Mechanism of bone loss after liver transplantation: A histomorphometric analysis.

Organ transplantation is associated with increased bone loss and high fracture risk, but the pathophysiological mechanisms responsible have not been established. We have performed a histomorphometric analysis of bone remodeling before and 3 months after liver transplantation in 21 patients (14 male, 7 female) aged 38-68 years with chronic liver disease. Eight-micrometer undecalcified sections of trans-iliac biopsies were assessed using image analysis. Preoperatively, bone turnover was low with a tendency toward reduced wall width and erosion depth. The bone formation rate increased from 0.021 +/- 0.016 (mean +/- SD) to 0.067 +/- 0.055 microm2/microm/day after transplantation (p < 0.0002) and activation frequency from 0.24 +/- 0.21/year-1 to 0.81 +/- 0. 67/year-1 (p < 0.0001). No significant change was observed in wall width, but there was a trend toward an increase in indices of resorption cavity size. There was a small increase in osteoid seam width postoperatively (p< 0.02) and decrease in mineralization lag time (p < 0.001). No significant changes in indices of cancellous bone structure were observed in the postoperative biopsies. These results demonstrate a highly significant and quantitatively large increase in bone turnover in the first 3 months after liver transplantation. Although no significant disruption of cancellous bone structure was demonstrated during the time course of the study, the observed changes in bone remodeling predispose to trabecular penetration and may thus result in long-term adverse effects on bone strength.

Effects of hormone replacement therapy on cancellous bone microstructure in postmenopausal women.

Menopausal bone loss is associated with disruption of cancellous bone architecture which has adverse mechanical effects and is believed to be irreversible. The aim of this study was to examine the effects of long-term hormone replacement therapy on cancellous bone structure in women with postmenopausal osteoporosis. Iliac crest biopsies from 22 women with osteopenia or osteoporosis were obtained before and after hormone replacement therapy (mean duration 23.5 months). Cancellous bone architecture was assessed by strut analysis, trabecular bone pattern factor, and marrow star volume. Post-treatment biopsies showed no significant changes in any of the structural indices assessed. Our results suggest that hormone replacement therapy preserves existing cancellous bone structure but provide no evidence that this treatment is able to reverse structural disruption in women with postmenopausal osteopenia or osteoporosis.

Assessment of cancellous bone structure: comparison of strut analysis, trabecular bone pattern factor, and marrow space star volume.

Cancellous bone architecture is an important determinant of bone strength. Recently, several approaches to the assessment of bone structure in histological sections have been described; however, no comparative studies of these different methods have been reported. We have developed computerized methods for the simultaneous assessment of several indices of bone structure, including trabecular bone pattern factor, marrow space star volume, node-to-terminus ratio, trabecular number, and trabecular separation. The relationships between these variables has been examined in iliac crest cancellous bone obtained from 41 healthy subjects, 17 male and 24 female, aged 20-80 years (mean 47.9 years). All structural indices assessed showed significant correlations with cancellous bone area (p < 0.0001). Values for trabecular bone pattern factor and marrow space volume were highly correlated (r = 0.789; p < 0.0001). A comparison of indices obtained by strut analysis with trabecular bone pattern factor and marrow space star volume also revealed significant relationships, especially for the terminus-to-terminus strut length (r = 0.704 and r = 0.634, respectively; p < 0.0001) and node to terminus ratio (r = -0.947 and r = -0.788, respectively; p < 0.0001). The node-to-terminus ratio and trabecular bone pattern factor showed significant relationships with age which were independent of sex, cancellous bone area and trabecular width (p < 0.01 and p < 0.005, respectively). Our results demonstrate strong correlations between the different two-dimensional indices of bone structure in cancellous bone from healthy subjects. Trabecular penetration is likely to be an important determinant of all these variables, which may therefore reflect connectivity; however, direct comparison of these methods with three-dimensional techniques is required to establish their true relationship to bone structure.

The effects of gonadotrophin-releasing hormone agonists on iliac crest cancellous bone structure in women with endometriosis.

The administration of gonadotrophin-releasing hormone (GnRH) analogs to premenopausal women causes hypoestrogenism and bone loss, but the effects on cancellous microstructure have not been determined. In this study we have assessed bone structure in transiliac biopsies obtained from women before and after treatment for endometriosis with GnRH analogs. Twenty-one premenopausal women were studied, paired biopsies being obtained in 13; five women received both GnRH analogs and Org OD 14 (Tibolone, Livial). Comparison of pre- and post-treatment biopsies in women treated only with GnRH analogs showed a reduction in indices related to connectivity (node-to-terminus ratio, node-to-loop strut length, p < 0.02) and increase in inversely related indices (terminus-to-terminus and node-to-terminus strut length, p < 0.03). No significant changes were seen in any of the structural indices in women receiving both GnRH and Org OD 14 therapy. Activation frequency and bone formation rate at tissue level increased in women treated with GnRH agonists alone, although this change was not statistically significant. Our results suggest that bone loss induced by GnRH analogs may be associated with adverse effects on cancellous microstructure which are unlikely to be reversed following cessation of therapy. Concurrent treatment with Org OD 14 appears to prevent these changes.

Structural mechanisms of trabecular bone loss in primary osteoporosis: specific disease mechanism or early ageing?

Osteoporosis is characterised by reduced bone mass and disruption of cancellous bone architecture; however, it is unknown whether these changes arise from a specific disease process or represent one extreme of physiological bone loss. We have quantitatively assessed cancellous structure in 35 patients with primary osteoporosis and 41 normal subjects. Cancellous microstructure was assessed by computerised strut analysis and by calculation of trabecular width, separation and number. Node to terminus ratio, node to node and node to loop strut length were significantly decreased in patients with osteoporosis when compared to normal subjects (P < 0.001), whereas terminus count and terminus to terminus strut length were significantly increased (P < 0.001). When two subgroups were matched for age these differences remained highly significant (P < 0.005). However, when two subgroups were matched for cancellous area, no significant differences were observed in any of the structural indices except terminus count (P < 0.05). Mean trabecular width and number were significantly lower and trabecular separation significantly higher in the patients with osteoporosis before and after age-matching but their differences disappeared after matching for cancellous area. Multiple regression analysis confirmed highly significant correlations between cancellous bone area and structural indices after adjustment for age, sex and disease status (P < 0.001). Our data demonstrate that for a given cancellous area, structural changes in primary osteoporosis are similar to those observed during age-related bone loss in normal subjects. These findings support the hypothesis that primary osteoporosis is the result of greater biological ageing rather than a specific disease process and are consistent with evidence from other sources that low bone mass is associated with increased mortality from diseases unrelated to osteoporosis.

Bone turnover in non-steroid treated rheumatoid arthritis.

OBJECTIVE: To examine whether changes in cancellous bone turnover and resorption cavity depth contribute to bone loss in patients with non-steroid treated rheumatoid arthritis. METHODS: Iliac crest biopsies were obtained from 37 patients with non-steroid treated rheumatoid arthritis, 13 male and 24 female, aged 37-71 years. Bone turnover and resorption cavity characteristics were quantitatively assessed using semiautomated computerised techniques. RESULTS: When compared with age- and sex-matched control values, there was a significant reduction in bone formation rate at tissue level and activation frequency (P < 0.001) in the patient group. The eroded perimeter, mean and maximum eroded depth and cavity area were also significantly reduced (P < 0.01, < 0.005, < 0.01 and < 0.005 respectively). CONCLUSION: These results demonstrate low bone turnover in non-steroid treated rheumatoid arthritis and indicate that the reduced bone mass in these patients is due mainly to a negative remodelling balance.

Effect of long-term tamoxifen therapy on cancellous bone remodeling and structure in women with breast cancer.

The effects of long-term tamoxifen therapy on bone remodeling were studied in 41 women with breast cancer, 22 treated with tamoxifen for a minimum of 15 months (mean 33) and 19 untreated. Transiliac crest bone biopsies were obtained and a comprehensive histomorphometric analysis performed using a semiautomatic image analysis system. There were no statistically significant differences between the two groups in bone area, osteoid perimeter and area, or osteoid width. Mineral appositional rate, adjusted appositional rate, and mineralization lag time were also similar in the two groups; however, tissue-based bone formation rate was significantly lower in the tamoxifen-treated women (p = 0.05) and the remodeling period significantly longer (p < 0.05). Mean and maximum resorption cavity depth and cavity area were significantly reduced in the tamoxifen-treated patients compared to the untreated patients (p < 0.01, p < 0.01, and p < 0.03, respectively). Calculated and directly measured indices of cancellous bone structure were similar in the two groups, although the data indicated a trend toward greater connectedness in the tamoxifen-treated group. These data indicate that tamoxifen does not exert an antiestrogenic effect on bone remodeling in the human and are consistent with a weak estrogenic effect.

Reduced bone formation in patients with osteoporosis associated with inflammatory bowel disease.

The pathophysiology of bone loss associated with inflammatory bowel disease has not been clearly defined. In this study we have performed a detailed histomorphometric analysis of iliac crest bone obtained from 19 patients with inflammatory bowel disease in whom a diagnosis of osteoporosis had been made. Eleven subjects were receiving prednisolone at the time of their biopsy. Comparison with control values demonstrated a highly significant reduction in trabecular bone area in the patient group (p < 0.001). Wall width, adjusted appositional rate and bone formation rate were all significantly reduced in the patient group (p < 0.001) and the formation period was significantly increased (p < 0.001). Resorption cavities were slightly smaller in the patient group, differences in maximum cavity depth and cavity length achieving statistical significance (p < 0.005 and p < 0.05 respectively). The mineral appositional rate was significantly reduced in the patients with inflammatory bowel disease (p < 0.001) and the mineralization lag time significantly increased (p < 0.001); however, osteoid area, perimeter and seam width were not significantly different from controls. These results demonstrate that osteoporosis associated with inflammatory bowel disease is characterized by reduced bone formation at the cellular and tissue level; the proportionately greater change in wall width than in resorption cavity depth is consistent with a negative remodelling balance. Although none of the patients had osteomalacia as defined by the criteria of increased osteoid seam width and mineralization lag time, the higher mineralization lag time in the patient group indicates a mild mineralization defect.

Characteristics of trabecular bone resorption cavities in patients with chronic renal failure.

Using a computerised technique, resorption cavity characteristics in iliac crest trabecular bone were assessed in 30 patients with chronic renal failure and compared with data obtained from healthy subjects. The mean and maximum cavity depth were significantly greater in the patient group (P less than 0.0001); in addition, cavity area, the percentage of bone being remodelled, the number of cavities per mm trabecular surface and the percentage eroded surface were all significantly greater than in controls (P less than 0.0001). However, the surface length of individual cavities in the patient group did not differ significantly from that of controls. In the patient group, serum intact parathyroid hormone concentrations showed a significant positive correlation with mean resorption cavity depth (r = 0.451, P less than 0.05). Our results demonstrate that the increase in bone resorption associated with hyperparathyroidism secondary to chronic renal failure is due to an increase both in the number and depth of cavities, although the surface extent of individual cavities is normal. These findings indicate that factors determining the length of trabecular surface eroded and the depth of individual resorption cavities are controlled by different mechanisms.

Combined inter-observer and inter-method variation in bone histomorphometry.

Manual methods for the measurement of bone biopsies have largely been superseded by semi-automatic computerised techniques. Histomorphometrists often use control data obtained by other observers using different methods, thus combining inter-observer and inter-method variation. We have examined the combined effect of inter-method and inter-observer variation on measurements of bone area, osteoid perimeter, and osteoid width in iliac crest biopsies from healthy subjects, one observer using the manual grid system and the other using a semi-automated technique. Inter-observer and inter-method variation were independently determined, and the proportion of each expressed as a percentage of combined error. Our results indicate that the combination of inter-method and inter-observer variation causes significant differences in the values obtained for osteoid perimeter, whereas inter-method variation is mainly responsible for differences in osteoid width values; differences in bone area are largely due to inherent sampling variation. These variations indicate that caution is required when comparison is made with control data from other sources, especially if different techniques are employed.

Age-related changes in resorption cavity characteristics in human trabecular bone.

The depth of resorption cavities in trabecular bone is an important determinant of bone structure and has implications relevant to the cellular pathophysiology of bone loss in osteoporosis. However, assessment of resorption depth has proved difficult and few data are available; in this study we report age-related changes in iliac crest trabecular bone obtained from 41 normal healthy subjects (21 female, 20 male) aged 22-80 years. Using 8-microns undecalcified sections stained with toluidine blue, resorption cavities were quantitatively assessed by a computerized technique in which the eroded bone surface is reconstructed and measurements made interactively. Maximum and mean cavity depth showed no significant correlation with age in either sex. The absolute length of eroded surface was unrelated to age but the eroded surface/BS (%) and the number of cavities/BS (/mm) showed a significant positive correlation with age (r = 0.384 and 0.386 respectively, p less than 0.05). No significant correlation was found between age and either cavity area or density. These results suggest that increased resorption depth does not contribute to age-related bone loss, although the possibility that deeper resorption cavities occur which result in trabecular penetration and are therefore unrecognizable cannot be discounted. The age-related increase in eroded surface/BS (%) reflects a decreased available trabecular surface and/or increased number of cavities rather than a greater surface length of individual cavities; alternatively it may indicate an increased resorption period. No evidence of increased resorption depth at the time of the menopause was found in this study.

The relationship between spinal trabecular bone mineral content and iliac crest trabecular bone volume.

The relationship between spinal trabecular bone mineral density and iliac crest trabecular bone volume has been studied in 84 patients, 23 with primary osteoporosis, 19 with osteoporosis secondary to inflammatory bowel disease, and 42 with nonsteroid-treated rheumatoid arthritis. Spinal trabecular bone mineral density was measured in the first three lumbar vertebrae by quantitative computed tomography, and iliac crest trabecular bone volume was assessed histomorphometrically in sections from trans-iliac biopsies using computerized techniques. In all 84 patients, there was a significant positive correlation between the two measurements (r = 0.60, P less than 0.001). However, when the three patient groups were analyzed separately, a significant correlation was found in the group with secondary osteoporosis (r = 0.65, P less than 0.01) but not in the patients with primary osteoporosis (r = 0.07) or rheumatoid arthritis (r = 0.19). These results indicate that the relationship between spinal trabecular bone mineral density and iliac crest trabecular bone volume differs according to the underlying disease process, these differences possibly reflecting variations in skeletal patterns of bone loss in different types of osteoporosis.

A computerised technique for the quantitative assessment of resorption cavities in trabecular bone.

A computerised technique is described for the quantitative assessment of resorption cavities in iliac trabecular bone. Using an Ibas II image analyser, the original bone surface, eroded by bone resorbing cells, is reconstructed using a curve fitting technique that maintains a smooth continuity with the trabecular bone on either side of the cavity. Resorption depths are measured using an interactive elastic circle; all identified cavities are measured regardless of whether or not resorption is complete, and the measurements made include mean and maximum cavity depth, cavity length, area, and adjacent trabecular widths. Results in 13 normal subjects are presented. The technique is reproducible, simple to operate, relatively rapid, and can be applied to less sophisticated image analysis systems.

The relationship between resorption depth and mean interstitial bone thickness: age-related changes in man.

The amounts of bone resorbed and formed in each bone remodelling unit are important determinants of bone mass. The mean wall thickness (MWT), which indicates the amount formed, decreases with age; less is known about changes in resorption depth although two studies have reported some evidence in favor of an age-related decrease. We have calculated mean intersitial bone thickness (MIBT), which is indirectly related to resorption depth, from measurements of MWT and mean trabecular plate thickness (MTPT) in 48 normal subjects, aged 19-80 years. No significant age-related changes in MIBT were found in either sex, or in both sexes grouped together, whereas MWT and MTPT showed a significant decrease with age (P less than 0.001 and less than 0.05 respectively). Detailed analysis of possible relationships between MIBT and resorption depth revealed that, assuming formation never exceeds resorption, a decreased MIBT may be associated with an increased, constant, or decreased resorption depth; an increased MIBT can only result from a decreased resorption depth. A constant MIBT may indicate either an unchanged or decreased resorption depth; in the former case, MTPT is unchanged whereas a decreased resorption depth is associated with a decreased MTPT. In view of the age-related reduction in MTPT, our results are therefore consistent with earlier reports of an age-related decrease in resorption depth. No evidence for an increased resorption depth during the menopause was found in this study.

Age-related changes in trabecular width and spacing in human iliac crest biopsies.

Age-related changes in trabecular width and spacing have been examined in iliac crest bone obtained from 96 normal subjects. The mean trabecular plate density and separation were calculated from values of trabecular bone volume and mean trabecular plate thickness. There was an age-related decrease in mean trabecular plate thickness with age in both sexes; this was statistically significant in males (P less than 0.02) but not in females. In females, there was a significant decrease in mean trabecular plate density (P less than 0.001) and increase in mean trabecular plate separation (P less than 0.001); similar but non-significant trends were observed in the males. These results demonstrate that trabecular thinning contributes to age-related bone loss in both sexes. Loss of trabeculae also occurs in both sexes, but to a greater extent in females than in males.

Structural mechanisms of trabecular bone loss in man.

The relationship between trabecular thinning and loss of connectedness of the trabecular bone pattern has been studied in iliac crest bone samples from 89 normal subjects in order to determine the structural mechanisms underlying age-related bone loss. Trabecular width and structure were quantitatively assessed using computerized techniques. Highly significant negative correlations were found between the mean trabecular plate thickness and number of free ends/mm2 both in males (r = -0.571) and in females (r = -0.667) (P less than 0.001). Mean trabecular plate thickness also showed significant negative correlations with other structural indices indicating reduced connectedness, whereas positive correlations were found with those indices representing preservation of connectedness. Examination of the relative frequency of trabecular widths less than 100 microns revealed that only 2-5% of the trabecular surface would be susceptible to erosion by a resorption cavity of normal depth. These results indicate that trabecular thinning and erosion are interdependent processes in age-related bone loss. Since only a small percentage of the trabecular surface is susceptible to erosion, and resorption cavities normally occupy only 1-5% of the total trabecular surface, these findings imply that the site of activation of new BMUs may not be randomly distributed but may instead be preferentially located at sites of lower trabecular width.