Association of vertebral endplate microstructure with bone strength in men and women

Journal Pre-proof Association of vertebral endplate microstructure with bone strength in men and women

MeiLissa McKay, Timothy M. Jackman, Amira I. Hussein, Ali Guermazi, Jingjiang Liu, Elise F. Morgan PII:

S8756-3282(19)30441-7

DOI:

https://doi.org/10.1016/j.bone.2019.115147

Reference:

BON 115147

To appear in:

Bone

Received date:

27 August 2019

Revised date:

23 October 2019

Accepted date:

5 November 2019

Please cite this article as: M. McKay, T.M. Jackman, A.I. Hussein, et al., Association of vertebral endplate microstructure with bone strength in men and women, Bone(2018), https://doi.org/10.1016/j.bone.2019.115147

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© 2018 Published by Elsevier.

Journal Pre-proof Association of Vertebral Endplate Microstructure with Bone Strength in Men and Women MeiLissa McKaya, Timothy M Jackmana, Amira I Husseina, Ali Guermazib, Jingjiang Liua, Elise F Morgana a

Department of Mechanical Engineering, 110 Cummington Mall, Boston University, Boston, MA 02215 USA. b Department of Radiology, Boston University School of Medicine, 820 Harrison Avenue, FGH Building, 3rd Floor, Boston, MA 02118, USA.

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Corresponding Author Elise Morgan [email protected] Department of Mechanical Engineering 110 Cummington Mall Boston University Boston, MA 02215 USA

Journal Pre-proof Keywords compressive strength, bone density, bony endplate, vertebral fracture, trabecular architecture, sex-associated differences

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Graphical Abstract

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Highlights ● Male vs. female vertebrae exhibit similar mean bone density and microstructure of the vertebral endplate and underlying subchondral trabecular bone. ● In male vertebrae, the density of the endplate region is only weakly associated with the average density of the vertebra. ● In female vertebrae, the density of the endplate region is not associated with the average density of the vertebra. ● Predictions of vertebral strength based on average density are improved when incorporating a measure of the endplate region density. ● Predictions of vertebral strength based on average density are not improved when incorporating measures of subchondral trabecular architecture.

Journal Pre-proof Abstract Epidemiological and biomechanical evidence indicates that the risk of vertebral fracture differs between men and women, and that vertebral fracture frequently involves failure of the endplate region. The goal of this study was to compare the bone microstructure of the endplate region—defined as the (bony) vertebral endplate and underlying subchondral trabecular bone— between sexes and to determine whether any such sex differences are associated with vertebral

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strength. The bone density (volume fraction, apparent density and tissue mineral density) of the

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superior-most 2 mm of the vertebra, and the bone density and trabecular architecture of the next

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5 mm were quantified using micro-computed tomography in human T8 (12 female, 16 male) and

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L1 (13 female, 12 male) vertebrae. Average density of the vertebra (integral bone mineral density (BMD)) was determined by quantitative computed tomography and compressive strength

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by mechanical testing. Few differences were found between male and female vertebrae in the

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density of the endplate region; none were found in trabecular architecture. However, whereas endplate volume fraction was positively correlated with integral BMD in male vertebrae

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(r=0.654, p<0.001), no correlation was found in the female vertebrae (r=0.157, p=0.455).

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Accounting for the density of the endplate region improved predictions of vertebral strength (p<0.034) and eliminated sex-specificity in the strength prediction that was based on integral BMD alone. These results suggest that the density of the endplate region influences vertebral fracture and that non-invasive assessment of this region’s density can contribute to predictions of vertebral strength in men and women.

Journal Pre-proof 1. Introduction Vertebral fractures are one of the most common complications of osteoporosis in both sexes, and their incidence is higher in women than in men. In those aged 50 years and older, the incidence of vertebral fractures is as much as two-fold higher in women [1-4]. Age is also a stronger risk factor for vertebral fracture in women than men [5-7]. Determining the mechanisms underlying the sex-associated differences in vertebral fracture may help reduce the large

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economic and human costs of these fractures.

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Prior studies have examined sex-associated differences in the loads the vertebra

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experiences as well as in factors intrinsic to the vertebra, such as bone density and size. In the

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elderly population, women have greater thoracic kyphosis, which is associated with increased compressive force on the vertebra [8], as compared to men [9-11]. Both sexes experience a

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decrease in volumetric and areal bone mineral density (BMD) with age, but the age-associated

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decline in BMD of both the entire trabecular compartment and the periphery of the compartment is greater in women [12-14]. Owing to these greater declines in BMD and the lack of sex

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differences in the change in vertebral cross-sectional area with age, it has been estimated that the

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decline in vertebral compressive strength with age is larger in women than in men [13]. Taken together, these results suggest that the factor of risk (the ratio of vertebral loading to vertebral strength) is higher in elderly women vs. elderly men [15]. Vertebral strength also depends on trabecular architecture, but evidence of sex-associated differences in architecture is mixed. Although some studies have reported differences in trabecular bone volume fraction (BV/TV), trabecular number (Tb.N*), and the proportion of vertical trabeculae between male and female vertebrae [16-19], others have found no differences in any microstructural parameters investigated [20-23]. These discrepancies in findings may be

Journal Pre-proof due in part to sampling, because the microstructure of the trabecular centrum is highly spatially heterogeneous [24-27]. An alternative approach is to examine whether differences between sexes exist in a region of the vertebra directly involved in vertebral fracture. Laboratory studies have indicated that deflection of the vertebral endplate, the thin, porous plate-like layer of bone between the cartilaginous endplate and trabecular centrum, is a principal mechanism by which vertebrae fail [28-32]. These results are consistent with the use of endplate depression,

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particularly in the central portion of the endplate, as a criterion in radiological classification of

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vertebral fracture [33]. Both the initiation and progression of endplate deflection are associated

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with the BV/TV of the vertebral endplate and the microstructure of the subchondral trabecular

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bone [28, 29]. Evidence also indicates that the location of initial endplate collapse [29] as well as the vertebral bone microstructure [22, 34-38] and stiffness [39] are associated with degeneration

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of the adjacent intervertebral disc. However, whether the bony microstructure of the endplate

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region differs in male vs. female vertebrae is unknown. The specific objectives of this study were: 1) to compare the bone density of the (bony)

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vertebral endplate and the bone density and trabecular architecture of the subchondral trabecular

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bone between male and female vertebrae; and 2) to identify associations between these microstructural properties and vertebral strength in the two sexes. 2. Materials and Methods 2.1 Specimen Preparation This study used thoracic (T8) and lumbar (L1) vertebrae from the fresh-frozen spines of 52 donors (28 men, 24 women) aged 35-91 years (Table 1). The donor medical records that were provided by the tissue banks (National Disease Research Interchange, Philadelphia, PA; Life Legacy Foundation, Tuscon, AZ) indicated the absence of disqualifying diseases and conditions

Journal Pre-proof such as osteosarcoma, Paget’s disease, hypo- or hyperparathoidism, or history of alcohol abuse, chronic kidney disease or chemotherapy. The T8 and L1 vertebrae were harvested as part of, respectively, T7-T9 and T12-L2 spine segments, such that the mechanical testing would be performed with adjacent discs attached. We acquired lateral radiographs of the spine segments ex vivo (Faxitron, Tuscon, AZ) to confirm the absence of prevalent fracture. During dissection, the anterior and posterior longitudinal ligaments and ligamentum flavum were left intact. The upper

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portion of the superior vertebra and inferior portion of the inferior vertebra of each spine

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segment were cut away so that upon embedding the remaining portions of these vertebrae in

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polymethyl methacrylate (PMMA), the PMMA infiltrated the trabecular centrum and provided

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secure fixturing for mechanical testing. The spine segments were kept hydrated at all times and, when not in use, wrapped in saline-soaked gauze, sealed in plastic bags, and stored at -20°C.

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2.2 Computed Tomography and Mechanical Testing

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Imaging of the spine segments with quantitative computed tomography (QCT) and micro-computed tomography (μCT), and subsequent mechanical testing, were performed in the

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course of prior studies [28, 40]. Briefly, each spine segment underwent a QCT scan (GE

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Lightspeed VCT; GE Healthcare, Milwaukee, WI; 0.3125x0.3125x0.625 mm/voxel) to compute integral volumetric bone mineral density (integral BMD), which is the volumetric BMD of both trabecular and cortical compartments of the vertebral body combined. Due to loss of the image files, integral BMD could not be calculated for one L2 specimen. The cross-sectional area of each T8 and L1 vertebra was defined as the cross-sectional area (CSA) of the largest elliptical cylinder that fit entirely within the trabecular centrum of the vertebra [24]. The thoracic spine segments were randomly assigned into a set for testing under axial compression (n = 14) and a set for testing under axial compression with anterior flexion (n = 14).

Journal Pre-proof The lumbar spine segments were tested under axial compression. At the time of testing, each spine segment was placed in a custom-built, radiolucent device for mechanical testing that had been filled with 60% saline and 40% of 50-proof ethanol [41]. The positioning of the segment was such that the middle vertebra was aligned with the vertical axis, and the superior and inferior vertebrae were angled according to the natural curvature presented by the spine segment. Each spine segment was imaged with μCT (μCT 80; Scanco Medical, Brüttisellen, Switzerland) at a

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nominal resolution of 37 μm/voxel prior to and during mechanical testing. The settings for

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voltage, current and integration time were 70 kVp, 114 mA, and 300 ms, respectively. Only the

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initial μCT scan was analyzed in the present study, and the mechanical test data from only the

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axial compression tests was used. The ultimate force was the force at the peak of the forcedisplacement curve, and the yield force was defined as the force at which the slope of the force-

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displacement curve decreased. Three of the lumbar segments were excluded because failure

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occurred in an adjacent level before the L1 yielded. For an additional two lumbar specimens, the ultimate force was not measured, because due to operator error the tests were halted following

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the yield point but prior to the ultimate point. Based on qualitative and quantitative observations

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of the μCT images [28,40] to identify regions with visibly large deformations and large displacement gradients as the likely locations of failure, the superior 2mm layer of the vertebra was the site of failure in ten of the 14 T8 specimens and 16 of the 19 L1 specimens. For the remaining four T8 specimens and three L1 specimens, the regions of identified failure were at the inferior endplate. No disc herniation was observed in any specimen. Failure was identified in the cortical shell in a minority of the specimens but was accompanied by failure in the superior 2mm layer. 2.3 Endplate Density and Subchondral Trabecular Architecture

Journal Pre-proof All measurements of the density and architecture of the endplate region were performed using the μCT scans (Scanco Medical). The analyses focused on the superior endplate region, because of the much higher failure rate of the superior vs inferior endplate region during vertebral fracture [42, 43]. Noise reduction in the μCT scans was achieved with a Gaussian filter (sigma = 0.8; support = 1). Conversion of native intensity values to mineral densities was made possible by a standard curve obtained from imaging a potassium hydroxyapatite phantom

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(Scanco Medical). A threshold of 263.7 mg HA/ccm, determined from an adaptive, iterative

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technique (Scanco Medical), was used to distinguish bone tissue from marrow, disc and other

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soft tissues, and background. To minimize the effect of air bubbles present within the cadaver

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vertebrae, we used an automated technique to replace the intensity values of voxels that are defined as air by the average intensity of the bone marrow.

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Endplate volume fraction (Ep.BV/TV) was calculated from the μCT scans by first

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defining the surface of the superior vertebral endplate, then calculating the bone volume fraction of the region that extends 2mm downward [72] from this surface (Figure 1). Ep.BV/TV was used

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in lieu of measuring endplate thickness, because the ambiguity in the exact location of the

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boundary between vertebral endplate and trabecular bone (particularly, but not only, in vertebrae with double endplates, endplate sclerosis, or endplate defects [19, 44]) made measurements of endplate thickness less repeatable and standardized [19, 45]. Tissue mineral density (Ep.TMD) and apparent bone mineral density (Ep.BMD) were also calculated for this 2-mm-tall layer. The cross-sectional area (transverse plane) of the endplate layer was calculated and denoted as Ep.CSA. The architecture of the subchondral bone was defined by analyzing the 5-mm-tall layer directly under the 2-mm-tall layer used to measure Ep.BV/TV (Figure 1). The following were

Journal Pre-proof quantified: bone volume fraction (Sub.BV/TV), trabecular separation (Tb.Sp*), trabecular number (Tb.N*), structure model index (SMI), degree of anisotropy (DA), connectivity density (ConnD), tissue mineral density (Sub.TMD) and apparent bone mineral density (Sub.BMD) [46]. The cross-sectional area (transverse plane) of the subchondral layer was calculated and denoted as Sub.CSA. These 2-mm-tall and 5-mm-tall layers were then divided into regions corresponding to

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the central endplate (defined as the region interior to the epiphyseal rim) and the epiphyseal rim.

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This partitioning was done because of evidence that, during the onset of vertebral fracture,

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depression and collapse of the central endplate occurs more frequently [30, 33, 47, 48].

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Ep.BV/TV, Ep.TMD, Ep.BMD, and all of the subchondral parameters were re-calculated for only the central endplate region. For one T8 specimen, the 2-mm and 5-mm data were not

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2.4 Scoring of Disc Degeneration

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available due to corruption of the image file.

Given the evidence that bone microstructure may be associated with degeneration of the

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adjacent intervertebral disc [22, 34-38], degeneration of the intervertebral discs adjacent to the

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superior T8 and L1 endplates was assessed according to two grading systems: apparent loss of disc integrity (ALDI) [49] and disc space narrowing (DSN) [50]. The ALDI grade, which ranges 0 (healthy) to 2 (degenerated), is based primarily on the clarity of the demarcation between the nucleus pulposus and annulus fibrosus in the mid-transverse QCT image and secondarily on the presence of osteophytes. Each disc was assigned an ALDI grade by two observers. DSN ranges 0 to 3 and was scored by a trained radiologist (AG). 2.5 Statistical Analyses Each measure of density, trabecular architecture, and vertebral strength was compared

Journal Pre-proof between male and female vertebrae using t-tests or Wilcoxon rank sum tests, depending on the normality of the data. These comparisons were carried out separately for the T8 and L1 vertebrae. Subsequently, multiple linear regression was used to test for differences between sexes in measures of density and architecture of the endplate and subchondral layers after adjusting for integral BMD. In the multiple linear regression models, the independent variables were integral BMD, sex, and level (T8 or L1), and the two- and three-way interaction terms were included. If

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the interaction between sex and integral BMD was found to be significant, the dependent

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variable was regressed against integral BMD for each sex separately.

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Multiple linear regression was also used to determine the dependence of each measure of

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vertebral strength on each of the following sets of variables: 1) Ep.BV/TV×Ep.CSA and integral BMD×CSA; 2) Ep.BMD×Ep.CSA and integral BMD×CSA; 3) Sub.BV/TV×Sub.CSA and

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integral BMD×CSA; 4) Sub.BMD×Sub.CSA and integral BMD×CSA; and 5) integral

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BMD×CSA. Each of the first four models was compared to Model 5 using a restricted vs. full F test; the purpose of these comparisons was to examine the additional predictive effect of

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accounting for the density (BV/TV or BMD) of the endplate region. In addition, we used

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stepwise linear regression to explore adding measures of the subchondral trabecular architecture to Models 3 and 4. In preparation for these stepwise analyses, pairwise correlations among the various measures of microstructure in the subchondral layer were determined so as to identify a smaller set of independent variables with reduced multicollinearity. The stepwise regression was then carried out to identify which variables within this smaller set best predicted vertebral strength. Finally, the best-performing models were then re-run but with the addition of sex as an independent variable as well as the interaction between sex and the other independent variables. Pearson chi-square analysis was carried out to test for associations between disc

Journal Pre-proof degeneration and sex. Analysis of variance (ANOVA) was used to test for differences in integral BMD and the microstructural measures of the endplate region among the different disc grades. All of the statistical analyses were repeated using the measurements of density and architecture for only the central portion of the endplate region (“Central”) rather than the entirety of the 2-mm or 5-mm layer (“Whole”). A significance level of 0.05 was used in all cases, though a Bonferroni correction was applied for the t-tests, Wilcoxon tests, and the ANOVAs on the disc

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grades on account of the large number of individual comparisons made in these tests.

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3. Results

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Few differences were found between male and female vertebrae in the density of the

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endplate region, and none were found in the trabecular architecture. The comparisons by t-tests and Wilcoxon rank sum tests (Table 1) showed that, following Bonferroni correction, only

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Central Sub.TMD in L1 was lower in male than female specimens (p<0.001). No other

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differences in Central or Whole measures were found (p>0.015 prior to Bonferroni correction). However, a divergent association in men vs. women was found between Central Ep.BV/TV and

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integral BMD (Figure 2A; p=0.023 for the interaction between sex and integral BMD): whereas

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Central Ep.BV/TV was positively correlated with integral BMD in male vertebrae (r=0.654, p<0.001), no correlation was found in the female vertebrae (r=0.157, p=0.455). No such divergence was present in the association between Central Sub.BV/TV and integral BMD (Figure 2B; p=0.946 for the interaction between sex and integral BMD) or in the association between any other measure of density or architecture of the endplate region and integral BMD (p>0.158). Accounting for the density of the endplate region improved predictions of vertebral strength. Models 1-4, which included one of these density measures as well as integral BMD

Journal Pre-proof produced higher coefficients of determination (R2-values) than Model 5, which had integral BMD as the only independent variable (R2=0.290-0.448 vs. R2=0.174 in the case of yield force, and R2=0.519-0.546 vs. R2=0.438 in the case of ultimate force; p<0.034; Table 2). These improvements were seen regardless of whether the endplate measures were for the Central or Whole portion. The only exception was that adding Central Sub.BMD×Sub.CSA to the regression model for ultimate force produced only a trend (p=0.082) towards improvement of the

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R2-value.

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In contrast with the improvements seen in strength prediction when accounting for the

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density of the vertebral endplate or subchondral bone, accounting for the subchondral trabecular

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architecture had little effect. For prediction of yield force using measures of Whole subchondral trabecular architecture in addition to the independent variables of Model 3 (i.e., Whole

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Sub.BV/TV×Sub.CSA and integral BMD×CSA), the stepwise regression analyses identified

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Tb.Sp* as an additional explanatory variable (p=0.004) that improved the coefficient of determination from R2=0.415 to R2=0.428. In all other cases, however, no architectural measures

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were added by the stepwise regression.

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Given that the majority of the best-performing regression models for prediction of vertebral strength were those in which the independent variables were a measure of the density of the endplate region (multiplied by the region CSA) and integral BMD×CSA (i.e., Models 14), we compared the effect of including sex as an additional independent variable in these models to that of adding sex to the model that previously had only integral BMD×CSA as the independent variable (Model 5). In all cases, interaction terms between sex and the other independent variable(s) were included. Whereas a trend towards a sex-specific association was seen between strength and integral BMD×CSA (p=0.063; Figure 3A), the inclusion of a density

Journal Pre-proof measure for the endplate region (i.e., Ep.BV/TV×Ep.CSA, Ep.BMD×Ep.CSA, Sub.BV/TV×Sub.CSA, or Sub.BMD×Sub.CSA) as an additional independent variable in the regression model eliminated any sex-specificity in the strength prediction (Figure 3B), regardless of whether the density measure was for the Central or Whole portion. For both grading schemes of disc degeneration, no difference in grades were found between the sexes (p=0.300 for ALDI; p=0.461 for DSN). Neither integral BMD (p=0.742 for

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ALDI; p=0.313 for DSN) nor any of the measures of density or trabecular architecture of the

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endplate region were found to differ among disc grades (0.026
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correction).

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4. Discussion

The goal of this study was to compare the bone microstructure of the endplate region—

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defined as the (bony) vertebral endplate and underlying subchondral trabecular bone—between

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sexes and to determine whether any such sex differences are associated with vertebral strength. Although little difference in mean values of density or trabecular architecture was found between

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the male and female specimens, the association between endplate bone volume fraction

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(Ep.BV/TV) and the average density of the vertebral body (integral BMD) differed between the sexes. Specifically, no association was found for the female vertebrae. This finding is noteworthy, given that, for both cortical and trabecular bone, BV/TV is the microstructural parameter most strongly associated with stiffness and strength [52-56]. Hence, measures of the average density of the vertebra such as integral BMD may have limited utility in predicting the strength of the endplate region in women. We further found that predictions of the compressive strength of the vertebra based on integral BMD were improved when the regression models also incorporated a measure of density of the endplate region. Together with the growing

Journal Pre-proof understanding of the role of the endplate region in the biomechanical mechanisms of vertebral fracture, these results indicate that non-destructive assessment of the density of this region can benefit predictions of vertebral strength in men and women. The strengths of this study largely pertain to the experimental design. Although prior studies have noted that regional measures of density and trabecular architecture are correlated with vertebral strength [26, 48, 57-59], in some cases even after adjustment for the average

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density of the vertebra [60], ours applies this general approach specifically to a region that is the

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frequent site of vertebral failure. Our findings therefore suggest that the association between the

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density of the endplate region and vertebral strength may be a true mechanistic relationship

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rather than a mere association. Hulme and colleagues found a strong association between vertebral strength and the density and architecture (Conn.D, SMI, Tb.N) of the trabecular bone in

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the quadrant of the centrum (superior-anterior, superior-posterior, inferior-anterior, inferior-

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posterior) closest to location where endplate failure was noted after mechanical testing was complete [48]. Zhao et al. found correlation between vertebra yield stress and the optical density

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of trabecular bone in the superior half [19]. Our results are consistent with this prior work and

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extend it to show, first, that this type of association exists even when the microstructural measurements pertain only to the endplate region (upper 2-7 mm) and second, that the association remains after adjusting for the average density of the entire vertebra. Another strength of the design of this study is that both discs adjacent to the vertebra of interest (T8 or L1) were present. Although inclusion of the discs likely causes more variability in the measurement of vertebral strength than if the discs were removed and the endplates potted in cement, we believe that particularly for studies of endplate mechanics it is crucial to load the vertebral body in a physiologically representative manner.

Journal Pre-proof This study also has limitations. The specimens were from donors of advanced age, and thus the results may not apply to younger populations. We used a very slow loading rate in the mechanical tests, which may also restrict the applicability of our findings to the slow-onset type of vertebral fractures common in the elderly as compared to vertebral fractures caused by trauma. The pooling of data from two different levels of the spine additionally warrants caution, since differences in features such as vertebral anatomy and spinal curvature between mid-

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thoracic and upper lumbar levels may affect failure mechanisms. However, it is interesting to

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note that incorporating measures of endplate density produced a more unified prediction of

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strength across both levels as compared to predictions made using only integral BMD (Figure 3). This result provides further evidence that, in the context of vertebral failure, the endplate region

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is functionally important in ways not fully described by the vertebra’s average density. We also

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examined whether our results depended on the definition of Finally, although we did not find any

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association between measures of disc degeneration and either vertebral strength or the density or architecture of the endplate region, the measures of disc degeneration that we used are relatively

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coarse. As assessed instead by magnetic resonance imaging (MRI) [61] and gross observation of

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mid-sections [62, 63], worsening disc degeneration has been associated with decreased indentation strength of the endplate. Ongoing work in our laboratory is incorporating MRI for both quantitative and semi-quantitative assessments of discs. Although the use of a fixed height, 2 mm, for definition of the “Ep” layer obviates the need to the exact boundary between endplate and trabecular bone, it is possible that this approach masks differences among specimens in how microstructure varies within this region. The female vertebrae are shorter than the male vertebra (Table 1), and the 2mm layer thus encompasses a larger portion of the vertebra for the former compared to the latter. We repeated the analyses

Journal Pre-proof using a layer height of 1 mm so as to test the specificity of the findings to the 2mm height. As expected, the values of Ep.BV/TV and Ep.BMD were higher for the 1mm-tall layer than the 2mm-tall layer. However, the results regarding associations with sex and vertebral strength were nearly unchanged. That is, the 1mm Ep.BV/TV and 1mm Ep.BMD did not differ between sexes (p>0.265), the 1mm Ep.BV/TV showed a positive association with integral BMD in male (p=0.035) but not female (p=0.869) vertebrae, and including either the 1mm Ep.BV/TV or 1mm

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Ep.BMD improved predictions of vertebral strength (p=0.009 for 1mm Ep.BV/TV×Ep.CSA;

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p=0.006 for 1mm Ep.BMD×Ep.CSA). The only main result that differed is that the sex-

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specificity in the association between vertebral strength and integral BMD remained even after

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including the 1mm Ep.BV/TV (p=0.032) or the 1mm Ep.BMD (p=0.039) in the regression model. This last result suggests that sex-specificity in the association between vertebral strength

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and integral BMD might be attributed to sex-linked differences in the mechanical behavior of the

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transitional zone between the very plate-like structure of the endplate and the trabecular centrum. Overall, the findings of our study could have important implications for prediction of

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fracture risk in the spine. Failure of the endplate region is common in vertebral fracture, and the

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mechanical properties of this region are expected to be more strongly related to the density of this region than to that of the entire vertebral body or vertebra. Thus, the weak association of Ep.BV/TV with integral BMD overall, and the lack of such association for women, may be one reason why integral BMD is insufficiently sensitive for predicting vertebral fracture risk [64]. In general, we found only weak to moderate associations between measures of density of the endplate region and integral BMD, and that inclusion of these endplate density measurements improved predictions of vertebral strength as compared to predictions made using only integral BMD. These results motivate study of the potential for standardizing measurements of endplate

Journal Pre-proof areal BMD by dual energy x-ray absorptiometry (DXA) or volumetric BMD by clinical CT, for the purpose of incorporating these measurements when estimating fracture risk in the spine. Although our results indicate the utility of endplate density for predicting vertebral strength in both sexes, it is important to note that we did not directly assess the mechanical behavior of the endplate region. Using microindentation tests, Dall’Ara et al. found that the elastic modulus of the mineralized tissue of the bony endplate is similar to that of the cortical

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shell and vertebral trabeculae, but no comparisons with respect to donor sex, age or spine BMD

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were performed [65]. A number of studies have performed indentation tests on the surface of the

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vertebral endplate. These studies have shown that indentation strength is moderately and

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positively correlated with measures of the average density of the vertebra [62, 66, 67] as well as with the density of only the endplate region [61, 67, 68]. These results support the idea that

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endplate density is mechanistically linked to vertebral strength and failure. However, since the

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primary motivation of these prior studies was the problem of subsidence of inter-body fusion implants, the sizes of the indenters, ~1.5-3 mm, were chosen based on the length scale of typical

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implant surface features. The characteristic length scale of spatial variations in stress in the intact

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disc-vertebra interface tends to be larger [69]. The mechanical behavior of the endplate region under loading conditions most relevant to vertebral fracture is not well characterized at present. Our study also raises the question of what factors can affect the density and mechanical behavior of the endplate region in ways not captured by measurement of spine BMD. During aging, bone density and trabecular architecture deteriorate more rapidly in the subchondral layer compared to the mid-transverse region of the centrum [25]. These inhomogeneous changes together with the overall greater rate of decline in vertebral bone density with age in women compared to men could explain why we found differences between the sexes in the association

Journal Pre-proof between bone density in the superior-most region of the vertebra and the average density of the vertebra. Degenerative changes and damage such as tidemark avulsions [77] and annular fissures [78] could also change the habitual distributions of stress and strain and hence density throughout the endplate region. These degenerative features are more common in the lower regions of the spine and could, along with differences in spinal curvature between the midthoracic and lumbar regions, be reasons why TMD of the subchondral layer is higher than that of

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the endplate layer in the L1 specimens (Table 1). Microstructural deterioration with age also

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appears to be more pronounced in the central portion of the subchondral layer as compared to the

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portion underlying the ring apophysis [34, 70]. These age-related changes could be one reason

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why prevalence of vertebral fracture increases with age even without a corresponding decrease in the BMD of the vertebra [71]. Interestingly, studies of changes with respect to age and disc

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degeneration in the thickness, porosity and density of the bony endplate specifically have

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produced mixed results [35, 37, 38, 48, 72, 73]. These conflicting results may be due to the difficulties in defining the boundary between the endplate and subchondral trabecular bone, to

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the existence of only minor, if any, changes in thickness with age, to differences in methods of

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assessing disc health, and to non-monotonic changes in endplate density and structure over the course of disc degeneration. A final note is that the robustness of the endplate region could be influenced as much by events during skeletal growth as in aging. The available evidence indicates that during skeletal development, the vertebral endplate is the last site within the vertebra to mineralize and is not fully formed until skeletal maturity [74-76]. Thus, factors such as nutrition, hormonal variations, and exercise during adolescence may affect acquisition of peak bone density and architectural form within the endplate region. In summary, few differences were found between male and female vertebrae in the

Journal Pre-proof density or trabecular architecture of the endplate region. In both sexes, accounting for the density, but not the trabecular architecture, of this region improved predictions of vertebral strength compared to predictions made only from integral BMD. This improvement, together with the weak association between the density of the endplate region and integral BMD, indicates that this region has an independent role in vertebral fracture. These results suggest future avenues of research into the prospect of using estimates of endplate density in clinical

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screening and into the relationship between the mechanical behavior of the endplate region and

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that of the vertebral as a whole.

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5. Acknowledgements

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Funding for this study was provided by the National Institutes of Health (NIH R01AR054620). This study was also made possible by the Micro-Computed Tomography Imaging

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core facility at Boston University and the equipment in this facility that was funded by the

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Loaiza.

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National Science Foundation (BES0521255). The authors would also like to thank Johnfredy

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when others (in the same spine) do not? Clin Anat 2015;28: 195-204.

Journal Pre-proof Table 1. By level and sex, mean values ± standard deviations of integral vBMD and each measure of density and microstructure of the central region of the endplate and subchondral layers. The p-values are from t-tests comparing the means, or from Wilcoxon rank sum tests if denoted with an asterisk (*), between sexes. Boldface font is used to identify instances in which differences between sexes remain even after Bonferroni correction, whereas italicized font is used to identify those in which the differences did not remain upon Bonferroni correction. The pvalues shown in the table are reported prior to Bonferroni correction. T8

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Female 12 80.8 ± 12.8 0.135 ± 0.026 0.244 ± 0.044 159.0 ± 26.7 503.4 ± 34.1 0.080 ± 0.022 57.1 ± 19.3 561.5 ± 24.1 1.53 ± 0.10 1.07 ± 0.42 1.03 ± 0.25 2.63 ± 0.32 2.22 ± 1.03 22.0 ± 2.0 1643 ± 688 1858 ± 705

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p n/a 1.00 0.443 0.015 0.038 0.252 0.258 0.500 0.385 0.083 0.212 0.225 0.196 0.370 0.052 0.203 0.125

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Male 16 70.8 ± 15.7 0.211 ± 0.041 0.264 ± 0.059 175.8 ± 27.6 472.8 ± 18.1 0.117 ± 0.033 100.4 ± 24.7 478.4 ± 14.5 1.29 ± 0.10 0.74 ± 0.10 1.44 ± 0.21 2.56 ± 0.45 5.14 ± 1.99 19.0 ± 1.8 2194 ± 704 2768 ± 888

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n Age (years) Integral BMD (g/cc) Ep.BV/TV (-) Ep.BMD (mgHA/cc) Ep.TMD (mgHA/cc) Sub.BV/TV (-) Sub.BMD (mgHA/cc) Sub.TMD (mgHA/cc) DA (-) Tb.Sp* (mm) Tb.N* (1/mm) SMI (-) ConnD (1/mm3 ) Vertebra Height (mm) Yield Strength (N)§ Ultimate Strength (N) §

Female 12 71.8 ± 12.5 0.197 ± 0.052 0.212 ± 0.043 152.4 ± 27.5 480.7 ± 17.0 0.104 ± 0.023 94.8 ± 18.0 483.8 ± 16.9 1.23 ± 0.72 0.81 ± 0.16 1.33 ± 0.22 2.77 ± 0.40 4.34 ± 2.45 17.7 ± 1.0 1698 ± 675 1995 ± 864

L1 Male 12 79.6 ± 8.5 0.142 ± 0.020 0.225 ± 0.038 150.6 ± 19.4 475.7 ± 18.7 0.070 ± 0.016 56.8 ± 11.3 521.3 ± 27.4 1.55 ± 0.17 1.00 ± 0.37 1.09 ± 0.30 2.68 ± 0.27 2.37 ± 1.23 23.5 ± 2.6 2260 ± 331 2394 ± 378

p n/a 0.242 0.393 0.257 0.374 0.020 0.242 0.959 <0.001 0.625* 0.668 0.576 0.650 0.752 0.043 0.045 0.172*

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* Comparison between sexes performed with Wilcoxon rank sum test rather than t-test § The sample sizes for yield strength (n = 7, 7, 11, 10 for Female T8, Male T8, Female L1, Male L1, respectively) and ultimate strength (n = 7, 7, 10, 9 for Female T8, Male T8, Female L1, Male L1, respectively) are smaller than those listed in the first row of the table, because half of the T8 specimens were not tested in compression, three of the L1 spine segments exhibited failure in the adjacent level before the L1 yielded, and because due to operator error the tests for two of the L1 specimens were halted following the yield point but prior to the ultimate point.

Journal Pre-proof Table 2. Coefficients of variation from multiple linear regression of vertebral compressive strength (yield force or ultimate force) against the independent variables listed for each of Models 1-4: The p-values correspond to restricted-vs-full F-tests that test the null hypothesis that the first independent variable listed for the model does not explain more of the variation in strength than integral BMD×CSA alone (i.e., that there is no difference between the given model and Model 5).

0.033

0.522

0.024

0.455

0.002

0.459

0.002

0.532

0.018

0.523

0.019

0.366

0.016

0.442

0.003

0.512

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0.032

0.513

0.031

0.343

0.026

0.495

<0.001

0.474

0.094

0.539

0.014

0.197

n/a

0.197

n/a

0.411

n/a

0.411

n/a

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0.510

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integral BMD×CSA

0.003

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Model 5

0.444

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Model 4

0.014

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Model 3

Ultimate Force Central Whole R2 p R2 p

0.374

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Model 2

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Model 1

Independent Variables Ep.BV/TV×Ep.CSA, integral BMD×CSA Ep.BMD×Ep.CSA, integral BMD×CSA Sub.BV/TV×Sub.CSA, integral BMD×CSA Sub.BMD×Sub.CSA, integral BMD×CSA

Yield Force Central Whole R2 p R2 p

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Figure 1. Definition and microstructural characterization of the endplate region: From the

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micro-computed tomography image of each vertebra, the superior-most 2-mm-tall layer and the 5-mm-tall layer immediately underneath were isolated. Note that the superior and inferior

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boundaries of each of these layers were not planar but rather followed the superior contour of the

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vertebral endplate. For the 2-mm-tall layer (“Ep”), the bone volume fraction (Ep.BV/TV), apparent bone mineral density (Ep.BMD), and tissue mineral density (Ep.TMD) were quantified.

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For the 5-mm-tall layer (“Sub”) these three density measures (Sub.BV/TV, Sub.BMD,

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Sub.TMD) as well as the trabecular architecture were quantified.

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Journal Pre-proof

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Figure 2. (A) The bone volume fraction of the superior-most 2 mm of the vertebra (Ep.BV/TV)

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is moderately correlated with integral BMD in male vertebrae (r=0.654, p<0.001) and not correlated with integral BMD in female vertebrae (r=0.157, p=0.455). In contrast, (B) no sexspecificity (p=0.946) was observed in the association (r=0.686, p<0.001) between the bone volume fraction of the subchondral trabecular bone (Sub.BV/TV) and integral BMD. The lack of correlation in (A) for the female vertebrae remains when the specimen with the lowest value of Ep.BV/TV (outlier) is excluded.

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Figure 3. Measured ultimate force plotted against the value of ultimate force predicted by (A)

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integral BMDxCSA (Model 5, Table 2), (B) Central Ep.BV/TV*Ep.CSA and integral

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BMDxCSA (Model 1, Table 2), and (C) Central Sub.BV/TV*Sub.CSA and integral BMDxCSA

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(Model 3, Table 2). Whereas a trend towards a sex-specific association was seen (A) between strength and integral BMD×CSA (p=0.063), the inclusion of (B) Ep.BV/TV×Ep.CSA, or (C)

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Sub.BV/TV×Sub.CSA to the regression model eliminated any sex-specificity in the strength

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prediction. The R2-value and associated p-value for each of the regression fits shown in the plots

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are provided next to the line of best fit.