Colin R. Dunstan

Effect of aluminum on normal and uremic rats: tissue distribution, vitamin D metabolites, and quantitative bone histology.

SummaryThe effects of intraperitoneal aluminum chloride (1.5 mg aluminum/kg/day for 9 weeks) were studied in normal and uremic rats. Parameters measured included tissue aluminum, serum vitamin D metabolites, and quantitative bone histology.Aluminum administration increased tissue concentrations of this metal in uremic and nonuremic animals. Bone aluminum concentrations were higher in uremic rats (121 ± 27 mg/kg compared to 47 ± 4), whereas liver values were higher in the nonuremic group (175 ± 47 mg/kg compared to 100 ± 36). Serum concentrations of 25-hydroxyvitamin D and 24,25-dihydroxyvitamin D were reduced in uremia, but aluminum was without apparent effect on any vitamin D metabolite.Aluminum, in the doses administered, caused no skeletal changes in nonuremic animals. Some uremic, non-aluminum-treated rats developed osteomalacia and marrow fibrosis. However, osteomalacia was more severe and the osteoclast count was higher in the uremic, aluminum-treated rats. In this group of animals the mineral apposition rate was reduced at the metaphyseal endosteum but increased at the periosteum, indicating different control mechanisms at the two sites.

Osteocyte death and hip fracture

SummaryThe viability of osteocytes can be demonstrated in sawn decalcified sections of bone by their lactate dehydrogenase activity. In the cancellous bone of the femoral head, the proportion of lacunae containing viable osteocytes decreased from 88 ± 7% (mean ± SD) at 10–29 years to 58 ±12% (P < 0.001) by 70–89 years. Viability in the second lumbar vertebra was 88 ± 3% in subjects aged 25–90 years and did not decrease with age. Mean osteocyte viability in the femoral head of 21 hip fracture patients aged 72–94 years was 58 ± 21%, similar to controls of a similar age, though there was greater variation and, in five patients, osteocyte viability was less than 25%. In hip, fracture patients, microfracture callus incidence correlated positively with osteocyte viability, with little or no fracture callus observed if the bone viability was low. Ultimate compressive strength did not correlate with osteocyte viability. In the femoral head there is gradual, age-related reduction in osteocyte viability that can be more pronounced in hip fracture. Osteocyte death may affect bone quality by impairing repair of fatigue damage.

The effect of age on bone composition and viability in the femoral head.

We studied the effect of age on bone composition and osteocyte viability in femoral heads from fifty-one subjects. The assessment included determination of: bone volume, ash weight, calcium, and phosphorus content; osteocyte viability in fresh sections stained for lactate dehydrogenase activity; microfractures in fresh sections after removal of marrow elements; bone area, the presence of metabolic bone disease, and the histology of microfractures in embedded calcified sections; and the extent of trabecular microfractures. Bone area and numbers of microfractures were also assessed in eight elderly hip-fracture patients. Bone volume decreased with age, but there was considerable variation in each age group, and no significant difference between men and women. Ash weight and the bone content of calcium and phosphorus also decreased with age, but were constant if corrected for bone volume. Almost all osteocytes were viable in subjects who were younger than twenty-five years, and thereafter viability progressively decreased to a mean of 74 per cent in the eighth decade of life. There was a significant negative correlation between osteocyte viability and age. There was no evidence of metabolic bone disease in any patient. The numbers of microfractures increased with age and correlated negatively with bone viability (r = -0.31, p less than 0.05); in simple linear correlation a relationship between bone area and microfractures could not be demonstrated but in multiple linear correlation, after the inclusion of bone viability, there was an additional negative correlation between numbers of microfractures and bone area (p less than 0.005). Bone area and numbers of microfractures in hip-fracture patients were similar to those in age-matched controls.

Bone death in hip fracture in the elderly

SummaryWe examined femoral head bone from 50 cadavers and from 21 patients who had suffered pathologic fracture of the femoral neck. We used a histochemical technique for lactate dehydrogenase (LDH) activity to demonstrate osteocyte viability. The femoral heads were removed within 36 hours of death or fracture, as LDH activity persists in the cytoplasm of viable cells for this time at 37° after interruption of the blood supply. In the controls, there was an age-related reduction in mean osteocyte viability, from 88±7% (mean±SD) at age 10–29 years to 58±12% at age 70–89 years. In the hip fracture patients, mean osteocyte viability was 58±21% but there was much variability in both osteocyte viability and bone mass. In 5 fracture patients, there was extensive osteocyte death, suggesting that most of the femoral head bone was nonviable; these patients had little microfracture callus. Others had predominantly viable bone which was usually osteoporotic, and their bone frequently showed microfracture callus. Osteomalacia was not seen in any patient. It is suggested that bone death, in addition to osteoporosis, may sometimes contribute to hip fracture in the elderly.

Effect of aluminum and parathyroid hormone on osteoblasts and bone mineralization in chronic renal failure

SummaryBone aluminum, quantitative bone histology, and plasma parathyroid hormone (PTH) were compared in 29 patients undergoing chronic hemodialysis. Histologic techniques included double tetracycline labeling and histochemical identification of osteoclasts and osteoblasts. Bone aluminum was measured chemically by flameless atomic absorption spectrophotometry, and histochemically. When measured chemically, the bone aluminum was 67±46 (SD) mg/kg dry weight (normal 2.4±1.2 mg/kg); histochemically, aluminum was present at 2.9±4.4% of trabecular surface. The biochemical and histochemical results agreed well (r=0.80,P<0.001). No double tetracycline labels were seen at the mineralization front where aluminum was deposited, indicating cessation of mineralization at these sites. The osteoblast surface correlated positively with plasma PTH (r=0.67,P<0.001) and negatively with bone aluminum level (r=−0.42,P<0.05). Multiple linear regression showed a correlation of aluminum with osteoblasts additional to that of PTH, consistent with a direct effect of aluminum in depressing osteoblast numbers. Though a relationship between PTH and chemically determined bone aluminum level could not be demonstrated, there was a negative correlation between osteoclast count and aluminum, and the nine patients with severe hyperparathyroid bone disease had lower chemically determined aluminum levels than the other patients. These results suggest that aluminum (a) directly inhibits mineralization, (b) is associated with decreased PTH activity and hence osteoblast numbers, and (c) directly reduces osteoblast numbers. In addition to inducing severe, resistant osteomalacia, aluminum appears to contribute to the mild osteomalacia commonly seen in renal failure, characterized by extensive thin osteoid and low tetracycline and osteoblast surfaces.

Bone death in transient regional osteoporosis

A 48-year-old man developed transient regional osteoporosis, with hip and later knee pain. He responded well to lumbar sympathectomy. The femur and tibia adjacent to the painful knee were osteoporotic, while the medial femoral condyle showed increased uptake in a bone scan. In the femoral condyle, bone histology showed areas of dead bone undergoing osteoclastic resorption, and increased bone formation. The tibial bone was histologically normal. The partial bone death in the distal femur suggests that the disorder may be related to both avascular necrosis of bone and reflex sympathetic dystrophy.

The Determination of Bone Viability: A Histochemical Method for Identification of Lactate Dehydrogenase Activity in Osteocytes in Fresh Calcified and Decalcified Sections of Human Bone

Summary We have developed a technique for assessing bone viability by the histochemical demonstration of lactate dehydro‐genase (LDH) activity in osteocytes. Fresh sawn and ground (75 mU) sections were prepared from femoral heads removed at operation from patients with osteoarthritis of the hip. the sections were decalcified overnight in cold 10% EDTA, pH 7.0. LDH activity was shown by the tetrazolium‐formazan reaction with nitroblue tetrazolium as indicator and lithium lactate as substrate. Osteocytes were regarded as viable if their cytoplasm stained dark blue, indicating LDH activity; lacunae containing non‐viable osteocytes could be identified by interference contrast illumination. Nearly all osteocytes were viable in the samples studied. Small trabecular fragments, such as could be obtained by needle biopsy, were also suitable for staining after grinding to approximately 50 p. the method should have application both in research and in diagnosis of ischemic bone disease.

Quantitative bone histology: A new method

&NA; A method is described for quantitative histology of undecalcified bone using an embedding medium suitable for histochemistry. Six normal subjects were given tetracycline markers 24 and 3 d prior to the biopsy which was taken from the posterior iliac spine using an 8 gauge Jamshidi needle. The tissue was embedded in a mixture of equal parts methylmethacrylate and hydroxyethylmethacrylate. Three consecutive sections were cut and (1) stained histochemically for acid phosphatase activity to identify osteoclast cytoplasm, (2) stained with pyronin to identify osteoblast cytoplasm, (3) mounted unstained for tetracycline fluorescence. Tracings of the 3 sections were superimposed using a camera lucida attachment to the microscope, and the tracings quantitated with an electronic digitizer. The major parameters were bone area (17.1 ± 4.4%) (mean ± SD), osteoblast or forming surface (5.9 ± 2.7%) and osteoclast or resorbing surface (1.0 ± 0.3%). Bone apposition rate was 1.0 ± 0.2 μm/dayand resorption velocity (calculated by assuming bone formation and resorption to be equal) 8.4 ± 4.1 μm/day. Mineralization was assessed by osteoid area (3.8 ± 2.5%), osteoid surface (13.9 ± 6.6%), tetracycline uptake atthemineralizingfront(62.3 ± 13.5%), and mineralization lag time (18.1 ± 2.5 days).

The Receptor Activator of Nuclear Factor-κB Ligand Inhibitor Osteoprotegerin Is a Bone-Protective Agent in a Rat Model of Chronic Renal Insufficiency and Hyperparathyroidism

Osteoprotegerin (OPG) acts by neutralizing the receptor activator of nuclear factor-κB ligand (RANKL), the primary mediator of osteoclast differentiation, function, and survival. We examined whether OPG could affect the bone loss associated with chronic kidney disease (CKD) in a rodent model of CKD and secondary hyperparathyroidism (SHPT). SHPT was induced in rats by 5/6 nephrectomy (5/6 Nx) and a 1.2% P/0.6% Ca2+ diet. Starting 1 week after 5/6 Nx, rats were treated with vehicle (veh) or OPG-Fc (3 mg/kg, intravenously) every 2 weeks for 9 weeks. At baseline, 3, 6, and 9 weeks, blood was taken and bone mineral density (BMD) and bone mineral content (BMC) were assessed by dual-energy X-ray absorptiometry. Serum parathyroid hormone (sPTH) levels reached 912 pg/ml in 5/6 Nx rats vs. 97 pg/ml in shams at 9 weeks. OPG-Fc had no effect on sPTH or Ca2+ levels throughout the 9-week study, indicating that SHPT was a renal effect independent of bone changes. At 3 weeks, 5/6 Nx-veh rats had osteopenia compared with sham-veh rats and 5/6 Nx-OPG-Fc rats had significantly higher percent changes in whole-body BMC, leg BMD, and lumbar BMD versus 5/6 Nx-veh rats. By 6–9 weeks, elevated sPTH was associated with reversal of bone loss and osteitis fibrosa in the proximal tibial metaphysis. OPG-Fc decreased this sPTH-driven high bone turnover, resulting in augmented thickness of proximal tibial trabeculae in 5/6 Nx rats. Thus, RANKL inhibition with OPG-Fc can block the deleterious effects of continuously elevated sPTH on bone, suggesting that RANKL may be an important therapeutic target for protecting bone in patients with CKD and SHPT.

The Effect of Long-Term Low-Dose Diphosphonate Treatment on Rat Bone

Weaning, male rats were given ethane-1-hydroxy 1,1-diphosphonate (EHDP) or dichloromethylene diphosphonate (Cl2MDP) 0.5 mgP/kg/day for 140 days. Samples prepared after sacrifice were: (1) thin ground (30 micrometers) transverse tibial sections, (2) methacrylate-embedded 5 micrometers sections of the proximal tibial metaphysis, and (3) ultrathin calcified metaphyseal sections for electron microscopy. Cl2MDP reduced the diaphyseal medullary cavity, and EHDP increased the osteoid width. Both drugs increased endosteal bone apposition (possible secondarily to the impaired resorption) and, perhaps as a result, periosteal apposition was increased. Consequently, diaphyseal bone area was unchanged. Metaphyseal bone area was increased and osteoclast numbers reduced, in contrast to the increased osteoclast numbers reported previously in animal experiments with diphosphonates. Acid phosphatase activity within osteoclasts was markedly reduced with EDHP and unchanged with Cl2MDP. The ultrastructural changes in osteoclasts, i.e., a deficiency of ruffled borders, reduced numbers of cytoplasmic vacuoles and the presence of crystals between bone and the osteoclastic plasmalemma, were more marked with EHDP than Cl2MDP.