Bone histological heterogeneity in postmenopausal osteoporosis: A sequential histomorphometric study

We studied sequential bone biopsies performed at 6 to 24 month intervals from 14 untreated osteoporotic women (64 ± 7). Subgroups were defined, respectively, by increased osteoclastic resorption surfaces and decreased osteoblastic surfaces ± 2 S.D. Normal values were obtained from bone biopsy of 23 normal women (61 ± 8). When patients were divided into subgroups according to the above criteria the first biopsy showed that 3 out of the 14 patients had high resorption surfaces and 6 had low osteoblastic surfaces. Eight patients spontaneously changed during the study. In 2 patients there was a change in resorption surfaces, in 3 in osteoblastic surfaces and in 3 a change in both osteoblastic and resorption surfaces was observed. Considering the first or second bone biopsy results the patient variance was higher than the control subject's variance; however the variance between the first and second bone biopsy of one patient was not different from the variance inside the group of patients. The average intraindividual variation of the parameters on sequential biopsies was of the same order as the one we previously observed on simultaneous bone biopsies of normal and hemodialyzed patients. We concluded that if osteoporosis is a heterogeneous disorder, subgroups cannot be definitively defined on the basis of cellular parameters of bone remodelling assessed on bone biopsies.     

Dose effects on ewe bone remodeling of short-term sodium fluoride administration — A histomorphometric and biochemical study

The early effects of two doses of sodium fluoride (NaF) on bone remodeling was studied in 14 ewes divided into two groups. Group I received orally 1 mg NaF/kg/day and group II received a five-fold greater dose. No calcium supplement was given. Transiliac bone biopsies and blood samples were taken before treatment (T₀) and after 45 (T₄₅) days of treatment. Bone fluoride content significantly increased in group II. In both groups, a significant decrease of serum calcium and phosphorus, and a slight but nonsignificant augmentation in serum parathyroid hormone were noted. Osteoid perimeter and area were significantly increased. The osteoid width significantly increased in both groups, but was twice higher in group II than I. At T₄₅, the osteoblast perimeter increased in both groups. Osteoid perimeter was significantly correlated with serum osteocalcin values (r = 0.74; p < 0.001) and bone fluoride content (r = 0.64; p < 0.01). The bone formation rate at tissue level tended to increase in both groups. Concerning the apposition rate, a decrease was noted which was 1.5-fold higher in group II than in I. The increased formation period resulted from a prolonged inactive period in group II. These results point out a stimulatory effect of fluoride on the birth rate of osteoblasts. However, fluoride prolonged the lifespan of osteoblasts that had reduced activity.     

Osteocalcin and bone morphometric parameters in adults without bone disease

Summary Serum bone Gla-protein (s-BGP or osteocalcin) and other serum biochemical parameters were measured in 19 subjects (8 women and 11 men, aged 20–82 years) without any bone disease. Each subject simultaneously underwent an iliac crest biopsy; tetracycline double-labeling was performed in 11 subjects, allowing correlations between s-BGP and bone histomorphometric parameters. s-BGP was significantly correlated with static bone parameters: trabecular bone volume (r=0.74;P<0.001), osteoid surfaces (r=0.69;P<0.001), osteoblastic surfaces (r=0.68;P<0.002); dynamic bone formation parameters: total labeled surfaces (r=0.72;P<0.01); and the bone formation rate (r=0.69;P<0.01). We conclude that s-BGP is a valuable marker for evaluating bone formation in healthy adult subjects.     

The influence of ibuprofen on fracture repair: biomechanical, biochemical, histologic, and histomorphometric parameters in rats

Ibuprofen is a widely used cyclo-oxygenase inhibitor in clinical practice. It has been demonstrated by others to have an inhibitory effect on fracture repair in animals. In the present study, we were unable to demonstrate any significant alterations in fracture biomechanics as measured by torsion testing and fracture stage in mature Sprague-Dawley rats treated with 30 mg/kg/day oral dose of ibuprofen, starting 3 days following fracture, over a 12-week time interval. Fracture histology and serum osteocalcin levels were no different in treated animals than control animals. Furthermore, histomorphometric parameters of bone remodeling, including bone volume and bone formation rate in the intact tail vertebrae of these animals with unilateral femur fractures, were no different between treated and control animals.     

Dynamic histomorphometric evaluation of human fetal bone formation

We have evaluated dynamic and static parameters of bone formation in femoral metaphyses collected from two human fetuses at 19 weeks of gestation. Tetracycline was administered to the mother at set intervals (2-5-2 day schedule) before interruption of pregnancy. Labels were distinct and sharply linear, suggesting a well organized calcification front at this early stage of mineralization. Mineral apposition rate (MAR) was fastest (4.1 ± 0.3 μm/d) in the periosteal (Ps) envelope, and about half that value in the endosteal envelopes (endocortical: 2.5 ± 0.1, cancellous 2.1 ± 0.1 μm/d). Because cellular activities may vary throughout the metaphyseal area, sections were arbitrarily separated in 0.75 mm layers starting from the growth plate. Three measured parameters decreased rapidly with increasing distance from the physis: Ps MAR: 4.9 to 2.3 μm/d, trabecular osteoid thickness: 5.9 to 1.2 μm, and cartilage volume (CgV/TV): 5.4% to 1.2%. Others did not vary significantly along the metaphysis. Comparison of several static parameters with those measured in five autopsy specimens from full-term infants showed that bone and cartilage volume, and trabecular thickness increased while osteoid thickness and parameters of resorption decreased in the second half of the gestation period. The study indicates that fetal bone matrix mineralization is already highly organized at mid-gestation, and validates the use of histomorphometry to assess bone maturation during early skeletal development.     

Decreased bone formation in osteoporotic patients compared with age-matched controls

Summary In order to study trabecular bone remodeling in postmenopausal osteoporosis we compared bone biopsies of 44 osteoporotic women aged 50–70 to those of 23 nonosteoporotic women, matched for age, who had a bone biopsy during anesthesia for knee arthritis. Trabecular bone volume, mean wall thickness, osteoblastic surfaces, labeled surfaces, and bone formation rate were decreased in osteoporotic women compared with control women. The osteoclast number and the osteoclastic surfaces were the same in the two groups. The normal distribution of the histomorphometric static parameters in osteoporotic patients did not allow the separation of subgroups. These data indicate that decreased bone formations is a major contributing factor leading to trabecular bone loss in postmenopausal osteoporosis.     

Trabecular and endocortical bone remodeling in postmenopausal osteoporosis: Comparison with normal postmenopausal women

To assess the bone turnover abnormalities which characterize postmenopausal osteoporosis with vertebral fractures (PMOp), a transiliac bone biopsy was performed after double labeling of the mineralizing front with tetracycline in 50 untreated PMOp patients who were compared with 13 healthy age-matched volunteer females. The analysis of bone remodeling and structure parameters demonstrated that PMOp is a disease affecting both the cancellous and the endocortical envelopes and characterized by increased resorption and by a marked decrease in the osteoblastic apposition rate due to a reduced duration of bone formation. This induces a decrease in the width of both individual osteons and trabeculae. In PMOp, the wide spectrum of bone turnover as compared with the controls, associated with the typical bimodal distribution of cancellous osteoid perimeter, allowed us to identify two subsets, one with normal turnover (NT) and one with high turnover (HT) representing 30% of the cases. When compared to NT, HT was characterized by increased osteoclast number, lower bone volume, thinner osteons, increased formation at the tissue-level and markedly decreased duration of formation. In HT the marked decrease in the duration of activity of osteoblasts and the markedly increased number of osteoclasts induced a greater decrease in bone volume, despite the increase of bone formation at the tissue level. These subsets could not be distinguished by any clinical or biochemical parameter except for serum bone gla protein (osteocalcin) which was significantly higher (as a group) in HT than in NT. The underlying cause for these two subsets is unknown. We conclude that PMOp affects the cancellous and the endocortical bone. Bone loss results from a wide spectrum of bone turnover abnormalities, with two distinct subsets, one with normal turnover and one with high turnover.     

Electromagnetic stimulation of bone repair: a histomorphometric study

The effect of pulsing electromagnetic fields (PEMFs) on bone repair was studied in principal metacarpal bones of eight adult male horses: Six horses were treated with PEMFs, and two horses were untreated. In treated horses, Helmholtz coils were applied during a 60-day period to the left metacarpal bones, bored with eight holes of equal diameter and depth, from the middiaphysis toward the distal metaphysis. Eight equal holes bored in the right metacarpal, surrounded by unactivated Helmholtz coils, were taken as controls. The two untreated horses were taken as additional control. The results of computer-assisted histomorphometric analysis indicate that (a) in diaphyseal levels, the amount of bone formed during 60 days is significantly greater (p less than 0.01) in PEMF-treated holes than in contralateral ones and those in control horses; (b) in metaphyseal levels, PEMF-treated holes are sometimes more closed, sometimes less, as compared with contralateral holes and those in control horses; in any case the statistical analysis indicates that the symmetry in the rate of hole repair, found between the two antimeres of control horses, is not appreciable at metaphyseal levels also; (c) there was no statistically significant difference between untreated holes in PEMF-treated horses and holes in control horses, neither at diaphyseal nor at metaphyseal levels. These preliminary findings indicate that PEMFs at low frequency influence the process of bone repair on both diaphysis and metaphysis, and seem to improve the process of bone repair in skeletal regions normally having a lower osteogenetic activity, i.e., in diaphyses as against metaphyses.     

Postoophorectomy bone loss is associated with reduced bone Gla protein serum levels: A possible effect of osteoblastic insufficiency

Summary To further investigate the relationship between oophorectomy (OF) and mineral bone loss, 15 women who underwent total hysterectomy and bilateral oophorectomy were studied for 12 months after surgery. Mineralometric and metabolic data were obtained before and after 3, 6, and 12 months. The women lost bone mineral content (measured by single photon absorptiometry) at the same rate they lost cortical and trabecular bone, suggesting that bone loss after OF is a generalized phenomenon. Our data also show that in increase in bone resorption takes place only in the first period after OF; the persistency of a negative bone balance up to 12 months, accompanied by a reduction of osteocalcin serum levels, may be dependent on a reduced bone formation, probably due to osteoblastic insufficiency.     

Absence of mouse pleiotrophin does not affect bone formation in vivo

Pleiotrophin (Ptn) is an extracellular matrix protein that regulates hippocampal synaptic plasticity and learning behavior in vivo. Since the overexpression of Ptn in transgenic mice leads to increased bone formation, we analyzed whether a deficiency in Ptn expression would have a negative effect on bone remodeling. Bones from Ptn-deficient mice and wild-type littermates were analyzed using radiography, μCT imaging and undecalcified histology. Biomechanical stability was determined in a three-point-bending assay. Cellular activities were assessed using dynamic histomorphometry and the determination of urinary collagen degradation products. Skeletons of Ptn-deficient mice have no gross abnormalities, displayed a normal size, and showed no differences in growth plate organization compared to wild-type littermates. There were no obvious differences in bone mass as determined by radiographic and μCT imaging. The absence of a bone remodeling phenotype in Ptn-deficient mice was further confirmed using static histomorphometry and biomechanical testing. Finally, the number, morphology, and function of osteoclasts, osteoblasts, and osteocytes were not altered in Ptn-deficient mice compared to wild-type littermates. The complete skeletal analysis of Ptn-deficient mice presented here demonstrates that the lack of Ptn in mice does not affect bone formation in vivo. Therefore, Ptn does not play a significant role in normal bone physiology.     

Can serum osteocalcin levels predict bone turnover in patients with osteoporosis?

The assessment of bone metabolism by biochemical markers remains difficult problem. Serum osteocalcin, synthesized in bone cells, is now becoming a sensitive indicator of bone turnover in patients with metabolic bone diseases. We measured serum osteocalcin levels by radioimmunoassay in 18 patients with osteoporosis and examined whether they reflect bone formation, resorption or both. We found that serum osteocalcin levels in biopsy-identified osteoporotics were widely ranged (1.8 – 18.8 ng/ml). Tetracycline double labeling method exibited two types of labeling pattern in the iliac bone, that is double labeled or no double labeled (impaired labeled) pattern. Serum concentrations of osteocalcin in patients with double labeling were significantly higher than those with impaired labeling (11.2±4.0 vs 4.1 ±2.1 ng/ml, P<0.01). Furthermore, serum osteocalcin levels showed a significantly positive linear correlation with the parameters reflecting bone formation, but not with bone resorption. These data indicate that serum osteocalcin levels reflect bone formation in osteoporotics and thereby could be a useful indicator of osteoblastic function in osteoporosis.     

Evaluation of bone turnover in type I osteoporosis using biochemical markers specific for both bone formation and bone resorption

The aims of the study were to evaluate the use of bone-specific biochemical markers of turnover in type I osteoporosis, to test for evidence of heterogeneity of bone turnover in this condition, and to attempt to devise an uncoupling index by using the relationship between bone-specific biochemical markers of bone formation and bone resorption. In women with type I osteoporosis (mean age 64 years, SD 5;n=63) the mean level of serum osteocalcin, a specific biochemical marker of bone formation, was 9.9 ng/ml (SD 2.0), which was higher than the level in normal postmenopausal women (mean age 65 years, SD 6;n=8.9 ng/ml (SD 2.0;p<0.01). The variance of serum osteocalcin levels in the two groups was similar. Compared with this 11% increase in the biochemical marker for bone formation, the markers of bone resorption, total urinary deoxypyridinoline (bone-specific), pyridinoline and hydroxyproline were increased by 40% (p<0.0001), 61% (p<0.0001) and 25% (p<0.01), respectively. Furthermore, these biochemical markers of bone resorption had greater variance in women in type I osteoporosis than in the normal postmenopausal women (p<0.001). The urinary excretion of the free crosslinks deoxypyridinoline, pyridinoline and glycosylated pyridinoline were increased by 26% (p<0.001), 17% (p<0.01) and 13% (NS) respectively. An uncoupling index was calculated for the difference between urinary deoxypyridinoline and serum osteocalcin using the results from the normal women and expressed asz-scores. We conclude that the pyridinium crosslinks of collagen enable better discrimination between normal and osteoporotic women than does hydroxyproline. In osteoporosis there appears to be heterogeneity of bone resorption. Finally, an uncoupling index indicated that in osteoporosis bone resorption was increased to a greater extent than bone formation as compared with normal postmenopausal women.     

Human fetal bone development: histomorphometric evaluation of the proximal femoral metaphysis

Quantitative data on metaphyseal bone histology during early human development are scarce. In the present study the proximal femoral metaphysis of 35 fetuses and newborns (gestational age 16–35 weeks) was analyzed by histomorphometry. Averaged over the entire metaphyseal area, the relative amount of bone and cartilage was higher in the third compared to the second trimester. Osteoid thickness increased with gestational age, whereas indices of bone resorption decreased. The relative amount of cartilage decreased with increasing distance from the growth plate, whereas the relative amount of bone increased. This was due to trabecular thickening, which occurred at an estimated rate of 3 μm/day in areas close to the growth plate. Despite this rapid rate of net bone gain, osteoid indices were relatively low, indicating that mineralization occurred very rapidly after bone deposition. These observations suggest that modeling, not remodeling, is the predominant mechanism responsible for the development of femoral metaphyseal cancellous bone in utero.     

Bone particles disturb new bone formation on the interface of the titanium implant after reaming of the marrow cavity

Histomorphometric studies were conducted in rats to determine whether bone particles would disturb new bone formation on the interface of titanium implants inserted after reaming of the marrow cavity. In eighty 10-week-old female Wistar rats, smooth-surfaced titanium alloy implants were inserted bilaterally into the marrow cavity after reaming in the distal femur. There were three experimental groups: in the irrigated femora, sterile saline was flushed through the medullary canal; in the particle femora, autologous bone particles were inserted into the intramedullary cavity; and in the reamed femora, the implant was inserted without procedures after reaming. The rats were sacrificed at one, two, four or eight weeks postoperatively, and Villanueva bone staining was applied for histomorphometric studies. The bone volume of new bone on the interface of the implant in the irrigated femora was greater than that in the particle or the reamed femora throughout the study period. The results suggest that clearance of bone particles by irrigation after reaming of the marrow cavity significantly facilitates new bone formation on the interface of implants by one week. The findings also suggest the potential clinical application of total canal irrigation prior to insertion of cementless femoral components as well as cemented prosthesis.     

Flurbiprofen-induced stimulation of periosteal bone formation and inhibition of bone resorption in older rats

The skeletal effects of flurbiprofen (Fb), a nonsteroidal antiinflammatory drug, was studied by histomorphometry in 9-month-old retired female breeder, Sprague-Dawley rats. Flurbiprofen was given subcutaneously at 0, 0.2, 0.1, 0.5, 2.5, or 5 mg/kg/d for 21 days. Flurbiprofen had no effect on longitudinal growth, but stimulated radial growth (+200%) over controls. In the tibial shaft, Fb stimulated the mineral apposition rate (+25%), mineral bone formation rate (+100%), and periosteal labeling length (+64%) at the 2.5 and 5.0 mg Fb/kg dose levels, and had no effect on marrow cavity size compared to controls. However, these changes were insufficient to increase cortical bone mass. In the proximal tibial metaphysis, Fb suppressed osteoclasts/mm² of metaphyseal tissue (-47%), osteoclasts/mm of bone surface (-46%), and the osteoclast/osteoblast ratio (-50%), increased the calcified cartilage core population (+100%), and had no effect on osteoblast numbers at all dose levels. There was an insignificant increase in metaphyseal cancellous bone mass. The current study leads to the conclusion that flurbiprofen-stimulated periosteal bone growth was due to direct stimulation of osteoblast recruitment and activity independent of longitudinal bone growth. Further, it confirms early findings in young rats that flurbiprofen induced depressed bone resorption without lowering bone formation. However, because of insufficient treatment time, the older rat did not accumulate bone as the young rats did.     

Acute effects of deflazacort and prednisone on rates of mineralization and bone formation

The aims of this study were to determine (1) whether acute suppression of bone formation could be evaluated after the administration of corticosteroids in man by quantitative bone histomorphometry; and (2) whether there were significant differences between the effects of prednisone and its analog deflazacort. Thirteen patients who needed high-dose corticosteroid therapy were randomly allocated to two groups of treatment (prednisone or deflazacort). Quantitative bone histomorphometry, using the technique of triple labeling, and biochemical measurements of bone turnover were studied. There were no differences in biochemical indices of bone turnover between prednisone and deflazacort at the beginning and end of the 15 days of treatment course. During corticosteroid treatment, there were no significant changes in biochemical indices of bone turnover but a significant decline in total alkaline phosphatase (P<0.01). Histomorphometric indices, as revealed by measurements of tetracycline interval and extent of labeling, showed no significant differences in either mineral apposition rate or bone formation rate in the two groups. We conclude that the acute glucocorticoid suppression of bone turnover by glucocorticoids is not detectable within the first 2 weeks of treatment by histomorphometric techniques. No differences in bone effects of prednisone and deflazacort were detected in this short-term study.     

Diet,bone mass,and osteocalcin: A cross-sectional study

To determine the relationships among nutrient intake, bone mass, and bone turnover in women we have investigated these issues in a population-based, crosssectional, observational study in one county in central Sweden. A total of 175 women aged 28–74 at entry to the study were included. Dietary assessment was made by both a semiquantitative food frequency questionnaire and by four 1-week dietary records. Dual energy X-ray absorptiometry was performed at five sites: total body, L2–L4 region of the lumbar spine, and three regions of the proximal femur. Serum concentrations of osteocalcin (an osteoblast-specific protein reflecting bone turnover) were measured by a radioimmunoassay. Linear regression models, with adjustment for possible confounding factors, were used for statistical analyses. A weak positive association was found between dietary calcium intake as calculated from the semiquantitative food frequency questionnaire and total body bone mineral density (BMD) among premenopausal women. No association emerged between dietary calcium intake and sitespecific bone mass, i.e., lumbar spine and femoral neck, nor was an association found between dietary calcium intake and serum osteocalcin. BMD at some of the measured sites was positively associated with protein and carbohydrates and negatively associated with dietary fat. In no previous studies of diet and bone mass have dietary habits been ascertained so carefully and the results adjusted for possible confounding factors. Neither of the two methods of dietary assessment used in this study revealed any effect of calcium intake on BMD at fracture-relevant sites among these healthy, mostly middle-aged women. A weak positive association was found between calcium intake estimates based on the food frequency questionnaire and total body BMD. In this study population the preventive effect of high dietary calcium on osteoporosis is probably very weak. The independent significance of protein, carbohydrates, and fat is uncertain.     

Insulin increases histomorphometric indices of bone formation In vivo

Recent clinical studies have established that bone density is related to both fat mass and circulating insulin levels. A direct action of insulin on the osteoblast may contribute to these relationships. Osteoblast-like cells have insulin receptors, and insulin has been shown to stimulate proliferation of these cells in vitro. However, it has not been possible to study the effects of insulin administration on bone in vivo because of the metabolic effects of insulin, particularly hypoglycemia. A model involving the local injection of insulin over one hemicalvaria of an adult mouse overcomes these difficulties and permits the histomorphometric study of insulin's action on bone. Insulin or vehicle was injected daily for 5 days over the right hemicalvariae of adult mice, and the animals were sacrificed 1 week later. All indices of bone formation were significantly increased in imsulin-treated hemicalvariae compared with the noninjected hemicalvariae. There was a 2.73±0.50-fold increase in osteoid area (P=0.0005), a 2.20±0.37-fold increase in osteoblast surface (P=0.021) and a 2.04±0.29-fold increase in osteoblast number (P=0.021). Indices of bone resorption tended to decline and mineralized bone area tended to increase in insulin-treated animals. The direct action of insulin on bone may contribute to the increased bone density seen in obesity and to the osteopenia of type I diabets, conditions associated with insulin excess and deficiency, respectively.     

Minocycline prevents the decrease in bone mineral density and trabecular bone in ovariectomized aged rats

In the current study, we examined the effects of minocycline, on the osteopenia of ovariectomized aged rats. Old female rats were randomly divided into five groups: sham, ovariectomized control and ovariectomized treated with minocycline, 17β-estradiol, or both agents. Bone samples were collected 8 wk after the treatment. Ovariectomy reduced bone mineral density of the whole femur and at the condylar, distal metaphyseal and head-neck-trochanter regions 10%–19% and the loss of bone density was prevented by treatment with minocycline or 17β-estradiol. Histomorphometric analysis of distal femur showed ovariectomy reduced the trabecular bone area, the trabecular bone number, trabecular bone thickness and increased the trabecular bone separation. The microanatomic structure of trabecular bone also showed that the number of nodes, node to node, cortical to node, node to free end was reduced by ovariectomy. Treatment with minocycline attenuated the effect of ovariectomy on trabecular bone in aged animals. In contrast, cortical bone was not affected by ovariectomy or minocycline treatment. The effect of minocycline on bone turnover was also examined. Minocycline increased osteoid surface, mineralizing surface, mineral apposition rate, bone formation rate and reduced eroded surface. We have therefore concluded that the modest increase in bone mineral density and the improvement in the trabecular bone status noted in minocycline treated ovariectomized aged rats is likely due to an increase in bone formation coupled with a decrease in bone resorption.     

Bone tissue response to four-month antiorthostatic bedrest: A bone histomorphometric study

Summary A histomorphometric analysis were made on iliac crest biopsies from eight healthy male volunteers submitted to a 4-month antiorthostatic bedrest. Bone mass and bone cell parameters, reflecting resorption and formation activities, were measured before and after the bedrest period. Trabecular bone volume and mean cortical thickness were not modified despite a decreased number of trabeculae and nonsignificant increase of the trabecular thickness; total and active resorption surfaces and the number of osteollast per mm² of trabecular surfaces do not vary significantly. Osteoid thickness does not vary but we found a reduced osteoid surface and a nonsignificant decreased osteoid volume. Our results suggest that bone architecture may be more affected by the reduction of mechanical forces than the bone mass. These modifications were supposed to be the result of an accelerated bone turnover in the early stage of immobilization. In this study, we failed to find disuse osteoporosis; however, we must point out that the new organization of the trabecutae could affect the bone mechanical properties.