行政院國家科學委員會補助專題研究計畫成果報告
（計畫名稱）
跳躍運動（起跳 v.s. 落地）對骨骼發展的影響
計畫類別：■ 個別型計畫 □ 整合型計畫 計畫編號：NSC 94-2314-B-006-033-
執行期間： 94 年 8 月 1 日至 95 年 7 月 31 日
計畫主持人：黃滄海
共同主持人：楊榮森、陳洵瑛
計畫參與人員：林欣仕、賴郁婷、黃苡瑋
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中 華 民 國 95 年 11 月 30 日
The Effects of Free Fall Landing on Bone Development Abstracts
The purpose of this study is to investigate the effects of free fall landing on bone development. In the present study, we designed a time course of free fall landing treatment. Animals were randomly assigned into two groups, which were free fall landing (FFL) group and control (CON) group. Animals of the FFL group receive 40cm high free fall landing ten times a day and five days a week.
During the four-week experimental period, no difference was shown between two groups. In histomophometric analysis, the FFL group was significantly higher in BV/TV(%) after 1 week as well as 4 weeks landing training (p< .05).
The FFL group was 10% thicker (Tb. Th.) and 6-16% denser (Tb. Sp.) in trabecular bone though the significance level did not attain. Our present study provide a new evidence that growing bone could response to high impact loading not just shown in molecular level but also in architecture level of trabecular bone. Further immunohistological staining and biomechanical analysis are ongoing in purpose of exposing more details of mechanisms that bone adapted to high impact loading.
Keywords: bone, mechanical loading, trabecular bone, free fall landing
中文摘要
本研究之目的旨在探討落地動作之刺激對骨骼發展的影響。本研究中，實驗動物
（大鼠）分為落地組（free fall landing group, FFL）及控制組（control group,
CON），其中落地組每日接受自 40 公分的高處落下之訓練，每天 10 次，每週訓
練五天。實驗動物分別在第一週及第四週結束後犧牲，並採集其骨骼組織樣本進 行各項分析。結果：不論是一週或四週訓練後，兩組實驗動物均無差異；在組織 型態學分析方面，訓練一週後，FFL 組即呈現骨量比率（bone volume ratio, %）
顯著高於控制組，此一骨量比率的增加幅度在四週的訓練後更為明顯。此外，
FFL 組的骨骼組織亦呈現骨小樑較 CON 組厚 10%，而骨小樑密度（間距）亦比 CON 組在一週及四週訓練後，分別高出 6 及 16%。這意指著一週的落地訓練，
對成長中之大鼠骨骼而言，不僅僅如先前研究所述會引致蛋白表現量的改變，甚 至在骨骼結構上的改變也已達可評估之水準。本研究仍有數項免疫染色及骨骼生 物力學分析正在進行中，務求提供更多有關骨骼適應高衝擊負荷的相關證據，以 期能對骨骼面對負荷的適應機制有進一步了解。
關鍵詞：骨骼、機械性負荷、骨小樑、落地
Introduction
Mechanical loading has been well proven for its contributive effects on bone metabolism. In animal studies, low frequency ( <2 Hz) free fall landing has been suggested to benefit bone formation (Honda et al., 2001; Welch et al., 2004). Among those investigations, local mechanical induced bone strain has been reported to be one of the important factors affecting bone metabolism (Burr et al., 2002). In addition, merely 10 repetitions per day of high enough free fall landing were suggested to favor bone formation activity. In human studies, intervention of daily free fall landing for 3-8 months was also reported to increase bone mineral density in elementary children (Fuchs et al., 2001;
Johannsen et al., 2003; Kontulainen et al., 2002; MacKelvie et al., 2002;
McKay et al., 2000; Petit et al., 2002). However, the mechanisms of how local mechanical loading affecting bone metabolism or bone development has not been clarified yet. Further investigations by using histomorphometric,
immunohistological and biomechanical analysis would be valuable for further understanding how mechanical loading affects bone metabolism and bone development.
Materials and Methods
Animal: Female Wistar rats (n=32, 3 weeks old) were purchased from National Cheng Kung University Animal Center and were kept under controlled
conditions which included a room temperature of 211C with a 12:12 hour light-dark cycle.d All animals were fed with standard Purina Rodent Chow 5001 (Labdiet®, Richmond, IN, U.S.A.) containing 0.95% calcium and 1.07 % phosphate (wt/wt of dry food) and distilled water ad libitum. The procedures of the animal study, including the raising, feeding, and the whole sacrificing processes followed theAPS’s “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).
Free Fall Landing: Animals were randomly assigned into free fall landing group (FFL group) and control group (CON group), respectively. Landing trail was begun as animals were 7-wk-old in age. Animals of FFL group received daily ten times passive free fall landing, which were performed by our
investigators. Animals were gently holding and release from 40 cm height and landing on a dry and flat surface. The CON rats were held and raising similarly
for ten times without free fall landing. Animals were received treatments mentioned above five days a week and were sacrificed after 1 and 4 weeks (table 1).
Table 1 experimental design and treatment for two groups
One week Four week
FFL group n=9 n=9
CON group n=9 n=9
Bone sample preparation: After the treatments were completed, the animals were sacrificed under deep anesthesia with sodium pentobarbital (65mg/kg).
Left femur, tibia, ulnae, radius and humerus of each rat were removed, fixed with a 4% neutral paraformaldehyde solution for 24-48 hour and decalcified with a 10% EDTA under 4°C 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 biomechanical measurement, similar bone tissue of right side were
removed, cleaned the soft tissue, wrap in 0.9% sodium chloride steeped gauze and aluminum foil, and then, stored under -20°C for future geometric and biomechanical testing.
Bone histomorphometric analysis:Serial sections (5m in thickness) of each sample were cutand stained by Mayer’s hematoxylin-eosin solution according to our previous studies (Huang et al. 2002). The quantitative study of the proximal metaphysis was performed by histomorphometric procedures described by Parfitt et al. (1987) with an image analysis software (Image Pro Plus 4.5 for Windows; Media Cybernetics, Silver Spring, Maryland).
Parameters measured in the present study were including 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., mm).
All parameters were quantified by using software Image-Pro Plus (version 6.1, Media Cybernetics, Inc., USA).
Statistics: All data were analyzed by student’s t-test for comparing the difference between the FFL group and the CON group. All data were expressed as the meanstandard error (SE), and differences were considered significant if p < 0.05.
Results
During the four-week experimental period, no difference was shown
between two groups. In histomophometric analysis, the FFL group was significantly higher in BV/TV(%) after 1 week as well as 4 weeks landing training (p< .05). The FFL group was thicker (Tb. Th.) and denser (Tb. Sp.) in trabecular bone though the significance level did not attain (Table 2).
0 50 100 150 200 250 300
0 1 2 3 4
Time (week)
Bodyweight(g)
FFL CON
Figure 1 Body weight changes during the experimental period.
Table 2 Histomorthometric analysis of trabecular bone
1 week 4 week
FFL CON p value FFL CON p value
BV/TV(%) 27.71.2 24.20.7 .026 30.01.2 24.71.9 .030 Tb. Th. (m) 65.92.6 59.02.6 .074 75.13.0 69.12.5 .122 Tb. Sp. (m) 157.112.0 166.66.8 .504 143.48.4 166.98.7 .069 Tb. N. (1/mm) 4.60.2 4.40.1 .497 4.60.2 4.20.1 .119 MeanSE. BV/TV (%), bone volume ratio; Tb. Th. (m ), mean thickness of trabecular bone; Tb. Sp. (m), trabecular separation; Tb. N., 1/mm, trabecular number.
Discussions
High impact loading on bone has been well proven for its benefits on bone formation and bone development. Among numerous types of body movements contained in high impact activity, free fall landing has been suggested to be more contributive in bone metabolism (Burr et al., 2002; Welch et al., 2004). In numerous previous studies, the effects of long term free fall landing on bone development or metabolism has been verified (Welch et al., 2004; Judex &
Zernicke, 2000). However, the mechanisms related to how bone adapted to
high impact loading need to be clarified further. In the present study, we designed a time course trial in purpose of monitoring more parameters, which would be helpful for further understanding the process of bone response to high impact loading. Interestingly, spongy bone showed a significant higher in bone volume ratio in the FFL group (FFL vs. CON: 27.71.2 vs. 24.2 0.7), which implied high turnover spongy bone could response to the stress of the present free fall landing as early as in the initial first week. This bone formation effects caused from landing even magnifying more after four weeks (FFL vs.
CON: 30.01.2 vs. 24.7 1.9). Although statistical difference was not shown, the FFL group was thicker and denser in trabecular bone. As compared to the previous studies, our present study provide a new evidence that growing bone could response to high impact loading not just shown in molecular level but also in architecture level of trabecular bone. Further immunohistological
staining and biomechanical analysis are ongoing in our laboratory for acquiring more evidences as well as clarifying further about the mechanisms that bone response to high impact loadings.
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