E.N. Ebbesen

Adipocyte tissue volume in bone marrow is increased with aging and in patients with osteoporosis

Aging of the human skeleton is characterized by decreased boneformation and bone mass and these changes are more pronounced inpatients with osteoporosis. As osteoblasts and adipocytes share a commonprecursor cell in the bone marrow, we hypothesized that decreased boneformation observed during aging and in patients with osteoporosis is theresult of enhanced adipognesis versus osteoblastogenesis from precursorcells in the bone marrow. Thus, we examined iliac crest bone biopsiesobtained from 53 healthy normal individuals (age 30–100) and 26patients with osteoporosis (age 52–92). Adipose tissue volumefraction (AV), hematopoietic tissue volume fraction (HV) and trabecularbone volume fraction (BV) were quantitated as a percentage of totaltissue volume fraction (TV) (calculated as BV + AV + HV) usingthe point-counting method. We found an age-related increase in AV/TV(r = 0.53, p < 0.001, n =53) and an age-related decline in BV/TV (r =−0.46, p < 0.001, n = 53) as well asin the HV/TV (r = −0.318, p <0.05, n = 53). There was an age-related inversecorrelation between BV/TV and AV/TV (r =−0.58, p < 0.001). No significant correlation betweenthe AV/TV and the body mass index (r = 0.06, n.s.,n = 52) was detectable. Compared with age-matchedcontrols, patients with osteoporosis exhibited an increased AV/TV(P < 0.05) and decreased BV/TV (P < 0.05)but no statistically significant difference in HV/TV. Our datasupport the hypothesis that with aging and in osteoporosis an enhancedadipogenesis is observed in the bone marrow and that these changes areinversely correlated to decreased trabecular bone volume. The cellularand molecular mechanisms mediating these changes remain to bedetermined.

Age-related changes in the biochemical properties of human cancellous bone collagen: relationship to bone strength.

Abstract. The metabolism of bone collagen has received little attention in relation to age-related loss of bone mass and strength. The aim of the present study was to analyze bone collagen content and metabolism in human bone with respect to age. The material consisted of iliac crest bone biopsies from 94 individuals: 46 women (ages 18–96, mean age 60.8 years) and 48 men (ages 23–92, mean age 59.5 years). Excluded from the study were all individuals with known osteoporotic lumbar vertebral fractures and renal, hepatic, or malignant diseases. Prior to collagen analysis the biopsies were scanned in a pQCT scanner for density assessment and then tested biomechanically. The results showed a decline in apparent bone density with age (P < 0.0001), a decline in maximum stress, Youngs modulus, and energy absorption with age (P < 0.001). Concomittantly, there was an age-related decline in the intrinsic collagen content with age (P < 0.001). However, there were no biochemical modifications of the bone collagen during aging. There were no significant differences between women and men in the slopes of the regressions-curves. When multiple regression analyses were performed, only apparent bone density came out as a significant contributor in the correlation to biomechanical properties. Nevertheless, the decrease in bone collagen content with age might indicate an increase in the mineralization degree (probably due to decreased bone turnover) and thereby a change in material properties of bone. In conclusion, the present study has shown that loss of bone mass plays the major role in loss of bone strength. However, there is also a change in bone composition during normal aging, leading to a decrease in collagen content and an increase in the degree of mineralization. At this skeletal site, in a normal population there was no change in the biochemical properties of bone collagen.

Hormonal replacement therapy reduces forearm fracture incidence in recent postmenopausal women — results of the Danish Osteoporosis Prevention Study

OBJECTIVES To study the fracture reducing potential of hormonal replacement therapy (HRT) in recent postmenopausal women in a primary preventive scenario. METHODS Prospective controlled comprehensive cohort trial: 2016 healthy women aged 45-58 years, from three to 24 months past last menstrual bleeding were recruited from a random sample of the background population. Mean age was 50. 8+/-2.8 years, and the number of person years followed was 9335.3. There were two main study arms: a randomised arm (randomised to HRT; n=502, or not; n=504) and a non-randomised arm (on HRT; n=221, or not; n=789 by own choice). First line HRT was oral sequential oestradiol/norethisterone in women with intact uterus and oral continuous oestradiol in hysterectomised women. RESULTS After five years, a total of 156 fractures were sustained by 140 women. There were 51 forearm fractures in 51 women. By intention-to-treat analysis (n=2016), overall fracture risk was borderline statistically significantly reduced (RR=0.73, 95% CI: 0.50-1.05), and forearm fracture risk was significantly reduced (RR=0.45, 95% CI: 0.22-0.90) with HRT. Restricting the analysis to women who had adhered to their initial allocation of either HRT (n=395) or no HRT (n=977) showed a significant reduction in both the overall fracture risk (RR=0.61, 95% CI: 0.39-0.97) and the risk of forearm fractures (RR=0.24, 95% CI: 0.09-0.69). Compliance with HRT was 65% after five years. CONCLUSIONS It is possible to reduce the number of forearm fractures and possibly the total number of fractures in recent postmenopausal women by use of HRT as primary prevention.

Age‐ and Gender‐Related Differences in Vertebral Bone Mass, Density, and Strength

This study was designed to evaluate age‐ and gender‐related differences in vertebral bone mass, density, and strength by dual‐energy X‐ray absorptiometry (DXA), quantitative computed tomography (QCT), peripheral QCT (pQCT), ash measurements, and biomechanical testing. The material comprised human lumbar vertebral bodies (L3) from 51 females and 50 males (age‐range: 18–96 years). The results showed that females had significantly lower vertebral body bone mass (ash weight) than males at any given age. The decline in bone mass with age was parallel for females and males. The different bone density measurements—cancellous ash density, total vertebral body ash density, DXA bone mineral density, QCT, and pQCT—showed no gender‐related difference concerning numeric value or changes with age. Morphometrical measurements showed that females had smaller vertebral bodies (volumes) than males. Hence the females had significantly smaller cross‐sectional area (CSA) of L3 than males (11.6 cm2 and 14.4 cm2, respectively). This led to females having lower maximum compressive load (N) than males at all ages, whereas maximum compressive stress (load/CSA) showed no gender‐related difference. In conclusion, females have lower vertebral body bone mass than males at any given age, due to smaller vertebral bodies. Hence, maximum compressive load (strength not corrected for size) was lower in females. Vertebral body cancellous bone density and total‐vertebral body density were equal when comparing genders, and no gender differences were found in the size‐corrected strength: maximum compressive stress. The decrease with age in vertebral body compressive strength decrease was twice as large as the age decrease in density.

Age-related differences between thinning of horizontal and vertical trabeculae in human lumbar bone as assessed by a new computerized method

To investigate whether vertical trabeculae undergo compensatory thickening with age in the human vertebral body, a new computerized method was developed that is able to distinguish between horizontal and vertical trabeculae on normal histological sections. Study subjects included 48 individuals (24 women aged 19-97 years, and 24 men aged 23-95 years). From each L-2, thick frontal sections of half of the vertebra were embedded undecalcified in methylmetacrylate and cut into 10-microm-thick sections. A simple method able to classify image pixels as belonging to either vertical or horizontal trabeculae was developed and implemented in a computer program. The parallel-plate model was modified so that it was able to determine trabecular thickness, number, and separation (Tb.Th, Tb.N, and Tb.Sp) for horizontal and vertical trabeculae separately. The histomorphometric parameters were measured in three different regions: whole section, mid-third, and sub-endplate, for both horizontal and vertical trabeculae. It was found that the trabecular thickness of vertical trabeculae was independent of age in all investigated regions. The thickness of the horizontal trabeculae, in contrast, decreased significantly with age in all these regions. Tb.N decreased significantly with age for both horizontal and vertical trabeculae in all regions. However, the relative loss of trabeculae per unit length was largest for the horizontal trabeculae, whereas the absolute loss of trabeculae per unit length was largest for the vertical trabeculae. Tb.Sp was found to increase significantly with age for both vertical and horizontal trabeculae in all regions. No significant gender-related differences were found. In conclusion, in this study we describe a new, simple method for separation of horizontal and vertical bone tissue. This method has been applied on frontal vertebral sections. Trabecular bone thickness measured with the parallel-plate model showed that the thickness of horizontal trabeculae decreases significantly with age, whereas the thickness of vertical trabeculae did not decrease significantly with age. Furthermore, although the relative loss of trabeculae was larger for horizontal trabeculae than for vertical trabeculae, the absolute loss of vertical trabeculae was higher than that of horizontal trabeculae.

Cytokines and bone loss in a 5-year longitudinal study : Hormone replacement therapy suppresses serum soluble interleukin-6 receptor and increases interleukin-1-receptor antagonist : The Danish Osteoporosis Prevention Study

The proinflammatory cytokines interleukin‐1β (IL‐1β) and IL‐6 may play a central role in the acceleration of postmenopausal bone loss, but observational studies have led to contradictory results. Estrogen‐dependent changes in the production of IL‐1 receptor antagonist (IL‐1ra) and the soluble IL‐6 receptor (sIL‐6R) potentially modify cytokine bioactivity. We therefore assessed the impact of menopause and hormone replacement therapy (HRT) on cytokines and activity modifiers in serum within a 5‐year longitudinal study. One hundred sixty perimenopausal women (age 50.1 ± 2.8 years) were randomized to HRT or no treatment. Serum IL‐6 increased with age (r = 0.16; p < 0.05), but cytokines did not correlate with baseline bone mineral density (BMD). HRT led to small increases in IL‐1ra (p < 0.001) and IL‐6 (p < 0.05), with a decrease in sIL‐6R (p < 0.01) and no change in IL‐1β. No changes were observed in the control group. IL‐1ra was inversely correlated with bone loss at the ultradistal forearm (r = 0.29; p < 0.05) and to a lesser degree at the spine (r = 0.20; p = 0.09). In addition, there was a weak positive correlation between sIL‐6R and bone loss at the ultradistal forearm (r = 0.26; p < 0.05). High IL‐6 levels were associated with slower bone loss (spine r = 0.31, p < 0.01) and controlling for age did not diminish this association. The percent change in sIL‐6R during HRT was correlated with the bone loss at the femoral neck (r = −0.29; p < 0.01) and weakly with bone loss in the spine (r = −0.16; p = 0.17). In conclusion, serum IL‐1ra and sIL‐6R are influenced by HRT and are associated with the rate of bone loss in perimenopausal women.

Predicting human vertebral bone strength by vertebral static histomorphometry

The study investigates the relationship between static histomorphometry and bone strength of human lumbar vertebral bone. The ability of vertebral histomorphometry to predict vertebral bone strength was compared with that of vertebral densitometry, and also with histomorphometry and bone strength of iliac crest bone biopsies. The material comprised matched sets of second lumbar vertebrae, third lumbar vertebrae, and two iliac crest bone biopsies from each of 21 women (19--96 years) and 24 men (23--95 years). One of the iliac crest biopsies and 9-mm-thick mediolateral slices of half of each of the entire vertebral bodies (L-2) were used for histomorphometry. The other iliac crest biopsies and the L-3 were destructively tested by compression. High correlation was found between BV/TV or Tb.Sp and vertebral bone strength (absolute value of r = 0.86 in both cases). Addition of Tb.Th significantly improved the correlation between BV/TV and bone strength, and the addition of bone space star volume significantly improved the correlation between Tb.Sp and bone strength (from absolute value of r = 0.86 to absolute value of r = 0.89 in both cases). Bone structure (connectivity density) was not capable of improving the prediction of bone strength of the vertebral body. The correlations between BV/TV of L-2 and bone strength of L-3 were comparable with the correlation obtained by quantitative computed tomography (QCT), peripheral QCT (pQCT), and dual-energy X-ray absorptrometry (DEXA) of L-3 and bone strength of L-3. The iliac crest was found to have low predictive power of vertebral bone strength (iliac BV/TV: r = 0.62; iliac bone strength: r = 0.67). No gender-related differences were found in any of the relationships. It was shown that trabecular bone volume BV/TV and mean trabecular plate separation Tb.Sp are good predictors of vertebral bone strength. The ability of histomorphometry to predict vertebral bone strength was comparable to that of densitometry. Bone structure assessed by connectivity density did not improve the correlation between static histomorphometric measures and vertebral bone strength. No gender-related differences were found in any of the relationships. Neither static histomorphometry nor biomechanical testing of iliac crest bone biopsies is a good predictor of vertebral bone strength.

Zone-dependent changes in human vertebral trabecular bone: clinical implications

We have previously shown that there are pronounced age-related changes in human vertebral cancellous bone density and microarchitecture. However, the magnitude of these changes seemed to be dependent on zone location in the vertebral body-the central third vs. the areas adjacent to the endplates. The aim of the present study was, therefore, to investigate whether such zone-specific differences could be identified by static histomorphometric measures. The material comprised 48 individuals (24 women aged 19-97 years, and 24 men aged 23-95 years). Three of the women had a known fracture of the L-2. From each L-2, thick frontal sections of half of the vertebra were embedded undecalcified in methylmethacrylate, cut into 10-microm-thick sections, and stained with aniline blue. The sections were scanned into a computer, and classic static histomorphometry was performed on the images. The histomorphometry was performed on both the whole section and on the separate zones (central and sub-endplate zone). The results showed that trabecular bone volume, trabecular number, and connectivity density decreased significantly faster with age, whereas marrow space star volume increased significantly faster with age in the zones adjacent to the endplates than in the central zone. The other histomorphometric measures showed no zone specificity in relation to aging. However, trabecular thickness and trabecular separation were both higher at all ages in the central zone than in the sub-endplate zone, although this was significant only for trabecular separation. The described differences might have significant clinical implications concerning quantitative computed tomography (QCT) scanning, X-ray analyses, and assessment of fracture liability in the human spine, but the underlying pathogenesis is still not known. This study shows that the human vertebral body can be described as two distinct zones with very specific age-related changes in density and microstructure. This zone-specificity is important for the correct interpretation of clinical data.

Static histomorphometry of human iliac crest and vertebral trabecular bone: a comparative study

We recently developed a new, rapid method for conducting static histomorphometry on large histologic sections. This method has now been applied on both iliac crest and lumbar vertebral bone to compare the age-related changes at these two skeletal sites and to investigate the correlation between the histomorphometric measures at the iliac crest and the vertebral body. The material comprised matched sets of unilateral transiliac crest bone biopsies and lumbar vertebral bodies (L-2) from 24 women (19-96 years) and 24 men (23-95 years) selected from a larger autopsy material. Three female subjects (80, 88, and 90 years) had a known vertebral fracture of L-2. The iliac crest biopsies and 9-mm-thick mediolateral slices of half the entire vertebral bodies were embedded in methylmetacrylate, stained with aniline blue, and scanned into a computer with a flatbed image scanner at a high resolution. With a custom-made computer program the following static histomorphometric measures were determined: trabecular bone volume; marrow and bone space star volume; node-strut analysis; trabecular bone pattern factor; trabecular thickness; trabecular number; trabecular separation; and anisotropy of bone and marrow phase. In addition, connectivity density was measured (ConnEulor method). The results showed that the age-related changes in the static histomorphometric measures are generally similar in the iliac crest and the vertebral body, and that these age-related changes are independent of gender. An exception, however, is connectivity density, where the age-related changes are similar for women and men in the vertebral body but significantly different in the iliac crest. Furthermore, the results showed that the histomorphometric measures were weakly intercorrelated between the iliac crest and the vertebral body, despite the generally similar pattern in age-related changes at these two skeletal sites. The highest correlation coefficient was found for trabecular separation (Tb.Sp; r = 0.63). Trabecular bone volume showed a correlation coefficient of r = 0.59. It is concluded that static histomorphometry performed on one skeletal site does not automatically predict static histomorphometric measures at another skeletal site. Therefore, it is recommended that static histomorphometry be performed at the skeletal site of interest-if at all possible.

A new method of comprehensive static histomorphometry applied on human lumbar vertebral cancellous bone

The aim of the present study was to assess age-related changes in the human spine by use of established static histomorphometry, and to determine how these static histomorphometric measures are interrelated in human cancellous bone tissue. The material comprised normal human lumbar vertebral bodies (L-2) from 12 women (19-96 years) and 12 men (23-91 years) selected from a larger autopsy material to give an even age and gender distribution. In addition, L-2 from three female subjects (80, 88, and 90 years) with a known vertebral fracture of L-2 were considered. Approximately 9-mm-thick frontal (mediolateral) slices were embedded in methylmetacrylate, stained with aniline blue, and scanned into a computer with a flatbed image scanner at a high resolution (2400 dpi). With a custom-made computer program the following static histomorphometric measures were determined: trabecular bone volume; marrow space star volume; bone space star volume; anisotropy of bone and marrow phase (star length distribution method); node-strut analysis (node:terminus ratio); trabecular thickness; trabecular number; trabecular separation; and trabecular bone pattern factor. In addition, connectivity density was determined (by the ConnEulor method). All 11 histomorphometric measures, except bone space star volume and the two measures of anisotropy, showed a significant correlation with age. Marrow space star volume (r = 0.82) and trabecular bone volume (r = -0.81) showed the highest correlation with age. Furthermore, it was found that all of the histomorphometric measures were correlated, to different degrees. Trabecular bone volume correlated significantly with all ten histomorphometric measures, whereas the two anisotropy measures were poorly correlated to the other measures. Finally, we found the histomorphometric values in this study to be in excellent accordance with various previously published results from studies of human trabecular vertebral bone, the sole exception being marrow space star volume, which was probably due to the small (artificial) region of interest (ROI) that was used in the earlier studies. In conclusion, the new method applied herein allows for easy assessment of age-related changes and also for assessment of relationships between histomorphometric measures in human vertebral cancellous bone.