Pre-treatment bone turnover does not influence the level of the response to alendronate in postmenopausal osteoporosis at the bone tissue level.

PURPOSE: The main effect of anti-resorptive agents such as bisphosphonates is a reduction of bone resorption, with a consequent marked decrease of bone turnover. This post-hoc analysis investigated the changes of histomorphometric parameters of bone turnover after alendronate (ALN), according to the baseline turnover. METHODS: Ninety postmenopausal women underwent a transiliac bone biopsy before and after 6 (n = 44) or 12 (n = 46) months of treatment with ALN (70 mg/week). The dynamic parameters reflecting the bone formation and bone turnover were mineralizing surface (MS/BS; %), bone formation rate (BFR/BS; µm3/µm2/d), and activation frequency (Ac.f; /yr). Biochemical markers sPINP and the sCTX were assessed before treatment and after 3, 6, and 12 months. Subjects were divided into quartiles based on the baseline values of BFR/BS. RESULTS: At baseline, MS/BS and Ac.f were significantly different (p < 0.0001) among the BFR quartiles. sCTX and sP1NP were not significantly different among quartiles. After ALN treatment, MS/BS was not significantly different among quartiles but Ac.f remained significantly lower in the first quartile compared to the third and fourth ones (p < 0.03). The absolute value of the difference between pre- and post-treatment significantly correlated with the baseline BFR/BS but when expressed in percent of the baseline value, the magnitude of the diminutions of MS/BS, Ac.f, sCTX, and sP1NP was similar in the four baseline BFR quartiles. CONCLUSION: The percentage response to ALN appeared independent of the baseline level of bone turnover. After treatment, the bone turnover tended to be similar in all BFR quartiles. This analysis investigated the influence of baseline turnover measured by bone histomorphometry on the effect of alendronate. When expressed in percent of pre-treatment values, the decreases of histomorphometric parameters and biochemical markers of bone turnover were independent of the baseline turnover.

Bone safety with risedronate: histomorphometric studies at different dose levels and exposure.

UNLABELLED: This report describes bone safety and histomorphometric data across different dose levels and dosing frequencies of risedronate. Normal bone structure and histomorphometric data were observed, with ongoing bone remodeling and mineralization regardless of dose. These data are reassuring and do not suggest compromised bone remodeling during treatment with established risedronate regimens. INTRODUCTION: The efficacy and bone safety of risedronate 5 mg daily were established in pivotal phase III randomized, placebo-controlled clinical studies. Histomorphometric analysis of paired biopsies demonstrated bone safety as reflected by presence of fluorescent tetracycline double-labels in all evaluable biopsies. This report describes bone safety and histomorphometric data across studies of various dose regimens of risedronate. METHODS: Bridging studies, with bone mineral density as the primary endpoint, demonstrated non-inferiority of risedronate 35 mg and 50 mg once a week, risedronate 150 mg once a month, and a risedronate 75-mg dose on two consecutive days a month versus risedronate 5 mg daily. The low oral bioavailability and known dosing limitations due to food interactions of bisphosphonates have led to development of an oral delayed-release dose form of risedronate 35 mg to be taken weekly, before or after breakfast. Bone biopsies were collected at 24 months in studies involving these risedronate dosing regimens; bone safety and histomorphometric data were evaluated. RESULTS: Qualitative bone histology showed normal mineralization of newly formed bone without evidence of pathological findings, such as osteomalacia, bone marrow dyscrasia, or bone marrow fibrosis. Importantly, ongoing bone remodeling, based on fluorochrome labeling, was observed in all patients regardless of dose and exposure. Key histomorphometric variables were comparable to those observed with the risedronate 5 mg daily dose and were within the range seen in healthy pre- and post-menopausal women. CONCLUSIONS: Overall, the results are reassuring with respect to bone safety and histomorphometric data, and do not suggest oversuppression of bone remodeling during treatment with these established risedronate regimens.

Insights into material and structural basis of bone fragility from diseases associated with fractures: how determinants of the biomechanical properties of bone are compromised by disease.

Minimal trauma fractures in bone diseases are the result of bone fragility. Rather than considering bone fragility as being the result of a reduced amount of bone, we recognize that bone fragility is the result of changes in the material and structural properties of bone. A better understanding of the contribution of each component of the material composition and structure and how these interact to maintain whole bone strength is obtained by the study of metabolic bone diseases. Disorders of collagen (osteogenesis imperfecta and Paget's disease of bone), mineral content, composition and distribution (fluorosis and osteomalacia); diseases of high remodeling (postmenopausal osteoporosis, hyperparathyroidism, and hyperthyroidism) and low remodeling (osteopetrosis, pycnodysostosis); and other diseases (idiopathic male osteoporosis, corticosteroid-induced osteoporosis) produce abnormalities in the material composition and structure that lead to bone fragility. Observations in patients and in animal models provide insights on the biomechanical consequences of these illnesses and the nature of the qualities of bone that determine its strength.

Is nitric oxide a mediator of the effects of low-intensity electrical stimulation on bone in ovariectomized rats?

Low-intensity electrical stimulation (LIES) may counteract the effects of ovariectomy (OVX) on nitric oxide synthase (NOS) expression, osteocyte viability, bone structure, and microarchitecture in rats (Lirani-Galvão et al., Calcif Tissue Int 84:502-509, 2009). The aim of the present study was to investigate if these effects of LIES could be mediated by NO. We analyzed the effects of NO blockage (by L-NAME) in the response to LIES on osteocyte viability, bone structure, and microarchitecture in OVX rats. Sixty rats (200-220 g) were divided into six groups: sham, sham-L-NAME (6 mg/kg/day), OVX, OVX-L-NAME, OVX-LIES, and OVX-LIES-L-NAME. After 12 weeks, rats were killed and tibiae collected for histomorphometric analysis and immunohistochemical detection of endothelial NOS (eNOS), inducible NOS (iNOS), and osteocyte apoptosis (caspase-3 and TUNEL). In the presence of L-NAME, LIES did not counteract the OVX-induced effects on bone volume and trabecular number (as on OVX-LIES). L-NAME blocked the stimulatory effects of LIES on iNOS and eNOS expression of OVX rats. Both L-NAME and LIES decreased osteocyte apoptosis. Our results showed that in OVX rats L-NAME partially blocks the effects of LIES on bone structure, turnover, and expression of iNOS and eNOS, suggesting that NO may be a mediator of some positive effects of LIES on bone.

Low-intensity electrical stimulation counteracts the effects of ovariectomy on bone tissue of rats: effects on bone microarchitecture, viability of osteocytes, and nitric oxide expression.

Low Intensity Electrical Stimulation (LIES) has been used for bone repair, but little is known about its effects on bone after menopause. Osteocytes probably play a role in mediating this physical stimulus and they could act as transducers through the release of biochemical signals, such as nitric oxide (NO). The aim of the present study was to investigate the effects of LIES on bone structure and remodeling, NOS expression and osteocyte viability in ovariectomized (OVX) rats. Thirty rats (200-220 g) were divided into 3 groups: SHAM, OVX, and OVX subjected to LIES (OVX + LIES) for 12 weeks. Following the protocol, rats were sacrificed and tibias were collected for histomorphometric analysis and immunohistochemical detection of endothelial NO synthase (eNOS), inducible NOS (iNOS), and osteocyte apoptosis (caspase-3 and TUNEL). OVX rats showed significant (p < 0.05 vs. SHAM) decreased bone volume (10% vs. 25%) and trabecular number (1.7 vs. 3.9), and increased eroded surfaces (4.7% vs. 3.2%) and mineralization surfaces (15.9% vs. 7.7%). In contrast, after LIES, all these parameters were significantly different from OVX but not different from SHAM. eNOS and iNOS were similarly expressed in subperiosteal regions of tibiae cortices of SHAM, not expressed in OVX, and similarly expressed in OVX + LIES when compared to SHAM. In OVX, the percentage of apoptotic osteocytes (24%) was significantly increased when compared to SHAM (11%) and OVX + LIES (8%). Our results suggest that LIES counteracts some effects of OVX on bone tissue preserving bone structure and microarchitecture, iNOS and eNOS expression, and osteocyte viability.

Histomorphometric assessment of the long-term effects of alendronate on bone quality and remodeling in patients with osteoporosis.

Treatment effects on bone quality and remodeling was assessed in postmenopausal women with osteoporosis treated with oral alendronate. One transiliac bone biopsy was obtained from 231 women at either 24 mo (n = 11) or 36 mo (n = 120) from the start of treatment with alendronate at doses of between 5 and 20 mg/d, or placebo. 64 biopsies at 24 mo (31 from the placebo group and 33 alendronate-treated patients) and 95 biopsies at 36 mo (40 from the placebo group and 55 alendronate-treated patients) provided adequate cancellous tissue, and were analyzed by histomorphometry. Mineral apposition rate was unaffected by treatment. At 24 and 36 mo, osteoid thickness, volume, and surface significantly decreased. At each of the doses studied, mineralizing surface and activation frequency significantly decreased at each time point (e.g., -92% and -87%, respectively, for the 10 mg daily dose after 2 yr). These diminutions were of the same magnitude for each dose at 24 mo, and for the two highest doses at 36 mo. A significant increase in wall thickness accompanied by a reduction in erosion depth was detected in biopsies obtained at 24 mo. These findings confirm that mineralization is normal, and trabecular bone turnover markedly decreased in patients receiving long-term dosing with alendronate. The findings also suggest that the observed increases in bone mineral density could result both from a reduction in the remodeling space due to a decreased activation frequency and a possible trend to a positive bone balance. In addition, further studies focused on a possible increase in the degree of mineralization of bone are required.

Trabecular bone microarchitecture after alendronate treatment of osteoporotic women.

OBJECTIVE: To compare the microarchitecture of iliac crest trabecular bone from women treated for two to three years with alendronate versus that of women treated with placebo. RESEARCH DESIGN AND METHODS: Three-dimensional micro-computed tomography (micro-CT; resolution 20 microm) and two-dimensional histomorphometry (resolution 5-7 microm) were used to examine trabecular bone from single transilial biopsies obtained at the completion of clinical trials. MAIN OUTCOME MEASURES: Microarchitectural variables, including bone volume, trabecular number, trabecular thickness, and trabecular spacing in specimens from alendronate- and placebo-treated women were examined. Three-dimensional images of trabecular bone from both groups were constructed from CT images. Correlations among variables and between techniques were also calculated. RESULTS: Eighty-eight specimens were suitable for evaluation by both techniques. As measured by two-dimensional histomorphometry, bone volume fraction (as a proportion of total volume) and trabecular thickness were significantly greater in alendronate specimens, 17.1 +/- 5.5% vs. 13.4 +/- 5.5% (p = 0.0043) and 127 +/- 29 microm vs. 109 +/- 28 microm (p = 0.0090), respectively, and trabecular spacing was significantly smaller, 729 +/- 227 microm vs. 862 +/- 338 microm (p = 0.005). Micro-CT yielded similar findings: bone volume and trabecular number were significantly greater in alendronate specimens: 19.4 +/- 6.2% vs. 16.2 +/- 6.3% (p = 0.0412) and 1.46(+/-) 0.32 vs. 1.31(+/-) 0.33 per mm (p = 0.0346). Two-dimensional and micro-CT measured characteristics correlated strongly with one another, with Pearson product moment correlation coefficients ranging from 0.60 (for trabecular thickness) to 0.83 (for bone volume). CONCLUSIONS: Trabecular microarchitecture of the ilium, whether studied by two- or three-dimensional methods, is better (greater bone volume, greater trabecular thickness, decreased trabecular spacing) after alendronate treatment than after two to three years of treatment with placebo. Bone volume in a trabecular region is strongly correlated to its microarchitecture, suggesting that bone quantity predicts values for these microarchitectural endpoints.

Excessive growth hormone expression in male GH transgenic mice adversely alters bone architecture and mechanical strength.

Patients with acromegaly have a higher prevalence of vertebral fractures despite normal bone mineral density (BMD), suggesting that GH overexpression has adverse effects on skeletal architecture and strength. We used giant bovine GH (bGH) transgenic mice to analyze the effects of high serum GH levels on BMD, architecture, and mechanical strength. Five-month-old hemizygous male bGH mice were compared with age- and sex-matched nontransgenic littermates controls (NT; n=16/group). Bone architecture and BMD were analyzed in tibia and lumbar vertebrae using microcomputed tomography. Femora were tested to failure using three-point bending and bone cellular activity determined by bone histomorphometry. bGH transgenic mice displayed significant increases in body weight and bone lengths. bGH tibia showed decreases in trabecular bone volume fraction, thickness, and number compared with NT ones, whereas trabecular pattern factor and structure model index were significantly increased, indicating deterioration in bone structure. Although cortical tissue perimeter was increased in transgenic mice, cortical thickness was reduced. bGH mice showed similar trabecular BMD but reduced trabecular thickness in lumbar vertebra relative to controls. Cortical BMD and thickness were significantly reduced in bGH lumbar vertebra. Mechanical testing of femora confirmed that bGH femora have decreased intrinsic mechanical properties compared with NT ones. Bone turnover is increased in favor of bone resorption in bGH tibia and vertebra compared with controls, and serum PTH levels is also enhanced in bGH mice. These data collectively suggest that high serum GH levels negatively affect bone architecture and quality at multiple skeletal sites.

Chronic administration of Glucagon-like peptide-1 receptor agonists improves trabecular bone mass and architecture in ovariectomised mice.

Some anti-diabetic therapies can have adverse effects on bone health and increase fracture risk. In this study, we tested the skeletal effects of chronic administration of two Glucagon-like peptide-1 receptor agonists (GLP-1RA), increasingly used for type 2 diabetes treatment, in a model of osteoporosis associated bone loss and examined the expression and activation of GLP-1R in bone cells. Mice were ovariectomised (OVX) to induce bone loss and four weeks later they were treated with Liraglutide (LIR) 0.3mg/kg/day, Exenatide (Ex-4) 10 µg/kg/day or saline for four weeks. Mice were injected with calcein and alizarin red prior to euthanasia, to label bone-mineralising surfaces. Tibial micro-architecture was determined by micro-CT and bone formation and resorption parameters measured by histomorphometric analysis. Serum was collected to measure calcitonin and sclerostin levels, inhibitors of bone resorption and formation, respectively. GLP-1R mRNA and protein expression were evaluated in the bone, bone marrow and bone cells using RT-PCR and immunohistochemistry. Primary osteoclasts and osteoblasts were cultured to evaluate the effect of GLP-1RA on bone resorption and formation in vitro. GLP-1RA significantly increased trabecular bone mass, connectivity and structure parameters but had no effect on cortical bone. There was no effect of GLP-1RA on bone formation in vivo but an increase in osteoclast number and osteoclast surfaces was observed with Ex-4. GLP-1R was expressed in bone marrow cells, primary osteoclasts and osteoblasts and in late osteocytic cell line. Both Ex-4 and LIR stimulated osteoclastic differentiation in vitro but slightly reduced the area resorbed per osteoclast. They had no effect on bone nodule formation in vitro. Serum calcitonin levels were increased and sclerostin levels decreased by Ex-4 but not by LIR. Thus, GLP-1RA can have beneficial effects on bone and the expression of GLP-1R in bone cells may imply that these effects are exerted directly on the tissue.

Bone effects of fluoride in animal models in vivo. A review and a recent study.

The effects of fluoride on bone in various animal models are reviewed. In these studies, the doses of fluoride varied from those equivalent to therapeutic doses to toxic doses, and the duration of the treatment was from 15 days to 33 months. No significant modification in serum calcium, phosphorus, and alkaline phosphatase was reported. An increased serum osteocalcin level was noted in ewes. Evidence for hyperparathyroidism was found in some but not all animal models. Studies performed in the rat reported that fluoride had different effects on the periosteal and endosteal bone. An increase in the extent of eroded surfaces was observed in all experimental studies, except one in the mouse. Increases in osteoid parameters and in the number of osteoblasts were noted in mouse, cat, pig, and ewe. Only one study, carried out in dogs, mentioned a decrease in osteoid parameters. Most of the authors reported a mineralization defect due either to a modification in the composition of the bone matrix or to a low calcium intake. The formation period was augmented during fluoride treatment but, at a fluoride dose equivalent to therapeutic doses, this augmentation was mainly due to an increased active formation period. In contrast, at a fivefold greater dose, it was due to an increased inactive formation period. The augmentation of bone volume after fluoride treatment was attributed to an unbalanced coupling between resorption and formation in favor of formation. All these experimental studies support the conclusion that fluoride induces a stimulation of the birthrate of osteoblasts, but at high doses decreases their activity.

Skeletal fluorosis: histomorphometric findings.

Histomorphometric analysis of undecalcified sections was performed in transiliac biopsy cores taken from 29 patients (16 men, 13 women, aged 51 +/- 17 years) suffering from skeletal fluorosis due to chronic exposure to fluoride. The origin of the exposure, known in 20 patients, was either by water (endemic or sporadic), or industrial, or in a few cases iatrogenic. Measured on calcified bone using a specific ion electrode, bone fluoride content was significantly high in each specimen (mean +/- SD: 0.79 +/- 0.36% of bone ash) as compared to control values (less than 0.10%). The radiologically evident osteosclerosis observed in each patient was confirmed by the significant increase of cancellous bone volume (40.1 +/- 11.2 vs. 19.0 +/- 2.8% in controls, p less than 0.0001). There were significant increases in cortical width (1292 +/- 395 vs. 934 +/- 173 microns, p less than 0.0001) and porosity (14.4 +/- 6.4 vs. 6.5 +/- 1.7%, p less than 0.002), but without reduction of cortical bone mass. Osteoid parameters were significantly increased in fluorotic patients. The increase in cancellous osteoid perimeter was almost threefold greater than that noted in cancellous eroded perimeter. The fluorotic group had a greater number of osteoblasts than controls, with a very high proportion of flat osteoblasts. In 15 patients doubly labeled with tetracycline, the mineral apposition rate was significantly decreased, while mineralization lag time significantly increased. Bone formation rate and adjusted apposition rate were significantly decreased in skeletal fluorosis. Cancellous wall width was normal in fluorosis but the formation period and active formation period were significantly increased.(ABSTRACT TRUNCATED AT 250 WORDS)

In vitro exposure to sodium fluoride does not modify activity or proliferation of human osteoblastic cells in primary cultures.

The anabolic effects of sodium fluoride (NaF) on trabecular bone mass in osteoporosis is now well established. In vivo histologic studies performed in humans and other animals have shown that fluoride induces an increase in osteoblast number at the tissue level. To determine the mechanisms of action of fluoride on osteoblasts, we studied the effects of NaF on short- and long-term cultures of human osteoblastic cells derived from bone explants obtained from 21 donors. In short-term experiments, bone-derived cells were exposed to NaF for 4 days. At doses ranging from 10(-11) to 10(-5) M, NaF did not modify the alkaline phosphatase (AP) activity or osteocalcin secretion. In long-term experiments, half the bone samples from 15 donors were cultured for 4 months in the presence of 10(-5) M NaF and the other half were maintained in NaF-free medium. Observations by light and electron microscopy disclosed no morphologic modification in bone explants after 4 months of exposure to NaF, despite an increase in the bone fluoride content. After the first month of culture, slight but not significant increases were noted in 6 of 10 cases for AP activity, 4 of 10 for osteocalcin secretion, and 5 of 7 for [3H]thymidine incorporation. After 4 months of culture in the presence of NaF, no change in AP activity or cell proliferation was noted. In contrast, the osteocalcin secretion significantly decreased (p < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of a new selective estrogen receptor modulator (MDL 103,323) on cancellous and cortical bone in ovariectomized ewes: a biochemical, histomorphometric, and densitometric study.

The aims of this study performed in ewes were: (1) to confirm in this animal model the effects on bone of ovariectomy (OVX) alone or associated with Lentaron (L), a potent peripheral aromatase inhibitor, used to amplify the effects of OVX and (2) to evaluate the effects of a new selective estrogen receptor modulator (SERM; MDL 103,323) on bone remodeling. Thirty-nine old ewes were divided into five groups: sham (n = 7); OVX (n = 8); OVX + L (n = 8); OVX + L + MDL; 0.1 mg/kg per day (n = 8); and OVX + L + MDL 1 mg/kg per day (n = 8). The animals were treated for 6 months. Biochemical markers of bone turnover (urinary excretion of type 1 collagen C-telopeptide [CTX], serum osteocalcin [OC], and bone alkaline phosphatase [BAP]) were measured each month. Bone biopsy specimens were taken at the beginning and after death at the end of the experiment. Bone mineral density (BMD) was measured by dual-energy X-ray absorptiometry (DXA) on the lumbar spine and femur. OVX induced a significant increase in biochemical markers. This effect was the highest after 3 months for CTX (+156% vs. sham) and after 4 months for OC and BAP (+74% and +53% vs. sham, respectively). L tended to amplify the effect of OVX on OC and BAP. OVX induced significant increases in the porosity, eroded, and osteoid surfaces in cortical bone but no effect was observed in cancellous bone. MDL treatment reduced the bone turnover as assessed by bone markers, which returned to sham levels as well as histomorphometry both in cortical and in cancellous bone. Cancellous osteoid thickness decreased by 27% (p < 0.05), mineralizing perimeter by 81% (p < 0.05), and activation frequency by 84% (p < 0.02) versus OVX + L. Femoral and spinal BMD were increased by MDL and tended to return to the sham values. The effects of OVX on bone turnover were different on cortical and cancellous bone. These effects on cortical bone were reflected by changes in biochemical markers. MDL markedly reduces bone turnover and increases BMD suggesting that this new agent may prevent postmenopausal bone loss.

Comparison of trabecular bone microarchitecture and remodeling in glucocorticoid-induced and postmenopausal osteoporosis.

Long-term treatment with glucocorticoids (GCs) leads to a rapid bone loss and to a greater risk of fractures. To evaluate the specific effects of this treatment on cancellous bone remodeling, structure, and microarchitecture, we compared 22 transiliac biopsy specimens taken in postmenopausal women (65 +/- 6 years) receiving GCs (> or = 7.5 mg/day, for at least 6 months) and 22 biopsy specimens taken in age-matched women with postmenopausal osteoporosis (PMOP), all untreated and having either at least one vertebral fracture or a T score < -2.5 SD. On these biopsy specimens, we measured static and dynamic parameters reflecting trabecular bone formation and resorption. Also, we performed the strut analysis and evaluated the trabecular bone pattern factor (TBPf), Euler number/tissue volume (E/TV), interconnectivity index (ICI), and marrow star volume (MaSV). Glucocorticoid-induced osteoporosis (GIOP), when compared with PMOP, was characterized by lower bone volume (BV/TV), trabecular thickness (Tb.Th), wall thickness (W.Th), osteoid thickness (O.Th), bone formation rate/bone surface (BFR/BS), adjusted mineral apposition rate/bone surface (Aj.AR/BS), and higher ICI and resorption parameters. After adjustment for BV/TV, the W.Th remained significantly lower in GIOP (p < 0.0001). The active formation period [FP(a+)] was not different. Patients with GIOP were divided into two groups: high cumulative dose GCs (HGCs; 23.7 +/- 9.7 g) and low cumulative dose GCs (LGCs; 2.7 +/- 1.2 g). HGC when compared with LGC was characterized by lower W.Th (p < 0.05), BV/TV (p < 0.001), Tb.Th (p < 0.05), trabecular number (Tb.N; p < 0.05), FP(a+)(p < 0.05), and nodes (p < 0.05), and higher E/TV (p < 0.05), ICI (p < 0.005), and TBPf (p < 0.05). When HGC was compared with PMOP, the results were similar except for the MaSV, which was significantly higher (p < 0.005). In summary, GIOP was characterized by lower formation and higher resorption than in PMOP, already present after LGC. With HGCs, these changes were associated with a more dramatic bone loss caused by a major loss of trabecular connectivity.

The assessment of bone formation and bone resorption in osteoporosis: a comparison between tetracycline-based iliac histomorphometry and whole body 85Sr kinetics.

Bone formation and resorption have been measured in patients with idiopathic osteoporosis by histomorphometry of 7.5-mm trephine biopsies and in the whole body by 85Sr radiotracer methodology and calcium balances. The studies were synchronized and most were preceded by double in vivo tetracycline labeling. Correlations between histological and kinetic bone formation indices were better when better when based on the extent of double tetracycline labels than on measurements of osteoid by visible light microscopy. Correction of the kinetic data for long-term exchange, using 5 months' serial whole body counting of retained 85Sr, improved the fit of the kinetic to the histological data. A statistical analysis of the measurement uncertainties showed that the residual scatter in the best correlations (between exchange-corrected bone formation rates and double-labeled osteoid surface indices) could be attributed to measurement imprecision alone. The exchange-corrected resorption rate correlated fairly well with iliac trabecular resorption surfaces, and using a volume referent rather than a surface referent for the histological index improved the statistical fit when patients with therapeutically accelerated bone turnover were included. A much better correlation was obtained by including osteoid volume acting as an independent predictor of bone resorption in a bivariate regression with a resorption surface index. The residual errors could then be accounted for by known measurement uncertainties. Whereas osteoid taking a double label closely predicted the kinetic rate of bone formation, further analysis suggested that osteoid that took no label or a single label was more closely related to bone resorption, presumably as a secondary result of the coupling of bone formation to bone resorption. The idea that continued bone loss in some patients is associated with defective osteoblastic bone formation is supported by the low rates found in some patients by both techniques. Heuristically these studies validate both in vivo tetracycline labeling for dynamic histomorphometry and corrections for long-term exchange in kinetic studies of bone formation, providing a quantitative framework for the design and analysis of future studies of bone remodeling in the osteoporoses.

Evidence for a toxic effect of aluminum on osteoblasts: a histomorphometric study in hemodialysis patients with aplastic bone disease.

To evaluate the potential role of aluminum (Al) in a subset of dialysis patients with aplastic bone disease, we have studied tetracycline-labeled bone biopsies of 32 patients (22 males and 10 females, 45-73 years) on maintenance hemodialysis. Selection criteria included normal resorption surfaces (RS) and osteoid thickness. Eleven patients (Group I) had no stainable bone Al (Al-; 61.7 +/- 7.2 years) and 21 (Group II) had stainable bone Al (Al+; 57.7 +/- 6.8 years). Serum Al was normal to slightly elevated in Group I, but significantly higher in Group II (p less than 0.01). Al surfaces (AlS), undetectable in Group I, were 67.8 +/- 17.9% in Group II. Bone Al content (BAC) was much lower in Group I than in Group II (14.8 +/- 3.7 vs. 113.8 +/- 100.2 micrograms/g, p less than 0.01), but higher in Group I than in controls (p less than 0.05). Extensive thin osteoid seams were present in Group II. AlS was correlated with OS (r = 0.56, p less than 0.001) and OV (r = 0.48, p less than 0.01). Labeled surfaces were decreased in both groups. Labeled osteoid surfaces (TLS/OS) were below 2 SD of the mean control values in 96% of patients and calcification rate (CR) was depressed below 0.20 micros/day in 44% of patients. Bone formation rate (BFR) was strikingly depressed, values being below one SD of the mean control value in 92-100% of patients at both levels and below 2 SD of the mean in 82% of patients at BMU levels. Mineralization lag time (OMP) was markedly prolonged above 2 SD of controls in 89% of patients.(ABSTRACT TRUNCATED AT 250 WORDS)

Influence of experimental conditions on osteoblast activity in human primary bone cell cultures.

Primary bone cell cultures were derived from human bone explants. Cellular activity was characterized by the alkaline phosphatase (AP) activity, osteocalcin, and type I and III collagen secretions in the supernatant. The determination of bone cell activity was performed in three different wells. No significant difference was noted between wells: the coefficient of variation was 8.0 +/- 2.9% for AP activity, 18.3 +/- 1.9% for osteocalcin secretion, and 22.5 +/- 14.3% for collagen. The AP activity and osteocalcin secretion significantly decreased with the number of passages: they were the highest after the first passage. Between each subject, the coefficient of variation was 85% for AP activity and osteocalcin secretion and 63 and 57% for type I and III collagen secretion, respectively. The AP activity did not differ with the age or sex of the donor. In contrast, osteocalcin secretion was significantly lower in females than in males. In males, osteocalcin significantly decreased with the age of the donor (r = -0.61; p less than 0.05). Cellular activity did not depend on the site of the biopsy. When bone explants from one donor were cultured in two different petri dishes, the activity of cells was similar in both dishes, except in one case. Primary cell cultures derived from human bone explants are the only model providing untransformed osteoblastlike cells of human origin. Because of the experimental conditions, some factors may have influenced the cellular activity and they must be taken into account to validate further in vitro studies.

Effects of alendronate on bone quality and remodeling in glucocorticoid-induced osteoporosis: a histomorphometric analysis of transiliac biopsies.

Effects of alendronate (ALN) on bone quality and turnover were assessed in 88 patients (52 women and 36 men aged 22-75 years) who received long-term oral glucocorticoid exposure. Patients were randomized to receive oral placebo or alendronate 2.5, 5, or 10 mg/day for 1 year and stratified according to the duration of their prior glucocorticoid treatment. Transiliac bone biopsies were obtained for qualitative and quantitative analysis after tetracycline double-labeling at the end of 1 year of treatment. As previously reported in glucocorticoid-induced osteoporosis, low cancellous bone volume and wall thickness were noted in the placebo group as compared with normal values. Alendronate treatment was not associated with any qualitative abnormalities. Quantitative comparisons among the four treatment groups were performed after adjustment for age, gender, and steroid exposure. Alendronate did not impair mineralization at any dose as assessed by mineralization rate. Osteoid thickness (O.Th) and volume (OV/BV) were significantly lower in alendronate-treated patients, irrespective of the dose (P = 0.0003 and 0.01, respectively, for O.Th and OV/BV); however, mineral apposition rate was not altered. As anticipated, significant decreases of mineralizing surfaces (76% pooled alendronate group; P = 0.006), activation frequency (-72%; P = 0.004), and bone formation rate (-71%; P = 0.005) were also noted with alendronate treatment. No significant difference was noted between the changes observed with each dose. Absence of tetracycline label in trabecular bone was noted in approximately 4% of biopsies in placebo and alendronate-treated groups. Trabecular bone volume, parameters of microarchitecture, and resorption did not differ significantly between groups. In conclusion, alendronate treatment in patients on glucocorticoids decreased the rate of bone turnover, but did not completely suppress bone remodeling and maintained normal mineralization at all alendronate doses studied. Alendronate treatment did not influence the osteoblastic activity, which is already low in glucocorticoid-induced osteoporosis.

Differential solubilization of osteoblastic alkaline phosphatase from human primary bone cell cultures.

Mineralization of cartilage and bone requires alkaline phosphatase activity. In order to study the enzymatic properties of bone alkaline phosphatase in bone disease and more particularly in patients with osteoporosis and osteoarthritis, we investigated the solubilization of alkaline phosphatase from primary bone cell cultures derived from human bone explants. To study the release of alkaline phosphatase from membranes, several detergents at a concentration above the critical micellar concentration and cholesterol were used. Solubilized alkaline phosphatase was characterized by enzymatic activity and electrophoresis analysis. Almost all the alkaline phosphatase was solubilized using non-ionic detergent as n-octylglucoside and hecameg. In comparison with initial membranous activity, the solubilized activity was increased by a factor, i.e. 2 +/- 0.05 (SEM, n = 3) (with n-octylglucoside), i.e. 2.1 +/- 0.05 (SEM, n = 3) (with Hecameg). With an ionic detergent (sodium dodecylsulfate), zwitterionic detergent ((cholamido propyl) dimethylammonio 1 propane sulfonate) and cholesterol, a fraction of alkaline phosphatase was resistant to solubilization. Electrophoresis studies showed that released alkaline phosphatase was a glycosylphosphatidylinositol protein (amphipatic form) with 140 kDa as apparent molecular weight. A hydrophilic form was obtained by treatment with a specific lipase. This study showed differential solubilization of osteoblastic alkaline phosphatase from human primary bone cell cultures. Better extractibility and higher activation of this membrane anchored enzyme were obtained with non-ionic detergents.

Serum bone Gla-protein in renal osteodystrophy: comparison with bone histomorphometry.

Serum bone Gla-protein (S-BGP) and other serum biochemical parameters, including alkaline phosphatase (S-AP) and immunoreactive PTH (S-iPTH), were measured in 42 patients undergoing chronic hemodialysis. Each patient also had a tetracycline-labeled transiliac bone biopsy, allowing correlations between the biochemical and trabecular bone histomorphometric parameters, S-BGP was markedly increased [64.0 +/- 74.8 (+/- SD) vs. 6.2 +/- 2.2 ng/ml in normal subjects] significantly correlated with S-AP (r = 0.53) and S-iPTH (r = 0.55) levels. S-BGP was significantly higher in the 14 patients with high turnover renal osteodystrophy (HT-ROD; S-BGP, 138.5 +/- 90.8 ng/ml) than in the 28 patients with low turnover (LT-ROD; S-BGP, 26.8 +/- 14.8 ng/ml). S-BGP was significantly correlated with the cellular parameters of bone resorption and formation (r = 0.57-0.69) and with the dynamic parameters of bone formation (r = 0.62-0.82). The extent of stainable bone aluminum was significantly negatively correlated with S-BGP (r = -0.51) and serum iPTH (r = -0.33), but not with S-AP. S-BGP measurement allowed better discrimination between LT-ROD and HT-ROD groups than did S-AP measurement. However, in the patients with LT-ROD, S-BGP did not discriminate between patients with or without osteomalacia. We conclude that S-BGP is a valuable marker for evaluating bone remodeling and, more specifically, the bone formation rate at the tissue level in hemodialyzed patients.