Bone mineralization density distribution in health and disease

Human cortical and trabecular bones are formed by individual osteons and bone packets, respectively, which are produced at different time points during the (re)modeling cycle by the coupled activity of bone cells. This leads to a heterogeneously mineralized bone material with a characteristic bone mineralization density distribution (BMDD) reflecting bone turnover, mineralization kinetics and average bone matrix age. In contrast to BMD, which is an estimate of the total amount of mineral in a scanned area of whole bone, BMDD describes the local mineral content of the bone matrix throughout the sample. Moreover, the mineral content of the bone matrix is playing a pivotal role in tuning its stiffness, strength and toughness. BMDD of healthy individuals shows a remarkably small biological variance suggesting the existence of an evolutionary optimum with respect to its biomechanical performance. Hence, any deviations from normal BMDD due to either disease and/or treatment might be of significant biological and clinical relevance. The development of appropriate methods to sensitively measure the BMDD in bone biopsies led to numerous applications of BMDD in the evaluation of diagnosis and treatment of bone diseases, while advancing the understanding of the bone material, concomitantly. For example, transiliacal bone biopsies of postmenopausal osteoporotic women were found to have mostly lower mineralization densities than normal, which were partly associated by an increase of bone turnover, but also caused by calcium and Vit-D deficiency. Antiresorptive therapy causes an increase of degree and homogeneity of mineralization within three years of treatment, while normal mineralization levels are not exceeded. In contrast, anabolic therapy like PTH decreases the degree and homogeneity of matrix mineralization, at least transiently. Osteogenesis imperfecta is generally associated with increased matrix mineralization contributing to the brittleness of bone in this disease, though bone turnover is usually increased suggesting an alteration in the mineralization kinetics. Furthermore, BMDD measurements combined with other scanning techniques like nanoindentation, Fourier transform infrared spectroscopy and small angle X-ray scattering can provide important insights into the structure-function relation of the bone matrix, and ultimately a better prediction of fracture risk in diseases, and after treatment.     

Annual intravenous zoledronic acid for three years increased cancellous bone matrix mineralization beyond normal values in the HORIZON biopsy cohort

The efficacy of 3 years of annual intravenous administration of zoledronic acid (ZOL) in reducing vertebral and nonvertebral fractures in postmenopausal osteoporosis has been shown by the HORIZON pivotal fracture trial. Histomorphometric analysis of transiliac bone biopsies from the HORIZON participants revealed significantly improved trabecular architecture and reduced bone remodeling for the ZOL‐treated versus placebo‐treated patients. The aim of our study was to evaluate the cancellous and cortical bone mineralization density distribution (BMDD) in these biopsies by quantitative backscattered electron imaging (qBEI). The study cohort comprised 82 patients on active treatment (ZOL, yearly doses of 5 mg) and 70 treated with placebo, and all received adequate Ca and VitD supplementation. Comparison of ZOL‐treated versus placebo‐treated cancellous (Cn.) and cortical (Ct.) BMDD‐derived variables resulted in significantly higher average (Cn.CaMean + 3.2%, Ct.CaMean + 2.7%) and mode calcium concentrations (Cn.CaPeak + 2.1%, Ct.CaPeak + 1.5%), increased percentages of highly mineralized bone areas (Cn.CaHigh + 64%, Ct.CaHigh + 31%), lower heterogeneity of mineralization (Cn.CaWidth ?14%, Ct.CaWidth ?13%), and decreased percentages of low mineralized bone areas (Cn.CaLow ?22%, Ct.CaLow ?26%) versus placebo (all p < 0.001). Cn. BMDD from the patients on active treatment also revealed a statistically significant shift to higher Ca concentrations when compared to a historical normal reference BMDD. These differences in BMDD from ZOL patients compared to the other groups were in line with the correlation of BMDD variables with previously determined cancellous mineralizing surface per bone surface (Cn. MS/BS, a primary histomorphometric index for bone turnover), showing that those with lower Cn. MS/BS had a higher degree of bone matrix mineralization. However, the differences in BMDD variables between the study groups remained when adjusted for Cn. MS/BS, suggesting that other factors in addition to reduced bone turnover might contribute to the higher bone matrix mineralization after ZOL treatment. © 2013 American Society for Bone and Mineral Research.     

Altered matrix mineralization in a case of a sclerosing osteosarcoma

Little is known about the tumor matrix mineralization of highly sclerotic osteosarcoma. We used quantitative backscattered electron imaging (qBEI) to determine the Bone mineralization density distribution (BMDD) of a highly sclerosing osteosarcoma of the proximal tibia as well as adjacent normal bone of a 10-year-old girl following chemotherapy according to the EURAMOS-1 protocol. Data were compared to recently published normative reference data for young individuals.Backscattered electron imaging of the tumor region revealed a dense accumulation of mineralized tumor bone matrix (up to 90% of the medullar space). The BMDD was shifted tremendously towards higher matrix mineralization (CaMean + 18.5%, CaPeak + 22.5%, CaHigh + 100 fold) compared to normal bone. Additionally the BMDD became much wider, indicating a higher heterogeneity in mineralization (CaWidth + 40%). In contrast to lamellar bone, which mineralizes via a mineralization front, the mineralization of the tumor matrix starts by randomly distributed spots of mineral clusters fusing together to a highly mineralized non-lamellar bone matrix. We also found an altered BMDD of the patient's normal bone when compared with the reference BMDD of young individuals.In conclusion this high radiodensity region of the sclerosing sarcoma is not only due to the high amount of tumor matrix but also to its high mineralization density. Chemotherapy may lead to altered matrix mineralization of normal bone due to suppression of bone turnover. The mechanism of matrix mineralization in a sclerosing osteosarcoma warrants further studies.     

Constant mineralization density distribution in cancellous human bone

The degree of mineralization of bone matrix is an important factor in determining the mechanical competence of bone. The remodeling and modeling activities of bone cells together with the time course of mineralization of newly formed bone matrix generate a characteristic bone mineralization density distribution (BMDD). In this study we investigated the biological variance of the BMDD at the micrometer level, applying a quantitative backscattered electron imaging (qBEI) method. We used the mean calcium concentration (Ca(Mean)), the most frequent calcium concentration (Ca(Peak)), and full width at half maximum (Ca(Width)) to characterize the BMDD. In none of the BMDD parameters were statistically significant differences found due to ethnicity (15 African-American vs. 27 Caucasian premenopausal women), skeletal site variance (20 ilium, 24 vertebral body, 13 patella, 13 femoral neck, and 13 femoral head), age (25 to 95 years), or gender. Additionally, the interindividual variance of Ca(Mean) and Ca(Peak), irrespective of biological factors, was found to be remarkably small (SD < 2.1% of means). However, there are significant changes in the BMDD in the case of bone diseases (e.g., osteomalacia) or following clinical treatment (e.g., alendronate). From the lack of intraindividual changes among different skeletal sites we conclude that diagnostic transiliac biopsies can be used to determine the BMDD variables of cancellous bone for the entire skeleton of the patient. In order to quantify deviations from normal mineralization, a reference BMDD for adult humans was calculated using bone samples from 52 individuals. Because we find the BMDD to be essentially constant in healthy adult humans, qBEI provides a sensitive means to detect even small changes in mineralization due to bone disease or therapeutic intervention.     

Low bone mass and high material bone density in two patients with Loeys-Dietz syndrome caused by transforming growth factor beta receptor 2 mutations

Loeys-Dietz syndrome (LDS) is a rare autosomal-dominant connective tissue disorder caused by heterozygous mutations in the genes encoding transforming growth factor beta receptor 1 or 2 (TGFBR1 or TGFBR2). Although an association between LDS and osteoporosis has been reported, the skeletal phenotype regarding bone mass is not well characterized. Here, we report on two LDS patients with mutations in TGFBR2. Patient 1 was a 24-year-old man who had a total of three fractures involving the left radius, the left metacarpal, and the right femur. At the age of 14 years, lumbar spine areal bone mineral density Z-score was -4.0 and iliac bone histomorphometry showed elevated bone turnover (bone formation rate per bone surface: 91 μm3/μm2/year; age-matched control values 37 [10], mean [SD]) and mildly low trabecular bone volume per tissue volume (17.2%; age-matched control values 25.7 [5.3]). Bone mineralization density distribution (BMDD) in trabecular bone was increased (Ca(Peak) 22.70 wt% Ca; age-matched control values 21.66 [0.52]). Patient 2, a 17-year-old girl, suffered from diffuse bone pain but had not sustained fractures. At 14 years of age, her lumbar spine areal bone mineral density Z-score was -3.4. Iliac bone histomorphometry at that age confirmed low bone mass (bone volume to tissue volume 10.1%, same control values as above) and high bone turnover (bone formation rate per bone surface 70 μm3/μm2/year). BMDD in trabecular bone was significantly shifted toward increased mineralization (Ca(Peak) 22.36 wt% Ca). Thus, it appears that LDS can be associated with low bone mass and high bone turnover but increased matrix mineralization of trabecular bone.     

DSPP effects on in vivo bone mineralization

Dentin sialophosphoprotein has been implicated in the mineralization process based on the defective dentin formation in Dspp null mice (Dspp-/-). Dspp is expressed at low levels in bone and Dspp-/- femurs assessed by quantitative micro-computed tomography (micro-CT) and Fourier transform infrared spectroscopic imaging (FTIRI) exhibit some mineral and matrix property differences from wildtype femurs in both developing and mature mice. Compared to wildtype, Dspp-/- mice initially (5 weeks) and at 7 months had significantly higher trabecular bone volume fractions and lower trabecular separation, while at 9 months, bone volume fraction and trabecular number were lower. Cortical bone mineral density, area, and moments of inertia in Dspp-/- were reduced at 9 months. By FTIRI, Dspp-/- animals initially (5 months) contained more stoichiometric bone apatite with higher crystallinity (crystal size/perfection) and lower carbonate substitution. This difference progressively reversed with age (significantly decreased crystallinity and increased acid phosphate content in Dspp-/- cortical bone by 9 months of age). Mineral density as determined in 3D micro-CT and mineral-to-matrix ratios as determined by 2D FTIRI in individual cortical and trabecular bones were correlated (r(2)=0.6, p<0.04). From the matrix analysis, the collagen maturity of both cortical and trabecular bones was greater in Dspp-/- than controls at 5 weeks; by 9 months this difference in cross-linking pattern did not exist. Variations in mineral and matrix properties observed at different ages are attributable, in part, to the ability of the Dspp gene products to regulate both initial mineralization and remodeling, implying an effect of Dspp on bone turnover.     

Bone Matrix Quality After Sclerostin Antibody Treatment

Sclerostin antibody (Scl‐Ab) is a novel bone‐forming agent that is currently undergoing preclinical and clinical testing. Scl‐Ab treatment is known to dramatically increase bone mass, but little is known about the quality of the bone formed during treatment. In the current study, global mineralization of bone matrix in rats and nonhuman primates treated with vehicle or Scl‐Ab was assayed by backscattered scanning electron microscopy (bSEM) to quantify the bone mineral density distribution (BMDD). Additionally, fluorochrome labeling allowed tissue age–specific measurements to be made in the primate model with Fourier‐transform infrared microspectroscopy to determine the kinetics of mineralization, carbonate substitution, crystallinity, and collagen cross‐linking. Despite up to 54% increases in the bone volume after Scl‐Ab treatment, the mean global mineralization of trabecular and cortical bone was unaffected in both animal models investigated. However, there were two subtle changes in the BMDD after Scl‐Ab treatment in the primate trabecular bone, including an increase in the number of pixels with a low mineralization value (Z₅) and a decrease in the standard deviation of the distribution. Tissue age–specific measurements in the primate model showed that Scl‐Ab treatment did not affect the mineral‐to‐matrix ratio, crystallinity, or collagen cross‐linking in the endocortical, intracortical, or trabecular compartments. Scl‐Ab treatment was associated with a nonsignificant trend toward accelerated mineralization intracortically and a nearly 10% increase in carbonate substitution for tissue older than 2 weeks in the trabecular compartment (p < 0.001). These findings suggest that Scl‐Ab treatment does not negatively impact bone matrix quality. © 2014 American Society for Bone and Mineral Research.     

Prophylactic Bisphosphonate Treatment Prevents Bone Fractures After Liver Transplantation

A randomized controlled prospective open-label single center trial was performed. At the time of transplantation patients were randomly assigned to one of two treatment arms: The study group of 47 patients received zoledronic acid (ZOL, 8 infusions at 4 mg during the first 12 months after LT), calcium (1000 mg/d) and vitamin D (800 IE/d). The control group consisted of 49 patients who received calcium and vitamin D at same doses (CON). The incidence of bone fractures or death was predefined as the primary endpoint. Secondary endpoints included bone mineral density (BMD), serum biochemical markers of bone metabolism, parameters of trabecular bone histomorphometry and mineralization density distribution (BMDD). Patients were followed up for 24 months. Analysis was performed on an intention-to-treat basis. The primary endpoint fracture or death was reached in 26% of patients in the ZOL group and 46% in the CON group (p = 0.047, log rank test). Densitometry results were different between the groups at the femoral neck at 6 months after LT (mean+/-SD BMD ZOL: 0.80 +/- 0.19 g/cm2 vs. CON: 0.73 +/- 0.14 g/cm2, p = 0.036). Mixed linear models of biochemical bone markers showed less increase of osteocalcin in the ZOL group and histomorphometry and BMDD indicated a reduction in bone turnover. Prophylactic treatment with the bisphosphonate zoledronic acid reduces bone turnover and fractures after liver transplantation.     

Fragility Fractures in Men with Idiopathic Osteoporosis Are Associated with Undermineralization of the Bone Matrix without Evidence of Increased Bone Turnover

The pathogenesis of primary osteoporosis in younger individuals is still elusive. An important determinant of the biomechanical competence of bone is its material quality. In this retrospective study we evaluated bone material quality based on quantitative backscattered electron imaging to assess bone mineralization density distribution (BMDD) in bone biopsies of 25 male patients (aged 18–61 years) who sustained fragility fractures but were otherwise healthy. BMDD of cancellous bone was compared with previously established adult reference data. Complementary information was obtained by bone histomorphometry. The histomorphometric results showed a paucity of osteoblasts and osteoclasts on the bone surface in the majority of patients. BMDD revealed a significant shift to lower mineralization densities for cancellous bone values: CaMean (weighted mean Ca content, –5.9%), CaPeak (mode of the BMDD, −5.6%), and CaHigh (portion of fully mineralized bone, −76.8%) were decreased compared to normative reference; CaWidth (heterogeneity in mineralization, +18.5%) and CaLow (portion of low mineralized bone, +68.8; all P < 0.001) were significantly increased. The shift toward lower mineral content in the bone matrix in combination with reduced indices of bone formation and bone resorption suggests an inherent mineralization defect leading to undermineralized bone matrix, which might contribute to the susceptibility to fragility fractures of the patients. The alteration in bone material might be related to osteoblastic dysfunction and seems fundamentally different from that in high bone turnover osteoporosis with a negative bone balance.     

PTH(1‐84) Administration in Hypoparathyroidism Transiently Reduces Bone Matrix Mineralization

Patients with hypoparathyroidism have low circulating parathyroid (PTH) levels and higher cancellous bone volume and trabecular thickness. Treatment with PTH(1‐84) was shown to increase abnormally low bone remodeling dynamics. In this work, we studied the effect of 1‐year or 2‐year PTH(1‐84) treatment on cancellous and cortical bone mineralization density distribution (Cn.BMDD and Ct.BMDD) based on quantitative backscattered electron imaging (qBEI) in paired transiliac bone biopsy samples. The study cohort comprised 30 adult hypoparathyroid patients (14 treated for 1 year; 16 treated for 2 years). At baseline, Cn.BMDD was shifted to higher mineralization densities in both treatment groups (average degree of mineralization Cn.Ca_Mean +3.9% and +2.7%, p < 0.001) compared to reference BMDD. After 1‐year PTH(1‐84), Cn.Ca_Mean was significantly lower than that at baseline (–6.3%, p < 0.001), whereas in the 2‐year PTH(1‐84) group Cn.Ca_Mean did not differ from baseline. Significant changes of Ct.BMDD were observed in the 1‐year treatment group only. The change in histomorphometric bone formation (mineralizing surface) was predictive for Cn.BMDD outcomes in the 1‐year PTH(1‐84) group, but not in the 2‐year PTH(1‐84) group. Our findings suggest higher baseline bone matrix mineralization consistent with the decreased bone turnover in hypoparathyroidism. PTH(1‐84) treatment caused differential effects dependent on treatment duration that were consistent with the histomorphometric bone formation outcomes. The greater increase in bone formation during the first year of treatment was associated with a decrease in bone matrix mineralization, suggesting that PTH(1‐84) exposure to the hypoparathyroid skeleton has the greatest effects on BMDD early in treatment. © 2015 American Society for Bone and Mineral Research.     

Normative data on mineralization density distribution in iliac bone biopsies of children, adolescents and young adults

Bone mineralization density distribution (BMDD) as assessed by quantitative backscattered electron imaging (qBEI) in iliac crest bone biopsies has become in the last years a powerful diagnostic tool to evaluate the effect of metabolic bone diseases and/or therapeutic interventions on the mineralization status of the bone material. However until now, normative reference data are only available for adults. The aim of the present study is to close this gap and establish normative data from children and compare them with reference BMDD data of adults.qBEI analyses were performed on bone samples from 54 individuals between 1.5 and 23 years without metabolic bone diseases, which were previously used as study population to establish normative histomorphometric standards.In the trabecular compartment, none of the BMDD parameters showed a significant correlation with age. The BMDD was shifted towards lower mineralization density (CaMean − 5.6%, p < 0.0001; CaPeak − 5.6%, p < 0.0001; CaLow + 39.0% p < 0.001; CaHigh − 80.7%, p < 0.001) and the inter-individual variation was higher compared to the adult population.The cortices appeared to be markedly less mineralized (CaMean − 3.1%, p < 0.0001) than cancellous bone due to higher amounts of low mineralized secondary bone. However, the cortical BMDD parameters showed a strong correlation (r = 0.38 to 0.85, with p < 0.001 to < 0.0001) with cancellous BMDD parameters.In conclusion, this study provides evidence that BMDD parameters in growing healthy subjects are relatively constant and that these data can be used as normative references in pediatrics osteology. The larger inter-individual variability compared to adults is most likely related to alterations of the bone turnover rate during growth.     

Bisphosphonates do not alter the rate of secondary mineralization

Bisphosphonates function to reduce bone turnover, which consequently increases the mean degree of tissue mineralization at an organ level. However, it is not clear if bisphosphonates alter the length of time required for an individual bone-modeling unit (BMU) to fully mineralize. We have recently demonstrated that it takes ~ 350 days (d) for normal, untreated cortical bone to fully mineralize. The aim of this study was to determine the rate at which newly formed trabecular BMUs become fully mineralized in rabbits treated for up to 414 d with clinical doses of either risedronate (RIS) or alendronate (ALN). Thirty-six, 4-month old virgin female New Zealand white rabbits were allocated to RIS (n = 12; 2.4 μg/kg body weight), ALN (n = 12; 2.4 μg/kg body weight), or volume-matched saline controls (CON; n = 12). Fluorochrome labels were administered at specific time intervals to quantify the rate and level of mineralization of trabecular bone from the femoral neck (FN) by Fourier transform infrared microspectroscopy (FTIRM). The organic (collagen) and inorganic (phosphate and carbonate) IR spectral characteristics of trabecular bone from undecalcified 4 micron thick tissue sections were quantified from fluorescently labels regions that had mineralized for 1, 8, 18, 35, 70, 105, 140, 210, 280, and 385 d (4 rabbits per time point and treatment group). All groups exhibited a rapid increase in mineralization over the first 18 days, the period of primary mineralization, with no significant differences between treatments. Mineralization continued to increase, at a slower rate up, to 385 days (secondary mineralization), and was not different among treatments. There were no significant differences between treatments for the rate of mineralization within an individual BMU; however, ALN and RIS both increased global tissue mineralization as demonstrated by areal bone mineral density from DXA. We conclude that increases in tissue mineralization that occur following a period of bisphosphonate treatment is a function of the suppressed rate of remodeling that allows for a greater number of BMUs to obtain a greater degree of mineralization.     

How can bone turnover modify bone strength independent of bone mass?

The amount of bone turnover in the skeleton has been identified as a predictor of fracture risk independent of areal bone mineral density (aBMD) and is increasingly cited as an explanation for discrepancies between areal bone mineral density and fracture risk. A number of mechanisms have been proposed to explain how bone turnover influences bone biomechanics, including regulation of tissue degree of mineralization, the disconnection or fenestration of individual trabeculae by remodeling cavities, and the ability of cavities formed during the remodeling process to act as stress risers. While these mechanisms can influence bone biomechanics, they also modify bone mass. If bone turnover is to explain any of the observed discrepancies between fracture risk and areal bone mineral density, however, it must not only modify bone strength, but must also modify bone strength in excess of what would be expected from the associated change in bone mass. This article summarizes biomechanical studies of how tissue mineralization, trabecular disconnection, and the presence of remodeling cavities might have an effect on cancellous bone strength independent of bone mass. Existing data support the idea that all of these factors may have a disproportionate effect on bone stiffness and/or strength, with the exception of average tissue degree of mineralization, which may not affect bone strength independent of aBMD. Disproportionate effects of mineral content on bone biomechanics may instead come from variation in tissue degree of mineralization at the micro-structural level. The biomechanical explanation for the relationship between bone turnover and fracture incidence remains to be determined, but must be examined not in terms of bone biomechanics, but in terms of bone biomechanics relative to bone mass.     

Impaired bone mineralization accompanied by low vitamin D and secondary hyperparathyroidism in patients with femoral neck fracture

Summary

Although it is well established that a decrease in bone mass increases the risk of osteoporotic fractures, the proportion of fractures attributable to areal bone mineral density (BMD) is rather low. Here, we have identified bone mineralization defects together with low serum 25-hydroxyvitamin D (25-(OH) D) levels as additional factors associated with femoral neck fractures.

Introduction

Osteoporotic fractures of the femoral neck are associated with increased morbidity and mortality. Although it is well established that a decrease in bone mass increases the risk of osteoporotic fractures, the proportion of fractures attributable to areal BMD is rather low. To identify possible additional factors influencing femur neck fragility, we analyzed patients with femoral neck fracture.

Methods

We performed a detailed clinical and histomorphometrical evaluation on 103 patients with femoral neck fracture including dual-energy X-ray absorptiometry, laboratory parameters, and histomorphometric and bone mineral density distribution (BMDD) analyses of undecalcified processed biopsies of the femoral head and set them in direct comparison to skeletal healthy control individuals.

Results

Patients with femoral neck fracture displayed significantly lower serum 25-(OH) D levels and increased serum parathyroid hormone (PTH) compared to controls. Histomorphometric analysis revealed not only a decreased bone volume and trabecular thickness in the biopsies of the patients, but also a significant increase of osteoid indices. BMDD analysis showed increased heterogeneity of mineralization in patients with femoral neck fracture. Moreover, patients with femoral neck fracture and serum 25-(OH) D levels below 12 μg/l displayed significantly thinner trabecular bone.

Conclusion

Taken together, our data suggest that impaired bone mineralization accompanied by low serum 25-(OH) D levels is of major importance in the etiology of femoral neck fractures. Therefore, balancing serum 25-(OH) D levels and thereby normalizing PTH serum levels may counteract pronounced mineralization defects and might decrease the incidence of femoral neck fractures.     

The degree of mineralization of bone tissue measured by computerized quantitative contact microradiography

Strength of bones depends on bone matrix volume (BMV), bone microarchitecture, but also bone mineralization, and we have recently shown in osteoporotic patients treated with alendronate that fracture risk and bone mineral density (BMD) were changed without modifications of BMV or bone microarchitecture. Mineralization of bone matrix implies two successive steps: a rapid primary mineralization on the calcification front followed by a slow process of secondary mineralization progressively adding about one-half of the mineral content on bone matrix. These two steps are clearly illustrated by microradiographs of compact and cancellous bone tissue from transiliac human biopsies. Our working hypothesis is based on the impact of changes in bone remodeling rate on the degree of mineralization of bone, i.e., on the BMD measured at the tissue level. Contact quantitative microradiography using a computerized microdensitometric method, is described and allows the measurement of the mean degree of mineralization of bone (MDMB). This parameter may be quantitatively evaluated by exposing an aluminum calibration step-wedge and a plane-parallel calcified tissue section simultaneously to the same beam of X-rays, then determining, from the resulting microradiograph, the thickness of aluminum that produces the same X-ray absorption as a given region of the bone tissue section. To be used as a control group, iliac bone samples were taken at necropsy from 43 subjects (30 women aged 48.4 +/- 3.7 years and 13 men aged 66.0 +/- 4.4 years) who died suddenly showing no apparent bone disease. A control MDMB, which does not change with age, and a control distribution of these values are thus established. These control values are necessary for interpreting the changes in MDMB observed in bone conditions untreated or treated.     

Treatment with eldecalcitol positively affects mineralization,microdamage, and collagen crosslinks in primate bone

Eldecalcitol (ELD), an active form of vitamin D analog approved for the treatment of osteoporosis in Japan, increases lumbar spine bone mineral density (BMD), suppresses bone turnover markers, and reduces fracture risk in patients with osteoporosis. We have previously reported that treatment with ELD for 6 months improved the mechanical properties of the lumbar spine in ovariectomized (OVX) cynomolgus monkeys. ELD treatment increased lumbar BMD, suppressed bone turnover markers, and reduced histomorphometric parameters of both bone formation and resorption in vertebral trabecular bone. In this study, we elucidated the effects of ELD on bone quality (namely, mineralization, microarchitecture, microdamage, and bone collagen crosslinks) in OVX cynomolgus monkeys in comparison with OVX-vehicle control monkeys. Density fractionation of bone powder prepared from lumbar vertebrae revealed that ELD treatment shifted the distribution profile of bone mineralization to a higher density, and backscattered electron microscopic imaging showed improved trabecular bone connectivity in the ELD-treated groups. Higher doses of ELD more significantly reduced the amount of microdamage compared to OVX-vehicle controls. The fractionated bone powder samples were divided according to their density, and analyzed for collagen crosslinks. Enzymatic crosslinks were higher in both the high-density (≥ 2.0 mg/mL) and low-density (< 2.0 mg/mL) fractions from the ELD-treated groups than in the corresponding fractions in the OVX-vehicle control groups. On the other hand, non-enzymatic crosslinks were lower in both the high- and low-density fractions. These observations indicated that ELD treatment stimulated the enzymatic reaction of collagen crosslinks and bone mineralization, but prevented non-enzymatic reaction of collagen crosslinks and accumulation of bone microdamage. Bone anti-resorptive agents such as bisphosphonates slow down bone remodeling so that bone mineralization, bone microdamage, and non-enzymatic collagen crosslinks all increase. Bone anabolic agents such as parathyroid hormone decrease bone mineralization and bone microdamage by stimulating bone remodeling. ELD did not fit into either category. Histological analysis indicated that the ELD treatment strongly suppressed bone resorption by reducing the number of osteoclasts, while also stimulating focal bone formation without prior bone resorption (bone minimodeling). These bidirectional activities of ELD may account for its unique effects on bone quality.     

Bone material properties in premenopausal women with idiopathic osteoporosis

Idiopathic osteoporosis (IOP) in premenopausal women is characterized by fragility fractures at low or normal bone mineral density (BMD) in otherwise healthy women with normal gonadal function. Histomorphometric analysis of transiliac bone biopsy samples has revealed microarchitectural deterioration of cancellous bone and thinner cortices. To examine bone material quality, we measured the bone mineralization density distribution (BMDD) in biopsy samples by quantitative backscattered electron imaging (qBEI), and mineral/matrix ratio, mineral crystallinity/maturity, relative proteoglycan content, and collagen cross‐link ratio at actively bone forming trabecular surfaces by Raman microspectroscopy and Fourier transform infrared microspectroscopy (FTIRM) techniques. The study groups included: premenopausal women with idiopathic fractures (IOP, n = 45), or idiopathic low BMD (Z‐score ≤ ?2.0 at spine and/or hip) but no fractures (ILBMD, n = 19), and healthy controls (CONTROL, n = 38). BMDD of cancellous bone showed slightly lower mineral content in IOP (both the average degree of mineralization of cancellous bone [Cn.Ca_Mean] and mode calcium concentration [Cn.Ca_Peak] are 1.4% lower) and in ILBMD (both are 1.6% lower, p < 0.05) versus CONTROL, but no difference between IOP and ILBMD. Similar differences were found when affected groups were combined versus CONTROL. The differences remained significant after adjustment for cancellous mineralizing surface (MS/BS), suggesting that the reduced mineralization of bone matrix cannot be completely accounted for by differences in bone turnover. Raman microspectroscopy and FTIRM analysis at forming bone surfaces showed no differences between combined IOP/ILBMD groups versus CONTROL, with the exceptions of increased proteoglycan content per mineral content and increased collagen cross‐link ratio. When the two affected subgroups were considered individually, mineral/matrix ratio and collagen cross‐link ratio were higher in IOP than ILBMD. In conclusion, our findings suggest that bone material properties differ between premenopausal women with IOP/ILBMD and normal controls. In particular, the altered collagen properties at sites of active bone formation support the hypothesis that affected women have osteoblast dysfunction that may play a role in bone fragility. © 2012 American Society for Bone and Mineral Research.     

Site-specific changes in bone microarchitecture, mineralization, and stiffness during lactation and after weaning in mice

Despite the dramatic bone loss that occurs during lactation, bone mineral density rapidly recovers after offspring are weaned and milk production stops. The goal of this study is to quantify site-specific changes in bone quantity and quality during and after lactation in a mouse model. We used micro computed tomography (μCT), individual trabecula segmentation (ITS), digital topological analysis (DTA)-based tissue mineral density (TMD) analysis, and micro finite element analysis (μFEA) to quantify the effects of lactation and weaning on bone microarchitecture, mineralization, and stiffness at the spine, tibia, and femur. We found a significant decrease in trabecular plate microarchitecture, tissue mineralization of the trabecular surface, trabecular central skeleton, and intervening envelopes, and whole bone stiffness in lactating versus nulliparous mice at all three sites. In recovered mice, all these different aspects of bone quality were comparable to nulliparous mice at the spine. In contrast, trabecular plate microarchitecture and whole bone stiffness at the tibia and femur in recovered mice were lower than nulliparous mice, as were central trabecular tissue mineralization and cortical structure at the femur. These findings are consistent with clinical observations of partial recovery of femoral bone mineral density BMD after lactation in humans. The observed differences in trabecular surface tissue mineralization in nulliparous, lactating, and recovered mice are consistent with prior observations that maternal bone turnover shifts from resorption to formation at the time of pup weaning. The significant differences in trabecular central tissue mineralization during these three states suggest that osteocytes may contribute to the reversible loss of mineral during and after lactation. Future studies are necessary to determine whether differing functions of various bone cells at individual skeletal sites cause site-specific skeletal changes during and after lactation.     

Altered bone matrix mineralization in a patient with Rett syndrome

Rett syndrome (RTT) is a common X-linked neurodevelopmental disorder caused by mutations in the coding region of methyl-CpG-binding 2 (MECP2) gene. Patients with RTT have a low bone mineral density and increased risk of fracture. However, very little is known if bone matrix mineralization is altered in RTT. A 17-year-old girl with a classical form of RTT with a heterozygous nonsense mutation in exon 3 in the MECP2-gene was treated in our hospital. Her femoral neck BMD is 43.3% below the 3rd percentile when compared to age and sex-matched controls. She underwent surgery for correction of her scoliosis, which provided a unique opportunity to obtain bone tissue to study bone matrix mineralization (Bone Mineralization Density Distribution—BMDD) using quantitative backscattered electron imaging (qBEI) and histomorphometry. BMDD outcomes were compared to recently published normative reference data for young individuals. qBEI analysis showed a significant shift to lower matrix mineralization despite histomorphometric indices indicate a low bone turnover. There was a reduction in CaMean (? 7.92%) and CaPeak (? 3.97%), which describe the degree of mineralization. Furthermore the fraction of low mineralized matrix (CaLow: + 261.84%) was dramatically increased, which was accompanied with an increase in the heterogeneity of mineralization (CaWidth: + 86.34%).Our findings show a significantly altered bone matrix mineralization of a typical patient with RTT. This may partly explain the low bone density seen in these patients. These results also warrant further studies on the molecular role of MECP2 in bone matrix mineralization.     

Increased Heterogeneity of Bone Matrix Mineralization in Pediatric Patients Prone to Fractures: A Biopsy Study

Idiopathic osteoporosis (IOP) in children is characterized by fragility fractures and/or low bone mineral density in otherwise healthy individuals. The aim of the present work was to measure bone mineralization density distribution (BMDD) based on quantitative backscattered electron imaging (qBEI) in children with suspected IOP. Entire cross‐sectional areas of transiliac bone biopsy samples from children (n = 24, 17 boys; aged 6.7–16.6 years) with a history of fractures (n = 14 with at least one vertebral fracture) were analyzed for cancellous (Cn) and cortical (Ct) BMDD. Outcomes were compared with normal reference BMDD data and correlated with the patients' clinical characteristics and bone histomorphometry findings. The subjects had similar average degree but significantly higher heterogeneity of mineralization in both Cn and Ct bone (Cn.CaWidth +23%, Ct.CaWidth +15%, p < 0.001 and p = 0.002, respectively), together with higher percentages of low mineralized cancellous (Cn.CaLow +35%, p < 0.001) and highly mineralized cortical bone areas (Ct.CaHigh +82%, p = 0.032). Ct.CaWidth and Ct.CaLow were positively correlated with mineralizing surface per bone surface (MS/BS; a primary histomorphometric determinant of bone formation) and with serum bone turnover markers (all p < 0.05). The correlations of the mineralization heterogeneity with histomorphometric and serum bone turnover indices suggest that an enhanced variation in bone turnover/formation contributes to the increased heterogeneity of mineralization. However, it remains unclear whether the latter is cause for, or the response to the increased bone fragility in these children with suspected IOP. © 2014 American Society for Bone and Mineral Research.