- Email: [email protected]
Counter-effect of constrained dynamic loading on osteoporosis in ovariectomized mice
Journal of Biomechanics 46 (2013) 1242–1247
Contents lists available at SciVerse ScienceDirect
Journal of Biomechanics journal homepage: www.elsevier.com/locate/jbiomech www.JBiomech.com
Counter-effect of constrained dynamic loading on osteoporosis in ovariectomized mice Hao Li, Rui-Xin Li, Zong-Ming Wan, Cheng Xu, Jian-Yu Li, Qing-Xin Hao, Yong Guo, Lu Liu, Xi-Zheng Zhang n Institute of Medical Equipment, Academy of Military Medical Sciences, Tianjin 300161, China
a r t i c l e i n f o
a b s t r a c t
Article history: Accepted 27 February 2013
In recent years, dynamic mechanical loading has been shown to effectively enhance bone remodeling. The current study attempted to research the counter-effect of constrained dynamic loading on osteoporosis (OP) in ovariectomized (OVX) mice. Female Kunming (KM) mice were randomly divided into 2 groups: SHAM and OVX. The right ulnas of the OVX mice were subjected to a 4-week constrained dynamic loading protocol, and the mechanical properties, trabecular micromorphology parameters and biochemical indices of osteogenesis were evaluated. We detected higher levels of tissue alkaline phosphatase (AKP) and serum bone gamma-carboxyglutamic-acid-containing proteins (BGPs), better trabecular micromorphology parameters and ulnar mechanical properties in the loading group than in the nonloading group. In summary, constrained dynamic loading could prevent ovariectomy-induced osteoporosis by facilitating osteogenesis, improving trabecular microstructure and enhancing bone mechanical properties. & 2013 Elsevier Ltd. All rights reserved.
Keywords: Osteoporosis Dynamic loading Counter-effect Osteogenesis Mechanical properties
1. Introduction Bones are mechanically sensitive tissues that operate throughout their life span in dynamic stress/strain conditions that require ‘‘balance’’ between structural, functional and mechanical properties. It is generally believed that the ‘‘mechanostat’’ acts as the mediator between mechanical stimulation and the bone biologic response and there accordingly should be thresholds of mechanical use, such as typical peak strain, that help determine where and how the bone (re)models (Frost, 1991, 1997). Postmenopausal women worldwide suffer most from osteoporosis, which is typically characterized by a lower bone density and a higher fracture risk (Karlsson et al., 2005; Marcus and Majumdar, 2001). Fortunately, increasing evidence suggests that loading forms, such as amplitude, frequency and intervals, play important roles in mechanics-induced bone remodeling. Broadly speaking, under the same or similar levels of strain distribution, the osteogenic capacity of bone weakens from dynamic loading to static loading, compression to tension, and loading with intervals to no intervals. Mechanical loading with a constrained amplitude and frequency (particularly 15–30 Hz) could cause obvious osteogenesis and improve the histomorphology and mechanical
behavior of cancellous bone (Kaspar et al., 2002; Lanyon, 1996; Robling et al., 2002; Skerry, 1997). In the present study, we hypothesized that dynamic loading producing 200 –3000 me of local peak strain in bone would be beneficial for bone formation based on the ‘‘mechanostat’’ theory (Frost, 1987a, 1987b). We built a 3D model of the mouse ulna using micro-CT, determined the loading conditions using a finite element analysis (FEA) to control the distribution of strain in the ulna and chose classical indices of osteogenesis and mechanical properties, including tissue AKP, serum BGP, trabecular microstructure parameters, fracture displacement, fracture stress and fracture energy (Brown et al., 1987; Ito et al., 2002; Nian et al., 2009; Pauchard et al., 2008; Zhao et al., 2011), to detect bone formation. The aim of this study was to evaluate the counter-effect of constrained dynamic loading during estrogen deficiency-induced OP formation using the mouse ovariectomy model of OP (Kalu, 1991; Peng et al., 1997; Rodgers et al., 1993).
2. Materials and methods 2.1. Animals and materials
n
Corresponding author. Tel.: þ86 02284656716. E-mail address: [email protected] (X.-Z. Zhang).
0021-9290/$ - see front matter & 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jbiomech.2013.02.016
Female KM mice were purchased from the laboratory animaI center of the Academy of Military Medical Sciences in Beijing. The Bicinchoninic Acid (BCA) Protein Assay Kit, Mouse OT/BGP ELISA Kit and AKP Assay Kit were obtained from the Nanjing Jiancheng Bioengineering Institute.
H. Li et al. / Journal of Biomechanics 46 (2013) 1242–1247
1243
2.2. Experimental design
3. Results
A total of 21 female 2-month-old KM mice were randomly divided into 2 groups: SHAM (7 mice) and OVX (14 mice). The mice in the former group were sham operated, while the latter were ovariectomized. Thereafter, the right ulnas of all the OVX mice were subjected to constrained dynamic loading (with a diaphyseal peak strain 1000 –3000 me and a frequency of 15 Hz) for 15 min every 3rd day over a period of 4 weeks. Finally, the ulnas were harvested and divided into 3 groups, SHAM (ulnas from the SHAM group), OVX þ Loading (right ulnas from the OVX group) and OVX (left ulnas from the OVX group), and their mechanical properties, micromorphology parameters, tissue AKP and serum BGP content were analyzed.
A 3D model of the mouse ulna was free meshed with elementtype Solid 92, 18,064 elements and 27,971 nodes in ANSYS 12.0. The integral strain–diaphyseal strain curve and the load–diaphyseal strain curve under limiting material constants (E1 ¼18.1 GPa, E2 ¼0.4 GPa, n1 ¼ n2 ¼0.3) are shown in Figs. 1 and 2. Using curve-fitting and theoretical calculations, we finally selected dynamic loading conditions as the sinusoidal displacement curve (y¼0.045 sin(30p t) (mm)) starting from an equilibrium position with a 0.5 N static preload because, under this condition, the 0.5 N preload and sinusoidal displacement with an amplitude of 0.045 mm will cause 220 me and 3000 me diaphyseal peak strain in the ulna, respectively. Fig. 3 shows the ideal and actual loading modes. In Fig. 4, we are clear of the strain distribution in the ulna at a glance. Considering the inevitable absorption of impact energy by the joints and muscle, the actual total diaphyseal peak strain should be less than 3220 me and is more likely to be 1000–3000 me, which is our favored scenario. Vaginal smears in the first 5 postoperative days are shown in Fig. 5. Mice in the SHAM group had a normal life with a regular estrous cycle (Fig. 5(b)), while mice in the OVX group experienced dioestrum (Fig. 5(c)), suggesting that the ovariectomy was successful. The HE-stained, non-decalcified slices in Fig. 6 qualitatively show the changes in the trabecular bone in each group after the 4-week dynamic loading procedure. The changes in the micromorphology parameters of cancellous bone were in accordance with the qualitative observations, as shown in Fig. 7. We defined the proximal and distal ends of the ulna and chose the sections of cancellous bone 3 mm from the proximal end and 1.5 mm from the distal end as our regions of interest, as shown in Fig. 7(a). The scanning parameters were set at a 50 kV voltage and 9 mm slices. The micromorphology parameters were calculated and are presented as histograms with statistical significance in Fig. 7(b) and (c). We found that the trabecular bone in the OVX group deteriorated with a smaller B.Ar/T.Ar, thinner Tb.Th, larger Tb.Sp and more Tb.N when compared with the SHAM group, while the microstructure of the trabecular bone in the OVXþLoading group improved with a higher B.Ar/T.Ar, thicker Tb.Th and smaller Tb.Sp when compared with the OVX group. We evaluated the mechanical properties of the ulna using a three-point bending test on an INSTRON 5865, and the mechanical
2.3. Identification of the constrained dynamic loading parameters Using FEA, loading parameters were identified that could avoid operational influences during classical strain measurement (strain gauge) in vivo. A 3D model of the mouse ulna obtained by micro-CT (SkyScan 1076, Belgium) was imported into ANSYS 12.0 and free meshed. Material properties were simplified to be isotropic and have linear elasticity under infinitesimal conditions, and we chose the modulus of cortical bone (E1 ¼18.1 GPa, n1 ¼ 0.3) and cancellous bone (E2 ¼0.4 GPa, n2 ¼ 0.3) (Brennan et al., 2011; Cory et al., 2010; Homminga et al., 2002; Ito et al., 2002; Pauchard et al., 2008; Zhang et al.,. 2008) as limiting boundaries. Increasing compressive displacement and static loads were applied along the axial direction to calculate the diaphyseal average von Mises strain, according to which we drew the integral strain–diaphyseal strain curve and the load–diaphyseal strain curve. Using curve-fitting, we took into account the attenuation effect when loading in vivo and determined the static preload and the amplitude of dynamic loading. A final verification of the strain distribution statistics by FEA was also performed.
2.4. Tests of ulnar osteogenic activity and mechanical behavior Tissue AKP was tested using a BCA Protein Assay Kit and an AKP Assay Kit. Serum BGP was tested using a Mouse OT/BGP ELISA Kit. Micromorphology parameters of ulnar cancellous bone, including percent trabecular area (B.Ar/ T.Ar), trabecular pattern factor (Tb.Pf), trabecular thickness (Tb.Th), trabecular separation (Tb.Sp) and trabecular number (Tb.N), were analyzed by the postprocessing of micro-CT images. The mechanical properties of the ulna were determined by the classical three-point bending test on an INSTRON 5865 (Instron Corporation, America) with the following parameters: 10 mm gauge length, 0.5 N preload and 3 mm/min loading rate, outputting fracture displacement/strain, fracture stress and fracture energy.
2.5. Statistical analysis All data were presented as the mean 7S.D. from at least three independent experiments. Significant differences were evaluated by a one-way analysis of variance (ANOVA) followed by the least significant difference (LSD)-t test. Significance was defined at p o 0.05.
Fig. 1. Integral strain–diaphyseal strain curve and linear fit of the ulna.
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H. Li et al. / Journal of Biomechanics 46 (2013) 1242–1247
Fig. 2. Load–diaphyseal strain curve and polynomial fit of the ulna.
Fig. 3. Ideal and actual loading modes of the mouse ulna.
property parameters are shown in Table 1. A significant reduction of the ulnar mechanical properties was detected in the OVX group compared with the SHAM group with lower fracture displacement, fracture strain, fracture load and fracture energy, while better mechanical properties were observed in the OVXþLoading group when compared with the OVX group. We also assayed tissue AKP and serum BGP, which reflected osteoblast activity. Table 2 shows that the ulnar osteogenic activities in the OVX þLoading group were significantly enhanced, with higher levels of tissue AKP and serum BGP compared with the OVX group.
4. Discussion The biological mechanisms and micromorphology changes of OP have gained increasing attention, with theories of Frost H.M. classic. The root cause of postmenopausal OP may be estrogen
deficiency, which raises the remodeling threshold, making bones less or not at all sensitive to normal mechanical stimulation as in the disuse model. This would induce continuous bone removal and deteriorating microstructure (Frost, 1987a), which is supported and demonstrated by our study. The results in Fig. 6(c), Fig. 7(b), Tables 1 and 2 demonstrate that the ulnas in the OVX group experienced active weakening of osteoblasts, microstructural breakage of cancellous bone and mechanical behavioral reduction 4 weeks after ovariectomy. In this study, we emphasized the counter-effect of constrained dynamic loading during estrogen deficiency-induced OP formation within 4 weeks after ovariectomy. It was exciting to find that the constrained dynamic loading did work well with certain indices of mechanical properties in Table 1, even no statistical difference vs. SHAM group. The ulnas in the OVXþ Loading group demonstrated a balanced and significant enhancement from molecular expression to macroscopic mechanical properties. The osteogenic activities were significantly enhanced [Table 2], the microstructure of
H. Li et al. / Journal of Biomechanics 46 (2013) 1242–1247
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Fig. 4. Distribution of von Mises strain in the ulna under peak loads.
Fig. 5. A mouse after ovariectomy (a). Postoperative vaginal smears show that (b) nucleate/anuclear keratinocytes and leukocytes (smaller) coexisted in the SHAM group, while (c) only leukocytes were present in the OVX group.
Fig. 6. HE-stained non-decalcified slices of ulna trabecular bone show no difference between (a) the SHAM group and (b) the OVX þLoading group, while obvious deterioration or breakage of trabecular bone existed in (c) the OVX group.
cancellous bone was improved (Figs. 6 and 7(b)), and better mechanical properties were also observed (Table 1). BGP is a type of stable protein secreted only by osteoblasts, and it directly reflects osteoblast activity and the rate of bone turnover. Patients suffering estrogen deficiency-induced highturnover osteoporosis have been reported to exhibit increased BGP content (Brown et al., 1987). However, in our study, we detected a lower BGP level in the OVX group compared with the SHAM group 4 weeks after ovariectomy, which could also be explained. One early effect of estrogen deficiency is to unbalance bone remodeling in favor of bone resorption and to sharply accelerate the rate of bone turnover, including bone formation, mineralization and especially desorption (Kanis, 1996). The significant increase in the tissue total protein concentration in
the OVX group vs. the SHAM group shown in Table 2 was powerful proof of this concept. Therefore, from a different perspective, the lower serum BGP content in the OVX group might be evidence of stronger bone resorption rather than slower bone formation itself. There is a synergistic effect between the cortical shell and trabecular bone in bone mechanical performance, while cancellous bone, occupying a minority of the total skeletal mass, is more sensitive to mechanical stimulation and more available for remodeling events than cortical bone (Kanis, 1996; Kinney et al., 2000; Kusuzaki et al., 2000; Lee et al., 2002). Previous research has also demonstrated that the stress is distributed primarily in the cortical shell in bone with a deteriorated trabecular microstructure (Ito et al., 2002). In our study, ovariectomy and dynamic loading
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H. Li et al. / Journal of Biomechanics 46 (2013) 1242–1247
Fig. 7. (a) Definition of the regions of interest. Comparisons between different groups of trabecular micromorphology parameters were analyzed in the (b) proximal and (c) distal ulna.
Table 1 Ulnar mechanical properties in a three-point bending test. GROUP
Fracture displacement (mm)
Fracture strain (%)
Fracture load (N)
Fracture energy (mJ)
SHAM OVX OVX þLOADING
1.37727 0.0453 1.2800 7 0.0319n 1.34167 0.0284nn
9.12577 0.6844 6.81747 0.5322n 8.0496 7 0.4446nn
6.14947 0.6347 2.71557 0.3939n 5.4077 7 0.4733nn
7.3637 71.5080 4.2954 70.5036n 5.7645 70.7497nn
n
p o 0.05 vs. SHAM. p o0.05 vs. OVX.
nn
appeared to have a greater influence on cancellous bone because we found that comparisons of trabecular micromorphology parameters between SHAM, OVX and OVXþLoading were less significant in Fig. 7(c) than those in Fig. 7(b) in which there was less trabecular bone despite the similar strain distribution. We were then enlightened that, ovariectomy-induced trabecular deterioration would dramatically increase the load-bearing bur den on the cortical shell, resulting in local strain over the minimum effect strain threshold of pathological changes (MESp) (Frost, 1987b), which would aggravate bone turnover and OP development.
Table 2 Tissue AKP and serum BGP. GROUP
Total tissue protein concentrations (lg/ ml)
SHAM 16.7287 3.818 OVX 19.7177 3.518n OVXþ LOADING 25.1637 3.792n/nn n
p o0.05 vs. SHAM. po 0.05 vs. OVX.
nn
Tissue AKP content (U/mg prot)
Serum BGP content (ng/ml)
5.487 7 0.259 3.824 7 0.808n 4.877 7 0.924nn
4.735 71.008 3.382 70.108n 6.171 70.216n/nn
H. Li et al. / Journal of Biomechanics 46 (2013) 1242–1247
In our study, the ovariectomized mice experienced a drastic estrogen reduction, which may limit the ability of this OP model to fully simulate the more gradual spontaneous menopause process. Despite this limitation, estrogen deficiency-induced osteoporosis can be prevented in the elderly by encouraging them to exercise properly with certain form, regularity and intensity. In addition, future studies are planned to evaluate osteoclast activities and related signaling pathways to understand the mechanisms of bone remodeling under mechanical stress. We sincerely hope that our research may be of clinical significance for the prevention or treatment of osteoporosis.
5. Conclusion Four weeks after ovariectomy, the OP model in OVX mice was successfully established. In the present study, the constrained dynamic load effectively prevented osteoporosis by facilitating osteogenesis, improving the microstructure of cancellous bone and increasing bone mechanical properties.
Conflict of interest statement There are no conflicts of interest to report.
Acknowledgements This research was supported by the Major Program of the National Natural Science Foundation of China (NO. 10832012) and the General Program of the National Natural Foundation of China (NO.11072266). References Brennan, O., Kennedy, O.D., Lee, T.C., Rackard, S.M., O’Brien, F.J., McNamara, L.M., 2011. The effects of estrogen deficiency and bisphosphonate treatment on tissue mineralisation and stiffness in an ovine model of osteoporosis. Journal of Biomechanics 44, 386–390. Brown, J.P., Delmas, P.D., Arlot, M., 1987. Active bone turnover of the corticoendosteal envelope in postmenopausal osteoporosis. Journal of Clinical Endocrinology & Metabolism 64 (5), 954–959. ¨ Cory, E., Nazarian, A., Entezari, V., Vartanians, V., Muller, R., Snyder, B.D., 2010. Compressive axial mechanical properties of rat bone as functions of bone volume fraction, apparent density and micro-ct based mineral density. Journal of Biomechanics 43, 953–960. Frost, H.M., 1987a. The mechanostat: a proposed pathogenetic mechanism of osteoporoses and the bone mass effects of mechanical and nonmechanical agents. Bone and Mineral 2 (2), 73–85. Frost, H.M., 1987b. Bone ‘‘mass’’ and the ‘‘mechanostat’’: a proposal. The Anatomical Record 219 (1), 1–9.
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Frost, H.M., 1991. A new direction for osteoporosis research: a review and proposal. Bone 12 (6), 429–437. Frost, H.M., 1997. Defining osteopenias and osteoporoses: another view (with insights from a new paradigm). Bone 20 (5), 385–391. Homminga, J., McCreadie, B.R., Ciarelli, T.E., Weinans, H., Goldstein, S.A., Huiskes, R., 2002. Cancellous bone mechanical properties from normals and patients with hip fractures differ on the structure level, not on the bone hard tissue level. Bone 30 (5), 759–764. Ito, M., Nishida, A., Koga, A., Ikeda, S., Shiraishi, A., Uetani, M., Hayashi, K., Nakamura, T., 2002. Contribution of trabecular and cortical components to the mechanical properties of bone and their regulating parameters. Bone 31 (3), 351–358. Kalu, D.N., 1991. The ovariectomized rat model of postmenopausal bone loss. Bone and Mineral 15 (3), 175–191. Kanis, J.A., 1996. Estrogens, the menopause, and osteoporosis. Bone 19 (5), 185S–190S. Karlsson, M., Obrant, C., Josefsson, P.O., 2005. In: Buchloz, R.W., Heckman, J.D., Court-Brown, C., Torentta, P., Koval, K.J., Wirth, M.A. (Eds.), Osteoporotic Fractures Rockwood and Green’s Fractures in Adults. Lippincott Williams & Wilkins. Kaspar, D., Seidl, W., Neidlinger-Wilke, C., Beck, A., Claes, L., Ignatius, A., 2002. Proliferation of human-derived osteoblast-like cells depends on the cycle number and frequency of uniaxial strain. Journal of Biomechanics 35, 873–880. Kinney, J.H., Haupt, D.L., Balooch, M., Ladd, A.J.C., Ryaby, J.T., Lane, N.E., 2000. Three-dimensional morphometry of the L6 vertebra in the ovariectomized rat model of osteoporosis: biomechanical implications. Journal of Bone and Mineral Research 15 (10), 1981–1991. Kusuzaki, K., Kageyama, N., Shinjo, H., Takeshita, H., Murata, H., Hashiguchi, S., Ashihara, T., Hirasawa, Y., 2000. Development of bone canaliculi during bone repair. Bone 27 (5), 655–659. Lanyon, L.E., 1996. Using functional loading to influence bone mass and architecture: objectives, mechanisms, and relationship with estrogen of the mechanically adaptive process in bone. Bone 18 (Suppl. 1), 37–43. Lee, K.C.L., Maxwell, A., Lanyon, L.E., 2002. Validation of a technique for studying functional adaptation of the mouse ulna in response to mechanical loading. Bone 31 (3), 407–412. Marcus, R., Majumdar, S., 2001. The nature of osteoporosis. In: Marcus, R., Feldman, D., Kelsey, J. (Eds.), Osteoporosis. Academic Press. Nian, H., Ma, M.-H., Nian, S.-S., Xu, L.-L., 2009. Antiosteoporotic activity of icariin in ovariectomized rats. Phytomedicine 16, 320–326. Pauchard, Y., Mattmann, C., Kuhn, A., Gasser, J.A., Boyd, S.K., 2008. European society of biomechanics S.M. Perren Award 2008: using temporal trends of 3D bone micro- architecture to predict bone quality. Journal of Biomechanics 41 (14), 2946–2953. ¨ anen, ¨ Peng, Z.-Q., Va¨ an H.K., Zhang, H.X., Tuukkanen, J., 1997. Long-term effects of ovariectomy on the mechanical properties and chemical composition of rat bone. Bone 20 (3), 207–212. Robling, A.G., Hinant, F.M., Burr, D.B., Turner, C.H., 2002. Improved bone structure and strength after long-term mechanical loading is greatest if loading is separated into short bouts. Journal of Bone and Mineral Research 17 (8), 1545–1554. Rodgers, J.B., Monier-Faugere, M.-C., Malluche, H., 1993. Animal models for the study of bone loss after cessation of ovarian function. Bone 14 (3), 369–377. Skerry, T.M., 1997. Mechnical loading and bone: what sort of exercise is beneficial to the skeleton? Bone 20 (3), 179–181. Zhang, J., Niebur, G.L., Ovaert, T.C., 2008. Mechanical property determination of bone through nano- and micro-indentation testing and finite element simulation. Journal of Biomechanics 41, 267–275. Zhao, X., Wu, Z.-X., Zhang, Y., Yan, Y.-B., He, Q., Cao, P.-C., Lei, W., 2011. Antiosteoporosis activity of cibotium barometz extract on ovariectomy-induced bone loss in rats. Journal of Ethnopharmacology 137, 1083–1088.
Contents lists available at SciVerse ScienceDirect
Journal of Biomechanics journal homepage: www.elsevier.com/locate/jbiomech www.JBiomech.com
Counter-effect of constrained dynamic loading on osteoporosis in ovariectomized mice Hao Li, Rui-Xin Li, Zong-Ming Wan, Cheng Xu, Jian-Yu Li, Qing-Xin Hao, Yong Guo, Lu Liu, Xi-Zheng Zhang n Institute of Medical Equipment, Academy of Military Medical Sciences, Tianjin 300161, China
a r t i c l e i n f o
a b s t r a c t
Article history: Accepted 27 February 2013
In recent years, dynamic mechanical loading has been shown to effectively enhance bone remodeling. The current study attempted to research the counter-effect of constrained dynamic loading on osteoporosis (OP) in ovariectomized (OVX) mice. Female Kunming (KM) mice were randomly divided into 2 groups: SHAM and OVX. The right ulnas of the OVX mice were subjected to a 4-week constrained dynamic loading protocol, and the mechanical properties, trabecular micromorphology parameters and biochemical indices of osteogenesis were evaluated. We detected higher levels of tissue alkaline phosphatase (AKP) and serum bone gamma-carboxyglutamic-acid-containing proteins (BGPs), better trabecular micromorphology parameters and ulnar mechanical properties in the loading group than in the nonloading group. In summary, constrained dynamic loading could prevent ovariectomy-induced osteoporosis by facilitating osteogenesis, improving trabecular microstructure and enhancing bone mechanical properties. & 2013 Elsevier Ltd. All rights reserved.
Keywords: Osteoporosis Dynamic loading Counter-effect Osteogenesis Mechanical properties
1. Introduction Bones are mechanically sensitive tissues that operate throughout their life span in dynamic stress/strain conditions that require ‘‘balance’’ between structural, functional and mechanical properties. It is generally believed that the ‘‘mechanostat’’ acts as the mediator between mechanical stimulation and the bone biologic response and there accordingly should be thresholds of mechanical use, such as typical peak strain, that help determine where and how the bone (re)models (Frost, 1991, 1997). Postmenopausal women worldwide suffer most from osteoporosis, which is typically characterized by a lower bone density and a higher fracture risk (Karlsson et al., 2005; Marcus and Majumdar, 2001). Fortunately, increasing evidence suggests that loading forms, such as amplitude, frequency and intervals, play important roles in mechanics-induced bone remodeling. Broadly speaking, under the same or similar levels of strain distribution, the osteogenic capacity of bone weakens from dynamic loading to static loading, compression to tension, and loading with intervals to no intervals. Mechanical loading with a constrained amplitude and frequency (particularly 15–30 Hz) could cause obvious osteogenesis and improve the histomorphology and mechanical
behavior of cancellous bone (Kaspar et al., 2002; Lanyon, 1996; Robling et al., 2002; Skerry, 1997). In the present study, we hypothesized that dynamic loading producing 200 –3000 me of local peak strain in bone would be beneficial for bone formation based on the ‘‘mechanostat’’ theory (Frost, 1987a, 1987b). We built a 3D model of the mouse ulna using micro-CT, determined the loading conditions using a finite element analysis (FEA) to control the distribution of strain in the ulna and chose classical indices of osteogenesis and mechanical properties, including tissue AKP, serum BGP, trabecular microstructure parameters, fracture displacement, fracture stress and fracture energy (Brown et al., 1987; Ito et al., 2002; Nian et al., 2009; Pauchard et al., 2008; Zhao et al., 2011), to detect bone formation. The aim of this study was to evaluate the counter-effect of constrained dynamic loading during estrogen deficiency-induced OP formation using the mouse ovariectomy model of OP (Kalu, 1991; Peng et al., 1997; Rodgers et al., 1993).
2. Materials and methods 2.1. Animals and materials
n
Corresponding author. Tel.: þ86 02284656716. E-mail address: [email protected] (X.-Z. Zhang).
0021-9290/$ - see front matter & 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jbiomech.2013.02.016
Female KM mice were purchased from the laboratory animaI center of the Academy of Military Medical Sciences in Beijing. The Bicinchoninic Acid (BCA) Protein Assay Kit, Mouse OT/BGP ELISA Kit and AKP Assay Kit were obtained from the Nanjing Jiancheng Bioengineering Institute.
H. Li et al. / Journal of Biomechanics 46 (2013) 1242–1247
1243
2.2. Experimental design
3. Results
A total of 21 female 2-month-old KM mice were randomly divided into 2 groups: SHAM (7 mice) and OVX (14 mice). The mice in the former group were sham operated, while the latter were ovariectomized. Thereafter, the right ulnas of all the OVX mice were subjected to constrained dynamic loading (with a diaphyseal peak strain 1000 –3000 me and a frequency of 15 Hz) for 15 min every 3rd day over a period of 4 weeks. Finally, the ulnas were harvested and divided into 3 groups, SHAM (ulnas from the SHAM group), OVX þ Loading (right ulnas from the OVX group) and OVX (left ulnas from the OVX group), and their mechanical properties, micromorphology parameters, tissue AKP and serum BGP content were analyzed.
A 3D model of the mouse ulna was free meshed with elementtype Solid 92, 18,064 elements and 27,971 nodes in ANSYS 12.0. The integral strain–diaphyseal strain curve and the load–diaphyseal strain curve under limiting material constants (E1 ¼18.1 GPa, E2 ¼0.4 GPa, n1 ¼ n2 ¼0.3) are shown in Figs. 1 and 2. Using curve-fitting and theoretical calculations, we finally selected dynamic loading conditions as the sinusoidal displacement curve (y¼0.045 sin(30p t) (mm)) starting from an equilibrium position with a 0.5 N static preload because, under this condition, the 0.5 N preload and sinusoidal displacement with an amplitude of 0.045 mm will cause 220 me and 3000 me diaphyseal peak strain in the ulna, respectively. Fig. 3 shows the ideal and actual loading modes. In Fig. 4, we are clear of the strain distribution in the ulna at a glance. Considering the inevitable absorption of impact energy by the joints and muscle, the actual total diaphyseal peak strain should be less than 3220 me and is more likely to be 1000–3000 me, which is our favored scenario. Vaginal smears in the first 5 postoperative days are shown in Fig. 5. Mice in the SHAM group had a normal life with a regular estrous cycle (Fig. 5(b)), while mice in the OVX group experienced dioestrum (Fig. 5(c)), suggesting that the ovariectomy was successful. The HE-stained, non-decalcified slices in Fig. 6 qualitatively show the changes in the trabecular bone in each group after the 4-week dynamic loading procedure. The changes in the micromorphology parameters of cancellous bone were in accordance with the qualitative observations, as shown in Fig. 7. We defined the proximal and distal ends of the ulna and chose the sections of cancellous bone 3 mm from the proximal end and 1.5 mm from the distal end as our regions of interest, as shown in Fig. 7(a). The scanning parameters were set at a 50 kV voltage and 9 mm slices. The micromorphology parameters were calculated and are presented as histograms with statistical significance in Fig. 7(b) and (c). We found that the trabecular bone in the OVX group deteriorated with a smaller B.Ar/T.Ar, thinner Tb.Th, larger Tb.Sp and more Tb.N when compared with the SHAM group, while the microstructure of the trabecular bone in the OVXþLoading group improved with a higher B.Ar/T.Ar, thicker Tb.Th and smaller Tb.Sp when compared with the OVX group. We evaluated the mechanical properties of the ulna using a three-point bending test on an INSTRON 5865, and the mechanical
2.3. Identification of the constrained dynamic loading parameters Using FEA, loading parameters were identified that could avoid operational influences during classical strain measurement (strain gauge) in vivo. A 3D model of the mouse ulna obtained by micro-CT (SkyScan 1076, Belgium) was imported into ANSYS 12.0 and free meshed. Material properties were simplified to be isotropic and have linear elasticity under infinitesimal conditions, and we chose the modulus of cortical bone (E1 ¼18.1 GPa, n1 ¼ 0.3) and cancellous bone (E2 ¼0.4 GPa, n2 ¼ 0.3) (Brennan et al., 2011; Cory et al., 2010; Homminga et al., 2002; Ito et al., 2002; Pauchard et al., 2008; Zhang et al.,. 2008) as limiting boundaries. Increasing compressive displacement and static loads were applied along the axial direction to calculate the diaphyseal average von Mises strain, according to which we drew the integral strain–diaphyseal strain curve and the load–diaphyseal strain curve. Using curve-fitting, we took into account the attenuation effect when loading in vivo and determined the static preload and the amplitude of dynamic loading. A final verification of the strain distribution statistics by FEA was also performed.
2.4. Tests of ulnar osteogenic activity and mechanical behavior Tissue AKP was tested using a BCA Protein Assay Kit and an AKP Assay Kit. Serum BGP was tested using a Mouse OT/BGP ELISA Kit. Micromorphology parameters of ulnar cancellous bone, including percent trabecular area (B.Ar/ T.Ar), trabecular pattern factor (Tb.Pf), trabecular thickness (Tb.Th), trabecular separation (Tb.Sp) and trabecular number (Tb.N), were analyzed by the postprocessing of micro-CT images. The mechanical properties of the ulna were determined by the classical three-point bending test on an INSTRON 5865 (Instron Corporation, America) with the following parameters: 10 mm gauge length, 0.5 N preload and 3 mm/min loading rate, outputting fracture displacement/strain, fracture stress and fracture energy.
2.5. Statistical analysis All data were presented as the mean 7S.D. from at least three independent experiments. Significant differences were evaluated by a one-way analysis of variance (ANOVA) followed by the least significant difference (LSD)-t test. Significance was defined at p o 0.05.
Fig. 1. Integral strain–diaphyseal strain curve and linear fit of the ulna.
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Fig. 2. Load–diaphyseal strain curve and polynomial fit of the ulna.
Fig. 3. Ideal and actual loading modes of the mouse ulna.
property parameters are shown in Table 1. A significant reduction of the ulnar mechanical properties was detected in the OVX group compared with the SHAM group with lower fracture displacement, fracture strain, fracture load and fracture energy, while better mechanical properties were observed in the OVXþLoading group when compared with the OVX group. We also assayed tissue AKP and serum BGP, which reflected osteoblast activity. Table 2 shows that the ulnar osteogenic activities in the OVX þLoading group were significantly enhanced, with higher levels of tissue AKP and serum BGP compared with the OVX group.
4. Discussion The biological mechanisms and micromorphology changes of OP have gained increasing attention, with theories of Frost H.M. classic. The root cause of postmenopausal OP may be estrogen
deficiency, which raises the remodeling threshold, making bones less or not at all sensitive to normal mechanical stimulation as in the disuse model. This would induce continuous bone removal and deteriorating microstructure (Frost, 1987a), which is supported and demonstrated by our study. The results in Fig. 6(c), Fig. 7(b), Tables 1 and 2 demonstrate that the ulnas in the OVX group experienced active weakening of osteoblasts, microstructural breakage of cancellous bone and mechanical behavioral reduction 4 weeks after ovariectomy. In this study, we emphasized the counter-effect of constrained dynamic loading during estrogen deficiency-induced OP formation within 4 weeks after ovariectomy. It was exciting to find that the constrained dynamic loading did work well with certain indices of mechanical properties in Table 1, even no statistical difference vs. SHAM group. The ulnas in the OVXþ Loading group demonstrated a balanced and significant enhancement from molecular expression to macroscopic mechanical properties. The osteogenic activities were significantly enhanced [Table 2], the microstructure of
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Fig. 4. Distribution of von Mises strain in the ulna under peak loads.
Fig. 5. A mouse after ovariectomy (a). Postoperative vaginal smears show that (b) nucleate/anuclear keratinocytes and leukocytes (smaller) coexisted in the SHAM group, while (c) only leukocytes were present in the OVX group.
Fig. 6. HE-stained non-decalcified slices of ulna trabecular bone show no difference between (a) the SHAM group and (b) the OVX þLoading group, while obvious deterioration or breakage of trabecular bone existed in (c) the OVX group.
cancellous bone was improved (Figs. 6 and 7(b)), and better mechanical properties were also observed (Table 1). BGP is a type of stable protein secreted only by osteoblasts, and it directly reflects osteoblast activity and the rate of bone turnover. Patients suffering estrogen deficiency-induced highturnover osteoporosis have been reported to exhibit increased BGP content (Brown et al., 1987). However, in our study, we detected a lower BGP level in the OVX group compared with the SHAM group 4 weeks after ovariectomy, which could also be explained. One early effect of estrogen deficiency is to unbalance bone remodeling in favor of bone resorption and to sharply accelerate the rate of bone turnover, including bone formation, mineralization and especially desorption (Kanis, 1996). The significant increase in the tissue total protein concentration in
the OVX group vs. the SHAM group shown in Table 2 was powerful proof of this concept. Therefore, from a different perspective, the lower serum BGP content in the OVX group might be evidence of stronger bone resorption rather than slower bone formation itself. There is a synergistic effect between the cortical shell and trabecular bone in bone mechanical performance, while cancellous bone, occupying a minority of the total skeletal mass, is more sensitive to mechanical stimulation and more available for remodeling events than cortical bone (Kanis, 1996; Kinney et al., 2000; Kusuzaki et al., 2000; Lee et al., 2002). Previous research has also demonstrated that the stress is distributed primarily in the cortical shell in bone with a deteriorated trabecular microstructure (Ito et al., 2002). In our study, ovariectomy and dynamic loading
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Fig. 7. (a) Definition of the regions of interest. Comparisons between different groups of trabecular micromorphology parameters were analyzed in the (b) proximal and (c) distal ulna.
Table 1 Ulnar mechanical properties in a three-point bending test. GROUP
Fracture displacement (mm)
Fracture strain (%)
Fracture load (N)
Fracture energy (mJ)
SHAM OVX OVX þLOADING
1.37727 0.0453 1.2800 7 0.0319n 1.34167 0.0284nn
9.12577 0.6844 6.81747 0.5322n 8.0496 7 0.4446nn
6.14947 0.6347 2.71557 0.3939n 5.4077 7 0.4733nn
7.3637 71.5080 4.2954 70.5036n 5.7645 70.7497nn
n
p o 0.05 vs. SHAM. p o0.05 vs. OVX.
nn
appeared to have a greater influence on cancellous bone because we found that comparisons of trabecular micromorphology parameters between SHAM, OVX and OVXþLoading were less significant in Fig. 7(c) than those in Fig. 7(b) in which there was less trabecular bone despite the similar strain distribution. We were then enlightened that, ovariectomy-induced trabecular deterioration would dramatically increase the load-bearing bur den on the cortical shell, resulting in local strain over the minimum effect strain threshold of pathological changes (MESp) (Frost, 1987b), which would aggravate bone turnover and OP development.
Table 2 Tissue AKP and serum BGP. GROUP
Total tissue protein concentrations (lg/ ml)
SHAM 16.7287 3.818 OVX 19.7177 3.518n OVXþ LOADING 25.1637 3.792n/nn n
p o0.05 vs. SHAM. po 0.05 vs. OVX.
nn
Tissue AKP content (U/mg prot)
Serum BGP content (ng/ml)
5.487 7 0.259 3.824 7 0.808n 4.877 7 0.924nn
4.735 71.008 3.382 70.108n 6.171 70.216n/nn
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In our study, the ovariectomized mice experienced a drastic estrogen reduction, which may limit the ability of this OP model to fully simulate the more gradual spontaneous menopause process. Despite this limitation, estrogen deficiency-induced osteoporosis can be prevented in the elderly by encouraging them to exercise properly with certain form, regularity and intensity. In addition, future studies are planned to evaluate osteoclast activities and related signaling pathways to understand the mechanisms of bone remodeling under mechanical stress. We sincerely hope that our research may be of clinical significance for the prevention or treatment of osteoporosis.
5. Conclusion Four weeks after ovariectomy, the OP model in OVX mice was successfully established. In the present study, the constrained dynamic load effectively prevented osteoporosis by facilitating osteogenesis, improving the microstructure of cancellous bone and increasing bone mechanical properties.
Conflict of interest statement There are no conflicts of interest to report.
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