs were shown. Since hind limbs are subjected to a higher impact during landing (18) and more similar to humans lower limb, the effects of free-fall landing on the histomorphometry of the hind limbs are worthy of investigation. In fact, trabecular bones can absorb the impact of a mechanical load via structural density. In a recent methodological study, scientists established a method for estimating biomechanical properties on proximal tibia (25), and

Corresponding author: Tsang-Hai Huang, Ph.D., Associate Professor, Institute of Physical Education, Health and Leisure Studies, National Cheng-Kung University, No. 1, University Rd. 701, Tainan, Taiwan, Republic of China. Tel: +886-6-2757575~81810, Fax: +886-6-2766427, E-mail: tsanghai@mail.ncku.edu.tw Received: September 17, 2011; Revised: November 3, 2011; Accepted: November 19, 2011. 2012 by The Society of Adaptive Science in Taiwan and Airiti Press Inc. ISSN : 2076-944X. http://www.sast.org.tw

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that study verified trabecular bones importance when an external loading was received. Based on the deduction above, we performed a time-serial (one and four weeks) study to investigate the effects of landing training on the trabecular bone of the proximal tibia in growing female rats. Methods of histomorphometry, geometry and tissue weight measurement were included for estimating how mechanical loading affects the spongy bone as well as the cortical bone.

hyde solution for 48 h and decalcified with a 10% Ethylenediaminetetraacetic acid (EDTA) solution (pH 7.4) under 4C for four weeks. After decalcification, each bone sample was incised longitudinally from the center portion and then, embedded with paraffin for further tissue section and histological staining. For geometric and tissue weight, right femora and tibiae were isolated, cleaned off the soft tissue, wrapt in 0.9% sodium chloride steeped gauze and aluminum foil, and then, stored under -20C for future geometric and tissue-weight measurements. Bone Histomorphometric Analysis

Materials and Methods

Animals Female Sprague-Dawley rats (n = 40, 3 weeks old) were purchased from National Cheng Kung University Animal Center and were kept under controlled conditions which included a room temperature of 21 1C with a 12:12 h light-dark cycle. All animals were fed with standard Purina Rodent Chow 5001 (Labdiet , Richmond, IN, USA) containing 0.95% calcium and 1.07% phosphate (wt/wt of dry food) and distilled water ad libitum. The procedures of the animal experimentation, including the raising, feeding, and the whole sacrificing processes followed the APSs Guiding Principles in the Care and Use of Animals and were approved by the Committee of Animal Study in National Cheng Kung University (Tainan, Taiwan, Document no. 940054). Free Fall Landing Animals were randomly assigned into one- or fourweek free-fall landing groups (1WLAND and 4WLAND groups) and two time-matched control groups (1WCON or 4WCON groups), respectively. Landing training was started at the age of 7-wk-old for all animals. For landing groups, female rats were subjected to free-fall landing (10 times/day, 5 days/ week) according to our previous study (18). Briefly, the animals were gently held and released from a height of 40 cm and landing on a dry and flat surface. Under such condition, the female rats hind limbs would be subjected to a ground reaction force at ~1015 times body weight (18). The CON rats were held and raised similarly for ten times per day without free-fall landing. Animals were sacrificed after 1 or 4-week experiment periods. Bone Sample Preparation After the treatments were completed, the animals were sacrificed under deep anesthesia with sodium pentobarbital (65 mg/kg). Left tibia of each rat were removed, fixed with a 3.7% neutral paraformaldeSerial sections (5 m in thickness) of each sample were made by a microtome (Accu-Cut SRM 100 Rotary Microtome, Sakura Finetek, Japan), and stained by Mayers hematoxylin-eosin solution according to our previous studies (12, 13). The quantitative study of the proximal metaphysis was performed by histomorphometric procedures described by Parfitt et al. (23) with an image analysis software (Image Pro Plus 6.1 for Windows; Media Cybernetics, Silver Spring, MD, USA). Parameters measured in the present study included the bone volume/tissue volume (BV/TV, %); mean thickness of the trabeculae (Tb.Th., m); trabecular number (Tb.N., 1/mm); trabecular separation (Tb.Sp., m). Tissue Weight Measurements Femora and tibiae were thawed at room temperature and weighed as wet weight (WW). Then, bone specimens were immersed in a solution (2 vol. of chloroform combined with 1 vol. of methanol) for 1 week and subsequently dried at 80C for 24 h (8, 10, 11). The fat-free dry weight (FFDW) of each femur was then measured. Geometry Measurements Long bone cross-sectional parameters, including total cross-sectional area, cortical bone area, bone marrow cavity area and cortical bone thickness were measured in the middle of tibiae and femora by using the software Image Pro Plus 6.1 for Windows (Media Cybernetics, Silver Spring, MD, USA). In addition, the cross-sectional moment of inertia (CSMI) was measured by the methods of Turner and Burr for irregular cross-sections (26), which is based on the assumption that a bone cross-section is made up of multiple rectangular elements (pixels) and the equation is shown as follows: I=

(wh3/12 + whdi2) i=1

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Table 1. Body Weight P-value 1WCON Pre-training Post-training 180.0 15.6 203.9 15.8 1WLAND 178.2 11.6 195.1 11.6 4WCON 173.6 11.9 229.3 16.1 4WLAND 173.4 8.2 227.2 9.5 landing 0.796 0.211 time 0.154 < 0.001 landing time 0.836 0.443

Data were presented as means SE.

A
40 CON LAND

B *
Tb.Th. (m)
landing: P = 0.002 time: P = 0.271 landing time: P = 0.463 250 200 150 100 50 0 1-Week 4-Week 1-Week 4-Week CON LAND * # landing: P = 0.018 time: P = 0.001 landing time: P = 0.909

30

BV/TV (%)

20

10

C
500 400 CON LAND landing: P = 0.082 time: P = 0.469 landing time: P = 0.448

D
2.5 2.0 CON LAND landing: P = 0.124 time: P = 0.698 landing time: P = 0.620

Tb.N. (1/mm)

Tb.Sp. (m)

300 200 100 0 1-Week 4-Week

1.5 1.0 0.5 0.0 1-Week 4-Week

Fig. 1. Histomorphometric analysis of trabecular bone of proximal tibiae. A. bone volume ratio (BV/TV, %); B. average thickness of trabeculae (Tb.Th., m); C. trabecular separation (Tb.Sp., m); D. trabecular number (Tb.N., 1/mm). Values were shown as means SE. *, significantly main effect of landing, landing training significantly increased BV/TV and trabecular thickness (P < 0.05); #, main effect of time, 4-week old rats were higher in trabecular thickness (P < 0.05).

where I is CMSI, n is the number of pixels, d is the distance from the center of the element of the area to a given axis on the cross-section. Statistics

In the main effect of time, the animals experienced a significant increase in body weight during the experimental period (Table 1). No difference was shown between the exercise groups and the control groups. Bone Histomorphometric Analysis

All data were presented as means standard error. Two-way (landing time) ANOVA ( = 0.05) was used for data analyses by using a statistical software (12.0 version, SPSS, Chicago, IL, USA).

Results
Body Weight

In the histomorphometric analysis, the main effect of exercise was shown as the exercise rats showed a significantly higher BV/TV (%) and trabecular thickness (m) (Fig. 1, A and B). The landing training groups was denser (Tb.Sp.) (P = 0.082) than the CON groups in the trabecular bone though no significance level was attained (Fig. 1C). In addition, the trabecular thickness

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Table 2. Tissue Weight Measurements P-value 1WCON F. WW T. WW F. FFDW T. FFDW 755.6 57.6 544.0 46.5 393.8 30.7 304.1 19.7 1WLAND 747.7 54.6 531.0 46.8 388.9 34.9 295.7 26.7 4WCON 859.2 58.4 583.9 40.4 478.6 34.4 354.3 27.1 4WLAND 870.9 43.5 602.0 43.8 466.1 22.0 361.5 20.4 landing 0.915 0.858 0.397 0.933 time < 0.001 < 0.001 < 0.001 < 0.001 landing time 0.581 0.289 0.709 0.320

Data were presented as means SE. F, femur; T, tibia; WW, wet weight (mg); FFDW, fat-free dry weight (mg). Table 3. Geometry Measurements P-value 1WCON T. CSMI T. TA T. CSA T. MA T. Th F. CSMI F. TA F. CSA F. MA F. Th 3.5 0.6 7.5 0.9 4.9 0.8 2.6 0.4 0.61 0.07 7.6 1.4 12.2 1.0 6.9 0.7 5.3 0.9 0.62 0.07 1WLAND 3.5 1.1 7.4 0.6 4.8 0.7 2.6 0.4 0.60 0.07 7.5 1.8 12.3 0.7 7.2 0.7 5.1 0.7 0.64 0.06 4WCON 3.7 7.4 4.8 2.6 0.60 7.8 12.1 6.9 5.3 0.61 0.5 0.8 0.6 0.6 0.07 1.4 1.0 0.7 0.7 0.06 4WLAND 3.5 1.2 7.0 0.8 4.5 0.7 2.5 0.4 0.60 0.06 7.5 1.3 11.5 1.8 7.1 0.7 4.4 1.8 0.65 0.06 landing 0.926 0.431 0.494 0.749 0.317 0.585 0.476 0.284 0.149 0.284 time 0.738 0.305 0.358 0.709 0.833 0.845 0.294 0.751 0.357 0.751 landing time 0.606 0.652 0.715 0.824 0.662 0.725 0.391 0.981 0.366 0.981

Data were presented as means SE. F, femur; T, tibia; CSMI, cross-sectional moment of inertia, (mm4); TA, total area, (mm2) ; CSA, cross-sectional area of cortical bone (mm2); MA. bone marrow area (mm2); Th, cortical thickness (mm).

showed the main effect of time as the Tb.Th. were significantly increased throughout the training period (Fig. 1B). Tissue Weight WW and FFDW of tibia and femur were shown in Table 2. Both tibia and femur showed increased WW and FFDW by time, as the 4-wk groups (11-wk-old) showed significantly higher tissue weight than the 1wk groups (8-wk-old). However, no noticeable effect of training was found in our current study. Geometry Measurements In geometry measurement, there was no significant difference shown among groups (Table 3).

Discussion
High impact loading on the bone has been well proven for its benefits on bone formation and bone development. Among numerous types of high impact activities, free-fall landing has been suggested to be one of the contributive modes for local bone metabolism (15, 18, 30, 31). In those previous studies, the effects

of long-term (30, 31) and short-term (18) free falllanding on bone development or metabolism were verified. In the present study, we designed a time course free-fall landing program in order to monitor the less investigated parameters (e.g. spongy bone architectures), which would be helpful for further understanding the process of spongy bone response to high impact loading. Significantly higher BV/TV in the LAND groups suggested high turnover spongy bone could respond to the stress of the present freefall landing. In addition, the LAND groups showed thicker and denser in trabecular bone, although the main effect of landing did not reach the level of significance in Tb.Sp. (P = 0.082) and Tb.N. (P = 0.124). As compared to previous studies (15, 18, 30, 31), our present study provides a new evidence that growing bone could respond to high impact loading not only in the cortical bone but also in the architectural level of the trabecular bone. Though it has been mentioned that free-fall landing could enhance the trabecular bone in rodents ulnae, we firstly initially verified a period of 5 days of free-fall landing does enhance the structure of the hind limb bone tissue of rats an area more similar to humans lower limb, and thus, more clinically applicable. In the present study, data regarding to whether

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free-fall landing can promote biomechanical properties in the trabecula were unavailable. However, based on previous studies (1, 5, 25), trabecular bone structure has been verified to correlate with biomechanical properties. In other words, improved trabecular microarchitecture could serve as an indirect demonstration of stronger spongy bone. Regarding to the size-related measurements, the results of the cortical bone area, the cortical bone thickness and CSMI of tibiae and femora were not affected by free-fall landing. Similar phenomenon was shown in a previous study (31). After a longer period free-fall landing training, young female rats showed higher spongy BMD in the proximal tibia without an increase in the cortical bone area and the cortical BMD of femoral diaphysis. Similarly, after a 3-every-other-day jumping training followed by ten days of rest, bone formation rate was promoted in the cross-section of tibial diaphysis, while no significant difference was revealed on in bone geometry (22). In this study, 1- and 4-week free-fall landing caused a significantly different adaptation behavior between the spongy bone and the cortical bone. Spongy bones in metaphysis showed beneficial effects in tissue volume and thickness of the trabeculae while no difference was shown in size-related measurements of the cortical bone. That could be explained by the inherent differences between the trabecular bone and the cortical bone. Due to the higher surface area-perunit volume, more bone cells are located within the trabecular bone than the cortical bone. And thus, when subjected to external mechanical loading, trabecular bones could respond faster and are more sensitive in turnover rates. Moreover, treadmill running training or short-term free-fall landing enhances post-yield energy or strength of bone tissue without changes in bone size or shape in the cortical bone (14, 16, 18, 29), suggesting that the way the cortical bone adapts to external mechanical loading might be more in qualityrelated changes than in size-related changes. Although the cellular (e.g. osteoblast/osteoclast activity or number) and densitometric (e.g. BMD, BMC) measurements were not available in the current study, it has been reported that high impact activity, such as free-fall landing training, could produce high strain rates on bone, and are more associated with promoting bone formation activity and suppressing in bone resorption activity (15, 21). Moreover, for growing rats, free-fall landing-enhanced spongy bone structure would further contribute to a site-specific bone density. After an eight-week free-fall landing training, spongy bone density measured by peripheral quantitative computed tomography (pQCT) in the proximal tibiae significantly increased as compared to the nontrained animals (31). Conversely, BMD in diaphysis of tibiae showed no difference between landing and

non-landing groups (31), which were different from human studies (2, 4). Perhaps, the different results shown between humans and rodents might be due to the training period. For cross-sectional human studies, exercise-trained subjects regularly participated in athletic training for several years (2, 4). For control trial studies, human subjects were usually trained by vertical jumping exercise for at least 6 monthes or longer to reveal significant enhancement in BMD or BMC (7). Although rodents have shorter absolute life cycle, eight-week landing training in animal studies could still be a relatively shorter period, and thus, the efficacy of free-fall landing in cortical BMD could not be shown. Though the densitometric data werent provided in the current study, tissue WW and FFDW (Table 2), again, indrectly verified that a relatively short-term (e.g. 1 or 4 weeks) free-fall landing could not show further promotion in total mineral accumulation in growing bones. Instead of densitometric meansurement, the histomorphometric analysis of trabeculae verified that the spongy bone maybe more sensitive to high impact loading, and we initially found that a free-fall landing training could enhance the spongy bone volume ratio and trabecular thickness. In conclusion, our study preliminarily showed that 1-week and 4-week landing training is benificial to the trabecular structure of the proximal tibia in rodents. For future study, it would be valuable to investigate whether such an ameliorated spongy bone structure caused by free-fall landing would further contribute to site-specific material properties.

Acknowledgments
This study was supported by a grant from the National Science Council (NSC-94-2314-B-006-033, Taiwan). Miss Jae Cody is appreciated for her editorial assistance in English.

Conflict of Interest
The current study has no conflict of interest.

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