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Quantitative associations between osteocyte density and biomechanics, microcrack and microstructure in OVX rats vertebral trabeculae
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Journal of Biomechanics 41 (2008) 1324–1332 www.elsevier.com/locate/jbiomech www.JBiomech.com
Quantitative associations between osteocyte density and biomechanics, microcrack and microstructure in OVX rats vertebral trabeculae Yu-Lin Ma, Ru-Chun Dai, Zhi-Feng Sheng, Yan Jin, Yu-Hai Zhang, Ling-Na Fang, Hui-Jie Fan, Er-Yuan Liao Institute of Metabolism and Endocrinology, The Second Xiang-Ya Hospital, Central South University, Changsha, 410011, Hunan, PR China Accepted 10 January 2008
Abstract Osteocytes actively regulate bone modeling and remodeling, direct skeletal mineralization, and regulate calcium/phosphate homeostasis and extracellular matrix metabolism; yet the specific role of osteocytes in maintaining bone structural integrity and strength is unknown. Studies have shown that the density of osteocytes decreases with age and estrogen deficiency, as seen in postmenopausal women. Here, we examined the relationships between osteocyte density and the related variables, including biomechanics, bone mineral density, microcrack and microstructure of vertebral trabeculae, in ovariectomized rats. We found that osteocyte density correlated with some of the parameters that determine the biomechanical quality of bone. Our findings suggest that osteocytes could play a crucial role in maintaining the mechanical quality of bone, and osteocyte density could be considered as an alternative index in assessing bone quality. r 2008 Elsevier Ltd. All rights reserved. Keywords: Osteocyte density; Microcrack; Bone mineral density; Microstructure; Biomechanics
1. Introduction Clinically, osteoporosis is defined as a measurement of bone mineral density (BMD) less than 2.5 standard deviations below the mean value of a young adult (T-score). However, BMD alone cannot account for all the variations in bone strength (Delmas and Seeman, 2004). This necessitates the measuring of other determinants of bone quality, such as bone architecture and micro-damage accumulation (Dai et al., 2004). Less well established is the obvious impact that reduced osteocyte viability might have on bone quality. A previous study in mice demonstrated that osteocyte death could reduce vertebral compression strength, independent of changes in bone mass or architecture (Weinstein et al., 2002). Manolagas et al. (2005) established that the strength of bone is significantly lower in 16-month-old female C57BL/6 mice compared with 8-month-old mice, without a detectable decrease in BMD. This was associated with an increased prevalence of Corresponding author. Tel./fax: +86 731 536 1472.
E-mail address: [email protected] (E.-Y. Liao). 0021-9290/$ - see front matter r 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.jbiomech.2008.01.017
osteocyte apoptosis. The presence of viable osteocytes in bone has been shown to be associated with the ability of bone to remodel efficiently, maintain normal levels of mineralization and repair accumulated microdamage (Qiu et al., 2003; Hernandez et al., 2004; Hazenberga et al., 2006). Therefore, osteocyte viability might represent an important and, to date, poorly understood determinant of bone quality. The causes of the aging-related loss of osteocytes remain unclear, but it has been established that osteocyte apoptosis is engendered by a wide variety of conditions including glucocorticoid excess (Kogianni et al., 2004; O’Brien et al., 2004), microdamage (Gu et al., 2005a), estrogen deficiency (Tomkinson et al., 1998) and oxidative stress (Mann et al., 2007). Estrogen receptors are abundantly expressed in osteocytes, but the expression is less in other cells of the osteoblast lineage, suggesting that osteocytes are more likely to be involved in the regulation of estrogen-mediated bone remodeling. In the trabecular iliac crest bone, osteocyte density decreases with aging and it differs in healthy subjects when compared with patients with osteoporosis (Qiu et al., 2003; Hernandez et al., 2004).
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Therefore, increased knowledge of osteocyte density would contribute to the assessment of osteoporosis. The purpose of the present study is to quantify osteocyte density, microcrack, microstructure and biomechanical parameters in ovariectomized rats, ovariectomized rats treated with 17b-estradiol and genistein, and sham-operation rats. We have also assessed whether the decline in biomechanical quality in estrogen-deficient rats is associated with fewer osteocytes and whether estrogen replacement treatment (ERT) can ameliorate these parameters. Based on the current understanding of the relationship between osteocyte density and bone quality (Manolagas et al., 2005; O’Brien et al., 2004; Tomkinson et al., 1998), we hypothesized that osteocyte density might be a potential determinant of the biomechanical quality of bone. 2. Materials and methods 2.1. Animal preparation All animal procedures were approved by the Institutional Animal Care and Use Committee of People’s Republic of China. Forty female Sprague–Dawley (7-month-old) rats were obtained from the Animal Center of the Second Xiang-Ya Hospital (Central South University, China). The rats were single-fed, housed at 23–25 1C with a 12 h light/dark cycle, and allowed free access to water. The rats underwent either ovariectomy (n ¼ 30, OVX) or a sham-operation (n ¼ 10, SHAM). The OVX rats were randomly divided into three groups: OVX and treated with 17b-estradiol (Sigma, Chemical, St. Louis, MO, USA, 10 mg/kg/day, EST) (Dai et al., 2004) and genistein (5 mg/kg/day, GEN) (Fanti et al., 1998). At 15 weeks post-operation, all the rats were anesthetized with phenobarbital (1 mg/kg, injected intraperitoneally) and sacrificed by bloodletting from the ventral aorta. The vertebrae were dissected and stored at 70 1C until micro-computed tomography (m-CT) scanning was performed.
2.2. Mechanical tests The L-5 vertebral body specimen was prepared as previously described (Dai et al., 2004). The vertebral cylinder samples were attached to the material-testing machine and analysis systems (CSS44100, Changchun Research Institute of Test Machines, Changchun, China). The vertebral height and diameters were measured. A compression force was applied in the craniocaudal direction using a steel disk (1.8 cm diameter) at a nominal deformation rate of 2 mm/min. Load–deformation curves were recorded continually in the computerized monitor linked to the tester. The ultimate compressive load was obtained directly from the load–deformation curves at the level of maximum load (ML). The elastic modulus (EM) was estimated using the following equation: EM ¼ stiffness (height/CSA), where stiffness is the slope of the fitting linear portion of the load/ displacement curve and CSA is the cross-sectional area calculated as rectangle area.
2.3. Micro-computed tomography (m-CT) (Sheng et al., 2007) The L-6 vertebral body specimens were scanned by a micro-CT specimen scanner (GE eXplore LocusSP Specimen Scanner; GE Healthcare Company, London, Canada), which is a cone-beam scanning system. The scanning protocol was 80 kV and 80 mA, with an isotopic resolution of 6.5 6.5 6.5 mm voxel size and an exposure time of 3000 ms per frame. Trabecular bone volume fraction (BV/TV), bone surface density (BS/BV), trabecular number (Tb.N), trabecular separation (Tb.Sp), trabecular thickness (Tb.Th), geometric degree of anisotropy (DA), connectivity
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density (Conn.D), structure model index (SMI) and trabecular volumetric BMD at both organ and tissue levels were determined.
2.4. Fatigue damage testing (Dai et al., 2004) After micro-CT scanning, the L-6 vertebra were treated in the same way as the L-5 vertebra. Fatigue damage tests were performed on the PLD 5010 bone fatigue damage testing machine (Changchun Research Institute of Test Machines, Changchun, China), with a loading frequency of 2.5 Hz, a loading number of 10,000 times for each vertebra, and a loading strength of 60 N. After the fatigue damage tests, the vertebral bodies were fixed in 70% ethanol, followed by en-bloc basic fuchsin staining, plastic embedding and sectioning.
2.5. En-bloc basic fuchsin staining The en-bloc basic fuchsin staining method as described above (Dai et al., 2004) was used to detect microcracks. After staining, basic fuchsinstained specimens were immersed in xylene for 1 day, followed by routine plastic embedding for bone histomorphometry analysis.
2.6. Observation of microcracks and quantification of osteocyte density (Dai et al., 2004; Da Costa Go0 mez et al., 2005) Two serial coronal sections of 50–80 mm were prepared for morphometric analysis using polarizing microscopy at 400 magnification and Leica DMLA microscope-image analysis system (Leica Corporation, Wetzla, Germany). The following histomorphometric variables were determined: microcrack density (Cr.Dn, ]/mm2); microcrack mean length (Cr.Le, mm); and microcrack surface density (Cr.S.Dn, mm/mm2). All the data were collected by a single observer. Blue-violet epifluorescent light (425–440 excitation and 475 nm barrier filter) at 400 magnification was used to count the number of osteocytes. The histomorphometric variable was osteocyte density (Ot.N/T.Ar, ]/ mm2).
2.7. Statistical analysis Differences among treatment groups were evaluated using a one-way analysis of variance (ANOVA). When a significant overall F-value (Po0.05) was present, differences between individual group means were tested using Fisher’s protected least-significant difference (PLSD) post-hoc test. A probability (P) value of o0.05 was considered significant. Correlation and regression analyses were also performed to study the relationships between BMD, bone mineral content (BMC), mechanical testing parameters, microcrack indices, microstructure properties and osteocyte density.
3. Results 3.1. Micro-CT analysis of trabecular bone Table 1 summarizes the data of micro-CT analysis of the vertebral trabecular bone. Volumetric BMD (vBMD) was significantly lower in the OVX (p ¼ 0.000), EST (p ¼ 0.004) and GEN (p ¼ 0.002) groups than in the SHAM group. BMC was significantly lower in the OVX group than in the SHAM groups (p ¼ 0.049). There were no significant differences with regard to the BMC values among the SHAM, EST and GEN groups. BV/TV and Tb.N were significantly lower in the OVX (p ¼ 0.001 and p ¼ 0.000), EST (p ¼ 0.005 and p ¼ 0.026) and GEN (p ¼ 0.002 and p ¼ 0.002) groups than in the SHAM
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Table 1 Values of BMD, microstructure, microcrack parameters and biomechanical properties at 15 weeks postovariectomy (means7SD) Parameters
SHAM
OVX
EST
GEN
VBMD (mg/mm3) TBMD (mg/mm3) BMC (mg) BV/TV (%) BS/BV (%) Tb.Th (mm) Tb.N (mm3) Tb.Sp (mm) SMI Conn.D (mm3) DA ML (N) EM (MPa) Cr.Dn (]/mm2) Cr.S.Dn (mm/mm2) Cr.Le (mm) Ot.N/T.Ar (]/mm2)
364.6738.0 739.3712.2 7.971.2 26.074.4 31.672.4 63.774.9 4.170.58 242.3754.2 0.6670.51 68.5718.6 1.6070.55 148.1715.1 630.07132.7 0.4670.13b 13.775.4b 24.278.1b 1760.87376.6b
304.3741.3a 751.8718.0 6.572.2a 19.774.2a 31.972.9 63.375.6 3.170.53a 335.9773.1a 1.1470.44a 47.2712.6a 1.8270.74 107.5720.2a 694.17194.0 2.0270.39 121.5724.0 58.176.8 1299.67352.8
312.8737.5a 720.4714.2 6.871.5 20.373.7a 34.772.2 57.973.6a,b 3.570.45a 277.1749.3b 1.2870.35a 56.7716.9 1.8370.73 138.3714.1b 717.97164.5 0.5070.12b 28.079.1b 36.579.7b 1550.97202.2b
316.2742.2a 738.3712.5 7.3172.2 20.874.1a 32.772.4 61.574.6 3.470.52a 295.6790.0 1.2570.48a 71.9714.9b,c 1.7970.41 125.6713.9a,b 540.0752.5 0.5170.12b 24.076.4b 28.577.5b 1550.77215.5b
vBMD, volumetric BMD; tBMD, tissue BMD; BV/TV, bone volume fraction; BS/BV, bone surface density; Tb.Th, trabecular thickness; Tb.N, trabecular number; Tb.Sp, trabecular separation; SMI, structure model index; Conn.D, connectivity; DA, degree of anisotropy; ML, maximum loading; EM, elastic modulus; Cr.Dn, microcrack density; Cr.S.Dn, microcrack surface density; Cr.Le, mean microcrack length; Ot.N/T.Ar, osteocyte density.Values are expressed as means7SD. a Po0.05, compared with SHAM rats. b Po0.05, compared with OVX rats. c Po0.05, compared with ERT rats.
group. There were no significant differences among the four groups with regard to the BS/BV and DA values. SMI was significantly lower in the SHAM group than in the OVX (p ¼ 0.01), EST (p ¼ 0.003) and GEN (p ¼ 0.001) groups. Tb.Sp was significantly greater in the OVX group than in the SHAM (p ¼ 0.002) and EST (p ¼ 0.043) groups. There were no significant differences with regard to the Tb.Th values among the SHAM, GEN and OVX groups; however, it was significantly lower in the EST group than in the SHAM (p ¼ 0.009) and OVX (p ¼ 0.006) groups. Conn.D was significantly lower in the OVX group than in the SHAM (p ¼ 0.001) and GEN (p ¼ 0.000) groups. Conn.D was also significantly lower in the EST group than in the GEN group (p ¼ 0.018). The OVX group showed a marked deterioration in the microstructure of vertebral trabeculae (Fig. 1). 3.2. Biomechanics, microcrack and osteocyte density All the data of biomechanics, microcrack and osteocyte density of vertebrae are summarized in Table 1. Cr.Dn, Cr.S.Dn and Cr.Le were increased significantly in the OVX group than in the SHAM (p ¼ 0.000, 0.000, 0.005, respectively), EST (p ¼ 0.000, 0.0023, 0.015, respectively) and GEN (p ¼ 0.000, 0.000, 0.005, respectively) groups. ML was significantly lower in the OVX group than in the SHAM (p ¼ 0.000), EST (p ¼ 0.000) and GEN (p ¼ 0.026) groups. ML was also significantly lower in the GEN group than in the SHAM group (p ¼ 0.001). There were no significant differences with regard to EM among the four groups. Ot.N/T.Ar decreased significantly in the OVX
group than in the SHAM (p ¼ 0.001), EST (p ¼ 0.046) and GEN (p ¼ 0.033) groups. In the OVX group, under a blueviolet epifluorescent light microscope, we found empty lacunae and oval osteocytes with more than two nucleuslike fragments, which were characteristic of apoptotic osteocytes (Fig. 2). 3.3. Correlations between Ot.N/T.Ar and BMD, BMC, biomechanics and microstructure parameters When data were pooled from all four groups, Ot.N/T.Ar positively correlated with ML and BMC, but not EM and BMD (Figs. 3 and 4). There was a significant negative correlation between Ot.N/T.Ar and Cr.Le (Fig. 5), but there were no significant correlations between Ot.N/T.Ar and other microcrack indices (Cr.Dn and Cr.S.Dn). Ot.N/T. Ar positively correlated with Conn.D and negatively correlated with Tb.Sp (Figs. 6 and 7). There were no statistically significant correlations between Ot.N/T.Ar and other microstructure parameters of vertebral trabeculae (BV/TV, BS/BV, Tb.Th, Tb.N, SMI and DA). 4. Discussion To our knowledge, the present study is the first to find a positive correlation between Ot.N/T.Ar and bone quality. The statistical results were similar when Ot.N/T.Ar were calculated as independent and dependent variables. Osteocyte density, in addition to microcracking, BMD, microstructure and biomechanical testing, is a useful parameter
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Fig. 1. Effect of ovariectomy (OVX) and sex steroid replacement treatment on 3D morphological changes in the spongiosa of the vertebrae at 15 weeks after ovariectomy. OVX induced a marked deterioration in microarchitecture of vertebral trabeculae (A), microarchitecture of vertebral trabeculae in SHAM (B), EST (C) and GEN rats (D).
Fig. 2. En-bloc basic fuchsin staining (A) showing microcrack (black arrow) in OVX rats. Blue-violet epifluorescent light microscope showing oval osteocytes (white arrow) with more than two nucleus-like fragments, empty lacunae (black arrow) and microcracks (dashed arrow) in OVX rats (B), round osteocytes (white arrow) in SHAM (C), EST (D) and GEN rats (E). Scale bars ¼ 50 mm.
for assessing the biomechanical quality of bone and the efficacy of ERT in a rat model of osteoporosis. The dendritic processes of osteocytes are linked by gap junctions with the processes of neighboring osteocytes, as well as with cells present on the bone surface. These cells include the lining cells, cellular elements of the bone
marrow, and the endothelial cells of the bone marrow vasculature. Fluid flow shear stress might stimulate the canaliculi to release chemical mediums via gap junctions to translate mechanical strain into biochemical signals and biological responses (Heino et al., 2002; Kurata et al., 2006; Taylor et al., 2007). Osteocytes have been shown to inhibit
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2500.00 y = 1658.7–3.6622x r 2 = 0.5738 P = 0.0272 SHAM
1500.00 y = 405.457+8.6198x r 2 = 0.7874 P = 0.0001 SHAM GEN EST
1000.00
100.00
120.00 140.00 160.00 Maximum loading (N)
EST
1500.00
OVX
1000.00
180.00
Fig. 3. Osteocyte density (Ot.N/T.Ar) positively correlated with maximum loading (ML).
y = 58.2966x+ 1076.62 r 2 = 0.1309 P = 0.0171 SHAM GEN EST
0.00
y = 2150.62–2265.87x r 2 = 0.3964 p = 0.001 SHAM GEN EST
2000.00
OVX
1000.00
20.00 40.00 60.00 80.00 100.00 120.00 Microcrack mean length (µm)
Fig. 5. Osteocyte density (Ot.N/T.Ar) negatively correlated with microcrack mean length (Cr.Le).
Osteocyte density (#/mm2)
Osteocyte density (#/mm2)
GEN
500.00 80.00
1500.00
2000.00
OVX
500.00
2000.00
Osteocyte density (#/mm2)
Osteocyte density (#/mm2)
2000.00
1500.00
OVX
1000.00
500.00 2.0000
4.0000 6.0000 8.0000 10.0000 Bone mineral content (g)
12.0000
Fig. 4. Osteocyte density (Ot.N/T.Ar) positively correlated with bone mineral content (BMC).
500.00 0.20
0.40 0.60 Trabecular separation (µm)
0.80
Fig. 6. Osteocyte density (Ot.N/T.Ar) negatively correlated with trabecular separation (Tb.Sp).
osteoclastic bone resorption (Gu et al., 2005a) through transforming growth factor (TGF)(Heino et al., 2002) and promote osteoclastic activity through macrophage colonystimulating factor (MCS-F) and receptor activator of nuclear factor kappa B ligand (RANKL) (Kurata et al., 2006). Osteocytes can also stimulate the proliferation of bone marrow mesenchymal stem cells and their differentiation into osteoblasts (Heino et al., 2004) and regulate osteoblastic activity via gap junctions (Taylor et al., 2007). More recently, osteocyte-ablated mice were observed to exhibit fragile bone with intracortical porosity and microfractures, osteoblastic dysfunction, and trabecular bone loss with microstructural deterioration and adipose tissue proliferation in the marrow space; all of which are hallmarks of the aging skeleton (Tatsumi et al., 2007). The osteocyte is not only the mechanosensor (Cherian et al., 2003; Tatsumi et al., 2007) in bone (re)modeling, but
also has the potential to direct skeletal mineralization, regulate calcium/phosphate homeostasis and extracellular matrix metabolism (Feng et al., 2006; Lane et al., 2006). Thus, adequate numbers of osteocytes may be essential for maintaining both cellular activity and strength of bone. It is not clear how osteocytes affect the biomechanical quality of bone. Recent studies have demonstrated that osteocyte density correlated with the initiation and propagation of microdamage (Qiu et al., 2005). Osteocyte lacunae can act as sites for microcracks initiation (Reilly, 2000) to dissipate energy (Vashishth, 2004) or act as barriers to cause crack arrest. Therefore, any increase in osteocyte density may affect the length of cracks and bone strength. Osteocyte lacunae density was observed to decline exponentially with aging in dogs and microcrack density increased simultaneously (Frank et al., 2002).
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Osteocyte density (#/mm2)
2000.00
1500.00
Y = 1191.00+5.2922x r 2 = 0.1153 P = 0.0299 SHAM GEN EST OVX
1000.00
500.00 20.00000
40.00000 60.00000 80.00000 100.00000 Trabecular connectivity (mm3)
Fig. 7. Osteocyte density (Ot.N/T.Ar) positively correlated with trabecular connectivity (Conn.D).
The likelihood of microdamage was 3.8 times higher in bone with an osteocyte lacuna density greater than 728/mm2 (Qiu et al., 2005). On the other hand, reduced numbers of osteocytes can result in the deterioration of canalicular fluid flow, decreased ability to detect microdamage and increased bone fragility (Qui et al., 2003; Burger et al., 2003). Conceivably, reduced fluid flow can alter the volumetric proportion of water and crystal within the mineral; i.e too little water increases bone brittleness (Currey, 1984) and too much water decreases bone stiffness (Hernandez et al., 2001). Such a mechanism might account for the acute effect of osteocyte death on reducing vertebral compression strength in mice, independent of changes in bone mass or architecture (Weinstein et al., 2002). Evidently, much more work is needed to explore these possibilities. In a study by Qiu et al., (2006),black American women were found to have higher BMD indices, higher osteocyte density and lower osteon density than white American women, and the former were not as susceptible to fracture . These data support the hypothesis that microstructural features such as osteocyte lacunae, osteons and Volkmann’s canals can act as barriers to crack propagation (Da Costa Go0 mez et al., 2005). The results of our present study are consistent with these data, i.e. they indicate that adequate numbers of osteocytes are essential to remove bone microdamage and osteocyte density correlates with the biomechanical quality of bone. The mechanism by which estrogen inhibits osteocyte apoptosis remains unclear. Recent data suggest that the anti-apoptosis action of estrogen (Mann et al., 2007) is by way of anti-oxidative stress, thus inducing the expression of glutathione, prostaglandin (PG)-E2, TGF-b and bcl-2, inhibiting the production of inflammatory cytokines, and directly effecting an antioxidant molecule. Furthermore, estrogen has been observed to protect primary osteocytes against glucocorticoid-induced apoptosis (Gu et al.,
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2005b); thus, sex-steroid deficiency would result in increased osteocyte apoptosis (Tomkinson et al., 1998; Kousteni et al., 2001). Our present study found that the osteocyte density of trabecular bone in OVX rats decreased significantly, and the ratio of empty lacunae and oval osteocytes, with the characteristics of apoptotic osteocytes of more than two nucleus-like fragments, increased simultaneously (Hamaya et al., 2002). This is not quantified in the present study and would need to be identified through further research. Estrogen deficiency also resulted in BMC loss and deteriorated microstructure and biomechanical quality. Interestingly, ERT maintained the biomechanical quality of bone and osteocyte viability without significantly changing the parameters of BMD and microstructure except for those of Conn.D and Tb.Sp. This is consistent with the results of other studies (O’Brien et al., 2004; Mullender et al., 2005; Collishaw et al., 2003). These data suggest that osteocytes might play a role in modulating bone quality. We found a weak but significant, positive correlation between Ot.N/T.Ar and ML, which is a direct indicator of bone mechanical strength. Though EM is also an important parameter for assessing the mechanical quality of bone, we did not find EM to be correlated with Ot.N/T.Ar. There may be an inaccuracy that cannot be ignored in calculating EM in our study. The cross-section of the vertebra is irregular; therefore, there is an inaccuracy in calculating the cross-sectional area using diameters of the vertebra. Earlier work by Dai et al. (2004) showed that the parameters of fatigue-damaged microcrack represented the changes of biomechanical quality of bone associated with ovariectomy in rats. Allen et al. (2006) found that, after 1 year of bisphosphonate treatment at clinical doses, a significant accumulation of microdamage was seen in the vertebra. However, this was offset by increases in bone volume and mineralization to the extent that there was no significant impairment of mechanical properties. Diab and Vashisth (2007) found that older men had fewer diffuse damages, yet more and longer linear microcracks than younger men. These data suggest that a certain degree of microdamage is essential for bone (re)modeling. Therefore, microcrack initiation does not always indicate decreased bone quality, but more so a mechanism of dissipating energy (Vashishth, 2004). We found no correlations between Ot.N/T.Ar and Cr.S.Dn or Cr.Dn, which is consistent with an earlier study by Frank et al. (2002). However, we did find a weak negative correlation between Ot.N/T.Ar and Cr.Le. Based on our findings and the current understanding of the relationships between osteocyte density (Manolagas et al., 2005; O’Brien et al., 2004; Tomkinson et al., 1998), microcrack (Diab and Vashishth, 2007) and the mechanical quality of bone, we hypothesize that osteocyte density and microcrack length might be potential determinants of bone quality. The effects of ERT are limited both in our OVX rats treated for 15 weeks and in postmenopausal women with osteoporosis treated for 6 years (Khastgir et al., 2001).
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In our present study ERT did not significantly influence most of the microstructure parameters except for Conn.D and Tb.Sp. We found a weak but significant negative correlation between Conn.D, Tb.Sp and Ot.N/T.Ar, but no correlations between Ot.N/T.Ar and other microstructure parameters (Tb.N, SMI, Tb.Th, BV/TV and BS/BV). This is inconsistent with the results of a previous study that Ot.N/T.Ar positively correlated with BA/TA in human cortical and cancellous bone (Vashishth et al., 2002). This may be due to a difference in subject number in the two studies. Sham surgery can potentially influence estradiol levels in subject rats, yet changes in estradiol levels were not monitored and this was a limitation of our study. Our results showed correlations between Ot.N/T.Ar and bone biomechanical quality; however, the correlations were weak, which indicates that the Ot.N/T.Ar is not the major determinant of bone quality. Further clarification of the relationships between Ot.N/T.Ar and other determinants of bone biomechanical quality, such as bone turnover, bone mineralization, bone matrix and mineral composition indices, are required (Compston, 2006). A recent study showed that there were statistically significant linear correlations between the remodeling indices (BMU activation frequency, densities of secondary osteons, forming osteon, and resorption cavities) and apoptotic osteocyte density, and a strongly positive correlation between the modeling indices (forming surface and surface MAR) and empty osteocyte lacunae, but not osteocyte density (Hedgecock et al., 2007). Additionally, osteocyte apoptosis following corticosteroid therapy is associated with reduced bone strength before any bone loss has occurred (O’Brien et al., 2004). The reasons for the loss of bone strength are unknown, but structural abnormalities in bone surrounding the apoptotic osteocyte may compromise bone strength (Lane et al., 2006). The phenomena of increased osteocyte apoptosis and decreased osteocyte density was also observed in weightlessness, disuse and glucocorticoid-induced osteoporosis (O’Brien et al., 2004; Kogianni et al., 2004; Aguirre et al., 2006.) In conclusion, these data indirectly reinforce the notion that osteocytes actively initiate and control bone (re)modeling and subsequent bone quality (Heino et al., 2002; Gu et al., 2005a). We found that the trabecular thickness did not decrease during the progression of bone loss induced by OVX. Even the EST rats had thinner trabeculae, which may be due to compensatory responses observed by other researchers (Waarsing et al., 2004; Sheng et al., 2007). The molecular mechanism of compensatory responses is unclear, and such a compensatory behavior did not overcome the mechanical impairments caused by OVX. Estrogen is known to act on osteoblasts depending on their stage of differentiation and estrogen receptor (ER) isoform expression (Hernandez et al., 2004). A previous study found that there was a fall in the proportion of osteocytes and osteoblasts expressing ER-a in postmenopausal women (Ireland et al., 2002). Additionally, they
demonstrated that type-I collagen synthesis was mainly regulated by ER-a. Since genistein has a similar chemical structure to estrogen, it is capable of modulating bone metabolism by activating the ERs. It is known that 17bestradiol has a higher affinity to ER-a than to ER-b. Contrary to that observed with 17b-estradiol, genistein has a higher affinity to ER-b than to ER-a. The present study demonstrated that genistein exerted a weaker effect than 17b-estradiol on the parameters of bone microstructure and biomechanics, which is likely to be due to the differences with regard to their binding affinities with the ER isoforms. Furthermore, the responses of osteocytes to both strain and estrogen involve ER-a, and only estrogen regulates the cellular concentration of ER-a (Zaman et al., 2006). This is consistent with the hypothesis that the decreased biomechanical quality associated with estrogen deficiency is a consequence of the reduced anabolic responses of osteocytes to strain. In summary, it has been shown in our present study that osteocyte density positively correlated with some parameters of the biomechanical quality of bone. We postulate that osteocyte density could be an alternative index for assessing bone quality. The mechanisms by which osteocytes orchestrate and control bone formation and resorption play a pivotal role in current bone research. Future advances in our understanding of the biology of the osteocyte might uncover new promising drug targets for osteoporosis. Conflict of interest All authors have no conflicts of interest. Drs. Ma and Dai contributed equally to this work. All authors were fully involved in the study and preparation of the manuscript, and the material within has not been and will not be submitted for publication elsewhere. Acknowledgement We gratefully acknowledge Grant no. 30400514 from the National Natural Science Foundation of China, Grant no. 06JJ50029 from the Hunan Province Natural Science Foundation of China and Grant no. 2004—468-50 from the Ministry of Health of PR China. References Aguirre, J.I., Plotkin, L.I., Stewart, S.A., Weinstein, R.S., Parfitt, A.M., Manolagas, S.C., Bellido, T., 2006. Osteocyte apoptosis is induced by weightlessness in mice and precedes osteoclast recruitment and bone loss. Journal of Bone and Mineral Research 21, 605–615. Allen, M.R., Iwata, K., Phipps, R., Burr, D.B., 2006. Alterations in canine vertebral bone turnover, microdamage accumulation, and biomechanical properties following 1-year treatment with clinical treatment doses of risedronate or alendronate. Bone 39, 872–879. Burger, E.H., Klein-Nulend, J., Smit, T.H., 2003. Strain-derived canalicular fluid flow regulates osteoclast activity in a remodelling osteon—a proposal. Journal of Biomechanics 36, 1453–1459.
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Hernandez, C.J., Majeska, R.J., Schaffler, M.B., 2004. Osteocyte density in woven bone. Bone 35, 1095–1099. Ireland, D.C., Bord, S., Beavan, S.R., Compston, G.E., 2002. Effects of estrogen on collagen synthesis by cultured human osteoblasts depend on the rate of cellular differentiation. Journal of Cell Biochemistry 86, 251–257. Khastgir, G., Studd, J., Holland, N., Alaghband-Zadeh, J., Fox, S., Chow, J., 2001. Postmenopausal women with osteoporosis: histomorphometric evidence in a longitudinal study. Journal of Clinical Endocrinology and Metabolism 86, 289–295. Kogianni, G., Mann, V., Ebetino, F., Nuttallc, M., Nijweided, P., Simpsona, H., Noble, B., 2004. Fas/CD95 is associated with glucocorticoid-induced osteocyte apoptosis. Life Science 75, 2879–2895. Kousteni, S., Bellido, T., Plotkin, L.I., O’Brien, C.A., Bodenner, D.L., Han, L., 2001. Nongenotropic, sex-nonspecific signaling through the estrogen or androgen receptors: dissociation from transcriptional activity. Cell 104, 719–730. Kurata, K., Heino, T.J., Higaki, H., Va¨a¨na¨nen, H.K., 2006. Bone marrow cell differentiation induced by mechanically damaged osteocytes in 3D gel-embedded culture. Journal of Bone and Mineral Research 21, 616–625. Lane, N.E., Yao, W., Balooch, M., Nalla, R.K., Balooch, G., Habelitz, S., Kinneyand, J.H., Bonewald, L.F., 2006. Glucocorticoid-treated mice have localized changes in trabecular bone material properties and osteocyte lacunar size that are not observed in placebo-treated or estrogen-deficient mice. Journal of Bone and Mineral Research 21, 466–476. Mann, V., Huber, C., Kogianni, ., Collins, F., Noble, B., 2007. The antioxidant effect of estrogen and selective estrogen receptor modulators in the inhibition of osteocyte apoptosis in vitro. Bone 40, 674–684. Manolagas, S.C., Almeida, M., Han, L., Weinstein, R., Jilka, R., Bellido, T., O’Brien, C., Kousteni, S., 2005. Decreased defense against reactive oxygen species: a common pathogenetic mechanism of the effects of aging and estrogen deficiency on bone. Journal of Bone and Mineral Research 20, S94 [abstract]. Mullender, M.G., Tan, S.D., Vico, L., 2005. Differences in osteocyte density and bone histomorphometry between men and women and between healthy and osteoporotic subjects. Calcified Tissue International 77, 291–296. O’Brien, C.A., Jia, D., Plotkin, L.I., Bellido, T., Powers, C.C., Stewart, S.A., Manolagas, S.C., Weinstein, R.S., 2004. Glucocorticoids act directly on osteoblasts and osteocytes to induce their apoptosis and reduce bone formation and strength. Endocrinology 145, 1835–1841. Qiu, S., Rao, D.S., Palnitkar, S., Parfitt, A.M., 2003. Reduced iliac cancellous osteocyte density in patients with osteoporotic vertebral fracture. Journal of Bone and Mineral Research 18, 1657–1663. Qiu, S., Rao, D.S., David, P., 2005. The morphological association between microcracks and osteocyte lacunae in human cortical bone. Bone 37, 10–15. Qiu, S., Rao, D.S., Palnitrar, S., Parfitt, A.M., 2006. Differences in osteocyte and lacunar density between Black and White American women. Bone 38, 130–135. Reilly, G.C., 2000. Observations of microdamage around osteocyte lacunae in bone. Journal of Biomechanics 33, 1131–1134. Sheng, Z., Dai, R., Wu, X., Fang, L., Fan, H., Liao, E., 2007. Regionallyspecific compensation for bone loss in the tibial trabeculae of estrogen deficient rats. Acta Radiologica 48, 531–539. Tatsumi, S., Ishii, K., Amizuka, N., Li, M., Kobayashi, T., Kohno, K., et al., 2007. Targeted ablation of osteocytes induces osteoporosis with defective mechanotransduction. Cell Metabolism 5, 464–475. Taylor, AF., Saundersm, M.M., Shinglem, D.L., Cimbalam, J.M., Zhou, Z., Donahue, H.J., 2007. Mechanically stimulated osteocytes regulate osteoblastic activity via gap junctions. American Journal of Physiology. Cell Physiology 292, C545–C552.
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Journal of Biomechanics 41 (2008) 1324–1332 www.elsevier.com/locate/jbiomech www.JBiomech.com
Quantitative associations between osteocyte density and biomechanics, microcrack and microstructure in OVX rats vertebral trabeculae Yu-Lin Ma, Ru-Chun Dai, Zhi-Feng Sheng, Yan Jin, Yu-Hai Zhang, Ling-Na Fang, Hui-Jie Fan, Er-Yuan Liao Institute of Metabolism and Endocrinology, The Second Xiang-Ya Hospital, Central South University, Changsha, 410011, Hunan, PR China Accepted 10 January 2008
Abstract Osteocytes actively regulate bone modeling and remodeling, direct skeletal mineralization, and regulate calcium/phosphate homeostasis and extracellular matrix metabolism; yet the specific role of osteocytes in maintaining bone structural integrity and strength is unknown. Studies have shown that the density of osteocytes decreases with age and estrogen deficiency, as seen in postmenopausal women. Here, we examined the relationships between osteocyte density and the related variables, including biomechanics, bone mineral density, microcrack and microstructure of vertebral trabeculae, in ovariectomized rats. We found that osteocyte density correlated with some of the parameters that determine the biomechanical quality of bone. Our findings suggest that osteocytes could play a crucial role in maintaining the mechanical quality of bone, and osteocyte density could be considered as an alternative index in assessing bone quality. r 2008 Elsevier Ltd. All rights reserved. Keywords: Osteocyte density; Microcrack; Bone mineral density; Microstructure; Biomechanics
1. Introduction Clinically, osteoporosis is defined as a measurement of bone mineral density (BMD) less than 2.5 standard deviations below the mean value of a young adult (T-score). However, BMD alone cannot account for all the variations in bone strength (Delmas and Seeman, 2004). This necessitates the measuring of other determinants of bone quality, such as bone architecture and micro-damage accumulation (Dai et al., 2004). Less well established is the obvious impact that reduced osteocyte viability might have on bone quality. A previous study in mice demonstrated that osteocyte death could reduce vertebral compression strength, independent of changes in bone mass or architecture (Weinstein et al., 2002). Manolagas et al. (2005) established that the strength of bone is significantly lower in 16-month-old female C57BL/6 mice compared with 8-month-old mice, without a detectable decrease in BMD. This was associated with an increased prevalence of Corresponding author. Tel./fax: +86 731 536 1472.
E-mail address: [email protected] (E.-Y. Liao). 0021-9290/$ - see front matter r 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.jbiomech.2008.01.017
osteocyte apoptosis. The presence of viable osteocytes in bone has been shown to be associated with the ability of bone to remodel efficiently, maintain normal levels of mineralization and repair accumulated microdamage (Qiu et al., 2003; Hernandez et al., 2004; Hazenberga et al., 2006). Therefore, osteocyte viability might represent an important and, to date, poorly understood determinant of bone quality. The causes of the aging-related loss of osteocytes remain unclear, but it has been established that osteocyte apoptosis is engendered by a wide variety of conditions including glucocorticoid excess (Kogianni et al., 2004; O’Brien et al., 2004), microdamage (Gu et al., 2005a), estrogen deficiency (Tomkinson et al., 1998) and oxidative stress (Mann et al., 2007). Estrogen receptors are abundantly expressed in osteocytes, but the expression is less in other cells of the osteoblast lineage, suggesting that osteocytes are more likely to be involved in the regulation of estrogen-mediated bone remodeling. In the trabecular iliac crest bone, osteocyte density decreases with aging and it differs in healthy subjects when compared with patients with osteoporosis (Qiu et al., 2003; Hernandez et al., 2004).
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Therefore, increased knowledge of osteocyte density would contribute to the assessment of osteoporosis. The purpose of the present study is to quantify osteocyte density, microcrack, microstructure and biomechanical parameters in ovariectomized rats, ovariectomized rats treated with 17b-estradiol and genistein, and sham-operation rats. We have also assessed whether the decline in biomechanical quality in estrogen-deficient rats is associated with fewer osteocytes and whether estrogen replacement treatment (ERT) can ameliorate these parameters. Based on the current understanding of the relationship between osteocyte density and bone quality (Manolagas et al., 2005; O’Brien et al., 2004; Tomkinson et al., 1998), we hypothesized that osteocyte density might be a potential determinant of the biomechanical quality of bone. 2. Materials and methods 2.1. Animal preparation All animal procedures were approved by the Institutional Animal Care and Use Committee of People’s Republic of China. Forty female Sprague–Dawley (7-month-old) rats were obtained from the Animal Center of the Second Xiang-Ya Hospital (Central South University, China). The rats were single-fed, housed at 23–25 1C with a 12 h light/dark cycle, and allowed free access to water. The rats underwent either ovariectomy (n ¼ 30, OVX) or a sham-operation (n ¼ 10, SHAM). The OVX rats were randomly divided into three groups: OVX and treated with 17b-estradiol (Sigma, Chemical, St. Louis, MO, USA, 10 mg/kg/day, EST) (Dai et al., 2004) and genistein (5 mg/kg/day, GEN) (Fanti et al., 1998). At 15 weeks post-operation, all the rats were anesthetized with phenobarbital (1 mg/kg, injected intraperitoneally) and sacrificed by bloodletting from the ventral aorta. The vertebrae were dissected and stored at 70 1C until micro-computed tomography (m-CT) scanning was performed.
2.2. Mechanical tests The L-5 vertebral body specimen was prepared as previously described (Dai et al., 2004). The vertebral cylinder samples were attached to the material-testing machine and analysis systems (CSS44100, Changchun Research Institute of Test Machines, Changchun, China). The vertebral height and diameters were measured. A compression force was applied in the craniocaudal direction using a steel disk (1.8 cm diameter) at a nominal deformation rate of 2 mm/min. Load–deformation curves were recorded continually in the computerized monitor linked to the tester. The ultimate compressive load was obtained directly from the load–deformation curves at the level of maximum load (ML). The elastic modulus (EM) was estimated using the following equation: EM ¼ stiffness (height/CSA), where stiffness is the slope of the fitting linear portion of the load/ displacement curve and CSA is the cross-sectional area calculated as rectangle area.
2.3. Micro-computed tomography (m-CT) (Sheng et al., 2007) The L-6 vertebral body specimens were scanned by a micro-CT specimen scanner (GE eXplore LocusSP Specimen Scanner; GE Healthcare Company, London, Canada), which is a cone-beam scanning system. The scanning protocol was 80 kV and 80 mA, with an isotopic resolution of 6.5 6.5 6.5 mm voxel size and an exposure time of 3000 ms per frame. Trabecular bone volume fraction (BV/TV), bone surface density (BS/BV), trabecular number (Tb.N), trabecular separation (Tb.Sp), trabecular thickness (Tb.Th), geometric degree of anisotropy (DA), connectivity
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density (Conn.D), structure model index (SMI) and trabecular volumetric BMD at both organ and tissue levels were determined.
2.4. Fatigue damage testing (Dai et al., 2004) After micro-CT scanning, the L-6 vertebra were treated in the same way as the L-5 vertebra. Fatigue damage tests were performed on the PLD 5010 bone fatigue damage testing machine (Changchun Research Institute of Test Machines, Changchun, China), with a loading frequency of 2.5 Hz, a loading number of 10,000 times for each vertebra, and a loading strength of 60 N. After the fatigue damage tests, the vertebral bodies were fixed in 70% ethanol, followed by en-bloc basic fuchsin staining, plastic embedding and sectioning.
2.5. En-bloc basic fuchsin staining The en-bloc basic fuchsin staining method as described above (Dai et al., 2004) was used to detect microcracks. After staining, basic fuchsinstained specimens were immersed in xylene for 1 day, followed by routine plastic embedding for bone histomorphometry analysis.
2.6. Observation of microcracks and quantification of osteocyte density (Dai et al., 2004; Da Costa Go0 mez et al., 2005) Two serial coronal sections of 50–80 mm were prepared for morphometric analysis using polarizing microscopy at 400 magnification and Leica DMLA microscope-image analysis system (Leica Corporation, Wetzla, Germany). The following histomorphometric variables were determined: microcrack density (Cr.Dn, ]/mm2); microcrack mean length (Cr.Le, mm); and microcrack surface density (Cr.S.Dn, mm/mm2). All the data were collected by a single observer. Blue-violet epifluorescent light (425–440 excitation and 475 nm barrier filter) at 400 magnification was used to count the number of osteocytes. The histomorphometric variable was osteocyte density (Ot.N/T.Ar, ]/ mm2).
2.7. Statistical analysis Differences among treatment groups were evaluated using a one-way analysis of variance (ANOVA). When a significant overall F-value (Po0.05) was present, differences between individual group means were tested using Fisher’s protected least-significant difference (PLSD) post-hoc test. A probability (P) value of o0.05 was considered significant. Correlation and regression analyses were also performed to study the relationships between BMD, bone mineral content (BMC), mechanical testing parameters, microcrack indices, microstructure properties and osteocyte density.
3. Results 3.1. Micro-CT analysis of trabecular bone Table 1 summarizes the data of micro-CT analysis of the vertebral trabecular bone. Volumetric BMD (vBMD) was significantly lower in the OVX (p ¼ 0.000), EST (p ¼ 0.004) and GEN (p ¼ 0.002) groups than in the SHAM group. BMC was significantly lower in the OVX group than in the SHAM groups (p ¼ 0.049). There were no significant differences with regard to the BMC values among the SHAM, EST and GEN groups. BV/TV and Tb.N were significantly lower in the OVX (p ¼ 0.001 and p ¼ 0.000), EST (p ¼ 0.005 and p ¼ 0.026) and GEN (p ¼ 0.002 and p ¼ 0.002) groups than in the SHAM
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Table 1 Values of BMD, microstructure, microcrack parameters and biomechanical properties at 15 weeks postovariectomy (means7SD) Parameters
SHAM
OVX
EST
GEN
VBMD (mg/mm3) TBMD (mg/mm3) BMC (mg) BV/TV (%) BS/BV (%) Tb.Th (mm) Tb.N (mm3) Tb.Sp (mm) SMI Conn.D (mm3) DA ML (N) EM (MPa) Cr.Dn (]/mm2) Cr.S.Dn (mm/mm2) Cr.Le (mm) Ot.N/T.Ar (]/mm2)
364.6738.0 739.3712.2 7.971.2 26.074.4 31.672.4 63.774.9 4.170.58 242.3754.2 0.6670.51 68.5718.6 1.6070.55 148.1715.1 630.07132.7 0.4670.13b 13.775.4b 24.278.1b 1760.87376.6b
304.3741.3a 751.8718.0 6.572.2a 19.774.2a 31.972.9 63.375.6 3.170.53a 335.9773.1a 1.1470.44a 47.2712.6a 1.8270.74 107.5720.2a 694.17194.0 2.0270.39 121.5724.0 58.176.8 1299.67352.8
312.8737.5a 720.4714.2 6.871.5 20.373.7a 34.772.2 57.973.6a,b 3.570.45a 277.1749.3b 1.2870.35a 56.7716.9 1.8370.73 138.3714.1b 717.97164.5 0.5070.12b 28.079.1b 36.579.7b 1550.97202.2b
316.2742.2a 738.3712.5 7.3172.2 20.874.1a 32.772.4 61.574.6 3.470.52a 295.6790.0 1.2570.48a 71.9714.9b,c 1.7970.41 125.6713.9a,b 540.0752.5 0.5170.12b 24.076.4b 28.577.5b 1550.77215.5b
vBMD, volumetric BMD; tBMD, tissue BMD; BV/TV, bone volume fraction; BS/BV, bone surface density; Tb.Th, trabecular thickness; Tb.N, trabecular number; Tb.Sp, trabecular separation; SMI, structure model index; Conn.D, connectivity; DA, degree of anisotropy; ML, maximum loading; EM, elastic modulus; Cr.Dn, microcrack density; Cr.S.Dn, microcrack surface density; Cr.Le, mean microcrack length; Ot.N/T.Ar, osteocyte density.Values are expressed as means7SD. a Po0.05, compared with SHAM rats. b Po0.05, compared with OVX rats. c Po0.05, compared with ERT rats.
group. There were no significant differences among the four groups with regard to the BS/BV and DA values. SMI was significantly lower in the SHAM group than in the OVX (p ¼ 0.01), EST (p ¼ 0.003) and GEN (p ¼ 0.001) groups. Tb.Sp was significantly greater in the OVX group than in the SHAM (p ¼ 0.002) and EST (p ¼ 0.043) groups. There were no significant differences with regard to the Tb.Th values among the SHAM, GEN and OVX groups; however, it was significantly lower in the EST group than in the SHAM (p ¼ 0.009) and OVX (p ¼ 0.006) groups. Conn.D was significantly lower in the OVX group than in the SHAM (p ¼ 0.001) and GEN (p ¼ 0.000) groups. Conn.D was also significantly lower in the EST group than in the GEN group (p ¼ 0.018). The OVX group showed a marked deterioration in the microstructure of vertebral trabeculae (Fig. 1). 3.2. Biomechanics, microcrack and osteocyte density All the data of biomechanics, microcrack and osteocyte density of vertebrae are summarized in Table 1. Cr.Dn, Cr.S.Dn and Cr.Le were increased significantly in the OVX group than in the SHAM (p ¼ 0.000, 0.000, 0.005, respectively), EST (p ¼ 0.000, 0.0023, 0.015, respectively) and GEN (p ¼ 0.000, 0.000, 0.005, respectively) groups. ML was significantly lower in the OVX group than in the SHAM (p ¼ 0.000), EST (p ¼ 0.000) and GEN (p ¼ 0.026) groups. ML was also significantly lower in the GEN group than in the SHAM group (p ¼ 0.001). There were no significant differences with regard to EM among the four groups. Ot.N/T.Ar decreased significantly in the OVX
group than in the SHAM (p ¼ 0.001), EST (p ¼ 0.046) and GEN (p ¼ 0.033) groups. In the OVX group, under a blueviolet epifluorescent light microscope, we found empty lacunae and oval osteocytes with more than two nucleuslike fragments, which were characteristic of apoptotic osteocytes (Fig. 2). 3.3. Correlations between Ot.N/T.Ar and BMD, BMC, biomechanics and microstructure parameters When data were pooled from all four groups, Ot.N/T.Ar positively correlated with ML and BMC, but not EM and BMD (Figs. 3 and 4). There was a significant negative correlation between Ot.N/T.Ar and Cr.Le (Fig. 5), but there were no significant correlations between Ot.N/T.Ar and other microcrack indices (Cr.Dn and Cr.S.Dn). Ot.N/T. Ar positively correlated with Conn.D and negatively correlated with Tb.Sp (Figs. 6 and 7). There were no statistically significant correlations between Ot.N/T.Ar and other microstructure parameters of vertebral trabeculae (BV/TV, BS/BV, Tb.Th, Tb.N, SMI and DA). 4. Discussion To our knowledge, the present study is the first to find a positive correlation between Ot.N/T.Ar and bone quality. The statistical results were similar when Ot.N/T.Ar were calculated as independent and dependent variables. Osteocyte density, in addition to microcracking, BMD, microstructure and biomechanical testing, is a useful parameter
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Fig. 1. Effect of ovariectomy (OVX) and sex steroid replacement treatment on 3D morphological changes in the spongiosa of the vertebrae at 15 weeks after ovariectomy. OVX induced a marked deterioration in microarchitecture of vertebral trabeculae (A), microarchitecture of vertebral trabeculae in SHAM (B), EST (C) and GEN rats (D).
Fig. 2. En-bloc basic fuchsin staining (A) showing microcrack (black arrow) in OVX rats. Blue-violet epifluorescent light microscope showing oval osteocytes (white arrow) with more than two nucleus-like fragments, empty lacunae (black arrow) and microcracks (dashed arrow) in OVX rats (B), round osteocytes (white arrow) in SHAM (C), EST (D) and GEN rats (E). Scale bars ¼ 50 mm.
for assessing the biomechanical quality of bone and the efficacy of ERT in a rat model of osteoporosis. The dendritic processes of osteocytes are linked by gap junctions with the processes of neighboring osteocytes, as well as with cells present on the bone surface. These cells include the lining cells, cellular elements of the bone
marrow, and the endothelial cells of the bone marrow vasculature. Fluid flow shear stress might stimulate the canaliculi to release chemical mediums via gap junctions to translate mechanical strain into biochemical signals and biological responses (Heino et al., 2002; Kurata et al., 2006; Taylor et al., 2007). Osteocytes have been shown to inhibit
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2500.00 y = 1658.7–3.6622x r 2 = 0.5738 P = 0.0272 SHAM
1500.00 y = 405.457+8.6198x r 2 = 0.7874 P = 0.0001 SHAM GEN EST
1000.00
100.00
120.00 140.00 160.00 Maximum loading (N)
EST
1500.00
OVX
1000.00
180.00
Fig. 3. Osteocyte density (Ot.N/T.Ar) positively correlated with maximum loading (ML).
y = 58.2966x+ 1076.62 r 2 = 0.1309 P = 0.0171 SHAM GEN EST
0.00
y = 2150.62–2265.87x r 2 = 0.3964 p = 0.001 SHAM GEN EST
2000.00
OVX
1000.00
20.00 40.00 60.00 80.00 100.00 120.00 Microcrack mean length (µm)
Fig. 5. Osteocyte density (Ot.N/T.Ar) negatively correlated with microcrack mean length (Cr.Le).
Osteocyte density (#/mm2)
Osteocyte density (#/mm2)
GEN
500.00 80.00
1500.00
2000.00
OVX
500.00
2000.00
Osteocyte density (#/mm2)
Osteocyte density (#/mm2)
2000.00
1500.00
OVX
1000.00
500.00 2.0000
4.0000 6.0000 8.0000 10.0000 Bone mineral content (g)
12.0000
Fig. 4. Osteocyte density (Ot.N/T.Ar) positively correlated with bone mineral content (BMC).
500.00 0.20
0.40 0.60 Trabecular separation (µm)
0.80
Fig. 6. Osteocyte density (Ot.N/T.Ar) negatively correlated with trabecular separation (Tb.Sp).
osteoclastic bone resorption (Gu et al., 2005a) through transforming growth factor (TGF)(Heino et al., 2002) and promote osteoclastic activity through macrophage colonystimulating factor (MCS-F) and receptor activator of nuclear factor kappa B ligand (RANKL) (Kurata et al., 2006). Osteocytes can also stimulate the proliferation of bone marrow mesenchymal stem cells and their differentiation into osteoblasts (Heino et al., 2004) and regulate osteoblastic activity via gap junctions (Taylor et al., 2007). More recently, osteocyte-ablated mice were observed to exhibit fragile bone with intracortical porosity and microfractures, osteoblastic dysfunction, and trabecular bone loss with microstructural deterioration and adipose tissue proliferation in the marrow space; all of which are hallmarks of the aging skeleton (Tatsumi et al., 2007). The osteocyte is not only the mechanosensor (Cherian et al., 2003; Tatsumi et al., 2007) in bone (re)modeling, but
also has the potential to direct skeletal mineralization, regulate calcium/phosphate homeostasis and extracellular matrix metabolism (Feng et al., 2006; Lane et al., 2006). Thus, adequate numbers of osteocytes may be essential for maintaining both cellular activity and strength of bone. It is not clear how osteocytes affect the biomechanical quality of bone. Recent studies have demonstrated that osteocyte density correlated with the initiation and propagation of microdamage (Qiu et al., 2005). Osteocyte lacunae can act as sites for microcracks initiation (Reilly, 2000) to dissipate energy (Vashishth, 2004) or act as barriers to cause crack arrest. Therefore, any increase in osteocyte density may affect the length of cracks and bone strength. Osteocyte lacunae density was observed to decline exponentially with aging in dogs and microcrack density increased simultaneously (Frank et al., 2002).
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Osteocyte density (#/mm2)
2000.00
1500.00
Y = 1191.00+5.2922x r 2 = 0.1153 P = 0.0299 SHAM GEN EST OVX
1000.00
500.00 20.00000
40.00000 60.00000 80.00000 100.00000 Trabecular connectivity (mm3)
Fig. 7. Osteocyte density (Ot.N/T.Ar) positively correlated with trabecular connectivity (Conn.D).
The likelihood of microdamage was 3.8 times higher in bone with an osteocyte lacuna density greater than 728/mm2 (Qiu et al., 2005). On the other hand, reduced numbers of osteocytes can result in the deterioration of canalicular fluid flow, decreased ability to detect microdamage and increased bone fragility (Qui et al., 2003; Burger et al., 2003). Conceivably, reduced fluid flow can alter the volumetric proportion of water and crystal within the mineral; i.e too little water increases bone brittleness (Currey, 1984) and too much water decreases bone stiffness (Hernandez et al., 2001). Such a mechanism might account for the acute effect of osteocyte death on reducing vertebral compression strength in mice, independent of changes in bone mass or architecture (Weinstein et al., 2002). Evidently, much more work is needed to explore these possibilities. In a study by Qiu et al., (2006),black American women were found to have higher BMD indices, higher osteocyte density and lower osteon density than white American women, and the former were not as susceptible to fracture . These data support the hypothesis that microstructural features such as osteocyte lacunae, osteons and Volkmann’s canals can act as barriers to crack propagation (Da Costa Go0 mez et al., 2005). The results of our present study are consistent with these data, i.e. they indicate that adequate numbers of osteocytes are essential to remove bone microdamage and osteocyte density correlates with the biomechanical quality of bone. The mechanism by which estrogen inhibits osteocyte apoptosis remains unclear. Recent data suggest that the anti-apoptosis action of estrogen (Mann et al., 2007) is by way of anti-oxidative stress, thus inducing the expression of glutathione, prostaglandin (PG)-E2, TGF-b and bcl-2, inhibiting the production of inflammatory cytokines, and directly effecting an antioxidant molecule. Furthermore, estrogen has been observed to protect primary osteocytes against glucocorticoid-induced apoptosis (Gu et al.,
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2005b); thus, sex-steroid deficiency would result in increased osteocyte apoptosis (Tomkinson et al., 1998; Kousteni et al., 2001). Our present study found that the osteocyte density of trabecular bone in OVX rats decreased significantly, and the ratio of empty lacunae and oval osteocytes, with the characteristics of apoptotic osteocytes of more than two nucleus-like fragments, increased simultaneously (Hamaya et al., 2002). This is not quantified in the present study and would need to be identified through further research. Estrogen deficiency also resulted in BMC loss and deteriorated microstructure and biomechanical quality. Interestingly, ERT maintained the biomechanical quality of bone and osteocyte viability without significantly changing the parameters of BMD and microstructure except for those of Conn.D and Tb.Sp. This is consistent with the results of other studies (O’Brien et al., 2004; Mullender et al., 2005; Collishaw et al., 2003). These data suggest that osteocytes might play a role in modulating bone quality. We found a weak but significant, positive correlation between Ot.N/T.Ar and ML, which is a direct indicator of bone mechanical strength. Though EM is also an important parameter for assessing the mechanical quality of bone, we did not find EM to be correlated with Ot.N/T.Ar. There may be an inaccuracy that cannot be ignored in calculating EM in our study. The cross-section of the vertebra is irregular; therefore, there is an inaccuracy in calculating the cross-sectional area using diameters of the vertebra. Earlier work by Dai et al. (2004) showed that the parameters of fatigue-damaged microcrack represented the changes of biomechanical quality of bone associated with ovariectomy in rats. Allen et al. (2006) found that, after 1 year of bisphosphonate treatment at clinical doses, a significant accumulation of microdamage was seen in the vertebra. However, this was offset by increases in bone volume and mineralization to the extent that there was no significant impairment of mechanical properties. Diab and Vashisth (2007) found that older men had fewer diffuse damages, yet more and longer linear microcracks than younger men. These data suggest that a certain degree of microdamage is essential for bone (re)modeling. Therefore, microcrack initiation does not always indicate decreased bone quality, but more so a mechanism of dissipating energy (Vashishth, 2004). We found no correlations between Ot.N/T.Ar and Cr.S.Dn or Cr.Dn, which is consistent with an earlier study by Frank et al. (2002). However, we did find a weak negative correlation between Ot.N/T.Ar and Cr.Le. Based on our findings and the current understanding of the relationships between osteocyte density (Manolagas et al., 2005; O’Brien et al., 2004; Tomkinson et al., 1998), microcrack (Diab and Vashishth, 2007) and the mechanical quality of bone, we hypothesize that osteocyte density and microcrack length might be potential determinants of bone quality. The effects of ERT are limited both in our OVX rats treated for 15 weeks and in postmenopausal women with osteoporosis treated for 6 years (Khastgir et al., 2001).
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In our present study ERT did not significantly influence most of the microstructure parameters except for Conn.D and Tb.Sp. We found a weak but significant negative correlation between Conn.D, Tb.Sp and Ot.N/T.Ar, but no correlations between Ot.N/T.Ar and other microstructure parameters (Tb.N, SMI, Tb.Th, BV/TV and BS/BV). This is inconsistent with the results of a previous study that Ot.N/T.Ar positively correlated with BA/TA in human cortical and cancellous bone (Vashishth et al., 2002). This may be due to a difference in subject number in the two studies. Sham surgery can potentially influence estradiol levels in subject rats, yet changes in estradiol levels were not monitored and this was a limitation of our study. Our results showed correlations between Ot.N/T.Ar and bone biomechanical quality; however, the correlations were weak, which indicates that the Ot.N/T.Ar is not the major determinant of bone quality. Further clarification of the relationships between Ot.N/T.Ar and other determinants of bone biomechanical quality, such as bone turnover, bone mineralization, bone matrix and mineral composition indices, are required (Compston, 2006). A recent study showed that there were statistically significant linear correlations between the remodeling indices (BMU activation frequency, densities of secondary osteons, forming osteon, and resorption cavities) and apoptotic osteocyte density, and a strongly positive correlation between the modeling indices (forming surface and surface MAR) and empty osteocyte lacunae, but not osteocyte density (Hedgecock et al., 2007). Additionally, osteocyte apoptosis following corticosteroid therapy is associated with reduced bone strength before any bone loss has occurred (O’Brien et al., 2004). The reasons for the loss of bone strength are unknown, but structural abnormalities in bone surrounding the apoptotic osteocyte may compromise bone strength (Lane et al., 2006). The phenomena of increased osteocyte apoptosis and decreased osteocyte density was also observed in weightlessness, disuse and glucocorticoid-induced osteoporosis (O’Brien et al., 2004; Kogianni et al., 2004; Aguirre et al., 2006.) In conclusion, these data indirectly reinforce the notion that osteocytes actively initiate and control bone (re)modeling and subsequent bone quality (Heino et al., 2002; Gu et al., 2005a). We found that the trabecular thickness did not decrease during the progression of bone loss induced by OVX. Even the EST rats had thinner trabeculae, which may be due to compensatory responses observed by other researchers (Waarsing et al., 2004; Sheng et al., 2007). The molecular mechanism of compensatory responses is unclear, and such a compensatory behavior did not overcome the mechanical impairments caused by OVX. Estrogen is known to act on osteoblasts depending on their stage of differentiation and estrogen receptor (ER) isoform expression (Hernandez et al., 2004). A previous study found that there was a fall in the proportion of osteocytes and osteoblasts expressing ER-a in postmenopausal women (Ireland et al., 2002). Additionally, they
demonstrated that type-I collagen synthesis was mainly regulated by ER-a. Since genistein has a similar chemical structure to estrogen, it is capable of modulating bone metabolism by activating the ERs. It is known that 17bestradiol has a higher affinity to ER-a than to ER-b. Contrary to that observed with 17b-estradiol, genistein has a higher affinity to ER-b than to ER-a. The present study demonstrated that genistein exerted a weaker effect than 17b-estradiol on the parameters of bone microstructure and biomechanics, which is likely to be due to the differences with regard to their binding affinities with the ER isoforms. Furthermore, the responses of osteocytes to both strain and estrogen involve ER-a, and only estrogen regulates the cellular concentration of ER-a (Zaman et al., 2006). This is consistent with the hypothesis that the decreased biomechanical quality associated with estrogen deficiency is a consequence of the reduced anabolic responses of osteocytes to strain. In summary, it has been shown in our present study that osteocyte density positively correlated with some parameters of the biomechanical quality of bone. We postulate that osteocyte density could be an alternative index for assessing bone quality. The mechanisms by which osteocytes orchestrate and control bone formation and resorption play a pivotal role in current bone research. Future advances in our understanding of the biology of the osteocyte might uncover new promising drug targets for osteoporosis. Conflict of interest All authors have no conflicts of interest. Drs. Ma and Dai contributed equally to this work. All authors were fully involved in the study and preparation of the manuscript, and the material within has not been and will not be submitted for publication elsewhere. Acknowledgement We gratefully acknowledge Grant no. 30400514 from the National Natural Science Foundation of China, Grant no. 06JJ50029 from the Hunan Province Natural Science Foundation of China and Grant no. 2004—468-50 from the Ministry of Health of PR China. References Aguirre, J.I., Plotkin, L.I., Stewart, S.A., Weinstein, R.S., Parfitt, A.M., Manolagas, S.C., Bellido, T., 2006. Osteocyte apoptosis is induced by weightlessness in mice and precedes osteoclast recruitment and bone loss. Journal of Bone and Mineral Research 21, 605–615. Allen, M.R., Iwata, K., Phipps, R., Burr, D.B., 2006. Alterations in canine vertebral bone turnover, microdamage accumulation, and biomechanical properties following 1-year treatment with clinical treatment doses of risedronate or alendronate. Bone 39, 872–879. Burger, E.H., Klein-Nulend, J., Smit, T.H., 2003. Strain-derived canalicular fluid flow regulates osteoclast activity in a remodelling osteon—a proposal. Journal of Biomechanics 36, 1453–1459.
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