Increased longitudinal growth in rats on a silicon-depleted diet.

Silicon-deficiency studies in growing animals in the early 1970s reported stunted growth and profound defects in bone and other connective tissues. However, more recent attempts to replicate these findings have found mild alterations in bone metabolism without any adverse health effects. Thus the biological role of silicon remains unknown. Using a specifically formulated silicon-depleted diet and modern methods for silicon analysis and assessment of skeletal development, we undertook, through international collaboration between silicon researchers, an extensive study of long-term silicon depletion on skeletal development in an animal. 21-day old female Sprague-Dawley rats (n=20) were fed a silicon-depleted diet (3.2 microg Si/g feed) for 26 weeks and their growth and skeletal development were compared with identical rats (n=10) on the same diet but with silicon added as Si(OH)(4) to their drinking water (53.2 microg Si/g water); total silicon intakes were 24 times different. A third group of rats, receiving a standard rodent stock feed (322 microg Si/g feed) and tap water (5 microg Si/g water), served as a reference group for optimal growth. A series of anthropometric and bone quality measures were undertaken during and following the study. Fasting serum silicon concentrations and especially urinary silicon excretion were significantly lower in the silicon-deprived group compared to the supplemented group (P=0.03 and 0.004, respectively). Tibia and soft-tissue silicon contents did not differ between the two groups, but tibia silicon levels were significantly lower compared to the reference group (P<0.0001). Outward adverse health effects were not observed in the silicon-deprived group. However, body lengths from week 18 onwards (P<0.05) and bone lengths at necropsy (P

Femoral neck cortical bone in female and male hip fracture cases: Differential contrasts in cortical width and sub-periosteal porosity in 112 cases and controls.

OBJECTIVES: To quantitate differences between cases of hip fracture and controls in cortical width around the mid-femoral neck in men and women. METHODS: Over 5 years, 64 (14 male) participants over age 55 (mean 79) years, who had never taken bone-active drugs and suffered intra-capsular hip fracture treated by arthroplasty, donated their routinely discarded distal intra-capsular femoral neck bone for histomorphometry. After embedding, complete femoral neck cross sections from the cut surface near the narrowest part of the neck were stained with von Kossa and cortical width was measured radially every 5 degrees of arc. Control material (n = 48, 25 male) was available through consented post mortems prior to the year 2000. Cortical widths were averaged for circumferential octants, each representing 45 degrees of arc. Divergence of individual cortical widths from their means was also examined. RESULTS: Because sections were required to have a complete cortex, sampling was biased towards cases with sub-capital versus trans-cervical fractures. Compared to sex- and age matched controls, male cases showed larger relative differences in cortical widths than female cases. Unexpectedly, cortical widths in female but not male cases also showed marked over-representation of extremely narrow (<0.1 mm) cortical widths, located mainly posteriorly. The numbers of these very narrow cortical widths observed per subject retrospectively predicted female fracture status in logistic regression independently of mean cortical width values. Together with mean cortical width differences, the numbers of measured cortical widths <0.1 mm (out of 72 measured) raised the sensitivity of predicting fracture status in women from 48 to 80% at 80% specificity. In almost all cases, very narrow cortical widths were identified in regions enclosing a cortical pore roofed on its endosteal surface by thin structural bone defined a priori as trabecular. CONCLUSIONS: Cortical widths <0.1 mm probably reflect zones where endosteal cortex has been trabecularised through expansion of an un-refilled sub-endosteal canal close to the periosteum. Persistent cortical defects occurring near the periosteal surface, where mechanical loading exerts its greatest stresses, are likely to result in extremes of localized concentrations of stress during a fall, unknown in young normal fallers. Such defects have the potential to help explain the excess of hip fractures among elderly women. Prevention of sub-periosteal tunnelling by osteoclasts might explain in part the additional benefits, beyond an increase in bone density, of treatments that reduce excessive bone resorption or else stimulate new bone formation on previously resorbed surfaces.

A single infusion of zoledronate prevents bone loss after stroke.

BACKGROUND AND PURPOSE: Stroke is a major risk factor for hip fracture. Patients with intermediate rather than severe or mild stroke deficits at the time of hospital discharge have the most fractures. This proof-of-concept study evaluated the efficacy of a single infusion of zoledronate, an intravenous bisphosphonate, in preserving hip bone density after stroke. METHODS: In a 1-year randomized, double-blind, placebo-controlled, clinical trial, 27 newly hemiplegic patients (6 females, 21 males) with acute stroke were assigned to receive 4 mg of the intravenous zoledronate (n=14) or placebo (n=13) within 35 days. Strict inclusion criteria were followed-up to ensure recruited patients were likely to have residual functional impairment. Both groups received calcium and vitamin D supplementation. The primary outcome measure was the change in bone mineral density (BMD; Lunar Prodigy) at the hemiplegic hip during the year of investigation. RESULTS: The treatment was generally well tolerated. Mean total hip BMD was unchanged in the hemiplegic hip of the zoledronate group (mean 0.0% change), whereas in the placebo group the total hip BMD changed by -5.5%, with the greatest bone loss observed in the trochanteric subregion (mean, -8.1%). On the unaffected side the mean change in total hip BMD was +1.0% with zoledronate versus a mean change of -2.7% without. Repeated measures ANOVA confirmed the significance of the differences between groups at both hips (hemiplegic, P<0.001; unaffected, P=0.002). CONCLUSIONS: Stroke patients were protected from the deleterious effects of hemiplegia on hip bone density for at least 1 year after a single infusion of zoledronate.

Infant growth influences proximal femoral geometry in adulthood.

UNLABELLED: The relationship between early growth and adult femoral geometry has not been studied previously. In 333 adults, we were able to show that infant weight predicts femoral width and cross-sectional moment of inertia but not femoral neck length. These results support the hypothesis that growth in early life leads to persisting differences in proximal femoral geometry. INTRODUCTION: Both femoral geometry and bone mass have been shown independently to predict both hip strength and fracture risk. Whereas growth during intrauterine and early postnatal life has been shown to influence adult bone mass, the relationship between growth in early life and adult femoral geometry has not been described previously. MATERIALS AND METHODS: We studied the relationship between growth during early life, adult hip geometry, and proximal femur bone mass in a sample of 333 men and women (60-75 years of age), for whom birth weight and weight at 1 year of age were recorded. Hip geometry was derived using Hip Structure Analysis software from proximal femur DXA scans (Hologic QDR 1000). RESULTS: There were significant (p < 0.002) relationships between weight at age 1 year and measures of femoral width as well as intertrochanteric (IT) cross-sectional moment of inertia (CSMI), but not with femoral neck length. The relationships with measures of femoral width but not CSMI remained after adjusting for adult body weight and were independent of proximal femoral BMC. CONCLUSIONS: These results support the hypothesis that different patterns of growth in utero and during the first year of life lead to persisting differences in proximal femoral geometry, thereby mediating in part the effects of early growth on risk of hip fracture in adulthood.

Reduced vitamin D in acute stroke.

BACKGROUND AND PURPOSE: Stroke leads to a reduction in bone mineral density, altered calcium homeostasis, and an increase in hip fractures. Vitamin D deficiency is well documented in long-term stroke survivors and is associated with post-stroke hip fractures. Less is known regarding levels in acute stroke. METHODS: We compared the serum 25-dihydroxyvitamin D levels of 44 patients admitted to an acute stroke unit with first-ever stroke with results obtained by measuring 96 healthy ambulant elderly subjects every 2 months for 1 year. Statistical Z scores of serum vitamin D were then calculated after seasonal adjustment for the month of sampling. RESULTS: The mean Z score of vitamin D in acute stroke was -1.4 SD units (95% CI, -1.7, -1.1), with 77% of patients falling in the insufficient range. CONCLUSIONS: Reduced vitamin D was identified in the majority of patients with acute stroke throughout the year and may have preceded stroke. Vitamin D is a potential risk marker for stroke, and the role of vitamin D repletion in enhancing musculoskeletal health after stroke needs to be explored.

Relation between age, femoral neck cortical stability, and hip fracture risk.

BACKGROUND: Hip fracture risk rises 100 to 1000-fold over 60 years of ageing. Loss of resistance to bending is not a major feature of normal ageing of the femoral neck. Another cause of fragility is local buckling or elastic instability. Bones adapt to their local experience of mechanical loading. The suggestion that bipedalism allows thinning of the underloaded superolateral femoral neck cortex arises from the failure of walking to transmit much mechanical load to this region. We aimed to measure whether elastic instability increases greatly with age since it might trigger hip fracture in a sideways fall. METHODS: We measured with computed tomography the distribution of bone in the mid-femoral neck of 77 proximal femurs from people who died suddenly aged 20-95 years. We then calculated the critical stress, from the geometric properties and density of the cortical zone most highly loaded in a sideways fall, as a threshold for elastic instability. FINDINGS: With normal ageing, this thin cortical zone in the upper femoral neck became substantially thinner. Relative to mean values at age 60 years, female cortical thickness declined by 6.4% (SD 1.1) per decade (p<0.0001), and critical stress by 13.2% (4.3) per decade (p=0.004) in the superoposterior octant compressed most in a sideways fall. Similar, but significantly smaller, effects were evident in men (p=0.004). This thinning compromised the capacity of the femur to absorb energy independently of osteoporosis. Patients with hip fracture had further reduced stability. INTERPRETATION: As women age, hip fragility increases because underloading of the superolateral cortex leads to atrophic thinning. Because walking does not sufficiently load the upper femoral neck, the fragile zones in healthy bones may need strengthening, for example with more well targeted exercise.

Sclerostin is a delayed secreted product of osteocytes that inhibits bone formation.

Osteocytes are the most abundant cells in bone and are ideally located to influence bone turnover through their syncytial relationship with surface bone cells. Osteocyte-derived signals have remained largely enigmatic, but it was recently reported that human osteocytes secrete sclerostin, an inhibitor of bone formation. Absent sclerostin protein results in the high bone mass clinical disorder sclerosteosis. Here we report that within adult iliac bone, newly embedded osteocytes were negative for sclerostin staining but became positive at or after primary mineralization. The majority of mature osteocytes in mineralized cortical and cancellous bone was positive for sclerostin with diffuse staining along dendrites in the osteocyte canaliculi. These findings provide for the first time in vivo evidence to support the concept that osteocytes secrete sclerostin after they become embedded in a mineralized matrix to limit further bone formation by osteoblasts. Sclerostin did not appear to influence the formation of osteocytes. We propose that sclerostin production by osteocytes may regulate the linear extent of formation and the induction or maintenance of a lining cell phenotype on bone surfaces. In doing so, sclerostin may act as a key inhibitory signal governing skeletal microarchitecture.

Osteocytic expression of constitutive NO synthase isoforms in the femoral neck cortex: a case-control study of intracapsular hip fracture.

UNLABELLED: NO is an osteocytic signaling molecule that can inhibit osteoclasts. The NO synthases eNOS and nNOS were expressed by >50% of osteonal osteocytes in controls. Hip fracture cases showed +NOS osteocytes only in deep osteonal bone, and 25-35% reduced expression overall. These data are consistent with increased osteonal vulnerability to deep osteoclastic attack. INTRODUCTION: Osteocytes may regulate the response to mechanical stimuli in bone through the production of local signaling molecules such as NO derived from the NO synthase eNOS. Because NO is inhibitory to osteoclastic resorption, it has been suggested that osteocytes expressing eNOS act as sentinels, confining resorption within single osteons. Recently, nNOS has been shown to be present in osteocytes of adult human bone. MATERIALS AND METHODS: Cross-sections of the femoral neck (eight female cases of intracapsular hip fracture and seven postmortem controls; age, 68-91 years) were analyzed by immunohistochemistry. The percentages of osteocytes expressing each of these two isoforms were calculated, and their distances to the nearest canal surface were measured. RESULTS: The percentage of +nNOS osteocytes was lower in the fracture cases than in the controls (cases: 43.12 +/- 1.49, controls: 56.68 +/- 1.45; p < 0.0001). Compared with nNOS, eNOS expression was further reduced (p = 0.009) in the cases but was not different in the controls (cases: 36.41 +/- 1.53, controls: 56.47 +/- 2.41; p < 0.0001). The minimum distance of +eNOS or +nNOS osteocytes to a canal surface was higher in the cases compared with controls (eNOS: controls; 44.4 +/- 2.2 microm, cases: 61.7 +/- 2.0 microm; p < 0.0001; nNOS: controls: 52.4 +/- 1.7 microm, cases: 60.2 +/- 2.1 microm; p = 0.0039). +eNOS osteocytes were closer to the canal surfaces than +nNOS osteocytes in the controls by 8.00 +/- 4.0 microm (p = 0.0012). CONCLUSION: The proportions of osteocytes expressing nNOS and eNOS were both reduced in the fracture cases, suggesting that the capacity to generate NO might be reduced. Furthermore, the reduction in NOS expression occurs in those osteocytes closest to the canal surface, suggesting that the ability of NO to minimize resorption depth might be impaired. Further studies are needed on the regulation of the expression and activity of these distinct NOS isoforms.

Bone marrow adipocytes: a neglected target tissue for growth hormone.

Bone marrow (BM) contains numerous adipocytes. These share a common precursor with osteoblasts and chondrocytes, but their function is unknown. It is unclear what regulates the differentiation of these three different cell types, though their subsequent metabolic activity is under hormonal regulation. GH and estrogen stimulate bone growth and mineralization, by direct effects on chondrocytes and osteoblasts. GH also stimulates lipolysis in subcutaneous and visceral adipocytes. However, adipocytes in BM have largely been ignored as potential targets for GH or estrogen action. We have addressed this by measuring BM adipocyte number, perimeter and area as well as bone area and osteoblast activity in GH-deficient dwarf (dw/dw), normal, or ovariectomized (Ovx) rats, with or without GH, IGF-1, PTH, or estrogen treatment or high fat feeding. Marrow adipocyte numbers were increased 5-fold (P < 0.001) in dw/dw rats, and cell size was also increased by 20%. These values returned toward normal in dw/dw rats given GH but not when given IGF-1. Cancellous bone area and osteoblast number were significantly (P < 0.005) lower in dw/dw rats, though alkaline phosphatase (ALP) activity in individual osteoblasts was unchanged. GH treatment increased % osteoblast covered bone surface without affecting individual cell ALP activity. Ovariectomy in normal or dw/dw rats had no affect on marrow adipocyte number nor size, although estrogen treatment in ovariectomized (Ovx) normal rats did increase adipocyte number. Ovx decreased tibial cancellous bone area in normal rats (64%; P < 0.05) and decreased osteoblast ALP-activity (P < 0.01) but did not affect the percentage of osteoblast-covered bone surface. Estrogen replacement reversed these changes. While treatment with PTH by continuous sc infusion decreased cancellous bone (P < 0.05) and high fat feeding increased the size of BM adipocytes (P < 0.01), they did not affect BM adipocyte number. These results suggest that GH has a specific action on BM adipocytes that is not simply due to altered bone or fat metabolism. We conclude that the marrow adipocyte lineage is an important and specific target for GH action. The inverse relationship between adipocyte number and osteoblast covered bone surface, together with the well-known effects of GH on epiphysial chondrocytes leads us to propose that GH plays two important roles on cells of all three lineages. During differentiation, it regulates the numbers of each cell type that are maintained from the common precursor lineage. Subsequently it has cell-specific effects on the metabolic activities of the differentiated cells. In the case of marrow adipocytes, GH-dependent lipolysis could provide an important hormonally regulated local high energy source in bone.

Ghrelin and des-octanoyl ghrelin promote adipogenesis directly in vivo by a mechanism independent of the type 1a growth hormone secretagogue receptor.

Ghrelin promotes fat accumulation, despite potent stimulation of the lipolytic hormone, GH. The function of the major circulating isoform of ghrelin, des-octanoyl ghrelin, is unclear, because it does not activate the GH secretagogue receptor (GHS-R1a) and lacks the endocrine activities of ghrelin. We have now addressed these issues by infusing ghrelin, des-octanoyl ghrelin, or synthetic GHS-R1a agonists into three rat models with moderate, severe, or total GH deficiency. We show that in the context of significant GH secretion, the adipogenic effect of systemic ghrelin infusion is pattern dependent. However, this adipogenic action is not mediated by the pituitary hormones. Using a novel unilateral local infusion strategy, we demonstrate that ghrelin promotes bone marrow adipogenesis in vivo by a direct peripheral action. Surprisingly, this effect was also observed with des-octanoyl ghrelin, whereas a potent synthetic GHS-R1a agonist was ineffective. Thus, these adipogenic effects are mediated by a receptor other than GHS-R1a. This is the first in vivo demonstration of a direct adipogenic effect of des-octanoyl ghrelin, a major circulating form of ghrelin that lacks GH-releasing activity. We suggest that the ratio of ghrelin and des-octanoyl ghrelin production could help regulate the balance between adipogenesis and lipolysis in response to nutritional status.

Bone mineralization density and femoral neck fragility.

The traditional view of osteoporotic fractures is that they result from a reduction in bone mass combined with alterations in the micro-architecture. Apart from the effects of bone remodeling, the material properties of the remaining bone are thought to be unaffected. To test this, we compared the degree of matrix mineralization in femoral neck biopsies taken from cases of intracapsular hip fracture with age- and sex-matched postmortem controls. Whole femoral neck biopsies from seven female hip fracture cases (72-90 years) and nine controls (68-94 years) were embedded in methylmethacrylate, and sections stained with Solochrome Cyanin R for analysis of osteoid. The blocks were then diamond micro-milled, carbon coated, and analyzed for the degree of matrix mineralization using halogenated dimethacrylate standards for quantitative backscattered electron (qBSE) imaging (20 kV, entire block face, sampling interval 5 microm). The BSE gray scale was adjusted such that 0 corresponds to an electron backscattering coefficient of 0.1159 (approximately 1.70 g/ml) and 255-0.1519 (approximately 2.18 g/ml). Remodeling and mineralization data were analyzed for both the whole biopsy face and on a regional (anterior; inferior, posterior, or superior) basis. Over the whole biopsy, the level of mineralization was lower in the cases than the postmortem controls (-2.8%, P < 0.05). In both cases and controls, cortical mineralization was higher in the inferior (compressive) region compared with superior (tensile) region (P < 0.05). Mineralization was lower in all regions of the cases (inferior: -3.3%; posterior: -3.1%; anterior: -2.7%; superior: -1.6%) compared to the controls. Mineralization density in cancellous bone was not regionally dependent but was lower in the fracture cases (-3.5%; P = 0.001). Although there were weak relationships between osteoid formation (%O.Ar/B.Ar) and the mean level of mineralization in both cortical (P = 0.068) and cancellous (P < 0.01) bone, adjustment for this did not markedly affect the case-control differences. In conclusion, this study has shown that in cases of intracapsular hip fracture, matrix mineralization is reduced in the femoral neck. Unexpectedly, in view of the likely role of mild to moderate vitamin D deficiency osteopathy in hip fracture, this decreased mineralization was independent of osteoid indices and therefore potentially independent of bone age. This raises the possibility that alterations in the bone matrix such as excessive glycation or changes in the composition of the collagen fibrils affect its mineralization in hip fracture cases.

Effects of gender, anthropometric variables, and aging on the evolution of hip strength in men and women aged over 65.

Although gender differences in fall rates may partly explain the higher prevalence of fractures in elderly women than men, male bones may also be intrinsically stronger or suffer less structural degradation with age than those of women. We used hip structural analysis (HSA) to study gender differences in hip geometry and bone mineral density (BMD) as they evolved over time in elderly white men and women with the aim of identifying candidate biological pathways leading to heightened risk of hip fracture. We recruited 443 women and 439 men aged 67-79 years from a diet and cancer prospective population-based cohort study to a study of hip bone loss. Hip BMD was measured on two occasions 2-5 years apart by dual-energy X-ray absorptiometry and HSA software used to derive BMD and structural parameters at the narrow neck (NN), the intertrochanter (IT), and the shaft (S) regions. Structural indices calculated in each region were cross-sectional area (CSA)-amount of bone surface area in the cross section after excluding soft tissue space; section modulus (Z)-an index of bending resistance, subperiosteal width, endocortical width, cortical thickness; and cortical buckling ratio (CBR)-a measure of cortical instability. Compared to men, women had lower values of BMD, CSA, Z, subperiosteal width, endocortical width, and cortical thickness in all regions, except S endocortical width, after adjusting for weight, height, and age (P < 0.0001). CBR was higher in women than in men (P < 0.0001) in all regions. Longitudinal analysis of rates of change revealed faster rates of BMD decline in women than in men at the Hologic total hip, Hologic femoral neck, and IT regions (P < 0.029). Women had faster rates of subperiosteal and endosteal expansion than men at the NN (P < 0.011) and IT (P < 0.049) and faster increase in Z at the NN (P = 0.029). At the IT region, cortical thinning was faster in women than in men (P = 0.037) and CBR increased at a faster rate in women (P = 0.011). In conclusion, Z is lower in women than in men and expansion of the proximal femur occurs in both sexes, being faster in women than in men. Z does not decline at the same rate as BMD, implying that part of the effect of aging on BMD is due to expansion of the bony envelope without loss of bone mineral content. Faster expansion in the female femoral neck may in turn lead to greater fragility if wider diameter and thinner cortices become locally unstable.

The fragile elderly hip: mechanisms associated with age-related loss of strength and toughness.

Every hip fracture begins with a microscopic crack, which enlarges explosively over microseconds. Most hip fractures in the elderly occur on falling from standing height, usually sideways or backwards. The typically moderate level of trauma very rarely causes fracture in younger people. Here, this paradox is traced to the decline of multiple protective mechanisms at many length scales from nanometres to that of the whole femur. With normal ageing, the femoral neck asymmetrically and progressively loses bone tissue precisely where the cortex is already thinnest and is also compressed in a sideways fall. At the microscopic scale of the basic remodelling unit (BMU) that renews bone tissue, increased numbers of actively remodelling BMUs associated with the reduced mechanical loading in a typically inactive old age augments the numbers of mechanical flaws in the structure potentially capable of initiating cracking. Menopause and over-deep osteoclastic resorption are associated with incomplete BMU refilling leading to excessive porosity, cortical thinning and disconnection of trabeculae. In the femoral cortex, replacement of damaged bone or bone containing dead osteocytes is inefficient, impeding the homeostatic mechanisms that match strength to habitual mechanical usage. In consequence the participation of healthy osteocytes in crack-impeding mechanisms is impaired. Observational studies demonstrate that protective crack deflection in the elderly is reduced. At the most microscopic levels attention now centres on the role of tissue ageing, which may alter the relationship between mineral and matrix that optimises the inhibition of crack progression and on the role of osteocyte ageing and death that impedes tissue maintenance and repair. This review examines recent developments in the understanding of why the elderly hip becomes fragile. This growing understanding is suggesting novel testable approaches for reducing risk of hip fracture that might translate into control of the growing worldwide impact of hip fractures on our ageing populations.

Fourier transform infrared imaging of femoral neck bone: reduced heterogeneity of mineral-to-matrix and carbonate-to-phosphate and more variable crystallinity in treatment-naive fracture cases compared with fracture-free controls.

After the age of 60 years, hip fracture risk strongly increases, but only a fifth of this increase is attributable to reduced bone mineral density (BMD, measured clinically). Changes in bone quality, specifically bone composition as measured by Fourier transform infrared spectroscopic imaging (FTIRI), also contribute to fracture risk. Here, FTIRI was applied to study the femoral neck and provide spatially derived information on its mineral and matrix properties in age-matched fractured and nonfractured bones. Whole femoral neck cross sections, divided into quadrants along the neck's axis, from 10 women with hip fracture and 10 cadaveric controls were studied using FTIRI and micro-computed tomography. Although 3-dimensional micro-CT bone mineral densities were similar, the mineral-to-matrix ratio was reduced in the cases of hip fracture, confirming previous reports. New findings were that the FTIRI microscopic variation (heterogeneity) of the mineral-to-matrix ratio was substantially reduced in the fracture group as was the heterogeneity of the carbonate-to-phosphate ratio. Conversely, the heterogeneity of crystallinity was increased. Increased variation of crystallinity was statistically associated with reduced variation of the carbonate-to-phosphate ratio. Anatomical variation in these properties between the different femoral neck quadrants was reduced in the fracture group compared with controls. Although our treatment-naive patients had reduced rather than increased bending resistance, these changes in heterogeneity associated with hip fracture are in another way comparable to the effects of experimental bisphosphonate therapy, which decreases heterogeneity and other indicators of bone's toughness as a material.

Extracellular matrix mineralization promotes E11/gp38 glycoprotein expression and drives osteocytic differentiation.

Osteocytes are terminally differentiated osteoblasts which reside in a mineralized extracellular matrix (ECM). The factors that regulate this differentiation process are unknown. We have investigated whether ECM mineralization could promote osteocyte formation. To do this we have utilised MLO-A5 pre-osteocyte-like cells and western blotting and comparative RT-PCR to examine whether the expression of osteocyte-selective markers is elevated concurrently with the onset of ECM mineralization. Secondly, if mineralization of the ECM is indeed a driver of osteocyte formation, we reasoned that impairment of ECM mineralization would result in a reversible inhibition of osteocyte formation. Supplementation of MLO-A5 cell cultures with ascorbic acid and phosphate promoted progressive ECM mineralization as well as temporally associated increases in expression of the osteocyte-selective markers, E11/gp38 glycoprotein and sclerostin. Consistent with a primary role for ECM mineralization in osteocyte formation, we also found that inhibition of ECM mineralization, by omitting phosphate or adding sodium pyrophosphate, a recognized inhibitor of hydroxyapatite formation, resulted in a 15-fold decrease in mineral deposition that was closely accompanied by lower expression of E11 and other osteocyte markers such as Dmp1, Cd44 and Sost whilst expression of osteoblast markers Ocn and Col1a increased. To rule out the possibility that such restriction of ECM mineralization may produce an irreversible modification in osteoblast behaviour to limit E11 expression and osteocytogenesis, we also measured the capacity of MLO-A5 cells to re-enter the osteocyte differentiation programme. We found that the mineralisation process was re-initiated and closely allied to increased expression of E11 protein after re-administration of phosphate or omission of sodium pyrophosphate, indicating an ECM mineralization-induced restoration in osteocyte formation. These results emphasise the importance of cell-ECM interactions in regulating osteoblast behaviour and, more importantly, suggest that ECM mineralization exerts pivotal control during terminal osteoblast differentiation and acquisition of the osteocyte phenotype.

Osteocyte recruitment declines as the osteon fills in: interacting effects of osteocytic sclerostin and previous hip fracture on the size of cortical canals in the femoral neck.

There is little information on the distribution of osteocytes within the individual cortical osteon, but using direct 3-D imaging in a single subject, Hannah et al. found a gradient with a two-fold higher density of cells adjacent to the cement line compared to near the canal. Since a limiting factor for bone formation might be the availability of osteoblasts due to their recruitment as osteocytes, we studied distributions of osteonal osteocytes in frozen sections of the femoral neck cortex. Osteocytes were stained with an anti-sclerostin antibody and counter-stained with toluidine blue. Adjacent sections were stained for alkaline phosphatase (ALP). Each osteonal osteocyte was categorised as being sclerostin-positive (scl+) or negative (scl-). ImageJ was used to measure the perimeter and area of each osteon and canal, while special purpose routines were used to measure the minimum distances of each osteocyte from the cement line and the canal. Canal area was strongly correlated with osteon area. Osteocytes were most dense close to the cement line; and their areal density within the matrix declined up to three-fold between the cement line and the canal, depending on osteon diameter. Large and small osteons had similar densities of osteocytes close to the cement line, but fractured neck of femur cases had significantly lower densities of osteocytes close to the canal. Higher osteocyte density close to the canal was associated with ALP expression. It is concluded that entombment of osteocytes newly drawn from the osteoblast pool into the mineralising matrix is independent of preceding bone resorption depth. As osteonal infilling proceeds, osteocyte formation declines more rapidly than matrix formation, leading to a progressive reduction in osteocyte density. A shrinking supply of precursor osteoblasts due to previous osteocyte recruitment, apoptosis, or both could produce this effect. In a statistically significant contrast, sclerostin negative osteocytes adjacent to the canal had the expected effect of reducing canal size in controls but this was not seen in hip fracture. This demonstrated the failure of osteonal osteoblasts to sustain bone formation through a complete remodelling cycle in osteoporosis, perhaps due to insufficient osteoblasts remaining capable of mineralized matrix formation. The failure of osteocytic sclerostin suppression to associate with bone formation in these osteons might alternatively be explained by downstream interference with sclerostin's effect on wnt signalling.

Patients with sclerosteosis and disease carriers: human models of the effect of sclerostin on bone turnover.

Sclerosteosis is a rare bone sclerosing dysplasia, caused by loss-of-function mutations in the SOST gene, encoding sclerostin, a negative regulator of bone formation. The purpose of this study was to determine how the lack of sclerostin affects bone turnover in patients with sclerosteosis and to assess whether sclerostin synthesis is decreased in carriers of the SOST mutation and, if so, to what extent this would affect their phenotype and bone formation. We measured sclerostin, procollagen type 1 amino-terminal propeptide (P1NP), and cross-linked C-telopeptide (CTX) in serum of 19 patients with sclerosteosis, 26 heterozygous carriers of the C69T SOST mutation, and 77 healthy controls. Chips of compact bone discarded during routine surgery were also examined from 6 patients and 4 controls. Sclerostin was undetectable in serum of patients but was measurable in all carriers (mean 15.5 pg/mL; 95% confidence interval [CI] 13.7 to 17.2 pg/mL), in whom it was significantly lower than in healthy controls (mean 40.0 pg/mL; 95% CI 36.9 to 42.7 pg/mL; p < 0.001). P1NP levels were highest in patients (mean 153.7 ng/mL; 95% CI 100.5 to 206.9 ng/mL; p = 0.01 versus carriers, p = 0.002 versus controls), but carriers also had significantly higher P1NP levels (mean 58.3 ng/mL; 95% CI 47.0 to 69.6 ng/mL) than controls (mean 37.8 ng/mL; 95% CI 34.9 to 42.0 ng/mL; p = 0.006). In patients and carriers, P1NP levels declined with age, reaching a plateau after the age of 20 years. Serum sclerostin and P1NP were negatively correlated in carriers and age- and gender-matched controls (r = 0.40, p = 0.008). Mean CTX levels were well within the normal range and did not differ between patients and disease carriers after adjusting for age (p = 0.22). Our results provide in vivo evidence of increased bone formation caused by the absence or decreased synthesis of sclerostin in humans. They also suggest that inhibition of sclerostin can be titrated because the decreased sclerostin levels in disease carriers did not lead to any of the symptoms or complications of the disease but had a positive effect on bone mass. Further studies are needed to clarify the role of sclerostin on bone resorption.

Changing structure of the femoral neck across the adult female lifespan.

The anatomic distribution of cortical and cancellous bone in the femoral neck may be critical in determining resistance to fracture. We investigated the effects of aging on femoral neck bone in women. In this cross-sectional study, we used clinical multidetector computed tomography (MDCT) of the hips to investigate aging effects in 100 female volunteers aged 20 to 90 years. We developed a clinically efficient protocol to measure cortical thickness (C.Th) and cortical, trabecular, and integral bone mineral density (CtBMD, TrBMD, and iBMD in mg/cm(3)) in anatomic quadrants of the femoral neck. We used a nested ANOVA to evaluate their associations with height, weight, location in the femoral neck, and age of the subject. Age was the principal determinant of both cortical thickness and BMD. Age had significantly different effects within the anatomic quadrants; compared with young women, elderly subjects had relative preservation of the inferoanterior (IA) quadrant but strikingly reduced C.Th and BMD superiorly. A model including height, weight, and region of interest (and their interactions) explained 83% of the measurement variance (p < .0001). There were marked C.Th and BMD differences between age 25 and age 85 in the already thin superior quadrants. At 25 years the predicted C.Th of the superoposterior quadrant was 1.63 mm, whereas at 85 years it was 0.33 mm [-1.33 mm, 95% confidence interval (CI) of difference over 60 years -1.69 to -0.95]. By contrast, at 25 years mean C.Th of the IA quadrant was 3.9 mm, whereas at 85 years it was 3.3 mm (-0.6 mm, 95% CI -0.83 to -0.10). CtBMD of the IA region was equivalent at 25 and 85 years. In conclusion, elderly women had relative preservation of IA femoral neck bone over seven decades compared with young women but markedly lower C.Th and BMD in the other three quadrants. The IA quadrant transmits mechanical load from walking. Mechanical theory and laboratory tests on cadaveric femurs suggest that localized bone loss may increase the risk of fracture in elderly fallers. It remains to be determined whether this MDCT technique can provide better prediction of hip fracture than conventional clinical dual X-ray absorptiometry (DXA).

Sclerostin and the regulation of bone formation: Effects in hip osteoarthritis and femoral neck fracture.

Remodeling imbalance in the elderly femoral neck can result in thin cortices and porosity predisposing to hip fracture. Hip osteoarthritis protects against intracapsular hip fracture. By secreting sclerostin, osteocytes may inhibit Wnt signaling and reduce bone formation by osteoblasts. We hypothesised that differences in osteocytic sclerostin expression might account for differences in osteonal bone-formation activity between controls and subjects with hip fracture or hip osteoarthritis. Using specific antibody staining, we determined the osteocytic expression of sclerostin within osteons of the femoral neck cortex in bone removed from subjects undergoing surgery for hip osteoarthritis (hOA: 5 males, 5 females, 49 to 92 years of age) or hip fracture fixation (FNF: 5 males, 5 females, 73 to 87 years of age) and controls (C: 5 males, 6 females, 61 to 90 years of age). Sclerostin expression and distances of each osteocyte to the canal surface and cement line were assessed for all osteonal osteocytes in 636 unremodeled osteons chosen from fields ( approximately 0.5 mm in diameter) with at least one canal staining for alkaline phosphatase (ALP), a marker of bone formation. In adjacent sections, ALP staining was used to classify basic multicellular unit (BMUs) as quiescent or actively forming bone (ALP(+)). The areal densities of scl(-) and scl(+) osteocytes (number of cells per unit area) in the BMU were inversely correlated and were strong determinants of ALP status in the BMU. In controls and hip fracture patients only, sclerostin-negative osteocytes were closer to osteonal surfaces than positively stained cells. Osteon maturity (progress to closure) was strongly associated with the proportion of osteonal osteocytes expressing sclerostin, and sclerostin expression was the chief determinant of ALP status. hOA patients had 18% fewer osteocytes per unit bone area than controls, fewer osteocytes expressed sclerostin on average than in controls, but wide variation was seen between subjects. Thus, in most hOA patients, there was increased osteonal ALP staining and reduced sclerostin staining of osteocytes. In FNF patients, newly forming osteons were similar in this respect to hOA osteons, but with closure, there was a much sharper reduction in ALP staining that was only partly accounted for by the increased proportions of osteonal osteocytes staining positive for sclerostin. There was no evidence for a greater effect on ALP expression by osteocytes near the osteonal canal. In line with data from blocking antibody experiments, osteonal sclerostin appears to be a strong determinant of whether osteoblasts actively produce bone. In hOA, reduced sclerostin expression likely mediates increased osteoblastic activity in the intracapsular cortex. In FNF, full osteonal closure is postponed, with increased porosity, in part because the proportion of osteocytes expressing sclerostin increases sharply with osteonal maturation.