Effect of testosterone treatment on the trabecular bone score in older men with low serum testosterone.

The trabecular bone score (TBS) is an indirect measure of vertebral bone microarchitecture. Our objective was to examine the effect of testosterone treatment on TBS. One hundred and ninety-seven hypogonadal men were randomized to testosterone or placebo. After 12 months, there was no difference in the changes in TBS by randomized group. INTRODUCTION: In the Bone Trial of the Testosterone Trials, testosterone treatment increased trabecular volumetric bone mineral density (vBMD) and increased estimated bone strength as determined by finite element analysis. The trabecular bone score (TBS) is an indirect measure of vertebral bone microarchitecture. TBS predicts fracture independent of lumbar spine areal (a) BMD. The objective of this study was to examine the effect of testosterone treatment on TBS compared to its effects on vBMD and aBMD. METHODS: Two hundred and eleven men were enrolled in the Bone Trial of the Testosterone Trials. Of these, 197 men had 2 repeat TBS and vBMD measurements; 105 men were allocated to receive testosterone, and 92 men to placebo for 1 year. TBS, aBMD, and vBMD were assessed at baseline and month 12. RESULTS: There was no difference in the percent change in TBS by randomized group: 1.6% (95% confidence intervals (CI) 0.2-3.9) in the testosterone group and 1.4% (95% CI -0.2, 3.1) in the placebo group. In contrast, vBMD increased by 6% (95% CI 4.5-7.5) in the testosterone group compared to 0.4% (95% CI -1.65-0.88) in the placebo groups. CONCLUSIONS: TBS is not clinically useful in monitoring the 1-year effect of testosterone treatment on bone structure in older hypogonadal men.

Bone density and strength from thoracic and lumbar CT scans both predict incident vertebral fractures independently of fracture location.

In a population-based study, we found that computed tomography (CT)-based bone density and strength measures from the thoracic spine predicted new vertebral fracture as well as measures from the lumbar spine, suggesting that CT scans at either the thorax or abdominal regions are useful to assess vertebral fracture risk. INTRODUCTION: Prior studies have shown that computed tomography (CT)-based lumbar bone density and strength measurements predict incident vertebral fracture. This study investigated whether CT-based bone density and strength measurements from the thoracic spine predict incident vertebral fracture and compared the performance of thoracic and lumbar bone measurements to predict incident vertebral fracture. METHODS: This case-control study of community-based men and women (age 74.6 ± 6.6) included 135 cases with incident vertebral fracture at any level and 266 age- and sex-matched controls. We used baseline CT scans to measure integral and trabecular volumetric bone mineral density (vBMD) and vertebral strength (via finite element analysis, FEA) at the T8 and L2 levels. Association between these measurements and vertebral fracture was determined by using conditional logistic regression. Sensitivity and specificity for predicting incident vertebral fracture were determined for lumbar spine and thoracic bone measurements. RESULTS: Bone measurements from T8 and L2 predicted incident vertebral fracture equally well, regardless of fracture location. Specifically, for predicting vertebral fracture at any level, the odds ratio (per 1-SD decrease) for the vBMD and strength measurements at L2 and T8 ranged from 2.0 to 2.7 (p < 0.0001) and 1.8 to 2.8 (p < 0.0001), respectively. Results were similar when predicting fracture only in the thoracic versus the thoracolumbar spine. Lumbar and thoracic spine bone measurements had similar sensitivity and specificity for predicting incident vertebral fracture. CONCLUSION: These findings indicated that like those from the lumbar spine, CT-based bone density and strength measurements from the thoracic spine may be useful for identifying individuals at high risk for vertebral fracture.

Biomechanical Computed Tomography analysis (BCT) for clinical assessment of osteoporosis.

The surgeon general of the USA defines osteoporosis as "a skeletal disorder characterized by compromised bone strength, predisposing to an increased risk of fracture." Measuring bone strength, Biomechanical Computed Tomography analysis (BCT), namely, finite element analysis of a patient's clinical-resolution computed tomography (CT) scan, is now available in the USA as a Medicare screening benefit for osteoporosis diagnostic testing. Helping to address under-diagnosis of osteoporosis, BCT can be applied "opportunistically" to most existing CT scans that include the spine or hip regions and were previously obtained for an unrelated medical indication. For the BCT test, no modifications are required to standard clinical CT imaging protocols. The analysis provides measurements of bone strength as well as a dual-energy X-ray absorptiometry (DXA)-equivalent bone mineral density (BMD) T-score at the hip and a volumetric BMD of trabecular bone at the spine. Based on both the bone strength and BMD measurements, a physician can identify osteoporosis and assess fracture risk (high, increased, not increased), without needing confirmation by DXA. To help introduce BCT to clinicians and health care professionals, we describe in this review the currently available clinical implementation of the test (VirtuOst), its application for managing patients, and the underlying supporting evidence; we also discuss its main limitations and how its results can be interpreted clinically. Together, this body of evidence supports BCT as an accurate and convenient diagnostic test for osteoporosis in both sexes, particularly when used opportunistically for patients already with CT. Biomechanical Computed Tomography analysis (BCT) uses a patient's CT scan to measure both bone strength and bone mineral density at the hip or spine. Performing at least as well as DXA for both diagnosing osteoporosis and assessing fracture risk, BCT is particularly well-suited to "opportunistic" use for the patient without a recent DXA who is undergoing or has previously undergone CT testing (including hip or spine regions) for an unrelated medical condition.

Treatment of bone loss in proximal femurs of postmenopausal osteoporotic women with AGN1 local osteo-enhancement procedure (LOEP) increases hip bone mineral density and hip strength: a long-term prospective cohort study.

This first-in-human study of AGN1 LOEP demonstrated that this minimally-invasive treatment durably increased aBMD in femurs of osteoporotic postmenopausal women. AGN1 resorption was coupled with new bone formation by 12 weeks and that new bone was maintained for at least 5-7 years resulting in substantially increased FEA-estimated femoral strength. INTRODUCTION: This first-in-human study evaluated feasibility, safety, and in vivo response to treating proximal femurs of postmenopausal osteoporotic women with a minimally-invasive local osteo-enhancement procedure (LOEP) to inject a resorbable triphasic osteoconductive implant material (AGN1). METHODS: This prospective cohort study enrolled 12 postmenopausal osteoporotic (femoral neck T-score ≤ - 2.5) women aged 56 to 89 years. AGN1 LOEP was performed on left femurs; right femurs were untreated controls. Subjects were followed-up for 5-7 years. Outcomes included adverse events, proximal femur areal bone mineral density (aBMD), AGN1 resorption, and replacement with bone by X-ray and CT, and finite element analysis (FEA) estimated hip strength. RESULTS: Baseline treated and control femoral neck aBMD was equivalent. Treated femoral neck aBMD increased by 68 ± 22%, 59 ± 24%, and 58 ± 27% over control at 12 and 24 weeks and 5-7 years, respectively (p < 0.001, all time points). Using conservative assumptions, FEA-estimated femoral strength increased by 41%, 37%, and 22% at 12 and 24 weeks and 5-7 years, respectively (p < 0.01, all time points). Qualitative analysis of X-ray and CT scans demonstrated that AGN1 resorption and replacement with bone was nearly complete by 24 weeks. By 5-7 years, AGN1 appeared to be fully resorbed and replaced with bone integrated with surrounding trabecular and cortical bone. No procedure- or device-related serious adverse events (SAEs) occurred. CONCLUSIONS: Treating femurs of postmenopausal osteoporotic women with AGN1 LOEP results in a rapid, durable increase in aBMD and femoral strength. These results support the use and further clinical study of this approach in osteoporotic patients at high risk of hip fracture.

Prediction of incident vertebral fracture using CT-based finite element analysis.

Prior studies show vertebral strength from computed tomography-based finite element analysis may be associated with vertebral fracture risk. We found vertebral strength had a strong association with new vertebral fractures, suggesting that vertebral strength measures identify those at risk for vertebral fracture and may be a useful clinical tool. INTRODUCTION: We aimed to determine the association between vertebral strength by quantitative computed tomography (CT)-based finite element analysis (FEA) and incident vertebral fracture (VF). In addition, we examined sensitivity and specificity of previously proposed diagnostic thresholds for fragile bone strength and low BMD in predicting VF. METHODS: In a case-control study, 26 incident VF cases (13 men, 13 women) and 62 age- and sex-matched controls aged 50 to 85 years were selected from the Framingham multi-detector computed tomography cohort. Vertebral compressive strength, integral vBMD, trabecular vBMD, CT-based BMC, and CT-based aBMD were measured from CT scans of the lumbar spine. RESULTS: Lower vertebral strength at baseline was associated with an increased risk of new or worsening VF after adjusting for age, BMI, and prevalent VF status (odds ratio (OR) = 5.2 per 1 SD decrease, 95% CI 1.3-19.8). Area under receiver operating characteristic (ROC) curve comparisons revealed that vertebral strength better predicted incident VF than CT-based aBMD (AUC = 0.804 vs. 0.715, p = 0.05) but was not better than integral vBMD (AUC = 0.815) or CT-based BMC (AUC = 0.794). Additionally, proposed fragile bone strength thresholds trended toward better sensitivity for identifying VF than that of aBMD-classified osteoporosis (0.46 vs. 0.23, p = 0.09). CONCLUSION: This study shows an association between vertebral strength measures and incident vertebral fracture in men and women. Though limited by a small sample size, our findings also suggest that bone strength estimates by CT-based FEA provide equivalent or better ability to predict incident vertebral fracture compared to CT-based aBMD. Our study confirms that CT-based estimates of vertebral strength from FEA are useful for identifying patients who are at high risk for vertebral fracture.

The associations between QCT-based vertebral bone measurements and prevalent vertebral fractures depend on the spinal locations of both bone measurement and fracture.

UNLABELLED: We examined how spinal location affects the relationships between quantitative computed tomography (QCT)-based bone measurements and prevalent vertebral fractures. Upper spine (T4-T10) fractures appear to be more strongly related to bone measures than lower spine (T11-L4) fractures, while lower spine measurements are at least as strongly related to fractures as upper spine measurements. INTRODUCTION: Vertebral fracture (VF), a common injury in older adults, is most prevalent in the mid-thoracic (T7-T8) and thoracolumbar (T12-L1) areas of the spine. However, measurements of bone mineral density (BMD) are typically made in the lumbar spine. It is not clear how the associations between bone measurements and VFs are affected by the spinal locations of both bone measurements and VF. METHODS: A community-based case-control study includes 40 cases with moderate or severe prevalent VF and 80 age- and sex-matched controls. Measures of vertebral BMD, strength (estimated by finite element analysis), and factor of risk (load:strength ratio) were determined based on QCT scans at the L3 and T10 vertebrae. Associations were determined between bone measures and prevalent VF occurring at any location, in the upper spine (T4-T10), or in the lower spine (T11-L4). RESULTS: Prevalent VF at any location was significantly associated with bone measures, with odds ratios (ORs) generally higher for measurements made at L3 (ORs = 1.9-3.9) than at T10 (ORs = 1.5-2.4). Upper spine fracture was associated with these measures at both T10 and L3 (ORs = 1.9-8.2), while lower spine fracture was less strongly associated (ORs = 1.0-2.4) and only reached significance for volumetric BMD measures at L3. CONCLUSIONS: Closer proximity between the locations of bone measures and prevalent VF does not strengthen associations between bone measures and fracture. Furthermore, VF etiology may vary by region, with VFs in the upper spine more strongly related to skeletal fragility.

A comparison of DXA and CT based methods for estimating the strength of the femoral neck in post-menopausal women.

UNLABELLED: The study goal was to compare simple two-dimensional (2D) analyses of bone strength using dual energy x-ray absorptiometry (DXA) data to more sophisticated three-dimensional (3D) finite element analyses using quantitative computed tomography (QCT) data. DXA- and QCT-derived femoral neck geometry, simple strength indices, and strength estimates were well correlated. INTRODUCTION: Simple 2D analyses of bone strength can be done with DXA data and applied to large data sets. We compared 2D analyses to 3D finite element analyses (FEA) based on QCT data. METHODS: Two hundred thirteen women participating in the Study of Women's Health Across the Nation (SWAN) received hip DXA and QCT scans. DXA BMD and femoral neck diameter and axis length were used to estimate geometry for composite bending (BSI) and compressive strength (CSI) indices. These and comparable indices computed by Hip Structure Analysis (HSA) on the same DXA data were compared to indices using QCT geometry. Simple 2D engineering simulations of a fall impacting on the greater trochanter were generated using HSA and QCT femoral neck geometry; these estimates were benchmarked to a 3D FEA of fall impact. RESULTS: DXA-derived CSI and BSI computed from BMD and by HSA correlated well with each other (R=0.92 and 0.70) and with QCT-derived indices (R=0.83-0.85 and 0.65-0.72). The 2D strength estimate using HSA geometry correlated well with that from QCT (R=0.76) and with the 3D FEA estimate (R=0.56). CONCLUSIONS: Femoral neck geometry computed by HSA from DXA data corresponds well enough to that from QCT for an analysis of load stress in the larger SWAN data set. Geometry derived from BMD data performed nearly as well. Proximal femur breaking strength estimated from 2D DXA data is not as well correlated with that derived by a 3D FEA using QCT data.

Relationship of femoral neck areal bone mineral density to volumetric bone mineral density, bone size, and femoral strength in men and women.

UNLABELLED: Using combined dual-energy X-ray absorptiometry (DXA) and quantitative computed tomography, we demonstrate that men matched with women for femoral neck (FN) areal bone mineral density (aBMD) have lower volumetric BMD (vBMD), higher bone cross-sectional area, and relatively similar values for finite element (FE)-derived bone strength. INTRODUCTION: aBMD by DXA is widely used to identify patients at risk for osteoporotic fractures. aBMD is influenced by bone size (i.e., matched for vBMD, larger bones have higher aBMD), and increasing evidence indicates that absolute aBMD predicts a similar risk of fracture in men and women. Thus, we sought to define the relationships between FN aBMD (assessed by DXA) and vBMD, bone size, and FE-derived femoral strength obtained from quantitative computed tomography scans in men versus women. METHODS: We studied men and women aged 40 to 90 years and not on osteoporosis medications. RESULTS: In 114 men and 114 women matched for FN aBMD, FN total cross-sectional area was 38% higher (P < 0.0001) and vBMD was 16% lower (P < 0.0001) in the men. FE models constructed in a subset of 28 women and 28 men matched for FN aBMD showed relatively similar values for bone strength and the load-to-strength ratio in the two groups. CONCLUSIONS: In this cohort of young and old men and women from Rochester, MN, USA who are matched by FN aBMD, because of the offsetting effects of bone size and vBMD, femoral strength and the load-to-strength ratio tended to be relatively similar across the sexes.

Validity of serial milling-based imaging system for microdamage quantification.

Understanding the three-dimensional distribution of microdamage within trabecular bone may help provide a better understanding of the mechanisms of bone failure. Toward that end, a novel serial milling-based fluorescent imaging system was developed for quantifying microscopic damage in three dimensions throughout cores of trabecular bone. The overall goal for this study was to compare two-dimensional (2D), surface-based measures of microdamage extracted from this new imaging system against those from more conventional histological section analyses. Human vertebral trabecular cores were isolated, stained en bloc with a series of chelating fluorochromes, monotonically loaded, and underwent microdamage quantification via the two methods. Bone area fraction measured by the new system was significantly correlated to that measured by histological point counting (p<0.001, R(2)=0.80). Additionally, the new system produced statistically equivalent (p=0.021) measures of damage fraction (mean+/-SD), Dx.AF=0.047+/-0.021, to that obtained from stereological point counting, Dx.AF=0.048+/-0.017, at a 10% difference level. These results demonstrate that this serial milling-based fluorescent imaging system provides a destructive yet practical alternative to more conventional histologic section analysis in addition to its ability to provide a better understanding of the three-dimensional nature of microdamage.

A biomechanical perspective on bone quality.

Observations that dual-energy X-ray absorptiometry (DXA) measures of areal bone mineral density cannot completely explain fracture incidence after anti-resorptive treatment have led to renewed interest in bone quality. Bone quality is a vague term but generally refers to the effects of skeletal factors that contribute to bone strength but are not accounted for by measures of bone mass. Because a clinical fracture is ultimately a mechanical event, it follows then that any clinically relevant modification of bone quality must change bone biomechanical performance relative to bone mass. In this perspective, we discuss a framework for assessing the clinically relevant effects of bone quality based on two general concepts: (1) the biomechanical effects of bone quality can be quantified from analysis of the relationship between bone mechanical performance and bone density; and (2) because of its hierarchical nature, biomechanical testing of bone at different physical scales (<1 mm, 1 mm, 1 cm, etc.) can be used to isolate the scale at which the most clinically relevant changes in bone quality occur. As an example, we review data regarding the relationship between the strength and density in excised specimens of trabecular bone and highlight the fact that it is not yet clear how this relationship changes during aging, osteoporosis development, and anti-resorptive treatment. Further study of new and existing data using this framework should provide insight into the role of bone quality in osteoporotic fracture risk.

Finite element models predict the location of microdamage in cancellous bone following uniaxial loading.

High-resolution finite element models derived from micro-computed tomography images are often used to study the effects of trabecular microarchitecture and loading mode on tissue stress, but the degree to which existing finite element methods correctly predict the location of tissue failure is not well characterized. In the current study, we determined the relationship between the location of highly strained tissue, as determined from high-resolution finite element models, and the location of tissue microdamage, as determined from three-dimensional fluoroscopy imaging, which was performed after the microdamage was generated in-vitro by mechanical testing. Fourteen specimens of human vertebral cancellous bone were assessed (8 male donors, 2 female donors, 47-78 years of age). Regions of stained microdamage, were 50-75% more likely to form in highly strained tissue (principal strains exceeding 0.4%) than elsewhere, and generally the locations of the regions of microdamage were significantly correlated (p<0.05) with the locations of highly strained tissue. This spatial correlation was stronger for the largest regions of microdamage (≥1,000,000µm(3) in volume); 87% of large regions of microdamage were located near highly strained tissue. Together, these findings demonstrate that there is a strong correlation between regions of microdamage and regions of high strain in human cancellous bone, particularly for the biomechanically more important large instances of microdamage.

The effect of impact direction on the structural capacity of the proximal femur during falls.

As with any structure, the structural capacity of the proximal femur depends on the applied loads and these can vary as a function of impact direction during a fall. However, despite its potential importance in hip fracture risk assessment, the relative importance of impact direction is unknown. To investigate the role of impact direction in hip fracture, we developed a detailed finite element model of the proximal femur. We analyzed four loading configurations that represent a range of possible falls on the greater trochanter. Our results indicate that a change in the angle between the line of action of the applied force and the axis of the femoral neck from 0 degrees (representing a direct lateral impact) to 45 degrees (representing a posterolateral impact) reduced structural capacity by 26%. This weakening of the femur with changes in impact direction is comparable to the weakening associated with 2-3 decades of age-related bone loss. Our result elucidates the independent contribution of fall mechanics to hip fracture risk by identifying an aspect of the fall (the direction of impact) that is an important determinant of fall severity. The results can also be incorporated into a refined clinical method for assessment of hip fracture risk that accounts for the complex interactions between fall severity and bone fragility.

Biomechanical effects of intraspecimen variations in trabecular architecture: a three-dimensional finite element study.

Trabecular architecture is considered important in osteoporosis and has been quantified by a variety of mean parameters characteristic of a whole specimen. Variations within a specimen, however, have been mostly ignored. In this study, the theoretical effects of these intraspecimen variations in architecture on predicted mechanical properties were investigated through a three-dimensional finite element parameter study that simulated variations in trabecular thickness in a controlled manner. An irregularly spaced lattice of different sized rods was used to simulate trabecular bone in three distinct volume fraction ranges, representing young, middle-aged, and elderly vertebral bone. Beta distributions (a type of non-normal distribution) of trabecular thickness with coefficients of variation of either 25%, 40%, or 55% were applied to the rods in each model, and 225 simulations of uniaxial compression tests were performed to obtain modulus values. Percent modulus reductions of 22% and 43% were predicted when the intraspecimen coefficient of variation in trabecular thickness was increased from 25% to 40% and from 25% to 55%, respectively, for models of equal volume fraction. Furthermore, this trend was predicted to be independent of volume fraction. We conclude, therefore, that consideration of the intraspecimen trabecular thickness variation in conjunction with volume fraction may improve the ability to predict trabecular modulus compared with use of volume fraction alone. Further, the model suggests that if age, disease, or drug treatments increase trabecular thickness variation, this may be detrimental to mechanical properties.

Direct and computed tomography thickness measurements of the human, lumbar vertebral shell and endplate.

Although the trabecular bone of the human vertebral body has been well characterized, the thin "cortical" shell and endplate that surround the trabecular centrum have not. In addition, the accuracy of estimating the thickness of the shell and endplate using computed tomography (CT) has not been evaluated directly. To address these issues, we measured the thickness of the vertebral shell and endplate in the mid sagittal plane of 16 human L1 vertebral bodies using direct and CT based methods. Specimens were assigned to four equal sized groups based on age (middle-aged, mean age = 49 years; old, mean age = 84) and gender. We investigated the dependence of the shell and endplate thicknesses on age, gender, and anatomic region. Our findings indicate that the shell and endplate in vertebrae over age 45 are porous and often irregular, with an average thickness of approximately 0.35 mm. However, when measured from CT images, the vertebral shell and endplate appear significantly thicker, indicating that measurements based on clinical CT scans overestimate the thickness by a factor of at least two. In addition, our data indicated that, in the midsagittal plane, the anterior shell is thicker than the posterior shell or either endplate. Although these data indicated that thickness did not depend on age or gender, these particular findings are inconclusive given the small and heterogeneous sample we examined. We conclude that the so-called cortical shell and endplate of the vertebral body are thin (less than one-half of a millimeter) and porous, and perhaps are better thought of as thin membranes of fused trabeculae than as true cortices.

Measurement of intraspecimen variations in vertebral cancellous bone architecture.

A three-dimensional technique was developed for the quantification of the number and cross-sectional geometry of individual trabeculae oriented along a given direction. As an example application, the number of vertical and horizontal trabeculae and their respective cross-sectional geometry were determined for a set of six vertebral cancellous bone specimens (L3-L4 female vertebral bodies; age range 39-63 years). Three-dimensional optical images at a spatial resolution of 20 microm were obtained using an automated serial milling technique. The thickness distributions were generally right skewed. The mean true thickness for both the vertically and horizontally oriented trabeculae showed a strong relationship with volume fraction (vertical: r2 = 0.86; p < 0.05; horizontal: r2 = 0.80; p < 0.05), and mean trabecular thickness (Tb.Th.) (vertical: r2 = 0.81; p < 0.05; horizontal: r2 = 0.72; p < 0.05). The horizontal trabeculae were greater in number and were thinner than the vertical trabeculae. The coefficient of variation of the intraspecimen vertical trabecular thicknesses ranged from 25% to 42%, and showed a weak, albeit insignificant, positive correlation with volume fraction (r2 = 0.46). The findings demonstrated substantial intraspecimen variations exist in trabecular thickness that are not related to volume fraction. Further studies are recommended to determine the potential role of such intraspecimen variations in architecture on biomechanical properties.

Impact of spatial resolution on the prediction of trabecular architecture parameters.

Although the efficacy of various measures for the assessment of trabecular bone architecture has been widely studied, the impact of spatial resolution on the estimation of these measures has remained relatively unexplored. In this study, ten cubes each of human trabecular bone from the femur and vertebral bodies were obtained from nine cadavers (four males and five females), aged 23-67 years (mean 42.3 years). These specimens were serially milled and imaged at a resolution of 40 microm to produce three-dimensional digitizations from which traditional morphometric and structural anisotropy measures could be computed based on a three-dimensional approach. The cubes were then artificially degraded to an in-plane resolution of 100 microm and an out-of-plane (slice) resolution of 100-1000 microm. These resolutions mimicked in vivo resolutions as seen using magnetic resonance (MR) imaging. All images, original and degraded, were individually segmented using a thresholding algorithm, and both the traditional morphometric and structural anisotropy measures were recomputed. The choice of slice direction was varied along the superior-inferior (axial), anterior-posterior (coronal), and medial-lateral (sagittal) directions to minimize the impact of the lower slice resolution on the architectural measures. It was found that traditional morphometric measures such as trabecular spacing and trabecular number showed weak resolution dependency; measures such as trabecular thickness, however, showed strong resolution dependency and required very high resolutions for precise measurement. In the case of the femur specimens, both structural anisotropy as well as the preferred orientation showed a strong resolution dependency. The resolution dependency of these parameters could be minimized for the femur and the vertebral body specimens if the slice direction was taken along the superior-inferior direction.

Three-dimensional imaging of trabecular bone using the computer numerically controlled milling technique.

Although various techniques exist for high-resolution, three-dimensional imaging of trabecular bone, a common limitation is that resolution depends on specimen size. Most techniques also have limited availability due to their expense and complexity. We therefore developed a simple, accurate technique that has a resolution that is independent of specimen size. Thin layers are serially removed from an embedded bone specimen using a computer numerically controlled (CNC) milling machine, and each exposed cross section is imaged using a low-magnification digital camera. Precise positioning of the specimen under the camera is achieved using the programmable feature of the CNC milling machine. Large specimens are imaged without loss of resolution by moving the specimen under the camera such that an array of field-of-views spans the full cross section. The images from each field-of-view are easily assembled and registered in the postprocessing. High-contrast sections are achieved by staining the bone black with silver nitrate and embedding it in whitened methylmethacrylate. Due to the high contrast nature and high resolution of the images, thresholding at a single value yielded excellent predictions of morphological parameters such as bone volume fraction (mean +/- SD percent error = 0.70 +/- 4.28%). The main limitations of this fully automated "CNC milling technique" are that the specimen is destroyed and the process is relatively slow. However, because of its accuracy, independence of image resolution from specimen size, and ease of implementation, this new technique is an excellent method for ex situ imaging of trabecular architecture, particularly when high resolution is required.

Mechanical and chemical characteristics of mineral produced by basic fibroblast growth factor-treated bone marrow stromal cells in vitro.

It has been shown that various organ and cell cultures exhibit increased mineral formation with the addition of basic fibroblast growth factor (bFGF) and phosphate ions in the medium. However, to date there has been no attempt to relate the chemical composition of mineral formed in vitro to a measure of its mechanical properties. This information is important for understanding the in vivo mineralization process, the development of in vitro models, and the design of tissue-engineered bone substitutes. In this study we examined the reduced modulus; hardness; and mineral-to-matrix, crystallinity, carbonate-to-mineral, and calcium-to-phosphorus ratios of mineral formed by bFGF-treated rat-derived bone marrow stromal cells in vitro. The cells were treated with 1 or 3 mM beta-glycerophosphate for 3 and 4 weeks. Both mechanical parameters, reduced modulus and hardness, increased with increasing beta-glycerophosphate concentration. The only chemical measure of the mineral composition that exhibited the same dependency was the mineral-to-matrix ratio. The values of crystallinity and carbonate fraction were similar to those for intact cortical bone, but the calcium-to-phosphorus ratio was substantially lower than that of normal bone. These data indicate that the mineral formed by bFGF-treated bone cells is mechanically and chemically different from naturally formed lamellar bone tissue after 4 weeks in culture. These results can be used to improve in vitro models of mineral formation as well as enhance the design of tissue-engineered bone substitutes.

Osteoblasts respond to pulsatile fluid flow with short-term increases in PGE(2) but no change in mineralization.

Although there is no consensus as to the precise nature of the mechanostimulatory signals imparted to the bone cells during remodeling, it has been postulated that deformation-induced fluid flow plays a role in the mechanotransduction pathway. In vitro, osteoblasts respond to fluid shear stress with an increase in PGE(2) production; however, the long-term effects of fluid shear stress on cell proliferation and differentiation have not been examined. The goal of this study was to apply continuous pulsatile fluid shear stresses to osteoblasts and determine whether the initial production of PGE(2) is associated with long-term biochemical changes. The acute response of bone cells to a pulsatile fluid shear stress (0.6 +/- 0.5 Pa, 3.0 Hz) was characterized by a transient fourfold increase in PGE(2) production. After 7 days of static culture (0 dyn/cm(2)) or low (0.06 +/- 0.05 Pa, 0.3 Hz) or high (0.6 +/- 0.5 Pa, 3.0 Hz) levels of pulsatile fluid shear stress, the bone cells responded with an 83% average increase in cell number, but no statistical difference (P > 0.53) between the groups was observed. Alkaline phosphatase activity per cell decreased in the static cultures but not in the low- or high-flow groups. Mineralization was also unaffected by the different levels of applied shear stress. Our results indicate that short-term changes in PGE(2) levels caused by pulsatile fluid flow are not associated with long-term changes in proliferation or mineralization of bone cells.

Relative roles of microdamage and microfracture in the mechanical behavior of trabecular bone.

Compared to trabecular microfracture, the biomechanical consequences of the morphologically more subtle trabecular microdamage are unclear but potentially important because of its higher incidence. A generic three-dimensional finite element model of the trabecular bone microstructure was used to investigate the relative biomechanical roles of these damage categories on reloading elastic modulus after simulated overloads to various strain levels. Microfractures of individual trabeculae were modeled using a maximum fracture strain criterion, for three values of fracture strain (2%, 8%, and 35%). Microdamage within the trabeculae was modeled using a strain-based modulus reduction rule based on cortical bone behavior. When combining the effects of both microdamage and microfracture, the model predicted reductions in apparent modulus upon reloading of over 60% at an applied apparent strain of 2%, in excellent agreement with previously reported experimental data. According to the model, up to 80% of the trabeculae developed microdamage at 2% apparent strain, and between 2% and 10% of the trabeculae were fractured, depending on which fracture strain was assumed. If microdamage could not occur but microfracture could, good agreement with the experimental data only resulted if the trabecular hard tissue had a fracture strain of 2%. However, a high number of fractures (10% of the trabeculae) would need to occur for this case, and this has not been observed in published damage morphology studies. We conclude therefore that if the damage behavior of trabecular hard tissue is similar to that of cortical bone, then extensive microdamage is primarily responsible for the large loss in apparent mechanical properties that can occur with overloading of trabecular bone.