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Keywords:

  • biomechanics;
  • bone density;
  • osteoporosis;
  • genetics

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

The inbred strains of mice C57BL/6J (B6) and C3H/HeJ (C3H) have very different femoral peak bone densities and may serve as models for studying the genetic regulation of bone mass. Our objective was to further define the bone biomechanics and microstructure of these two inbred strains. Microarchitecture of the proximal femur, femoral midshaft, and lumbar vertebrae were evaluated in three dimensions using microcomputed tomography (μCT) with an isotropic voxel size of 17 μm. Mineralization of the distal femur was determined using quantitative back-scatter electron (BSE) imaging. μCT images suggested that C3H mice had thicker femoral and vertebral cortices compared with B6. The C3H bone tissue also was more highly mineralized. However, C3H mice had few trabeculae in the vertebral bodies, femoral neck, and greater trochanter. The trabecular number (Tb.N) in the C3H vertebral bodies was about half of that in B6 vertebrae (2.8−1 ± 0.1 mm−1 vs. 5.1−1 ± 0.2 mm−1; p < 0.0001). The thick, more highly mineralized femoral cortex of C3H mice resulted in greater bending strength of the femoral diaphysis (62.1 ± 1.2N vs. 27.4 ± 0.5N, p < 0.0001). In contrast, strengths of the lumbar vertebra were not significantly different between inbred strains (p = 0.5), presumably because the thicker cortices were combined with inferior trabecular structure in the vertebrae of C3H mice. These results indicate that C3H mice benefit from alleles that enhance femoral strength but paradoxically are deficient in trabecular bone structure in the lumbar vertebrae.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

ALTHOUGH MANY environmental factors (i.e., diet and exercise) affect bone accumulation during growth, studies in twins have determined that about 70% of the variability in bone density is genetically based.(1–3) Most researchers have assumed that variation in bone density is influenced by multiple genes, but there may be a set of genes that disproportionately influence peak bone density.(4) Inbred strains of mice make useful models for studies of genetic effects on bone structure. Beamer et al.(5) showed a large variation in the femoral bone mineral content and density among 11 inbred strains of mice. The largest difference was between C3H/HeJ (C3H) with 27.48 mg of femoral mineral and C57BL/6J (B6) with 18.62 mg. Although the C3H femoral lengths and volumes were very similar to B6, the C3H had substantially thicker cortices. Consequently, the total bone mineral densities (BMDs) of the femora, determined using peripheral quantitative computed tomography (CT), were 0.691 mg/mm3and 0.450 mg/mm3, respectively, for C3H and B6 females at 4 months of age.

The C3H and B6 mouse strains appear to be very good models for high and low bone mass, respectively. However, it is unclear how the single characteristic of femoral BMD reflects the bone mass at clinically important sites such as the femoral neck and lumbar vertebrae. One might presume that genes that control BMD have equal influence throughout the skeleton. However, this presumption has not been tested directly. Recently, Kline et al.(6) reported the inbred mouse strain DBA/2 had significantly lower whole body areal BMD by dual-energy X-ray absorptiometry than that in B6. Conversely, Beamer et al.(5) reported that femoral volumetric BMD in DBA/2 was significantly greater than that in B6. These contradictory results suggest that differences in phenotypic assessment may complicate studies of the genetic influence on skeletal BMD.

In the current study, we evaluated the bone microstructure and biomechanical properties of several regions of the skeleton in inbred mouse strains B6 and C3H. We hypothesized that for these inbred strains, phenotypic trends in bone structure and strength will vary with anatomic location.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

Animal care

Twenty-five female C3H mice and 25 female B6 mice were raised to 16 weeks of age at The Jackson Laboratory. These mice were group-housed in polycarbonate cages. Water was acidified with HCl to achieve a pH of 2.8–3.2 and was freely available. The diet used for all mice was pasteurized NIH 31 6% fat diet (vitamin and mineral fortified; Purina Mills, St. Louis, MO, U.S.A.) and was freely available. Use of mice in this research project was reviewed and approved by the Institutional Animal Care and Use Committee of The Jackson Laboratory.

Biomechanical tests

We measured bone strength at the midshaft of the femur and at two clinically relevant sites: the femoral neck and the lumbar spine. Femora were tested in three-point bending at room temperature. Load was applied midway between two supports that were 5 mm apart. Load-displacement curves were recorded at a crosshead speed of 0.5 mm/s using a microforce materials testing machine. Data were stored on a microcomputer. Ultimate force (Fu), stiffness (S), and work to failure (U) were calculated from the load-displacement curve as described elsewhere.(7)Fu reflects the strength of the bone, while S reflects the rigidity, and U is the energy necessary to cause a fracture. After the femur was fractured, cortical thickness was measured at the midshaft using digital calipers accurate to 0.01 mm, with a precision of ±0.005 mm (Mitutoyo, Aurora, IL).

L5 vertebrae were dissected free and the posterior elements were removed using a small clipper. The end plates of the vertebral body were cut parallel using a diamond wafering saw (Isomet, Buehler, Lake Bluff, IL, U.S.A.). Mechanical tests were performed in compression using a servohydraulic materials testing machine (810, MTS Corp., Minneapolis, MN, U.S.A.). All tests were done with the specimen submerged in 37°C saline using a displacement rate of 1 mm/s. From the resulting load-displacement curves Fu, S, and U were determined. There was some variation in the specimen height after the vertebral end plates were made parallel. Because stiffness and work to failure are affected by specimen height, these parameters were normalized by dividing U by specimen height and multiplying S by specimen height.

The proximal femora were mounted vertically in aluminum cylinders and fixed in place with cyanacrylate cement. Load was applied to the femoral head until fracture of the femoral neck occurred. Load-displacement curves were recorded at a crosshead speed of 0.5 mm/s using a microforce materials testing machine.

Microcomputed tomography

A subset of intact bone samples (multisegment vertebrae or whole femur) were measured using desktop microcomputed tomography (μCT; μCT 20, Scanco Medical AG, Bassersdorf, Switzerland). A microfocus X-ray tube with a focal spot of 10 μm was used as a source. To perform a measurement, the specimen was mounted on a turntable that could be shifted automatically in the axial direction. Six hundred projections were taken over 216° (180° plus half the fan angle on either side). A standard convolution-backprojection procedure with a Shepp and Logan filter was used to reconstruct the CT images in 1024 × 1024 pixel matrices. For each sample, a total of 100–200 microtomographic slices, with a slice increment of 17 μm, were acquired. Measurements were stored in three-dimensional (3D) image arrays with an isotropic voxel size of 17 μm. A constrained 3D Gaussian filter was used to suppress partly the noise in the volumes. The bone tissue was segmented from marrow using a global thresholding procedure.(8) In addition to the visual assessment of structural images, morphometric indices were determined from the microtomographic data sets. Cortical and trabecular bone were separated using a semiautomated contour tracking algorithm to detect the outer and inner boundaries of the cortex. In trabecular bone, basic structural metrics including bone volume density (BV/TV), bone surface density (BS/TV), trabecular number (Tb.N), trabecular thickness (Tb.Th), and trabecular spacing (Tb.Sp), were measured in three dimensions using direct 3D morphometry.(9) Previous studies have shown trabecular structural metrics measured using μCT to closely correlate with those measured using standard histomorphometry.(10, 11)

Quantitative back-scattering electron imaging

A subset of intact distal femoral samples were dehydrated in ascending grades of acetone and infiltrated in ascending grades of plastic/acetone (Spurr). Specimens were then embedded in Spurr resin and placed in a 60°C oven for 48 h to polymerize. The Spurr epoxy blocks were polished to 1 μm finish, mounted on a stub, and carbon coated. Multiple samples mounted on a large specimen holder were examined in a Hitachi S2500 scanning electron microscope (SEM, Hitachi, Japan) operated at 20 kV and at a 15-mm working distance. To collect back-scattering electron (BSE) images, a Link Tetra BSE detector (Oxford Instruments, U.K.) was calibrated of quantitative image analysis using Al, Al2O2, and C standards. Changes in beam current were corrected by remeasuring against the Al standard. Images were collected at magnification ×40. Images were separated into six equal bins of increasing intensity (gray levels) representing the mineralization level of the tissue and the percentage area of the specimen cross-section that fell in each bin was recorded. Comparison between the mineralization profiles was done using a cumulative logit function.(12)

Statistical tests

Comparisons between B6 and C3H strains were made using an unpaired t-test implemented by Statview software (Abacus Concepts, Berkeley, CA, U.S.A.).

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

Data from biomechanical tests are summarized in Table 1 and Fig. 1. C3H mice had greater femoral strength and cortical thickness compared with B6. Fu, S, and U for the femoral shaft were 2.2-fold, 1.6-fold, and 2.2-fold greater than B6, respectively (Table 1). The femoral neck of C3H mice was significantly stiffer than B6 (+31%; p < 0.01) but also was significantly weaker (−15%; p < 0.001) and absorbed less energy before fracture (−55%; p < 0.001). There was no difference between the two strains in vertebral strength (Fu), while stiffness (S) was 43% greater in C3H (p < 0.01) but work to failure (U) was 31% lower (p < 0.01).

Table Table 1.. Biomechanical Measurements for C57BL/6J (B6) and C3H/HeJ (C3H) Mice (Mean ± SEM)
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Figure FIG. 1.. Average load-displacement curves from mechanical tests at the different skeletal sites.

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The μCT images showed the substantially thicker femoral cortices in the C3H mice (Fig. 2A). μCT images of the femoral neck showed a thinning of the neck diameter distal to the capsule attachment in the C3H strain (Fig. 2B). This anatomical feature, which clearly weakened the femoral neck in C3H mice, was not present in B6 mice (Fig. 2B). The μCT further showed a lack of 3D trabecular bone structure in the lumbar vertebrae of C3H mice, compared with B6 mice (Fig. 2C). Femoral cortical thickness for C3H mice was 88% greater than in B6 mice, but the vertebral Tb.N was about 2-fold greater in B6 mice (Table 2). This reduction in Tb.N also was reflected in BS density, which was about 2-fold greater in B6 mice, and Tb.Sp, which was about 2-fold greater in C3H mice. Thus, C3H mice had ample cortical bone but were deficient in trabecular bone structure at the sites examined. Also, femoral cortical thickness was well correlated with femoral strength (r = 0.94; p < 0.0001) but had no correlation with vertebral strength (r = 0.1; p = 0.48).

Table Table 2.. Morphological Measurements for C57BL/6J (B6) and C3H/HeJ (C3H) Mice (Mean ± SEM)
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Figure FIG. 2.. μCT section of the (A) midshaft of the femur, (B) femoral neck, and (C) L5 vertebral body for C57BL/6J (B6) and C3H/HeJ (C3H) mice. The images were measured in 3D providing a 17-μm isotropic voxel size.

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The distal femoral bone of C3H mice was more highly mineralized than that from B6 mice (p < 0.001). The C3H mineralization profile determined by BSE was shifted 15% to the right (higher mineralization; Fig. 3).

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Figure FIG. 3.. Mineralization of the femoral cortical bone from C57BL/6J (B6) and C3H/HeJ (C3H) mice determined using quantitative BSE imaging. BSE images were separated into six equal bins of increasing intensity (gray levels) representing the mineralization level of the tissue and the percentage area of the specimen cross-section that fell in each bin was recorded to create a mineralization profile. The shift to the right of the C3H data indicates a significant increase in mineralization.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

The results support our hypothesis that the genetic influence on skeletal bone structure and strength is site specific and complex. The enhanced cortical structure in C3H bone was coupled with inferior trabecular structure in the lumbar spine and proximal femur. It is possible that the C3H mice have a unique combination of alleles for improved cortical bone and impaired trabecular bone. Perhaps C3H mice have a defect in endochondral ossification so there is a failure of the normal development of spongiosa. Alternatively, the lack of trabecular bone structure in C3H mice could have resulted through a mechanical adaptation to thick cortices, that is, the enhanced cortical bone structure probably carries the majority of the mechanical load, which could cause a stress shielding effect and subsequent resorption of the trabecular bone. Further studies of the early development of trabecular bone structure in C3H and B6 mice are needed to provide more definitive conclusions about the biological mechanism for the skeletal heterogeneity. Regardless of mechanism, it is clear that the observation of enhanced femoral BMD does not necessarily imply improved bone strength at clinically relevant sites. This was particularly evident for the femoral neck of C3H mice for which the mechanical properties clearly were inferior to B6 although C3H has greater femoral BMD.(5) The reason for this discrepancy appears to be an anatomical feature in the distal region of the C3H femoral neck. The C3H neck diameter distal to the capsule attachment was reduced greatly, compared with B6, resulting in a weakened structure. In addition, there was no correlation between the femoral cortical thickness and the vertebral strength, suggesting that femoral BMD is not related to vertebral mechanical properties. These findings suggest that the genes affecting BMD at a given skeletal site may not exert the most important regulation over bone strength or fragility at other skeletal sites.

An interesting finding of this study was the relative genetic influences of cortical and trabecular bone structure. The skeleton of the C3H mice seemed to be made up predominantly of cortical bone, whereas the B6 mice had a more typical balance of trabecular and cortical bone volume. As a result, C3H mice had superior mechanical properties of the femoral midshaft, which is predominantly cortical bone, yet did not have greater vertebral bone strength although the C3H vertebral stiffness was greater. In fact, the vertebral properties of C3H mice could be considered inferior to B6 because of the reduced work to fracture. This finding suggests that the C3H vertebral bone structure was more brittle. Consequently, it may be advantageous mechanically to have a greater proportion of trabecular bone mass in vertebral bodies because this increases the work to fracture, thus reducing vertebral fragility, and reduces vertebral stiffness. Also, load distribution through trabecular bone may allow more efficient stress transfer to the soft vertebral discs and consequently throughout the whole spine.

This study employed a relatively new and powerful μCT imaging technique. In fact, this represents the first use of this technique for analysis of inbred mouse strains. The results were very encouraging. The high-resolution images provided structural explanations for the measured biomechanical properties. This was best illustrated for the femoral neck, where the observation of a narrowing of the distal femoral neck explained the inferior C3H biomechanical properties. The μCT images of the C3H lumbar vertebrae illustrated a lack of trabecular structure, which explained why the “high bone mass” C3H mice did not have enhanced vertebral strength.

Beamer et al.(5) reported total femoral BMD in C3H mice was significantly greater than in B6 mice. They also reported that the femoral cortical tissue density was over 30% greater in C3H mice, suggesting that C3H cortical bone tissue was more highly mineralized, compared with B6. However, the XCT 960M peripheral quantitative CT instrument used by Beamer et al.(5) has partial volume averaging errors associated with measuring the middiaphyseal endosteal edge. This leads to inaccurate assessment of cortical bone shell volume and cortical density (W.G. Beamer and J. Wergedal, unpublished results, 1999). Our new findings using BSE, show that C3H femoral bone tissue is 15% more highly mineralized compared with B6. Consequently, the increased femoral BMD in C3H mice results from the combination of increased cortical thickness and increased mineralization.

In summary, the results from this study suggest the following:

(1) The elevated femoral cortical thickness in the C3H mice resulted in superior mechanical properties of the femoral shaft.

(2) The improved femoral cortical thickness, mineralization, and strength in C3H mice was combined with inferior vertebral trabecular bone microstructure. Thus, strength of the lumbar vertebrae, a predominantly trabecular bone site, was similar in C3H mice and B6 mice.

In addition, we found a difference in femoral neck strength that mainly was caused by an anatomical feature in the C3H femoral neck, rather than differences in bone mass between the strains.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

The authors thank Douglas Holmyard for technical assistance with the BSE imaging. This work was supported in part by the U.S. Public Health Service (USPHS) National Institutes of Health grants AR43618 (W.G.B.) and AR43730 (C.H.T.) and by the U.S. Army grant DAMD17–96–1-6306 (D.J.B.).

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES
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