Do Ultrasound Measurements Reflect Bone Microarchitecture Rather than Bone Mass?

An In Vitro Study of the Rat Femur with the Use of Ultrasound, Densitometry, and Histomorphometry

Abstract

Rico H, Hernández ER, Páez E, et al. Do ultrasound measurements reflect bone microarchitecture rather than bone mass? An in vitro study of the rat femur with the use of ultrasound, densitometry, and histomorphometry. Invest Radiol 2001;36:323–326.

rationale and objectives.  

In an experimental study in 40 rat femurs, the authors correlated the amplitude-dependent speed of bone ultrasound (Ad-SOS) with the bone mineral content and density and with the bone trabecular connectivity: trabecular bone volume, trabecular number, trabecular thickness, and trabecular separation to evaluate and compare the usefulness of the Ad-SOS to determine bone quantity and/or quality.

methods. 

Bone mineral content and density were determined with dual-energy x-ray absorptiometry. Trabecular connectivity was determined with histomorphometric techniques.

results. 

There was a strong correlation between the Ad-SOS and the other parameters studied, with a particularly high positive correlation with trabecular bone volume and trabecular thickness, and an inverse correlation with trabecular separation. The correlation was weaker with the bone mineral content and bone mineral density and with the trabecular number. For the trabecular separation, the correlation was significant in all cases, but it was negative.

conclusions. 

Bone ultrasound, in this case Ad-SOS, defines the quality of the bone in terms of trabecular architecture rather than bone density; however, this conclusion is valid only for the rat femur model that the authors used.

The early skepticism about the role of ultrasound in osteoporosis is decreasing as a result of the growing body of evidence, particularly from prospective studies, that quantitative ultrasound is useful for bone assessment. ¹ The basic principle of ultrasound measurements of the skeleton is that the speed at which ultrasound propagates in bone is determined by the mass density and by the “elastic modulus” (inherent material quality) of the bone. ² Structure is one of the qualities of bone that ultrasound is purported to measure, in contrast to dual-energy x-ray absorptiometry (DXA), which measures bone mineral content and density only. Bone structure can be defined in terms of connectivity, porosity, and anisotropy. ³

Trabecular architecture can be measured by histomorphometry, ⁴ and a good reflection of this parameter is found in the trabecular bone volume, trabecular number, trabecular thickness, and trabecular separation. ^4–6 Hans et al ⁷ and Glüer et al ⁸ have studied the relationship between ultrasound and histomorphometric parameters that reflect the microarchitecture of the bone. Glüer et al showed a significant association between ultrasound parameters and the microarchitecture of the bone; Hans et al failed to do so. However, the fact that Hans et al studied the calcaneus after amputation in humans and Glüer et al studied bovine specimens may explain this discrepancy.

The rat is a species that displays remodeling patterns that can be superimposed on the human model. Ovariectomy or natural menopause in the rat induces changes similar to those observed in menopausal women. ⁹ Another important aspect is the similar response to bone treatment in rats and humans, as reported by Abe et al. ¹⁰ This means that data on bone mass in rats can be, in part, extrapolated to humans.

The objective of this study, conducted in rat femurs, was to establish a correlation between the values from amplitude-dependent speed-of-bone ultrasound (Ad-SOS), with bone mineral content and density values obtained with DXA, and trabecular architecture (trabecular bone volume, number, thickness, and separation) with histomorphometry in an effort to clarify the discrepancies that surround the comparison of these values, and to assess the usefulness of the Ad-SOS as a way to show the trabecular quantity and quality of the bone.

Methods

Rats

The study was conducted in a sample of 40 Wistar rat femurs; all rats were 150 days old and had a mean weight of 343 ± 37 g. The rats were supplied by the animal laboratory of the University of Alcalá. They all came from litters considered to be normal and were bred in the same habitat and living conditions. All were given free access to water and Panlab type A04 feed (Panlab, Barcelona, Spain), containing 7.1 g/kg calcium and 5 g/kg phosphorous; the energy content of the feed was 3100 kcal/kg. Living conditions (12 hours of light and 12 hours of darkness; mean room temperature 22° C and 50% environmental humidity), habitat, and diet met current European Union guidelines. The experimental procedure that was used complied with the guiding principles for the Care and Use of Animals and was approved by the appropriate institutional animal care and surveillance committee. Initial weight was measured by using a biomedical balance with precision digital electronic weighing scales.

Densitometric and Morphometric Studies

All of the rats were killed by exsanguination from the abdominal aorta after receiving 4 mg/100 g sodium pentothal. The femur of each rat was dissected and all soft tissue was removed. Weight was determined with a precision balance with precision digital electronic scales.

The bone mineral content and density of the whole right femur were measured by using techniques described elsewhere ¹¹ with a Norland XR-26 densitometer equipped with a special program for the study of small animals and samples (Norland Co., Fort Atkinson, WI). The instrument was calibrated daily.

Ultrasound Studies

The quantitative ultrasound study (Ad-SOS) was performed by using techniques described elsewhere ¹² with a DBM Sonic 1200R machine (Emsor, S.A., Madrid, Spain). The machine was equipped with a caliper that closes tangentially on the diaphysis of the femur to measure its thickness at the same location where the histomorphometric measurement would be carried out. This value is used together with the transit time to calculate the speed of sound through the femur. The instrument transmits at a frequency of 1.25 MHz with 22 W power. Coupling is achieved by using standard ultrasound gel. The DBM Sonic was calibrated daily with a Plexiglas phantom supplied by Emsor.

Histomorphometric Studies

After bone densitometry and Ad-SOS, the femurs were processed by using previously published techniques ^13–15 for the histomorphometric study of nondecalcified bone. The specimens were sectioned longitudinally into 7-μm slices on a Microm microtome (Microm, Heidelberg, Germany) and were stained with Goldner stain. After being stained, histomorphometric measurements of bone were made on a Videoplan (Zeiss, Heidelberg, Germany) in two representative regions. Cancellous bone measurements were made 0.50 mm from the distal angle of the growth cartilage to exclude primary cancellous bone. Trabecular bone measurements were made on a strip 0.5 mm wide that covered the entire width of the femoral diaphysis; trabecular bone measurements consisted of trabecular bone volume, number, thickness, and separation.

Statistical Analysis

The results of descriptive statistics are presented as mean ± standard deviation. Precision (coefficient of variation [CV]) was calculated with root-mean-square. Correlation (Fisher r to z test) and simple linear regression analysis were used as appropriate to examine relations between the parameters studied. Simple linear regression was used to generate a representative scattergram of some data. The data were processed on a Macintosh computer using the StatView 4.02 statistical package (Abacus Concepts, Berkeley, CA).

Results

Values corresponding to the parameters studied are shown in Table 1. Table 2 lists the correlations observed between the different parameters. The CV for bone mineral content and density of the whole right femur, determined from six measurements made in three rat femurs at intervals of 3 to 4 days, was 0.8%. The CVs for histomorphometric measurements, determined from six sets of measurements made on six rat femurs at intervals of 3 to 4 days, were less than 4.5%. The precision of the Ad-SOS was determined from five measurements taken in eight femurs at time intervals not exceeding 21 days; the CV was 0.5%.

Table 1: Bone and Ultrasound Characteristics of Femurs of Female Wistar Rats

Table 2: Correlation for Fisher’s r to z Test Between Study Parameters

The Ad-SOS was correlated significantly with all other parameters, with a positive and strongly significant correlation with trabecular bone volume and thickness (r = 0.87 and 0.73, respectively) and a negative correlation with trabecular separation (r = 0.69) (P < 0.0001 in all cases). The correlation was weaker but still significant with trabecular number (r = 0.47, P < 0.0005), bone mineral content (r = 0.38, P < 0.05), and bone mineral density (r = 0.46, P < 0.0005).

Positive correlations were established between the remaining parameters, with the following exceptions (see Table 2): bone mineral content and density with trabecular thickness (r = 0.17 and 0.11, respectively, P = NS) and trabecular number with trabecular thickness (r = 0.13, P = NS). For trabecular separation the correlation was significant in all cases, but it was negative (r = −0.37 to −0.75, P < 0.05–0.0001).

Discussion

Ultrasound exploration is a new noninvasive technique for evaluating bone strength. In theory, ultrasound parameters vary with bone density, elasticity, and microstructure. ² Quantitative ultrasound (QUS) exploration of bone may make it possible to assess bone properties that currently cannot be evaluated with densitometry techniques. ^1,16 Studies have indicated that QUS variables may be influenced by the mechanical properties of bone, ¹⁷ which in turn are determined by the bone’s material and structural properties. There is growing interest in determining the true value of QUS measurements as they define density, elasticity, and other specific properties of bone. ^1,2,16,18 Nonetheless, few studies have analyzed the relationship between QUS measurements and the corresponding histomorphometric parameters. ¹⁸

The equipment used in this study evaluated Ad-SOS and is widely used to study bone mass in humans. ^19–21 When the same measurement techniques were used in rats, the results showed a significant correlation between the trabecular microarchitecture of the rat femur and the parameters describing trabecular architecture (trabecular bone volume, number, thickness, and separation) ⁴ and to a lesser degree, bone mineral content and density measured by DXA. In vitro studies have shown that QUS reflects trabecular orientation independently of bone mineral density. ²² Trabecular architecture is an indicator of bone quality and is an important measurement in the production of osteoporosis ^6,23 : it has been shown to be the most significant and discriminatory parameter when differentiating between normal women and subjects with osteoporosis. ²³ Trabecular architecture was also correlated with bone resistance, and Bouxsein and Radloff ²⁴ observed a strong correlation between QUS and the mechanical properties of calcaneal trabecular bone. A study by Nicholson et al ²⁵ in human vertebral cancellous bone found that the ability of ultrasound to reflect aspects of trabecular structure is strongly dependent on the direction in which ultrasound measurements are made; their results provided only qualified support for the hypothesis that ultrasound reflects cancellous bone structure independently of bone density.

In histological studies of trabecular bone in rats, Baron et al ²⁶ found that trabecular bone remodeling in female rats of advanced age was similar to that observed in the trabecular bone from the iliac crest in elderly women; trabecular bone findings were similar in young rats and children. These similarities make it possible (within logical limitations, of course) to extrapolate our results on the trabecular bone to humans. Hans et al ⁷ initially found that ultrasound measurements appeared to reflect bone quantity rather than bone microarchitecture. The conclusion was negative concerning the possibility of using ultrasound to assess structural characteristics. Our sample size, however, was not large enough to generate statistically significant correlations. In addition, the calcaneus is an anisotropic structure, whereas ultrasound interaction in bone is a three-dimensional phenomenon. More recent reports, ²⁷ however, indicate that QUS mainly reflects bone density and elasticity. Glüer et al ⁸ found a significant association showing that ultrasound velocity is influenced by the trabecular separation and that attenuation is affected by the separation and connectivity and is also dependent on the orientation of the trabeculae.

Our results corroborate those published by Glüer et al ⁸ but do not dispute those of Hans et al, ⁷ because the studies were performed with different methods and materials. In previous studies, we had observed in rats a strong correlation between Ad-SOS and bone mass as determined by DXA. ¹⁴ Other authors, among them Prins et al, ²² have reported the same correlation in humans, a correlation that Laugier et al ²⁸ expressed as r = 0.94. These data show that ultrasound can reflect aspects of trabecular bone structure and provide qualified support for the hypothesis that ultrasound measurements reflect cancellous bone structure, better and independently, rather than bone mineral content or density.