Age- and gender-dependent changes in three-dimensional microstructure of cortical and trabecular bone at the human femoral neck.

UNLABELLED: We investigated age- and gender-related variation of both cortical and trabecular microstructure in human femoral neck. We found that age-related change of cortical porosity is more noticeable than that of trabecular parameter. Our data may help to gain more insight into the potential mechanism of osteoporotic femoral neck fractures. INTRODUCTION: Variations in the microstructure of cortical and trabecular bone contribute to decreased bone strength. Age- and gender-related changes in cortical and trabecular microstructure of femoral neck is unclear. The aim of this study was to identify three-dimensional (3D) microstructural changes of both cortical and trabecular bone simultaneously in human femoral neck with age and gender, using micro-computed tomography (micro-CT). We hypothesized that there would be differences in age-related changes of cortical and trabecular bone for both women and men. METHODS: We used 56 femoral necks of 28 women and men (57-98 years of age) from a Japanese population. The subjects were chosen to give an even age and gender distribution. Both women and men were divided into three age groups: middle (57-68 years), old (72-82 years), and elderly (87-98 years) groups. We examined cortical bone specimen from the inferior sector of femoral neck and trabecular bone specimen from the middle of femoral neck using micro-CT and 3D bone analysis software. RESULTS: Cortical thickness (Ct.Th) decreased by 10-15%, cortical porosity (Ca.V/TV) almost doubled, and canal diameter (Ca.Dm) increased by 65-77% between the middle-aged and elderly groups for both women and men. The trabecular bone volume fraction (BV/TV) decreased by around 20%; trabecular thickness (Tb.Th), trabecular number (Tb.N), and connectivity density (Conn.D) decreased; and trabecular separation (Tb.Sp) and structure model index (SMI) increased with age for both women and men. As compared with women, men had higher Ct.Th and BV/TV and lower Ca.V/TV and Ca.Dm among three age groups. There was a significant inverse correlation between Ca.V.TV and BV/TV for both women and men. CONCLUSION: Our findings indicate that Ct.Th and BV/TV decreased, and Ca.V/TV and Ca.Dm increased in femoral neck with age for both women and men. The most obvious age-related change is the increase of Ca.V/TV. The decrease of BV/TV with age is more noticeable than that of Ct.Th. This is the first study that has provided both cortical and trabecular microstructural data simultaneously in a Japanese sample. These data may help us to gain more insight into the potential mechanism of osteoporotic femoral neck fractures.

Effect of intermittent treatment with human Parathyroid Hormone 1-34 in SAMP6 senescence-accelerated mice.

We examined trabecular and cortical bone in the senescence-accelerated mouse prone 6 (SAMP6) murine model of senile osteoporosis after treatment with human PTH 1-34. Sixteen-week-old female SAMP6 mice were assigned to control and PTH groups. PTH (20 microg/kg) was administered sc 3 times a week for 12 weeks. The control mouse strain, senescence-accelerated mouse resistant 1 (SAMR1), was used for comparison. The femoral metaphysis and diaphysis were used to measure bone mineral density (BMD), analyze the trabecular and the cortical structure by micro-computed tomography, and for conducting the bone strength test. PTH significantly attenuated the loss of BMD, improved the trabecular bone microstructure, and increased the bone strength in the femoral metaphysis. We did not find any differences in the bone strength of the femoral diaphysis after PTH treatment, although the cortical bone volume and cortical thickness were improved. Although the cortical thickness increased, the cortical bone density decreased, likely because of the increase of cortical porosity in the distal metaphysis after administration of PTH.

Regional variations of vertebral trabecular bone microstructure with age and gender.

UNLABELLED: The vertebral trabecular bone has a complex three-dimensional (3D) microstructure, with inhomogeneous morphology. A thorough understanding of regional variations in the microstructural properties is crucial for evaluating age- and gender-related bone loss of the vertebra, and may help us to gain more insight into the mechanism of the occurrence of vertebral osteoporosis and the related fracture risks. INTRODUCTION: The aim of this study was to identify regional differences in 3D microstructure of vertebral trabecular bone with age and gender, using micro-computed tomography (micro-CT) and scanning electron microscopy (SEM). METHODS: We used 56 fourth lumbar vertebral bodies from 28 women and men (57-98 years of age) cadaver donors. The subjects were chosen to give an even age and gender distribution. Both women and men were divided into three age groups, 62-, 77- and 92-year-old groups. Five cubic specimens were prepared from anterosuperior, anteroinferior, central, posterosuperior and posteroinferior regions at sagittal section. Bone specimens were examined by using micro-CT and SEM. RESULTS: Reduced bone volume (BV/TV), trabecular number (Tb.N) and connectivity density (Conn.D), and increased structure model index (SMI) were found between ages 62 and 77 years, and between ages 77 and 92 years. As compared with women, men had higher Tb.N in the 77-year-old group and higher Conn.D in the 62- and 77-year-old groups. The central and anterosuperior regions had lower BV/TV and Conn.D than their corresponding posteroinferior region. Increased resorbing surfaces, perforated or disconnected trabeculae and microcallus formations were found with age. CONCLUSION: Vertebral trabeculae are microstructurally heterogeneous. Decreases in BV/TV and Conn.D with age are similar in women and men. Significant differences between women and men are observed at some microstructural parameters. Age-related vertebral trabecular bone loss may be caused by increased activity of resorption. These findings illustrate potential mechanisms underlying vertebral fractures.

Ultrastructure of the water-clear cell in the parathyroid gland of SAMP6 mice.

Although the parathyroid water-clear cell is very rare, it has clinical significance because of its association with parathyroid hyperplasia or adenoma. SAMP6, a substrain of senescence-accelerated mouse, was developed as an animal model for senile osteoporosis. We investigated the morphology of the parathyroid glands in SAMP6 and age-matched normal mouse SAMR1. The parathyroid water-clear cells, which contained numerous vacuoles and the crystalloid inclusions, were found in SAMP6 mice at 5, 8 and 12 months of age. It was noted that the number of water-clear cells increased with aging, which are fairly consistent with the change of the serum parathyroid hormone (PTH) level. We did not find any water-clear cells in the parathyroid glands of SAMR1 mice. The existence of water-clear cells may represent hyperfunction of the parathyroid glands in SAMP6.

Morphological changes of skeletal muscle, tendon and periosteum in the senescence-accelerated mouse (SAMP6): a murine model for senile osteoporosis.

SAMP6, a substrain of senescence-accelerated mouse, was developed as an animal model for senile osteoporosis. Previously we observed age-related changes of the bone in SAMP6. In the present study, we investigated the morphology of the skeletal muscle, tendon and periosteum in SAMP6 and age-matched normal mouse SAMR1. We did not find any significant differences between SAMR1 and SAMP6 at 1 and 2 months of age. As compared with SAMR1, the cross-sectional area of type I and type II muscle fibers of the soleus muscle were significantly low in SAMP6 at 8 months of age. The projections in the interface of the muscle-tendon junctions were significantly decreased in SAMP6 at 8 months of age. The number of fibroblasts and the diameter of the tendon collagen fibers in Achilles fiber were significantly reduced in SAMP6 at 8 months of age. The diameter of Sharpey's fiber reduced in SAMP6 at 5 and 8 months of age. Some chondrocytes in the insertions of Achilles tendon and some osteogenic cells in the periosteum showed degenerative changes in SAMP6 at 5 and 8 months of age. The pronounced degenerative changes were detected in the skeletal muscle, muscle-tendon junction, tendon, tendon-bone interface and periosteum in SAMP6 with age. These findings indicated the atrophy of skeletal muscle, degeneration of tendon and periosteum in SAMP6, which may be involved in the bone loss for senile osteoporosis.

Ultrastructural changes in bones of the senescence-accelerated mouse (SAMP6): a murine model for senile osteoporosis.

SAMP6, a substrain of senescence-accelerated mice, was developed as an animal model for senile osteoporosis. In the present study, we investigated the bone morphology, together with serum calcium and bone mineral density (BMD) in SAMP6 and age-matched normal mice SAMR1. We did not find any significant differences between SAMR1 and SAMP6 at 1 month of age with regard to the serum compositions and bone morphology. As compared with SAMR1, BMD, the femoral weight, femoral calcium and phosphorus levels were significantly reduced in SAMP6 at 2 and 5 months of age. The number of osteoblasts in trabecular bones was also significantly reduced. Swollen mitochondria and myelin-like structures were found in osteoblasts and osteocytes of SAMP6 mice at 2 and 5 months of age. There was a greater proportion of resting surface and less forming surface in the femoral endosteal surfaces of SAMP6 mice. The amount of trabecular bone in the lumbar vertebra and the distal metaphysis of the femur was reduced. The number of the mast cells in bone marrow of the tibia significantly increased in SAMP6 mice. These findings indicate that the lower bone mass in SAMP6 was due to the reduction in osteoblast formation and suggested that mast cells in bone marrows play a role in the pathogenesis of senile osteoporosis.

Immunocytochemical localization of parathyroid hormone in rabbit parathyroid glands.

Immunocytochemical localization of parathyroid hormone was examined in the rabbit parathyroid gland by means of protein A-gold technique. Protein A-gold particles were observed on the secretory granules and the large secretory granules thought to be storage granules. No protein A-gold particles were observed on cisternae of the endoplasmic reticulum and the Golgi apparatus.

Effects of isoproterenol on the fine structure of the hamster parathyroid gland.

Ultrastructural changes of the parathyroid glands of isoproterenol-treated golden hamsters were investigated. Many chief cells in the parathyroid glands after 5 and 10 minutes of administration of isoproterenol contain well-developed Golgi complexes and granular endoplasmic reticulum, numerous prosecretory granules, and many secretory granules in the peripheral cytoplasm as compared with the control animals. Many chief cells in the parathyroid glands after 1, 3, 6 and 12 hours of administration have poorly-developed Golgi complexes, granular endoplasmic reticulum, many secretory granules and numerous lipid droplets as compared with the control animals. The morphology of the parathyroid gland after 30 minutes and 24 hours of administration resembles that of the control animals. It is considered that isoproterenol affects the secretory activity of the parathyroid gland.

Effect of low or high dietary calcium on the morphology of the rat femur.

The present study compared the effect of a calcium deficit or surfeit on femurs. Young female rats were fed with the normal (1.18%), low (0.05%), or high (2.00%) calcium diet for 3, 7, 15 or 30 days. Two groups received the low calcium diet for the first 15 days and then were followed by the normal (L-N) or high calcium diets (L-H) for the sequential 15 days. The morphology of the femur was studied together with serum calcium, parathyroid hormone (PTH), calcitonin and bone mineral density (BMD). We did not find any significant changes in the serum PTH level and bone morphology in the high calcium group. In the low calcium group, the serum PTH level increased, BMD of the whole body, the femoral weight and the femoral trabecular bone decreased as compared with the normal calcium group. There was a greater proportion of resorbing surface, less resting surface and larger vascular canal openings in the femoral endosteal surfaces in the low calcium group. In the L-N or L-H group, the femoral trabecular bone increased and the femoral resorbing surface decreased as compared with those of the low calcium group. These findings suggest that high calcium intakes do not affect the bone mass, and low calcium intakes have a deleterious effect on bone status, which may be related to vascular alternations of the bone. Reversing the low income calcium intake by a higher calcium diet can partially improve the bone alternations induced by low calcium intake.

Electron microscopic pathological patterns of alveolar septum in acute dextran-induced and alloxan-induced pulmonary edema in dogs.

We studied the incidence of electron microscopic pathological patterns of the alveolar septum observed 30 min after induction of pulmonary edema by dextran-70 infusion (6 dogs, dextran group) and by alloxan injection (6 dogs, alloxan group). For comparable amounts of extravascular lung water in both dextran and alloxan groups, which were twice as much as control group (6 dogs), we characterized the pathological changes. The incidence of the electron microscopic pathological patterns that appeared in dextran group compared with that in control group was significantly high in terms of the widening of the interstitial space, dispersion and disarray of collagen fibrils, and erythrocytes in the interstitial space. The incidence in alloxan group compared with that in control group was significantly high in terms of the swelling of epithelial cells and endothelial cells as well as the widening of the interstitial space, and dispersion and disarray of collagen fibrils. We conclude that dextran causes interstitial changes exclusively and alloxan causes cellular changes primarily coupled with secondary interstitial changes in acute pulmonary edema.

Ultrastructural study on the effects of hypophysectomy on the golden hamster parathyroid gland.

The ultrastructure of the parathyroid glands of hypophysectomized golden hamsters was studied. In the parathyroid glands of hypophysectomized animals the Golgi complexes and secretory granules were significantly decreased and large vacuolar bodies were significantly increased compared with those of the control animals. In addition, the chief cells contained a few prosecretory granules in the Golgi areas and a few secretory granules were present in the peripheral cytoplasm. These results suggest that the synthesis and release of parathyroid hormone may be suppressed in the parathyroid glands of the hypophysectomized animals.

Effects of melatonin on the ultrastructure of the golden hamster parathyroid gland.

Ultrastructural changes of the parathyroid glands of melatonin-treated golden hamsters were studied. Many chief cells in the parathyroid glands after 1 hour of administration of melatonin contained poorly-developed Golgi complexes associated with a few prosecretory granules and numerous lipid droplets as compared with those of the control animals. The morphology of the parathyroid glands after 5 hours of administration resembled that of the control animals. Many chief cells in the parathyroid glands after 24 hours of administration had well-developed Golgi complexes and cisternae of the granular endoplasmic reticulum, numerous prosecretory granules, a few lipid droplets and many secretory granules in the peripheral cytoplasm as compared with those of the control animals. The ultrastructure of the parathyroid glands after 48 hours of administration was almost similar to that of the control animals. It is considered that melatonin affects the secretory activity of the parathyroid gland.

Ultrastructure of mass of floccular substance in the parathyroid gland of golden hamster.

Mass of floccular substance was observed in the parathyroid glands of fetal, newborn and infantile golden hamsters. Mass composed of floccular substances was spherical with no limiting membrane around it. It was located near the nucleus and the Golgi area, but was also observed in the peripheral cytoplasm. No cell organelles were detected within area of mass.

Effects of short-term treatment with CaCl2 or EDTA on the parathyroid glands in pregnant golden hamsters, with special reference to large vacuolar bodies.

The large vacuolar bodies in the parathyroid glands of pregnant golden hamsters after administration of CaCl2 or EDTA were investigated. In the parathyroid glands of the pregnant animals 15 min after administration of CaCl2, the mean serum calcium concentration was significantly high when compared to that of the control animals. In the parathyroid glands of the pregnant animals 15, 30 and 60 min after administration of EDTA, the mean serum calcium concentration was significantly low when compared to that of the control animals. In the parathyroid glands of the pregnant animals 15 min after administration of CaCl2, the percentage area occupied by large vacuolar bodies was significantly increased when compared to that of the control animals. In the parathyroid glands of the pregnant animals 15 min after administration of EDTA, the percentage area occupied by large vacuolar bodies was significantly decreased when compared to that of the control animals.

Effects of ethanol on the ultrastructure of the hamster thyroid C-cell.

The morphology of the thyroid C-cells in golden hamsters after short- and long-term treatment with ethanol was studied. Immunohistochemistry was applied to examine the distribution of the C-cells in the thyroid gland. In the short-term experimental animals, the Golgi complexes and the granular endoplasmic reticulum were well developed and the number of the secretory granules was decreased as compared with those of the control animals. These findings suggest that the cellular activity of the thyroid C-cell is stimulated after short-term treatment with ethanol. The morphology of the thyroid C-cells of the long-term experimental animals was similar to that of the controls. It is conceivable that long-term treatment with ethanol does not affect the function of the C-cell.

Effects of prostaglandin E2 on the ultrastructure of the golden hamster parathyroid gland.

The effects of different ages on large vacuolar bodies in the parathyroid glands of golden hamsters after administration of prostaglandin E2 (PGE2) were investigated. In the parathyroid glands of the young and senile animals 15 min and the senile animals 60 min after administration of PGE2, the mean serum calcium concentration was significantly higher when compared to that of the young and senile control animals, respectively. In the experimental adult animals 60 min after administration of PGE2, the serum calcium concentration was seen to increase. In the parathyroid glands of the young animals 15 min and the adult and senile animals 60 min after administration of PGE2, the percent area occupied by large vacuolar bodies was significantly increased when compared to that of the young, adult and senile control animals, respectively. These findings suggest that the percent area occupied by large vacuolar bodies is increased in response to hypercalcemia induced by PGE2. It is thought that in the parathyroid glands suppressed by hypercalcemia there is a relationship between the percent area occupied by large vacuolar bodies and aging.

Effects of progesterone on the ultrastructure of the golden hamster parathyroid gland.

The ultrastructure of the parathyroid glands in golden hamsters after administration of progesterone was investigated. In the parathyroid glands of the female hamsters after administration of progesterone, the mean serum calcium concentration was significantly higher when compared to that of the control hamsters. In the male experimental hamsters, the percentage area occupied by Golgi complexes and lipid droplets was significantly increased when compared to that of the control hamsters, respectively. In the female hamsters after administration of progesterone, the percentage area occupied by large vacuolar bodies was significantly decreased when compared to that of the control hamsters. In the male and female experimental hamsters, the mean number of secretory granules per 100 microns2 of cytoplasm showed a significant increase compared with that of the male and female control hamsters, respectively. These findings suggest that the secretory activity of the parathyroid glands may be stimulated in response to hypocalcemia induced by progesterone.

Effects of ethanol on the ultrastructure of the hamster femur.

Several previous studies have indicated that chronic ingestion of ethanol exerts harmful effects on bones. However, few data are available concerning the effects of ethanol on the ultrastructure of bone. To further elucidate the effects of ethanol on bone, we studied the morphology of femur in golden hamsters after long-term treatment with ethanol. Six-week-old male hamsters were divided into 4 groups. Ethanol-treated animals were given ethanol at a concentration of 7% with food and water freely available, whereas the pair-fed animals (weight-matched to ethanol hamsters) had tap water available as the only drinking fluid. The femur weight, blood ethanol and serum calcium concentrations were determined after 3 and 5 months. The bone mineral density (BMD) of the whole body was measured before and after the experiment. Femurs of both sides were dissected and processed for morphometric measurement, light microscopy, scanning and transmission electron microscopy. In the ethanol-treated hamsters, BMD of the whole body and the weight of femur tended to decrease when compared with those of the controls. Light microscopy and scanning electron microscopy showed that the trabecula in the distal end of the femur from ethanol-treated hamsters were thinner than those of the controls. We also observed the disrupted swollen mitochondria of the femoral osteoblasts and osteocytes in the ethanol-treated hamsters. No significant difference in serum calcium levels and femoral osteoclasts was found. These results indicate that long-term treatment with ethanol results in disruption of femoral osteoblasts and reduction of bone mass in trabecular bone.

Morphological identification of the lipid-storing cells in golden hamster parathyroid glands after vitamin A treatment.

We investigated hamster parathyroid glands of different ages using electron microscopy and found a new cell type in young, adult and senile hamsters. Theses special cells were located in interstitial tissues and invariably contained several lipid droplets within the cytoplasm. The cells showed an elongated spindle with some cell processes. The cells contained small Golgi complexes and moderate cisternae of the granular endoplasmic reticulum. The morphological characteristics of these cells were mostly the same as those of lipid-storing cells in other organs (Yamada and Hirosawa, 1976). After vitamin A administration, the lipid droplets in these cells markedly increased in number and also in volume density. The other morphological features of these cells resembled those of the control animals. We called these cells parathyroid lipid-storing cells. They may incorporate and store vitamin A within the lipid droplets. They can be classified as one of the cellular components in hamster parathyroid gland.

Effects of cigarette smoke exposure on the ultrastructure of the golden hamster parathyroid gland.

Cigarette smoking has been identified as one of the risk factors to induce osteoporosis. However, we find no study on the morphology of the parathyroid gland under smoking exposure. We studied the ultrastructure of the parathyroid gland, lung and femur of the golden hamster exposed to cigarette smoke. Four-week-old male hamsters were housed in a plastic case (48x31x30 cm) and were exposed to cigarette smoke for 12 weeks, 5 minutes exposure, 4 times a day, 4 days a week. There were no differences in serum calcium level and the whole bone mineral density between the control and the smoke-exposed groups. In the parathyroid gland of the smoke-exposed animals, the Golgi complexes associated with many prosecretory granules were well developed and many secretory granules were located near the plasma membrane. Large lipid-like inclusion bodies were observed in the alveolar macrophages of the smoke-exposed animals. The femur morphology showed a wider area of resorbing surface in the smoke-exposed group than in the control group. From these findings, it is conceivable that the secretory activity of the parathyroid gland was stimulated with cigarette smoke exposure.