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Duchenne Muscular Dystrophy (DMD) is a progressive muscle disorder caused by genetic mutations of the dystrophin encoding gene. In the absence of functional dystrophin, DMD patients suffer from muscle inflammation and wasting, as well as compromised bone health with increased risk of fracture. The use of high dose glucocorticoids (GC) as the standard therapy also contributes to bone fragility. This study examined the effects of intermittent, daily administered parathyroid hormone (iPTH), an approved bone anabolic therapy, on growing bone and dystrophic muscle in the presence and absence of prednisone treatment using the Mdx mouse model of DMD. Five-weeks of prednisone treatment in Mdx mice decreased cortical bone thickness and area (p < 0.001), with a large increase in endocortical osteoclasts that were significantly improved by PTH treatment (p < 0.001). GC-induced decreases in cortical bone toughness and modulus were improved with iPTH therapy (p < 0.05). Mdx mice showed significantly less bone mass in trabecular compartments of lumbar vertebrae and iPTH treatment, with or without glucocorticoids, significantly improved structural and material properties of this bone. Prednisone improved grip strength and endurance of treadmill running, which were maintained and further improved, respectively, in co-treated Mdx mice. Altogether, our study demonstrates that iPTH therapy significantly ameliorated GC-induced bone loss and maintained or further enhanced the positive effects of GCs on dystrophic muscle function. These findings give insight into the potential for use of teriparatide to treat growing bone in children with DMD., (Copyright © 2019. Published by Elsevier Inc.)

Reconstruction of large skeletal defects is a significant and challenging issue. Tissue banks often use γ-irradiation (15-35 kGy) to sterilize bone allografts, which, however, drastically impairs the post-yield mechanical properties. In previous studies, we reported the development of a method that protects human bone collagen connectivity through ribose crosslinking while still undergoing γ-irradiation. Given these promising results, the next step was to determine if the presence of ribose within the bone tissue would interfere with the effectiveness of the γ-irradiation sterilization against bacteria. This study had two stages. The aim of the first stage was to assess the protective effect of ribose in solution using a Bacillus pumilus spore strip model. The aim of the second stage was to assess the protective effect of ribose (R) on spores within a human cortical bone model in comparison to conventionally irradiated bone (I). Treatment of B. pumilus spore strips with ribose in solution led to temperature-dependent effects on spore viability versus spore strips treated with PBS alone. Ribose solution at 60 °C led to a notable two logs decrease in spore count relative to PBS at 60 °C. In the human bone model, the number of spores in the I and R groups were greatly decreased in comparison to the non-irradiated N group. No spore colonies were detected in the R group (n = 4) whereas two of the four plates of group I formed colonies. This study provides evidence that the method of pre-treating bone with ribose crosslinking prior to irradiation sterilization, while improving irradiation sterilized bone allograft quality, also may improve the effectiveness of the sterilization process.

Strontium ranelate and fluoride salts are therapeutic options to reduce fracture risk in osteoporosis. Incorporation of these elements in the physiological hydroxyapatite matrix of bone is accompanied by changes in bone remodeling, composition, and structure. However, a direct comparison of the effectiveness of strontium and fluoride treatment in human cortical bone with a focus on the resulting mechanical properties remains to be established. Study groups are composed of undecalcified specimens from healthy controls, treatment-naïve osteoporosis cases, and strontium ranelate or fluoride-treated osteoporosis cases. Concentrations of both elements were determined using instrumental neutron activation analysis (INAA). Backscattered electron imaging was carried out to investigate the calcium content and the cortical microstructure. In comparison to osteoporotic patients, fluoride and strontium-treated patients have a lower cortical porosity indicating an improvement in bone microstructure. Mechanical properties were assessed via reference point indentation as a measure of bone's resistance to deformation. The strontium-incorporation led to significantly lower total indentation distance values compared to osteoporotic cases; controls have the highest resistance to indentation. In conclusion, osteoporosis treatment with strontium and fluoride showed positive effects on the microstructure and the mechanical characteristics of bone in comparison to treatment-naïve osteoporotic bone. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 433-442, 2017., (© 2016 Wiley Periodicals, Inc.)

The objective of the study was to determine the in vivo role of Filamin A (FLNA) in osteoclast generation and function, through the assessment of trabecular bone morphology, bone turnover, and the resulting changes in mechanical properties of the skeleton in mice with targeted deletion of FLNA in pre-osteoclasts. Using a conditional targeted knockdown of FLNA in osteoclasts, we assessed bone characteristics in vivo including micro-computed tomography (micro-ct), histomorphometric analyses, and bone mechanical properties. These parameters were assessed in female mice at 5 months of age, in an aging protocol (comparing 5-month-old and 11-month-old mice) and an osteoporosis protocol [ovariectomized (OVX) at 5 months of age and then sacrificed at 6 and 11 months of age]. In vivo bone densitometry, mechanical and histomorphometric analyses revealed a mild osteoporotic phenotype in the FLNA-null 5-month and aging groups. The WT and FLNA-KO bones did not appear to age differently. However, the volumetric bone mineral density decrease associated with OVX in WT is absent in FLNA-KO-OVX groups. The skeleton in the FLNA-KO-OVX group does not differ from the FLNA-KO group both in mechanical and structural properties as shown by mechanical testing of femora and vertebrae and histomorphometry of vertebrae. Additionally, FLNA-KO femora are tougher and more ductile than WT femora. The result of this study indicates that while FLNA-KO bones are weaker than WT bones, they do not age differently and are protected from estrogen-mediated post-menopausal osteoporosis.

The benefit of glucosamine (GlcN) in bone and joint disorders remains controversial. N-acetylation and other N-acylations of GlcN alter its biological properties fundamentally. We have shown previously that N-butyryl glucosamine (GlcNBu) preserved strikingly the subchondral bone structure in a destructive arthritis rat model. Here, we examine whether GlcNBu preserves bone in the ovariectomized (OVX) rat, a model for postmenopausal osteoporosis. Rats were randomized into 4 groups: group 1, sham OVX glucose (Glc) fed; group 2, sham OVX GlcNBu fed; group 3, OVX Glc fed; and group 4, OVX GlcNBu fed. A single, oral, 200-mg/kg dose of GlcNBu or Glc was administered daily for 6 months. Bone mineral content (BMC) and bone mineral density, and biomechanical properties of the femurs and spines were determined by standardized techniques. Two-way analysis of variance with a Bonferroni post hoc test was used for statistical analysis. Ovariectomy in group 3 resulted either in significant or highly significant effects in a number of the tests. For spinal BMCs the interaction between GlcNBu and OVX was significant. For the femurs, this interaction was also seen in energy to failure, and ultimate displacement and ultimate strain tests. In general, ovariectomy was necessary to show significant preventive effects of GlcNBu on mineral content and some biomechanical properties. We conclude that GlcNBu feeding in the OVX rat preserves bone mineral and some biomechanical properties. Translationally, GlcNBu can be positioned between nutriceuticals and pharmaceuticals for the prevention and treatment of osteoporosis. Advantages include low production costs and a favorable safety profile., (Copyright © 2013 Mosby, Inc. All rights reserved.)

Non-enzymatic glycation (NEG) and advanced glycation endproducts (AGEs) may contribute to bone fragility in various diseases, ageing, and other conditions by modifying bone collagen and causing degraded mechanical properties. In this study, we sought to further understand how collagen modification in an in vitro non-enzymatic ribation model leads to loss of cortical bone toughness. Previous in vitro studies using non-enzymatic ribation reported loss of ductility in the cortical bone. Increased crosslinking is most commonly blamed for these changes; however, some studies report positive correlations between measures of total collagen crosslinking and work-to-fracture/toughness measurements whilst correlations between general NEG and measures of ductility are often negative. Fifteen bone beam triplets were cut from bovine metatarsi. Each provided one native non-incubated control, one incubated control and one ribated specimen. Incubation involved simulated body fluid±ribose for fourteen days at 37°C. Pentosidine and pyridinoline crosslinks were measured using HPLC. Three-point bending tests quantified mechanical properties. Fracture surfaces were examined using scanning electron microscopy. The effects of ribation on bone collagen molecular stability and intermolecular connectivity were investigated using differential scanning calorimetry and hydrothermal isometric tension testing. Ribation caused increased non-enzymatic collagen modification and pentosidine content (16mmol/mol collagen) and inferior post-yield mechanical behaviour, especially post-yield strain and flexural toughness. Fracture surfaces were smoother with less collagen fibril deformation or tearing than observed in controls. In the ribated group only, pentosidine content and thermomechanical measures of crosslinking were positively correlated with measures of strain accommodation and energy absorption before failure. Non-enzymatic ribation and the resulting modifications reduce cortical bone pseudo-plasticity through a reduced capacity for post-yield strain accommodation. However, the positive correlations we have found suggest that increased crosslinking may not provide a complete explanation for this embrittlement., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Excessive systemic exposure to fluoride (F) can lead to disturbances in bone homeostasis and dental enamel development. We have previously shown strain-specific responses to F in the development of dental fluorosis (DF) and in bone formation/mineralization. The current study was undertaken to further investigate F responsive variations in bone metabolism and to determine possible relationships with DF susceptibility. Seven-week-old male mice from FVB/NJ, C57BL/6J, C3H/HeJ, A/J, 129S1/SvImJ, AKR/J, DBA/2J, and BALB/cByJ inbred strains were exposed to NaF (0 or 50 ppm as F(-)) in drinking water for 60 days. Sera were collected for F, Ca, Mg, PO(4), iPTH, sRANKL, and ALP levels. Bone marrow cells were subjected to ex vivo cell culture for osteoclast potential and CFU colony assays (CFU-fibroblast, CFU-osteoblast, CFU-erythrocyte/granulocyte/macrophage/megakaryocyte, CFU-granulocyte/macrophage, CFU-macrophage, and CFU-granulocyte). Femurs and vertebrae were subjected to micro-CT analyses, biomechanical testing, and F, Mg, and Ca content assays. DF was evaluated using quantitative fluorescence and clinical criteria. Strain-specific responses to F were observed for DF, serum studies, ex vivo cell culture studies, and bone quality. Among the strains, there were no patterns or significant correlations between DF severity and the actions of F on bone homeostasis (serum studies, ex vivo assays, or bone quality parameters). The genetic background continues to play a role in the actions of F on tooth enamel development and bone homeostasis. F exposure led to variable phenotypic responses between strains involving dental enamel development and bone metabolism., (Copyright © 2011 S. Karger AG, Basel.)

Municipal water fluoridation has notably reduced the incidence of dental caries and is widely considered a public health success. However, ingested fluoride is sequestered into bone, as well as teeth, and data on the long-term effect of exposure to these very low doses of fluoride remain inconclusive. Epidemiological studies suggest that effects of fluoride on bone are minimal. We hypothesized that the direct measurement of bone tissue from individuals residing in municipalities with and without fluoridated water would reveal a relationship between fluoride content and structural or mechanical properties of bone. However, consonant with the epidemiological data, only a weak relationship among fluoride exposure, accumulated fluoride, and the physical characteristics of bone was observed. Analysis of our data suggests that the variability in heterogenous urban populations may be too high for the effects, if any, of low-level fluoride administration on skeletal tissue to be discerned.

Kidney stone patients often have a decrease in BMD. It is unclear if reduced BMD is caused by a primary disorder of bone or dietary factors. To study the independent effects of hypercalciuria on bone, we used genetic hypercalciuric stone-forming (GHS) rats. GHS and control (Ctl) rats were fed a low Ca (0.02% Ca, LCD) or a high Ca (1.2% Ca, HCD) diet for 6 wk in metabolic cages. All comparisons are to Ctl rats. Urine Ca was greater in the GHS rats on both diets. GHS fed HCD had reduced cortical (humerus) and trabecular (L(1)-L(5) vertebrae) BMD, whereas GHS rats fed LCD had a reduction in BMD similar to Ctl. GHS rats fed HCD had a decrease in trabecular volume and thickness, whereas LCD led to a approximately 20-fold increase in both osteoid surface and volume. GHS rats fed HCD had no change in vertebral strength (failure stress), ductibility (failure strain), stiffness (modulus), or toughness, whereas in the humerus, there was reduced ductibility and toughness and an increase in modulus, indicating that the defect in mechanical properties is mainly manifested in cortical, rather than trabecular, bone. GHS rat cortical bone is more mineralized than trabecular bone and LCD led to a decrease in the mineralization profile. Thus, the GHS rats, fed an ample Ca diet, have reduced BMD with reduced trabecular volume, mineralized volume, and thickness, and their bones are more brittle and fracture prone, indicating that GHS rats have an intrinsic disorder of bone that is not secondary to diet.

Current clinical tools for evaluating fracture risk focus only on the mineral phase of bone. However, changes in the collagen matrix may affect bone mechanical properties, increasing fracture risk while remaining undetected by conventional screening methods such as dual energy x-ray absorptiometry (DXA) and quantitative ultrasound (QUS). The mechanical response tissue analyzer (MRTA) is a non-invasive, radiation-free potential clinical tool for evaluating fracture risk. The objectives of this study were two-fold: to investigate the ability of the MRTA to detect changes in mechanical properties of bone as a result of treatment with 1 M potassium hydroxide (KOH) and to evaluate the differences between male and female bone in an emu model. DXA, QUS, MRTA and three-point bending measurements were performed on ex vivo emu tibiae before and after KOH treatment. Male and female emu tibiae were endocortically treated with 1 M KOH solution for 1-14 days, resulting in negligible collagen loss (0.05%; by hydroxyproline assay) and overall mass loss (0.5%). Three-point bending and MRTA detected significant changes in modulus between days 1 and 14 of KOH treatment (-18%) while all values measured by DXA and QUS varied by less than 2%. This close correlation between MRTA and three-point bending results support the utility of the MRTA as a clinical tool to predict fracture risk. In addition, the significant reduction in modulus contrasted with the negligible amount of collagen removal from the bone after KOH exposure. As such, the significant changes in bone mechanical properties may be due to partial debonding between the mineral and organic matrix or in situ collagen degradation rather than collagen removal. In terms of sex differences, male emu tibiae had significantly decreased failure stress and increased failure strain and toughness compared to female tibiae with increasing KOH treatment time.

Introduction: Shwachman-Diamond syndrome (SDS) is an autosomal recessive disorder characterized by exocrine pancreatic insufficiency and bone marrow dysfunction. These result in malabsorption and hematological abnormalities. A skeletal dysplasia is also an integral feature of SDS. The present study assessed prevalence and determinants of osteopenia and osteoporosis in patients with SDS and disease-causing mutations in the SBDS gene., Materials and Methods: Eleven patients (8 males) aged from 5 to 37 years (median 16.7 years) with a genetically confirmed diagnosis of SDS were assessed for fracture history, bone mineral content (BMC), lean tissue mass (LTM) and bone mineral density (BMD) (Hologic Discovery A), osteoporotic vertebral changes, and for blood biochemistry and hematological parameters. Iliac crest bone biopsies were obtained from four patients for histology and histomorphometry., Results: The main findings were: (1) markedly reduced BMD Z-scores at the lumbar spine (median -2.1, range -4.4 to -0.8), proximal femur (median -1.3, range -2.2 to -0.7) and, whole body (median -1.0, range -2.8 to +0.6), and reduced Z-scores for height-adjusted BMC/LTM ratio (median -0.9, range -3.6 to +1.1); (2) vertebral compression fractures in three patients; and (3) blood biochemistry suggestive of mild vitamin D and vitamin K deficiency. Bone biopsies in four patients showed significant low-turnover osteoporosis with reduced trabecular bone volume, low numbers of osteoclasts and osteoblasts, and reduced amount of osteoid., Conclusions: The results suggest that in addition to the skeletal dysplasia, SDS is associated with a more generalized bone disease characterized by low bone mass, low bone turnover and by vertebral fragility fractures. Osteoporosis may result from a primary defect in bone metabolism, and could be related to the bone marrow dysfunction and neutropenia.

Fractography has not been fully developed as a useful technique in assessing failure mechanisms of bone. While fracture surfaces of osteonal bone have been explored, this may not apply to conventional mechanical testing of mouse bone. Thus, the focus of this work was to develop and evaluate the efficacy of a fractography protocol for use in supplementing the interpretation of failure mechanisms in mouse bone. Micro-computed tomography and three-point bending were performed on femora of two groups of 6-month-old mice (C57BL/6 and a mixed strain background of 129SV/C57BL6). SEM images of fracture surfaces were collected, and areas of "tension", "compression" and "transition" were identified. Percent areas of roughness were identified and estimated within areas of "tension" and "compression" and subsequently compared to surface roughness measurements generated from an optical profiler. Porosity parameters were determined on the tensile side. Linear regression analysis was performed to evaluate correlations between certain parameters. Results show that 129 mice exhibit significantly increased bone mineral density (BMD), number of "large" pores, failure strength, elastic modulus and energy to failure compared to B6 mice (p<0.001). Both 129 and B6 mice exhibit significantly (p<0.01) more percent areas of tension (49+/-1%, 42+/-2%; respectively) compared to compression (26+/-2%, 31+/-1%; respectively). In terms of "roughness", B6 mice exhibit significantly less "rough" areas (30+/-4%) compared to "smooth" areas (70+/-4%) on the tensile side only (p<0.001). Qualitatively, 129 mice demonstrate more evidence of bone toughening through fiber bridging and loosely connected fiber bundles. The number of large pores is positively correlated with failure strength (p=0.004), elastic modulus (p=0.002) and energy to failure (p=0.041). Percent area of tensile surfaces is positively correlated with failure strength (p<0.001), elastic modulus (p=0.016) and BMD (p=0.037). Percent area of rough compressive surfaces is positively correlated with energy to failure (p=0.039). Evaluation of fracture surfaces has helped to explain why 129 mice have increased mechanical properties compared to B6 mice, namely via toughening mechanisms on the compressive side of failure. Several correlations exist between fractography parameters and mechanical behavior, supporting the utility of fractography with skeletal mouse models.

Introduction: The influence of genetic background on bone architecture and mechanical properties is well established. Nevertheless, to date, only few animal studies explore an underlying genetic basis for extrinsic factors effect such as fluoride effect on bone metabolism., Materials and Methods: This study assessed the effect of increasing fluoride doses (0 ppm, 25 ppm, 50 ppm, 100 ppm) on the bone properties in 3 inbred mouse strains that demonstrate different susceptibilities to developing enamel fluorosis (A/J a "susceptible" strain, 129P3/J a "resistant" strain and SWR/J an "intermediate" strain). Fluoride concentrations were determined in femora and vertebral bodies. Bone mineral density was evaluating through DEXA. Finally, three-point bend testing of femora, compression testing of vertebral bodies and femoral neck-fracture testing were performed to evaluate mechanical properties., Results: Concordant with increasing fluoride dose were significant increases of fluoride concentration in femora and vertebral bodies from all 3 strains. Fluoride treatment had little effect on the bone mineral densities (BMD) in the 3 strains. Mechanical testing showed significant alterations in "bone quality" in the A/J strain, whereas moderate alterations in "bone quality" in the SWR/J strain and no effects in the 129P3/J strain were observed., Conclusion: The results suggest that genetic factors may contribute to the variation in bone response to fluoride exposure and that fluoride might affect bone properties without altering BMD.

Vanadium-based drugs lower glucose by enhancing the effects of insulin. Oral vanadium drugs are being tested for the treatment of diabetes. Vanadium accumulates in bone, though it is not known if incorporated vanadium affects bone quality. Nine- to 12-month-old control and streptozotocin-induced diabetic female Wistar rats were given bis(ethylmaltolato)oxovanadium(IV) (BEOV), a vanadium-based anti-diabetic drug, in drinking water for 12 weeks. Non-diabetic rats received 0, 0.25 or 0.75 mg/ml BEOV. Groups of diabetic rats were either untreated or treated with 0.25-0.75 mg/ml BEOV as necessary to lower blood glucose in each rat. In diabetic rats, this resulted in a Controlled Glucose group, simulating relatively well-managed diabetes, and an Uncontrolled Glucose group, simulating poorly managed diabetes. Plasma insulin, glucose and triglyceride assays assessed the diabetic state. Bone mineral density (BMD), mechanical testing, mineral assessment and histomorphometry measured the effects of diabetes on bone and the effects of BEOV on non-diabetic and diabetic bone. Diabetes decreased plasma insulin and increased plasma glucose and triglycerides. In bone, diabetes decreased BMD, strength, mineralization, bone crystal length, and bone volume and connectivity. Treatment was effective in incorporating vanadium into bone. In all treated groups, BEOV increased osteoid volume. In non-diabetic bone, BEOV increased cortical bone toughness, mineralization and bone formation. In controlled glucose rats, BEOV lowered plasma glucose and improved BMD, mechanical strength, mineralization, bone crystal length and bone formation rate. In poorly controlled rats, BEOV treatment slightly lowered plasma glucose but did not improve bone properties. These results suggest that BEOV improves diabetes-related bone dysfunction primarily by improving the diabetic state. BEOV also appeared to increase bone formation. Our study found no negative effects of vanadium accumulation in bone in either diabetic or non-diabetic rats at the dose given.

Skeletal fluorosis and dental fluorosis are diseases related to fluoride (F) ingestion. Bone is the largest storage site of F in our body. Therefore, bone F concentrations are considered biomarkers for total F body burden (exposure). However, difficult accessibility limits its use as a biomarker. Thus, a more accessible tissue should be considered and analyzed as a biomarker for total F body burden. The objective of this study, which was divided into two parts, was to evaluate teeth as a biomarker for skeletal F exposure. In part 1 of the study, 70 mice of three different strains (SWR/J, A/J and 129P3/J) were exposed to different levels of water fluoridation (0, 25, 50 and 100 ppm). Bone (femora and vertebrae) and teeth from these mice were then analyzed for F concentration using Instrumental Neutron Activation Analysis (INAA). In part 2 of the study, human teeth (enamel and dentin) and bone from 30 study subjects were collected and analyzed for F concentration using INAA. Study subjects lived in areas with optimum levels of water fluoridation (0.7 and 1 ppm) and underwent therapeutic extraction of their unerupted third molars. The values of bone and teeth F concentration were correlated for parts 1 and 2 of this study. The results showed that in the animal model, where animals were exposed to a wide range of F in their drinking water, tooth [F] correlated with bone [F]. However, no correlation was seen between bone and enamel F concentrations or between bone and dentin F concentrations in the human samples. Therefore, teeth are not good biomarkers for skeletal F exposure in humans when exposure is confined to optimum levels of F in the drinking water.

B7-H3, a member of the B7 family of the Ig superfamily proteins, is expressed on the surface of the antigen-presenting cells and down-regulates T cell functions by engaging an unknown counterreceptor on T cells. Although B7-H3 is ubiquitously expressed, its potential nonimmune functions have not been addressed. We found that B7-H3 is highly expressed in developing bones during embryogenesis and that its expression increases as osteoblast precursor cells differentiate into mature osteoblasts. In vitro bone formation by osteoblastic cells was inhibited when B7-H3 function was interrupted by the soluble recombinant protein B7-H3-Fc. Analysis of calvarial cells derived from neonatal B7-H3 knockout (KO) mice revealed normal numbers of osteoblast precursor cells possessing a normal proliferative capacity. However, the B7-H3-deficient calvarial cells exhibited impaired osteogenic differentiation, resulting in decreased mineralized bone formation in vitro. These results suggest that B7-H3 is required for the later phase of osteoblast differentiation. Although B7-H3 KO mice had no gross skeletal abnormalities, they displayed a lower bone mineral density in cortical (but not trabecular) bones compared with WT controls. Consistent with the reduced bone mineral density, the femurs of B7-H3 KO mice were more susceptible to bone fracture compared with those of WT mice. Taken together, these results indicate that B7-H3 and its unknown counterreceptor play a positive regulatory role in bone formation. In addition, our findings identified B7-H3 as another molecule that has a dual role in the bone-immune interface., (Copyright 2004 The National Academy of Sciencs of the USA)

Introduction: Trace elements are known to influence bone metabolism; however, their effects may be exacerbated in renal failure because dialysis patients are unable to excrete excess elements properly. Our study correlated bone quality in dialysis patients with levels of bone fluoride, magnesium, and aluminum. A number of studies have linked trace elements, including fluoride, magnesium, and aluminum, to the development of renal osteodystrophy (ROD). However, little is known about the relationship between trace elements and changes in bone quality in ROD patients. The purpose of this study was to examine bone quality in ROD patients, and correlate differences in bone quality to trace element concentrations in bone. Bone quality encompasses parameters that contribute to the mechanical integrity of the bone., Methods: One hundred fifty-three anterior iliac crest bone biopsies from patients with ROD were examined and subdivided into five groups based on the pathological features. Parameters contributing to bone quality, such as bone structure and remodeling, connectivity, mineralization, and microhardness, were assessed and correlated to bone chemical composition. In addition, clinical symptoms of ROD were assessed and correlated with bone composition., Results and Conclusions: There were no differences in bone architecture between the different ROD bone groups; however, differences in bone mineralization and microhardness were observed. Increase in bone fluoride was associated with increased osteoid parameters and decreased bone microhardness. Bone mineralization and microhardness decreased with increasing bone magnesium content and intact parathyroid hormone (PTH) level. Moreover, bone magnesium increased with intact PTH levels. The relationship between PTH, bone magnesium, mineralization, and microhardness was primarily observed in aplastic bone disorder. Furthermore, bone magnesium and aluminum contents were positively associated with bone pain and proximal myopathy in these patients. Most importantly, fluoride, magnesium, and aluminum showed significant correlations with one another. These results suggested that in ROD, bone fluoride may diminish bone microhardness by interfering with mineralization. Magnesium may be involved in the suppression of PTH secretion, lowering bone turnover thus leading to an increase in bone mineralization profile and microhardness in aplastic bone disorder. The effects of fluoride and magnesium on bone quality may be exacerbated by their interaction with aluminum.

Several proteins in the bone matrix have been implicated in the regulation of mineral crystal formation and growth. To investigate the relationships between these proteins and the mineral phase at various stages of mineral maturation, fetal porcine calvariae and long bones were fragmented and the particles (20 microm) separated by density gradient sedimentation into fractions of increasing density (1.8 to >2.2 g/cm3). Samples from each fraction were analyzed by X-ray diffraction to obtain the average crystal size/strain and chemical composition. Other samples were sequentially extracted, first with 4.0 mol/L guanidium hydrochloride (GuHCl) (G1), then with 0.5 mol/L ethylene-diamine tetraacetic acid (EDTA) (E), and again with 4.0 mol/L Gu-HCI (G2), for analysis of proteins in different tissue compartments. Based on the mineral density distribution and crystal size, fetal porcine bone protein content was determined for tissue residue and each extract and the protein composition analyzed by sodium dodecyl-polyacrylamide gel electrophoresis (SDS-PAGE). Although the insoluble organic matrix decreased with mineral density the collagen and protein content remained fairly constant, representing approximately 10% of the tissue weight, except in the highest density fraction. Whereas the total extractable protein, representing predominantly noncollagenous proteins, did not show density-related differences, differences were observed for individual proteins on SDS-PAGE. Consistent with their presence in osteoid, the content of bone sialoprotein (BSP), tyrosine-rich acidic matrix protein (TRAMP), and a series of small proteins with cell attachment properties in the G1 extract decreased with mineral density, whereas TRAMP and BSP were increased in G2 extracts. Mineral-associated proteins, including alpha2HS-glycoprotein, BSP, osteopontin (OPN), and osteocalcin, increased with mineral density, whereas secreted protein acidic and rich in cysteine (SPARC)/osteonectin, and some minor proteins, appeared to decrease. Differences of individual proteins within and between the calvarial and long bones could be related to the role of these proteins in the formation and maturation of hydroxyapatite crystals. Collectively, these studies demonstrate mineral density-associated changes in protein composition that reflect a rapid maturation of mineral crystals in embryonic porcine bones.

The purpose of this study was to evaluate the effects of ovariectomy on the mechanical properties of bone in the aged Sprague-Dawley rat model of osteoporosis. Eight female rats were sacrificed at the start of the study, at the age of four months. Twenty-four remaining rats were then bilaterally ovariectomized (OVX), and another twenty-four served as controls. Eight rats from each group were sacrificed at five, ten or fifteen weeks. The mean density of L1 vertebral bodies from OVX rats was lower than in their control counterparts, as was the compressive modulus, the ultimate compressive stress and the toughness. The second lumbar vertebra from each rat was cored to remove the bulk of the cancellous bone and tested in compression to failure. Ovariectomy did not affect the mechanical properties of the cored vertebrae, suggesting that the mechanically relevant changes take place in the cancellous bone. The gravimetric density and bone mineral density (measured by dual-energy x-ray absorptiometry) of whole femora were lower in OVX rats than control rats. However, the gravimetric density of the cortical bone was unchanged, and when femora were tested to fracture in three-point bending, the mechanical properties of the midshaft were found to be unaffected. This study suggests some limitations to the mature ovariectomized rat model of osteoporosis: mechanically significant loss of cancellous bone occurs in vertebrae much more rapidly than suggested by other studies. Further, there is little evidence of mechanically important bone loss in cortical bone within four months of ovariectomy.

The goal of this study was to determine the effects of chronically elevated blood androstenedione and estrone levels on the quality and quantity of both cancellous (trabecular) and cortical bone in a young (mean age 9.4 years) female primate model (M. fascicularis). Thirteen intact female monkeys received continuous androstenedione/estrone supplementation via subcutaneous implants over a 24-month period to simulate the human condition known as polycystic ovarian disease (PCOD). A group of 16 untreated intact age-matched female monkeys served as controls. Lumbar spine and whole body bone mineral density (BMD) status was determined mid-study by dual photon absorptiometry (DPA); subsequent analysis of the bone related to data obtained following the 2-year treatment period without further BMD measurement. Bone markers, including serum acid phosphatase, total bone alkaline phosphatase, bone gla protein and tartrate-resistant acid phosphatase were measured at the end of the study. At necropsy, the lumbar vertebrae and femora were recovered in order to analyze the bone mineral quality and quantity of cancellous and cortical bone respectively and to compare these with the control group. Mineralization profiles of the vertebrae and femora were obtained using the density fractionation technique. Chemical analysis of the three largest fractions retrieved by density fractionation was performed to evaluate differences in %Ca, %P, Ca/P ratio and mineral content (%Ca + %PO4) between control and experimental groups. In addition, unfractionated bone powder was examined by X-ray diffraction to identify any changes in crystal size. Coronal sections of vertebrae were analyzed for structural parameters using histomorphometry and image analysis. Cross-sections taken at the midshaft diaphyseal femora were analyzed for structural macroscopic and intracortical parameters. There was a significant increase in BMD at the L2-L4 region in the treatment group compared with the control groups (p < 0.005) as measured at 1 year into the trial. Serum acid phosphatase was significantly lower (p < 0.05) in the treatment group compared with the controls near study termination. A nonsignificant shift in the mineralization profile of the vertebrae towards less dense bone was observed in the treatment group, while there was a significant shift in the mineralization profile towards more dense bone in the treated femora compared with controls (p < 0.05) after a 2-year period. There was no difference between treatment and control groups in terms of size/strain of the cortical or cancellous bone crystal as detected by X-ray diffraction. There was a significant increase in cancellous bone area (B.Ar.) (p < 0.02) and a significant increase (p < 0.05) in mean trabecular width with a corresponding decrease in trabecular separation (p < 0.03) in the experimental group compared with the controls. There were no significant changes in osteoid parameters (perimeter, area or width) or eroded perimeter measurements in the experimental group compared with the controls. In the experimental group, trabecular strut analysis showed a significant increase in the number of nodes (p < 0.02) and in the total strut length (p < 0.003) compared with the controls. There was also a significant increase in the node to node (p < 0.04) and node to terminus (p < 0.004) strut length in the treatment group compared with the controls. A significant increase in B.Ar. without concurrent indices of ongoing remodelling differing from controls suggests that cancellous bone of the vertebral body in the treated young female primate had been receptive to the anabolic stimulus of androstenedione/estrone supplementation over the 2-year period. In contrast, macroscopic parameters of cortical bone such as perimeter, area and width were preserved over the 2-year course, while intracortical remodeling was evident with increased percent porosity (p < 0.001), osteonal bone (p < 0.01) and osteonal density (p < 0.01) observed in the treatment group compared with the controls. The endocrine profile of both elevated androstenedione and estrone levels in an intact female primate of reproductive age may identify differential effects of the condition known as polycystic ovarian disease on the skeletal compartments.

We hypothesized that fluoride partly acts by changing the levels of circulating calcium-regulating hormones and skeletal growth factors. The effects of oral fluoride on 24 female, Dutch-Belted, young adult rabbits were studied. The rabbits were divided into two study groups, one control and the other receiving about 16 mg fluoride/rabbit/day in their drinking water. After 6 months of fluoride dosing, all rabbits were euthanized and bone and blood samples were taken for analyses. Fluoride treatment increased serum and bone fluoride levels by over an order of magnitude (P < 0.001), but did not affect body weight or the following serum biochemical variables: urea, creatinine, phosphorus, total protein, albumin, bilirubin, SGOT, or total alkaline phosphatase. No skeletal fluorosis or osteomalacia was observed histologically, nor did fluoride affect serum PTH or Vitamin D metabolites (P > 0.4). BAP was increased 37% (P < 0.05) by fluoride; serum TRAP was increased 42% (P < 0.05); serum IGF-1 was increased 40% (P < 0.05). Fluoride increased the vertebral BV/TV by 35% (P < 0.05) and tibial ash weight by 10% (P < 0.05). However, the increases in bone mass and bone formation were not reflected in improved bone strength. Fluoride decreased bone strength by about 19% in the L5 vertebra (P < 0.01) and 25% in the femoral neck (P < 0. 05). X-ray diffraction showed altered mineral crystal thickness in fluoride-treated bones (P < 0.001), and there was a negative association between crystal width and fracture stress of the femur (P < 0.02). In conclusion, fluoride's effects on bone mass and bone turnover were not mediated by PTH. IGF-1 was increased by fluoride and was associated with increased bone turnover, but was not correlated with bone formation markers. High-dose fluoride treatment did not improve, but decreased, bone strength in rabbits, even in the absence of impaired mineralization.

Therapeutic efficacy of bisphosphonates depends on how much drug is present in bone. Therefore, it is important to measure the amount of bone bisphosphonate in toxicological and pharmacological studies. We analyzed pamidronate content of bone samples from two previously published long-term dose studies. In the first study, mature beagle dogs were given oral pamidronate at doses of 0, 2.5, 12.5 and 25 mg/kg per day for 1 year. The dogs in the second study received the same dosages for 1 year followed by 1 year without drug. In both studies, the amount of pamidronate measured was dependent upon dose in bone samples of ilium, sternum, and vertebral body. After 1 year of treatment, vertebral bone had more pamidronate than sternum or iliac bone on a permilligram basis. After 2 years, there was significantly less pamidronate in the vertebral body, sternum, and ilium than there had been at 1 year. The fall in pamidronate content was largest in vertebral body and least in the ilium. The higher uptake of pamidronate by the vertebral body during the 1 year study, and its greater loss of pamidronate after a year without drug treatment, would reflect the higher turnover of trabecular bone in the vertebral body vs. cortical bone in the ilium. The percent difference in bone pamidronate content between the 1 and 2 year dogs varied with dose. The largest percent loss occurred in the intermediate-dose group. These data suggest that, after 1 year without the drug, bone turnover rates in dogs treated with the highest dose of pamidronate were lower than at the intermediate dose. Thus, the time required for bone turnover rates to return to pretreatment levels is probably dose-dependent.

The goal of this study was to evaluate the effects of chronically-elevated male levels of the potent androgen testosterone on the quality and quantity of both cancellous and cortical bone in a young (mean age 8.0 years), nonhuman female primate model (M. fascicularis). Thirteen intact female monkeys received continuous testosterone supplementation via subcutaneous implants over a 24-month period. A group of 16 untreated, intact, age-matched female monkeys served as controls. At sacrifice, the lumbar vertebrae and femora were recovered in order to analyze the bone mineral quality and quantity of cancellous and cortical bone, respectively, and compared to the control group. Mineralization profiles of the vertebrae and femora were obtained using the density fractionation technique. Chemical analysis of the three largest fractions retrieved by density fractionation was performed to evaluate differences in %Ca, %P, Ca/P ratio, and mineral content (%Ca + %PO4) between the control and experimental groups. In addition, unfractionated bone powder was examined by X-ray diffraction to identify any changes in crystal size. Coronal sections of vertebrae were analyzed for structural parameters using histomorphometry and image analysis. Cross sections taken at the midshaft diaphyseal femora were analyzed for structural macroscopic and intracortical parameters. A nonsignificant shift in the mineralization profile of the vertebrae was observed whereas there was a significant shift in the mineralization profile towards more dense bone in the treated femora as compared with controls (P < 0.05). There was no difference in terms of size/strain of the cortical or cancellous bone crystal as detected by X-ray diffraction. There was a trend towards an increase in cancellous bone area (B.Ar.) in the testosterone-treated vertebrae (P = 0.08) as compared with controls. The architecture of the cancellous bone remained nonsignificantly different between the treatment and control groups as evaluated by image analysis. There was a decrease in osteoid perimeter (P = 0.05) in the experimental group as compared with controls. There was a significant decrease in eroded perimeter measurements in the experimental group as compared with controls (P < 0.03). Although there was a trend towards an increase in cancellous bone area, mineralization was not significantly different in the vertebrae of testosterone-treated female monkeys, indicating that the newly-formed bone tissue became relatively normally mineralized over the two-year period. An increase in bone area, with indices of an overall decreased remodelling pattern as compared with controls, suggests that cancellous bone in the young, nonhuman female primate had been receptive to supraphysiologic levels of testosterone supplementation over the two-year period. There was a trend for an increase in cortical bone area and width with an increased periosteal perimeter in the testosterone-treated group as compare with controls. There was an increase in intracortical remodelling activity with a significant increase in percent porosity (P < 0.05), osteonal bone (P < 0.05), and mean wall width (P < 0.05) in the testosterone-treated group. In conclusion, the cancellous bone from female monkeys appeared to respond to the antiresorptive stimulus of male levels of testosterone with significantly diminished turnover parameters in this compartment. In contrast, the cortical bone compartment responded by displaying significant intracortical remodelling over a two-year period.

Background: The study of osteoporosis in past populations offers valuable insight into the patterns and prevalence of the disease in both the past and in the present., Methods and Results: A review is made of different paleopathological studies better to understand bone loss in past populations and to examine the contribution such studies can make to our knowledge of osteoporosis in modern populations. The review includes studies of bone mass in past populations from different geographic regions. Nutritionally based hypotheses, traditionally used to explain bone loss in past populations, are reviewed and assessed against the current clinical and epidemiological findings. In general, the various studies have revealed different degrees of low bone mass in past populations; however, the pattern of bone loss and fragility seen in age-related and postmenopausal osteoporosis today is not evident in the past. Bone loss in earlier populations is often found in both sexes, whereas significant bone loss in females occurs often among the young-age categories. In addition, a prevalence of osteoporotic fracture is absent., Conclusions: We suggest that, despite influences that may have reduced bone mass in past populations, a protection of bone quality may have occurred, reducing the likelihood of bone fragility and fracture typically seen in modern osteoporotics. It seems evident that, although reduced bone mass may be prevalent in past history, clinically recognized osteoporosis is not.

Feral adult female cynomolgus monkeys were divided into three groups and treated for two years: (1) normal controls; (2) weak androgenic treatment (androstenedione+estrone); and (3) strong androgenic treatment (testosterone). The tibiae and the trabecular bone of femoral head from each group were tested mechanically. There were no significant changes in the elastic modulus and shear modulus of the tibiae, measured by three point bending and torsion tests, among the three groups. Significant increases in energy absorption capacity (+45% for testosterone) and maximum shear stress (+19.4% for androstenedione and +39% for testosterone) of the tibiae, measured by torsion tests, and the cortical bone density (+5.5% for androstenedione and +8.7% for testosterone), were observed. Testosterone treatment significantly increased torsional rigidity (+23%) and bending stiffness (+15%) of the tibiae while androstenedione did not change any of these structural properties. The results of compression tests of the trabecular bone samples indicated significant increases in their elastic modulus after androstenedione (+88%) or testosterone (+107%) treatment. The maximum compressive stress of the testosterone treated samples was significantly higher than those of both normal (+28%) and androstenedione treated groups (+26%). The trabecular bone density increased after both androgenic treatments. This increase was significant for the testosterone treated group (+8.6%). We conclude that in the young cynomolgus monkey, long-term androgenic treatment significantly improves some of the mechanical properties of both cortical and trabecular bones, increases bone density, and the stronger the androgen, likely, the more pronounced is the effect.

The mechanical properties of cortical and trabecular bones from beagles treated with the bisphosphonate pamidronate (administered intermittently 1 week every month for 3 months, at a dosage of 0.45 mumol/kg/day) were assessed. The mechanical properties of cortical bone were measured by four-point bending tests on femoral quadrants, in order to measure their elastic modulus and ultimate stress. The structural properties of whole tibias were measured in torsion to determine the torsional stiffness and failure torque. The elastic modulus and maximum compressive stress of the trabecular bone samples were measured by compression tests of trabecular cores. Intermittent treatment with pamidronate did not change the pattern of mechanical properties that occurs naturally around the femur or the torsional stiffness and failure torque of the tibias. By contrast, pamidronate did significantly increase the modulus of elasticity (by 19%) and maximum compressive stress (by 33%) of vertically aligned cylindrical trabecular specimens taken from the vertebrae of the beagles.

To evaluate the accuracy of bone mineral composition determination by electron microprobe analysis (EDX) the measurements have been compared to instrumental neutron activation analysis (INAA) and chemical analysis (ICPES). Bone specimens from five femoral heads were used. The trabecular content of calcium (Ca) and phosphorus (P) was analyzed by the three different methods. The EDX method allows for a microstructural analysis of intact, methylmetacrylate-embedded, undecalcified bone and the measuring points can thus be distinctly identified centrally in each trabecula. The analysis yielded 25.8 +/- 0.7 wt % Ca and 10.5 +/- 0.1 wt % P, compared with 22.2 +/- 0.5 and 23.0 +/- 1.0 wt % Ca, and 9.83 +/- 0.21 and 10.02 +/- 0.44 wt % P for INAA and ICPES, respectively. The EDX analysis was calibrated by consecutive measurements of a hard, pressed tablet of hydroxyapatit of known content. The mean Ca content deviated with -0.38 wt % from the given content and P with -0.89 wt %. We could not verify any particular interference from the embedding procedure, however, it is possible that the relatively lower P content still may reflect this. The magnesium (Mg) concentration was 0.31 +/- 0.02 wt % by EDX and 0.26 +/- 0.02 wt % by INAA. The EDX analytical method provides a useful tool for simultaneous elemental quantification in bone. It has the advantage of permitting the use of regular bone biopsy material and thus allowing for a unique microstructural evaluation of the degree of mineralization.(ABSTRACT TRUNCATED AT 250 WORDS)

Feral adult female cynomolgus monkeys were divided into two groups: normal controls and ovariectomized. Tibiae and trabecular bones from the femoral head, from each group, were tested using a materials testing machine. The bending stiffness of the tibiae was measured by nondestructive three-point bending tests and their maximum torque capacity by destructive torsion tests. The compressive strength of the trabecular bones was measured by compression tests. Ovariectomy caused significant decreases in elastic modulus of the tibiae (p < 0.008), measured by three-point bending tests, and in shear modulus (p < 0.015), failure shear stress (p < 0.01), and failure torque (p < 0.001) of the tibiae, measured by torsion tests. It caused a significant decrease in cortical bone density (p < 0.005), but no significant changes in tibial cross-sectional area and in cortical shaft external and internal diameters. The differences in elastic modulus, maximum compressive strength, and density of femoral trabecular bone samples between the two groups were not significant.

In vitro cultured neonatal mouse calvariae release calcium and buffer the medium proton concentration in response to a decrease in the medium pH caused by a reduction in bicarbonate concentration ([HCO3-]), a model of metabolic acidosis, but not to an equivalent decrease in pH caused by an increase in the partial pressure of carbon dioxide (PCO2), a model of respiratory acidosis. We have postulated that the medium is in equilibrium with the carbonated apatite in bone. To determine whether bone carbonate is depleted during models of acidosis, we cultured calvariae in control medium (pH approximately 7.4, PCO2 approximately 43, [HCO3-] approximately 26) or in medium in which the pH was equivalently reduced by either a decrease in [HCO3-] (metabolic acidosis, pH approximately 7.1, [HCO3-] approximately 13) or an increase in PCO2 (respiratory acidosis, pH approximately 7.1, PCO2 approximately 86) and determined net calcium flux (JCa) and bone carbonate content. We found that compared with control, after 3, 24, and 48 h there was a decrease in bone carbonate content during metabolic but not during respiratory acidosis. Compared with control, at 3 h JCa increased with both respiratory and metabolic acidosis; however, at 24 and 48 h JCa increased only with metabolic acidosis. JCa was correlated inversely with percent bone carbonate content in control and metabolic acidosis at all time periods studied (r = -0.809, n = 23, P < 0.001). Thus a model of metabolic acidosis appears to increase JCa from bone, perhaps due to the low [HCO3-] inducing bone carbonate dissolution.(ABSTRACT TRUNCATED AT 250 WORDS)

Bone mineralization changes with age and disease. The distribution of mineral particles in a given bone (mineralization profile) has been studied using density fractionation as well as microradiography and electron backscattering imaging. The biological determinant of mineralization is the rate of turnover. During rapid growth and periods of high remodeling, mineralization is shifted towards lower mineral density (hypomineralization). During aging and periods of low remodeling, mineralization is shifted towards higher mineral densities (hypermineralization). Chemicals can also influence the mineralization profile of bone. Fluoride induces hypermineralization by stabilizing the apatite lattice and reducing bone mineral solubility, whereas strontium induces hypomineralization by loosening the apatite lattice and increasing bone mineral solubility. Drugs such as bisphosphonates induce hypermineralization by inhibiting resorption and acting as crystal poison. Finally, mineralization can be impaired by defects as in rickets and osteomalacia or made excessive by continuous accretion of mineral without resorption as in osteopetrosis.

The goal of this study was to evaluate the effect of 1 year of APD administration on bone mineralization and bone mineral chemistry in the dog. Disodium pamidronate (APD) was given orally by gavage to mature beagle dogs at doses of 0, 2.5, 12.5 and 25.0 mg/kg per day (0.1% concentration for 12 months) as part of a long-term toxicity study. The os ilium and a vertebra were used to determine the mineralization profile and, subsequently, each density fraction was analysed chemically. The ribs were used to determine lattice parameters of the apatite crystal size using X-ray diffraction. The sternum was used to determine selected morphometric parameters using image analysis of specimen X-ray films and subsequently to determine mechanical properties using velocity-of-sound techniques. We found that for both the ilium and the vertebrae there was a significant shift of the mineralization profile towards greater density in a dose-related manner. This effect levelled off with the highest dose because the shift in mineralization profile correlated better with the square root of the dose than with the dose. Together with data on crystal size, which show an increase in lattice parameters and a decrease in crystal size with dose, our data lead us to believe that long-term administration of APD leads to an increase in bone mineralization without major changes in bone chemistry of Ca, Mg, and P and with a decrease in bone apatite crystal size. The image analysis shows a dose-related increase in trabecular bone volume and thickness.(ABSTRACT TRUNCATED AT 250 WORDS)

In order to investigate the effect of fluoride on bone mineral crystals, we gave groups of female rats 8 mM NaF/L water and distilled water to control groups. The rats were sacrificed at six weeks, three months, and six months. The fluor content of the bone was determined by neutron activation. X-ray diffraction showed no difference in bone crystal size/strain with fluoride treatment. Fourier transform Infrared Spectroscopy (FTIR) showed an increased crystallinity in fluoride-fed animals, which seems to be associated with a decrease of labile phosphate environment. Three carbonate bands have been found in fluoridated and normal bone samples. The distribution of carbonate ions on type A and B sites is strongly affected by fluoride. Type A carbonate is always present in bone, but decreases with increasing bone fluoride content. A carbonate band found at 866 cm-1 may correspond to a fluoride interaction with type B carbonate ions. Lastly, phosphate bands have been found to be shifted towards high wave number, which is probably related to the change in unit cell size induced by the fluoride ion. All these changes induced by fluoride reduce the solubility of bone crystals by direct incorporation of fluoride ions in the apatite lattice and by decreasing the labile phosphate environments.

Fluoride is known to have biological effects on bone cells as well as physicochemical effects on bone crystals. This review concentrates on the latter. Fluoride increases the stability of the apatite lattice and decreases the solubility of the apatite crystals. In bone mineral, this ion has been shown to affect bone crystal structure by increasing crystallinity and reducing specific surface area. These changes in turn lead to changes in the chemistry of bone mineral. Bone mineral deposition is delayed by fluoride. This ion does not diffuse into bone already formed, but is incorporated during mineralization. Subsequently fluoride tends to accumulate in the most highly mineralized bone. Bone treated with fluoride has been shown to be more resistant to acid dissolution than normal bone, which would explain the reduced rate of resorption of fluoridated bone. The distribution of fluoride in bone is not uniform, but its net effect is to increase bone mineral density probably by an increased packing of bone crystals. Finally, there is a debate as to whether fluoride produces a bone of different quality. Whether these changes in the quality of bone will prove to be helpful or harmful remain to be determined.

Strontium (Sr) has been shown to increase bone mass when given at low doses. In this study, the diets of rats containing 0.50% calcium were supplemented with Sr (0.19 and 0.40% of SrCl2 orally) for periods of four and eight weeks. Long bones and vertebrae were studied by density fractionation and each fraction was analyzed chemically. X-ray diffraction was used to determine crystal size. Static and dynamic histomorphometric parameters of bone formation were also measured. We found a shift towards lower density in the mineralization profiles of Sr supplemented rats (0.40%), as well as a decrease in bone crystal size at the larger dose of Sr. The CO3 content and the Ca/Sr ratio of the bone decreased with increasing Sr content. We found an increase in the vertebral trabecular bone volume together with an increase in osteoid volume in Sr supplemented rats. This study shows that Sr at the larger dose induces bone hypomineralization as well as an increase in bone mass in rats fed a relatively low calcium diet.

The effects of castration on cortical bone structure and chemistry were studied in male Wistar rats of two age groups, 8 and 24 months. Some rats were castrated and some were sham operated (control) in each group. All animals were killed after 4 months. Although no changes in serum chemistry, bone chemistry, or bone histology could be found, the young castrated animals versus controls showed less density of bone mineral as determined by the density fractionation technique and smaller crystallite size of mineral particles as determined by x-ray diffraction line-broadening analysis. These changes, indicative of less mature bone formation, were not observed in mature castrated or control rats despite a decreased amount of bone compared with young rats. In this model, castration appears to affect bone mineral quality in young but not in old animals.

We have reexamined the question of whether brushite (CaHPO4 X 2H2O) occurs in embryonic bone. On the basis of the experiments reported here, and a reexamination of data previously obtained in this laboratory, we have concluded that crystalline brushite as a separate phase identifiable by X-ray diffraction does not occur in embryonic chick or bovine bone. Crystalline brushite previously identified in this laboratory in the less-mineralized fractions of embryonic chick bone was apparently formed artifactually during the fractionation process, possibly from preexisting domains of HPO4(-2) groups in a noncrystalline brushite-like configuration.

The size of hydroxyapatite (HAP) crystals in calcified cartilage and cortical bone of the rat has been studied and compared with that of synthetic poorly crystalline hydroxyapatite (PCHA). Crystal size was determined by X-ray diffraction and selected-area dark field imaging, and their elemental compositions were determined by emission spectroscopy. The crystal size of cartilage, bone, and PCHA were found to be between 120 and 170 A in length by 50 A in width as determined by both X-ray diffraction and dark field imaging. Cartilage had a lower ash weight than bone but both have the same Ca/P ratio of 1.6. These findings, though in agreement with the X-ray diffraction literature, differ from observations made by conventional bright field electron microscopy. We conclude that mineral sites in cartilage and bone, which exist as platelike structures, are in fact aggregates of small 120-170 X 50 A HAP crystals. The consequences of these findings was discussed in view of crystal relationships with collagen and other macromolecules.

The status of bone mineral and osteocalcin in the young adult Rhesus monkey mandible was assessed following a 14-day period of postcranial immobilization, and after 7- and 28-day recovery periods. Specimens of cortical bone taken from the compact bone at the inferior border of the jaws were ground in liquid nitrogen and sieved to a particular size below 20 micron. The bone powder was then fractionated in a bromoform-toluene density gradient to determine its mineralization profile (Ca, P, CO3, and osteocalcin), and X-ray diffraction was used to determine apatite crystal size in some fractions. There was no change in the chemistry of the mandibular bone from the immobilized animals. However, the mineralization profile in that group showed a significant shift toward the higher density fractions, indicating the presence of a greater than normal content of mature well-mineralized bone. While this trend was accentuated in the jaws following a 7-day postimmobilization recovery period, partial recovery of the normal profile was observed after a 28-day recovery period. The osteocalcin profile shifted like the mineralization profile during the immobilization and recovery periods. X-ray diffraction analyses showed that the shift in the mineralization profile during the immobilization period was associated with a decrease in apatite crystal size.

55 femora of free ranging rhesus monkeys from the Caribbean Primate Research Center ranging in age from 2-25 years were analyzed using histology, density fractionation and chemical analysis and correlated with morphometric data. The vertebral density was determined by photon absorptiometry. 119 caudal vertebrae from the skeletal collection were analyzed chemically. Cortical mineral density and porosity increased with age, and vertebral density and cortical area increased with weight. There was significant sexual dimorphism in many histological variables reflecting size differences and cortical porosity was greater in males. The chemical analysis showed no influence of the diet on bone mineral but that Ca increase with age in females but not in males. Finally a subgroup of aged, animals had hypermineralized, and more porous bone. This subpopulation may prove to be a valuable model for senile osteoporosis.

The crystallinity of bone mineral at different stages of maturation has been measured by quantitative X-ray diffraction methods. Crystallinity measurements were made on tibial mid-diaphyses from 17-day embryonic chicks, newly-formed periosteal bone from embryonic chicks, and density-fractionated bone from post-hatch chickens from 5 weeks to 2 years of age. For a given animal age and degree of mineralization, crystallinity increases with animal age, indicating that changes in bone mineral occur even after mineralization is complete or nearly complete.

The relation between bone mineralization and glycosaminoglycans (GAG) was investigated in newborn and mature rabbit diaphyseal bone. Using the density fractionation technique, the bone was separated into fractions of increasing density from 1.4 to 2.3 grams/ml. Each fraction was analyzed by X-ray diffraction to determine the average crystal size. The GAG content of each fraction and of the unfractionated bone was determined by direct extraction and on a few fractions by sequential extraction in guanidine hydrochloride and guanidine/EDTA. There was a decrease in the GAG content with animal age and with increasing fraction density in the newborn rabbit. In one overlapping fraction (2.0-2.1 grams/ml), the GAG content was twice as high in the newborn as in the mature animal. Finally, the crystal size substantially increased from newborn to mature rabbits. Therefore, calcification and maturation of bone is associated with a decrease in the proteoglycan content of the organic matrix.

The maturation profiles of calcium, phosphorus, magnesium and hydroxyproline in the femoral trabecular and cortical bone and in the thoracic vertebrae from male rats flown on the 7-day 'Spacelab-3 Mission' were measured by density gradient analysis. In rats exposed to a spaceflight environment, profiles of the matrix and mineral moieties were shifted toward both the lower (vertebrae) and higher density fractions (femurs and vertebrae), patterns indicating a decrease in bone growth/turnover. X-Ray diffraction of vertebrae indicated that spaceflight is associated with a decrease in apatite crystal size/perfection.

This paper reviews the changes in the quality of bone mineral with age and in disease. After a brief review of morphological changes with aging in mammalian bones, microradiography is compared to backscattered electron imaging and their use in bringing out subtle changes in bone mineralization outlined. Changes in the quality of bone with disease is described using osteoporosis as an example. Chemical changes in the skeleton are then discussed and related to morphological changes. Finally, some examples of localized and generalized changes in bone mineral are given. This paper emphasizes that understanding the nature of the mineral phase in bone as well as its heterogeneity and its changes with age and in disease is essential to the elucidation of skeletal physiology and pathology.

X-ray diffraction radial distribution function analysis was used to determine if a significant amount of an amorphous solid phase of calcium phosphate exists in bone, and if so, whether the amount varies as a function of age and maturation. Unfractionated cortical bone from embryonic and posthatch chicks of various ages and a low-density fraction of embryonic bone were studied. No evidence was found for the presence of an amorphous solid phase of calcium phosphate in any of the samples studied, including the recently deposited bone mineral of the low density fraction of embryonic bone. As little as 12.5% of synthetic amorphous calcium phosphate (ACP) added to bone was readily detected by the radial distribution function technique used. The results clearly indicate that the concept that ACP is the initial solid mineral phase deposited in bone, and the major mineral constituent of young bone is no longer tenable. The concept does not provide an accurate description of the nature of the initial bone mineral deposited, or the changes that occur with maturation, nor can it account for the compositional and X-ray diffraction changes that the mineral component undergoes during maturation and aging.