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J Appl Physiol 99: 1951-1957, 2005. First published July 14, 2005; doi:10.1152/japplphysiol.00401.2005
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Mechanical loading-induced gene expression and BMD changes are different in two inbred mouse strains

Chandrasekhar Kesavan,1 Subburaman Mohan,1,2 Susanna Oberholtzer,1 Jon E. Wergedal,1,2 and David J. Baylink1,2

1Musculoskeletal Disease Center, Veterans Affairs Loma Linda Healthcare System, and 2Department of Medicine, Loma Linda University, Loma Linda, California

Submitted 11 April 2005 ; accepted in final form 12 July 2005


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Our goal is to evaluate skeletal anabolic response to mechanical loading in different age groups of C57B1/6J (B6) and C3H/HeJ (C3H) mice with variable loads using bone size, bone mineral density (BMD), and gene expression changes as end points. Loads of 6–9 N were applied at 2 Hz for 36 cycles for 12 days on the tibia of 10-wk-old female B6 and C3H mice. Effects of a 9-N load on 10-, 16-, and 36-wk-old C3H mice were also studied. Changes in bone parameters were measured using peripheral quantitative computed tomography, and gene expression was determined by real-time PCR. Total volumetric BMD was increased by 5 and 15%, respectively, with 8- and 9-N loads in the B6, but not the C3H, mice. Increases of 20 and 12% in periosteal circumference were reflected by dramatic 44 and 26% increases in total area in B6 and C3H mice, respectively. The bone response to bending showed no difference in the three age groups of B6 and C3H mice. At 2 days, mechanical loading resulted in significant downregulation in expression of bone resorption (BR), but not bone formation (BF) marker genes. At 4 and 8 days of loading, expression of BF marker genes (type I collagen, alkaline phosphatase, osteocalcin, and bone sialoprotein) was increased two- to threefold and expression of BR marker genes (matrix metalloproteinase-9 and thrombin receptor-activating peptide) was decreased two- to fivefold. Although expression of BF marker genes was upregulated four- to eightfold at 12 days of training, expression of BR marker genes was upregulated seven- to ninefold. Four-point bending caused significantly greater changes in expression of BF and BR marker genes in bones of the B6 than the C3H mice. We conclude that mechanical loading-induced molecular pathways are activated to a greater extent in the B6 than in the C3H mice, resulting in a higher anabolic response in the B6 mice.

bone density; mice; bone size; bone formation; bone resorption

IT IS WELL ESTABLISHED that maintenance of bone mass and development of skeletal architecture are dependent on mechanical stimulation. A number of studies have shown that mechanical loading promoted bone formation (BF) in the modeling skeleton and that removal of this stimulus resulted in a reduction in bone mass (1, 9, 13, 28, 29). In addition, recent studies have also shown that the increase in bone mass was variable in different subjects subjected to the same amount of mechanical stress, with some exhibiting a robust osteogenic response and others responding more modestly (8, 24, 26). We and others found evidence that this variation in response to mechanical loading is, in large part, genetically determined (12, 13, 19). Accordingly, we have identified two inbred mouse strains that differ in peak bone density and exhibit considerable differences in their bone response to immobilization and mechanical loading. In our studies, we found a greater loss of bone in response to hindlimb immobilization by sciatic neurectomy in the C57Bl/6J (B6) than in the C3H/HeJ (C3H) mouse strain. Furthermore, studies by Umemura et al. (28) and Kodama et al. (13), as well as four-point bending studies by Akhter et al. (1), revealed that an identical amount of mechanical force applied to both mouse models produced a greater increase in BF (i.e., periosteal and endosteal formation) parameters in B6 than in C3H mice.

In a number of in vitro studies that employed mechanical stimulation using various models in mouse and human cells, several signaling pathways, including mitogen-activated protein kinase (16, 17, 31), focal adhesive kinase (4, 18, 30), and nitric oxide (3, 11, 15, 32), were found to mediate the effects of mechanical loading in bone. However, the genetic mechanisms that contribute to any variations in anabolic response to loading remain unclear. One approach often used to identify the genetic factors or genes that contribute to differences in phenotypic variation is the quantitative trait loci (QTL) technique. In the QTL approach, two inbred mouse strains exhibiting a phenotypic difference of interest are crossed, and any genetic loci that cosegregate with the phenotype are identified. A successful QTL approach requires an optimized in vivo loading model, valid end points for measurement of difference in bone anabolic response, and an optimal age that shows the greatest difference in the phenotype in response to mechanical loading. In this study, we proposed that the magnitude of skeletal anabolic response in B6 and C3H mice is dependent on the age of the mouse and the amount of load applied, which is reflected by changes in expression levels of BF and/or bone resorption (BR) marker genes. To test this hypothesis, our initial objectives were 1) to evaluate the response of bone to different loads applied by four-point bending, 2) to determine whether any differences in the response of bone to mechanical strain between B6 and C3H mice can be accurately quantitated by peripheral quantitative computed tomography (pQCT) and/or expression levels of BF marker genes, and 3) to evaluate whether the bone response to four-point bending varies in different age groups in B6 and/or C3H mice.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Animals

Female B6 and C3H inbred mice of different ages (10, 16, and 36 wk) were purchased from Jackson Laboratory (Bar Harbor, ME) and housed at the Animal Research Facility, Jerry L. Pettis Memorial Veterans Affairs Medical Center (Loma Linda, CA) under appropriate conditions. Animal procedures performed in this study were approved by the Animal Studies Subcommittee of the Jerry L. Pettis Medical Center. Body weights were as follows: 18.8 ± 0.1, 20.7 ± 0.3, and 26.2 ± 0.5 g for the B6 mice and 19.1 ± 0.1, 20.7 ± 0.5, and 27.8 ± 0.4 g for the C3H mice at 10, 16, and 36 wk of age, respectively.

In Vivo Loading Model/Regimen

We used the four-point bending method developed by Akhter et al. (1) as our in vivo loading regimen. Briefly, the four-point bending device (Instron, Canton, MA) consists of two upper vertically movable points covered with rubber pads, which are 4 mm apart, and two 12-mm lower nonmovable points covered with rubber pads. During bending, the two upper pads touch the lateral surface of the tibia through overlaying muscle and soft tissue, and the lower pads touch the medial surface of the proximal and distal parts of the tibia. The loading protocol consists of a 6- to 9-N load at a frequency of 2 Hz for 36 cycles, and the training is performed once per day. The right tibia is used for the loading test and the left tibia as an internal control. After the mice were anesthetized, the ankle of the tibia was positioned on the second lower immobile points of the bending device (Instron), such that the region of the tibia that was loaded did not vary in different mice. The mice were anesthetized with 95% oxygen-5% halothane for 2–3 min, and mechanical loading was performed while the mice were anesthetized. The mice were trained for 6 days/wk with 1 day of rest for 2 wk. On the 15th day, 48 h after the last loading regimen, the mice were killed, and tibias were collected and stored at 4°C until pQCT.

pQCT Densitometry

To determine whether there was a significant change in the geometric properties of loaded and unloaded tibias, we used the pQCT system (Stratec XCT Research, Stratec Medizintechnik, Berlin, Germany). The instrument was specifically modified for use on small bone specimens to measure bone mineral content, periosteal and endosteal circumferences, total area, and total content. Routine calibration was performed daily with a defined standard containing hydroxyapatite crystals embedded in Lucite. Scanning was performed using the manufacturer-supplied software program, which was designed to analyze the data and generate the values for the change in bone parameters. The X-ray attenuation data were analyzed on the basis of the software-defined threshold. We set up two thresholds for our analysis: 180–730 mg/cm3 to measure total area, total mineral content, periosteal circumference, and endosteal circumference in the loaded vs. unloaded bones and 730–730 mg/cm3 to measure cortical thickness and total volumetric and material bone mineral density (vBMD and mBMD, respectively).

To minimize the measurement errors caused by positioning of the tibia for pQCT, we used the tibia-fibular junction as the reference line. We selected four slices, 1 mm apart, beginning 3 mm proximal from the tibia-fibular junction for pQCT measurement. This region corresponds to the loading zone.

Strain Measurement

The differences in the amount of mechanical strain (µ{epsilon}) on the loaded region produced by different loads were measured in the B6 and C3H mice by the strain-gauge technique (2, 7, 7a). To measure the strain, we used only the loaded region of the tibia, which, according to our pQCT analysis, was the area most affected by 4-mm vertical movable points. Briefly, a P-3500 portable strain indicator and a strain gauge of a specific range (EP-XX-015DJ-120) were used to measure the amount of mechanical strain produced by different loads. Initially, the ends of the strain gauge circuits were soldered to copper wire and glued on the medial side of the tibia, 2.09 mm from the tibia-fibular junction, to provide a consistent position on the 4-mm loading zone. The copper wires were connected to the indicator, and the amounts of strain produced by the loads on the loading zone were recorded. The strain gauge data from four individual mice were averaged for each load.

Microcrack Detection by En Bloc Staining

The 10-wk-old B6 and C3H mice were subjected to four-point bending with a 9-N load for 10 days, and 2 days after the last load was applied, tibias were collected and stored in 10% formalin. One thick section was obtained from the loaded and unloaded bones, and both sides were measured for microcracks (bone area, mm2) by the fuchsin staining method (5).

RNA Extraction

A lipid extraction kit (Qiagen, Valencia, CA) was used to extract RNA from bones with the following modification. After the animals were euthanized, tissues were removed and immediately transferred to liquid nitrogen and stored at –80°C until RNA extraction. A mortar and pestle with liquid nitrogen were used to grind bones into fine powder. Trizol (~1 ml) was added to each sample, and the samples were ground to a fine powder and then transferred to fresh 1.5-ml RNase-free tubes. Chloroform (200 µl) was added to each sample, which was vortexed for 15 s and incubated at room temperature for 3 min. The samples were then centrifuged at 12,000 g for 15 min, and an aqueous layer was removed to a fresh tube after centrifugation. Ethanol (~700 µl) was added to the fresh samples, which were then vortexed for 15 s. The samples were transferred to a spin column, and the RNA was purified according to the manufacturer's instructions. Quality and quantity of RNA were analyzed using Bio-analyzer and Nano-drop instrumentation (Agilent).

Reverse Transcriptase Real-Time PCR

Expression of mRNA was quantitated according to the manufacturer's instructions (ABI Prism) using the SYBR green method on sequence detection systems (model 7900, Applied Biosystems). Briefly, purified total RNA (200 ng/µl) was used to synthesize the first-strand cDNA by reverse transcription using random hexamers and Superscript II reverse transcriptase according to the manufacturer's instructions (Invitrogen, Carlsbad, CA). This first-strand cDNA reaction (1 µl) was subjected to real-time PCR amplification using gene-specific primers (IDT-DNA), which were designed according to the ABI Primer Express instructions using Vector NTI software. Approximately 25 µl of reaction volume were used for the real-time PCR assay, which consisted of 1x (12.5 µl) Universal SYBR green PCR master mix (SYBR green dye, reaction buffers, dNTP mix, and Hot Start Taq polymerase; Applied Biosystems), each primer at 50 nM, 24 µl of water, and 1 µl of template. The thermal conditions consisted of an initial denaturation at 95°C for 10 min followed by 40 cycles of denaturation at 95°C for 15 s, annealing and extension at 60°C for 1 min, and a final-step melting curve of 95°C for 15 s, 60°C for 15 s, and 95°C for 15 s. All reactions were carried out in duplicate to reduce variation. The data were analyzed using SDS software (version 2.0), and the results were exported to Microsoft Excel for further analysis. The endogenous control ({beta}-actin) was used to normalize the data, and the normalized values were subjected to a 2–{Delta}{Delta}Ct (where C+ is contraction threshold) formula to calculate the fold change between the control and experimental groups. The formula and its derivations were obtained from the sequence detection system user guide (ABI Prism 7900).

Statistical Analysis

Values are means ± SE. Regression analysis, ANOVA (Bonferroni's post hoc test), and standard t-test were used to compare differences from loading between the strains using the percentage obtained from loaded vs. unloaded bones. We used STATISTICA software for our analysis, and the results were considered significantly different at P < 0.05.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Bone Anabolic Response to Loading

Total bone mineral content.   Four-point bending caused an increase in total bone mineral content in the B6 and C3H mice. The magnitude of the increase varied depending on the load between the B6 and C3H mice (Table 1). At 9 N, the percent increase in total bone mineral content in response to four-point bending was significantly greater in the B6 (48%) than in the C3H (19%) mice (Fig. 1A).


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  		Table 1. Changes in bone parameters in response to loads applied by four-point bending in 10-wk old female B6 and C3H mice

 


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  		Fig. 1. A: changes in bone geometrical parameters after 12 days of 4-point bending at 9 N in 10-wk-old C57Bl/6J (B6) and C3H/HeJ (C3H) mice in vitro measured by peripheral quantitative computed tomography. TC, total content; TA, total area; PC, periosteal circumference; CT, cortical thickness; EC, endosteal circumference; TD, total density; CD, cortical density. Values are means ± SE (n = 6). aP < 0.05; cP < 0.001 vs. C3H. B: changes in total volumetric bone mineral density (BMD) in response to loads applied by 4-point bending in 10-wk-old female B6 and C3H mice in vitro. Values are means ± SE (n = 6). aP < 0.05; cP < 0.001 vs. corresponding unloaded bones.

 
Area.   Total area increased in response to four-point bending in the B6 and C3H mice. Dramatic increases of 44 and 26% in total area were seen after 12 days of four-point bending in the B6 and C3H mice, respectively (Table 1). The increase in total area was greater in the B6 (P < 0.05) than in the C3H mice at 9 N, in contrast to other loads (Table 1, Fig. 1A).

Periosteal circumference.   The periosteal circumference increased by 20 and 12% in the B6 and C3H mice, respectively, in response to four-point bending (Table 1). The B6 mice showed a greater increase in periosteal circumference than the C3H mice (P < 0.05) at 9 N compared with other loads (Table 1, Fig. 1A).

Total vBMD.   Four-point bending caused a dose-dependent increase in total bone density in the B6 (regression analysis, P < 0.01), but not in the C3H mice (Table 1). The B6 mice showed 5 and 15% greater density at 8 and 9 N, respectively, after 12 days of loading, whereas the C3H mice exhibited no change (Table 1, Fig. 1B).

Cortical density.   Cortical density increased by 4% in the B6, but not in the C3H, mice at 9 N after 12 days of four-point bending (Fig. 1A).

Cortical thickness.   Four-point bending increased cortical thickness in the B6 and C3H mice after 2 wk of mechanical loading at 9 N. The magnitude of the increase was much greater in the B6 (27%) than in the C3H (7%) mice (Fig. 1A).

Endosteal circumference.   Endosteal circumference in response to four-point bending increased by 23 and 18% in the B6 and C3H mice (Table 1). The increase in endosteal circumference at 8 N was greater in the C3H than in the B6 mice (P < 0.05).

Strain Measurement

Because the B6 and C3H mice differ in bone geometric properties, we next evaluated whether the difference in bone anabolic response to different loads in the two strains of mice could be explained on the basis of a difference in the amount of mechanical strain produced by the loads. Therefore, using the strain gauge technique, we measured the amount of mechanical strain produced by various loads applied by four-point bending on the tibia of the 10-wk-old B6 and C3H mice. The results (Table 2) revealed an increase in mechanical strain with an increase in mechanical load in the B6 and C3H mice. Mechanical strains at all loads were slightly higher in the C3H than in the B6 mice, and the differences were statistically significant (P < 0.01, ANOVA; Table 2).


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  		Table 2. Mechanical strain produced by load applied by four-point bending in tibia of 10-wk-old B6 and C3H mice measured by strain gauge

 
Microcrack Detection

To rule out the possibility that the changes in bone parameters induced by a 9-N load are due to microcrack-induced healing, we measured microcracks by histological analysis. The microcrack/area was not significantly different between the loaded and unloaded bones in the B6 (0.82 ± 0.05 vs. 0.60 ± 0.04/mm2) or C3H (0.87 ± 0.07 vs. 0.95 ± 0.02/mm2) mice. Furthermore, the microcrack/area was not significantly different between the two strains in the loaded or unloaded bone.

Mechanical Loading in the B6 and C3H Mice at Various Ages

To study the response of bone to a 9-N load as a function of age, we subjected the 10-, 16-, and 36-wk-old (retired breeder) B6 and C3H mice to four-point bending with a loading regimen similar to that described above. The results indicate that four-point bending caused significant increases in the bone parameters at 10, 16, and 36 wk of age in both strains of mice when measured by pQCT. Changes in the total mineral content, total area, periosteal circumference, and total volumetric and cortical density in all three age groups were greater in the B6 than in the C3H mice (Tables 3 and 4, Fig. 2). Using Bonferroni's post hoc test, we found no statistical difference in the bone responses between the age groups of the B6 and C3H mice.


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  		Table 3. Changes in bone (tibia) geometric parameters in response to 12 days of four-point bending at 9 N in B6 mice at different ages

 

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  		Table 4. Changes in bone (tibia) geometric parameters in response to 12 days of four-point bending at 9 N in C3H mice at different ages

 


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  		Fig. 2. Changes in total area (A) and volumetric BMD (vBMD, B) in response to 12 days of 4-point bending in 10- to 36-wk-old female B6 ({blacklozenge}) and C3H ({blacksquare}) mice in vitro. Values are means ± SE (n = 6 for 10 wk, n = 9 for 16 and 36 wk) aP < 0.05 vs. unloaded control. bP < 0.05 vs. C3H.

 
Gene Expression Changes in Response to Four-Point Bending

To evaluate the involvement of osteoblast and osteoclast cell function in producing the optimal response to a given skeletal load, we used real-time PCR to measure changes in gene expression after 2, 4, 8, and 12 days of mechanical loading in the 10-wk-old female B6 mice. In the B6 mice, 2 days of four-point bending significantly decreased expression of BR genes but had no significant effect on expression of BF genes in the loaded tibia compared with the unloaded tibia (Table 5). In addition, expression of type I collagen (ColaI) and bone sialoprotein (BSP) was increased twofold and expression of matrix metalloproteinase-9 (MMP-9) and thrombin receptor-activating peptide (TRAP) was downregulated three- and fourfold, respectively, at 4 days of loading. No change was found in expression of osteocalcin (OC) and alkaline phosphatase (ALP; Table 5). Eight days of loading caused a threefold increase in expression of ColaI, BSP, ALP, and OC and a threefold downregulation of TRAP. No change in expression of MMP-9 was found between loaded and unloaded bones after 8 days of loading (Table 5). Prolongation of loading (up to 12 days) resulted in significant changes in expression of BF (4.1-, 7.8-, 6-, and 4-fold for ColaI, BSP, ALP, and OC, respectively) and BR (7.5- and 12.2-fold for MMP-9 and TRAP, respectively) marker genes (Table 5). Expression of receptor activator of NF-{kappa}B ligand (RANKL) was increased fivefold after 12 days of training in loaded bone compared with unloaded bone.


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  		Table 5. Fold change in mRNA expression of BF and BR genes in response to four-point bending in 10-wk-old female B6 mice

 
Difference in Expression of Genes Between the B6 and C3H Mice

Although expression levels of BF and BR marker genes were increased in both strains, the increases were significantly greater in the B6 than in the C3H mice. The most significant difference between the two mouse strains was in expression of BSP (Table 6).


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  		Table 6. Fold change in mRNA expression of BF and BR marker genes in response to 12 days of four-point bending in 10-wk-old female B6 and C3H mice

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
The salient features of the present study are as follows. 1) Four-point bending increased vBMD and mBMD significantly in the B6, but not in the C3H, mice. 2) Age (10–36 wk) had no effect on bone response to four-point bending. 3) Four-point bending caused acute changes in BR and BF gene expression. 4) Four-point bending-induced changes in expression were greater in the bones of the B6 than the C3H mice.

One of the major findings of this study is a striking 15% increase in total vBMD in the tibia of the B6 mice as a result of 12 days of mechanical loading at 9 N using the four-point bending technique. vBMD was increased by 4.5% at 8 N in the B6 mice. In contrast to the response observed in the B6 mice, none of the four loading regimens (i.e., 6, 7, 8, and 9 N) produced a significant increase in total vBMD in the tibia of the C3H mice. Interestingly, this lack of significant change in vBMD cannot be explained by inadequate mechanical strain, because in the tibia of the C3H mouse, a 9-N load produced a strain of 3,865 µ{epsilon}, which is above the physiological range (300–3,000 µ{epsilon}) and, notably, is higher than the mechanical strain produced in the tibia of the B6 mouse. Furthermore, a mechanical load of 9 N caused a significant increase in total area and periosteal circumference in the C3H mice, suggesting that the increase in the vBMD response in the tibia of C3H mice is not caused by a lack of mechanosensitivity.

In terms of the rapid increase in total vBMD observed in the B6 mice in response to four-point bending, we found that cortical thickness was increased by 27%. Consistent with this increase in cortical thickness, bone area increased significantly as well in the response of the B6 mice to mechanical loads of 9 N. The increase in bone area and cortical thickness can be explained by the ~20% increase in periosteal circumference, which results in a ~50% increase in total area in the loaded tibia compared with the unloaded tibia after 12 days of four-point bending. In contrast to the increases observed in the B6 mice, the magnitude of increase in periosteal circumference, total area, and cortical thickness was substantially less in the C3H mice. Consistent with these data, Akhter et al. (1) found a greater increase in the periosteal BF response in B6 than in C3H mice after four-point bending. Similarly, we found a significantly greater increase in expression of BF marker genes in the loaded tibia of the B6 than the C3H mice (Table 6). On the basis of these data, we have concluded that a greater increase in periosteal bone response in the B6 mouse contributes, in part, to the observed increase in total vBMD in the tibia of loaded bones of B6 mice.

Our findings demonstrate for the first time that mechanical loading results in a significant increase in mBMD, which also contributes to an increase in total vBMD. In this regard, we consider the increase in cortical density to represent changes in mBMD, because the volume of the vascular canal, as determined by histological analysis, was too low in the loaded bones to account for the increase in cortical BMD. Therefore, we believe that a mechanical load of 9 N caused a maximum mineralization and an increase in bone maturation (age) in the tibia of the B6 mouse. Consistent with this interpretation, we found that 2 and 4 days of four-point bending caused an acute downregulation of expression of BR marker genes. Thus the loading-induced decrease in remodeling could contribute to an increase in the rate of mineralization and, thereby, to the increase in mBMD and total vBMD in the tibia of the B6 mouse.

Our dose-response studies with different mechanical loads revealed that a significant bone anabolic response to loading was observed only at 8- and 9-N loads, which produced mechanical strains slightly above the physiological range (500–3,000 µe). Earlier studies (17a) demonstrated that mechanical strain produced by loads above the physiological range may lead to an accumulation of microcracks in the loaded bone. To determine whether the bone anabolic response observed after the 8- and 9-N loads was due to microcracks, we performed histological analysis on the cross-section-loaded region of the tibia of the B6 and C3H mice after 12 days of loading to identify potential microcracks. We did not observe microcracks in the B6 or C3H mice at the highest (i.e., 9-N) loads. These findings imply that differences in bone anabolic response between the C3H and B6 mice cannot be ascribed to a difference in the number of microcracks as evaluated by the method used in this study.

To include the newly formed bone, which may not have been fully mineralized, we used a threshold of 180–730 mg/cm3 for evaluation of loading-induced changes in total area, total mineral content, and periosteal and endosteal circumference. Thus it is possible that the dramatic changes in mineral content and bone size after 2 wk of loading may represent woven bone in addition to mature lamellar bone. Further studies are needed to evaluate the relative contribution of woven and lamellar bone to loading-induced increases in bone size and total mineral content.

Surprisingly, we found no difference in the mechanical strain-induced bone response regardless of age in the 10-, 16-, and 36-wk-old B6 or C3H mice. In contrast to our report, other studies on rats, turkeys, and humans (10, 14, 20, 27) showed that the bone response to mechanical stimuli declines with age. There are a number of potential explanations for the discrepancy between our data and the observations of previous studies. 1) Age-related impairment in bone anabolic response may be seen in >36-wk-old mice. 2) Aging may have a greater effect on the bone response to loading in some inbred strains of mice than in others. 3) The bone response to mechanical loading may vary with age at lower, but not higher, loads.

Another interesting finding from our study was that mechanical load caused an acute inhibition of BR, as evidenced by downregulation of MMP-9 and TRAP. This finding is consistent with the previous in vitro study in which mechanical stress reduced the expression of RANKL, inhibiting osteoclast formation and activation (21–23). However, 12 days of prolonged loading induced expression of BR marker genes (Table 5). The increase in BR 12 days after loading may be a consequence of remodeling in response to increased BF. Accordingly, endosteal circumference is increased after 12 days of loading. Furthermore, expression of RANKL, a key regulator of BR, was increased fivefold after 12 days of loading, suggesting that any loading-induced increase in BR at the endosteum may be mediated via an increase in production of RANKL.

In this study, we compared gene expression changes between the B6 and C3H mice to test the hypothesis that the difference in the bone response between these strains in pQCT can be observed in the expression levels of BF and/or BR marker genes. As anticipated, the B6 and C3H mice showed increased expression of BF and BR marker genes after 12 days of loading. However, the magnitude of the increases in the expression phenotypes was significantly greater in the B6 than the C3H mice (Table 6). This finding is consistent with our pQCT data, which showed a greater increase in the bone parameters in the B6 than in the C3H mice after 12 days of a 9-N load. Thus, using BMD changes and gene expression changes as end points, we have convincingly shown that the skeletal response to mechanical loading is in part genetically determined.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
ACKNOWLEDGMENTS
 REFERENCES
 
This work was supported by Department of Defense Assistance Award DAMD17-01-1-0074; the US Army Medical Research Acquisition Activity (Fort Detrick, MD) is the awarding and administering acquisition office. All work was performed in facilities provided by the Department of Veterans Affairs.


   ACKNOWLEDGMENTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
REFERENCES
 
We thank James Dekeyser for valuable technical support in setting up the four-point bending device and strain-gauge measurement.

The information in this publication does not necessarily reflect the position or the policy of the US Government, and no official endorsement should be inferred.


   FOOTNOTES
 

Address for reprint requests and other correspondence: S. Mohan, Loma Linda Univ., Musculoskeletal Disease Center (151), Jerry L. Pettis Memorial VA Medical Center, 11201 Benton St., Loma Linda, CA 92357 (e-mail: Subburaman.Mohan{at}med.va.gov)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 

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