Combination of nanoindentation and quantitative backscattered electron imaging revealed altered bone material properties associated with femoral neck fragility.

Osteoporotic fragility fractures were hypothesized to be related to changes in bone material properties and not solely to reduction in bone mass. We studied cortical bone from the superior and inferior sectors of whole femoral neck sections from five female osteoporotic hip fracture cases (74-92 years) and five nonfractured controls (75-88 years). The typical calcium content (Ca(Peak)) and the mineral particle thickness parameter (T) were mapped in large areas of the superior and inferior regions using quantitative backscattered electron imaging (qBEI) and scanning small-angle X-ray scattering, respectively. Additionally, indentation modulus (E) and hardness (H) (determined by nanoindentation) were compared at the local level to the mineral content (Ca(Ind)) at the indent positions (obtained from qBEI). Ca(Peak) (-2.2%, P = 0.002), Ca(Ind) (-1.8%, P = 0.048), E (-5.6%, P = 0.040), and H (-6.0%, P = 0.016) were significantly lower for the superior compared to the inferior region. Interestingly, Ca(Peak) as well as Ca(Ind) were also lower (-2.6%, P = 0.006, and -3.7%, P = 0.002, respectively) in fracture cases compared to controls, while E and H did not show any significant reduction. T values were in the normal range, independent of region (P = 0.181) or fracture status (P = 0.551). In conclusion, it appears that the observed femoral neck fragility is associated with a reduced mineral content, which was not accompanied by a reduction in stiffness and hardness of the bone material. This pilot study suggests that a stiffening process in the organic matrix component contributes to bone fragility independently of mineral content.

Effects of physical activity on evolution of proximal femur structure in a younger elderly population.

INTRODUCTION: For a fixed weight, a wider bone of standardised length is stiffer. But moving the cortices away from the centre of mass risks creating structural (elastic) instability, and hip fractures have been postulated to occur as a consequence of buckling of the thinned supero-lateral femoral neck cortex during a fall. We hypothesised that stereotyped physical activity (e.g., walking) may help conserve bending resistance (section modulus, Z) through redistribution of bone tissue, but it might be at the expense of supero-lateral cortical stability. METHODS: Hip structural analysis (HSA) software applied to DXA scans was used to derive measurements of section modulus and distances of a cross-section's centre of mass from the supero-lateral cortical margin (lateral distance, in cm). DXA scans were obtained on 1361 men and women in the EPIC-Norfolk population-based prospective cohort study. Up to 4 repeat DXA scans were done in 8 years of follow-up. Weight, height and activities of daily living were assessed on each occasion. A detailed physical activity and lifestyle questionnaire was administered at baseline. The lateral distance was measured on three narrow cross-sections with good precision: narrow neck (NN, coefficient of variation 2.6%), intertrochanter (IT) and shaft (S). A linear mixed model was used to assess associations with predictors. RESULTS: Ageing was associated with medial shifting of the centre of mass, so that lateral distance increased. Both greater weight and height were associated with greater lateral distance (P<0.0001). Among physical activity-related variables, walking/cycling for >1 h/day (P=0.025), weekly time spent on moderate impact activity (P=0.003), forced expiratory volume in 1 s (NN and IT, P<0.026) and lifetime physical activity (IT, P<0.0001) were associated with higher lateral distance. However, after adjusting for these variables, activities of daily living scores (NN, P<0.0001) and weekly time spent on low impact hip flexing activities were associated with shorter lateral distance (P=0.001). Greater baseline lateral distance was significantly associated with increased risk of subsequent hip fracture (n=26) in females (P<0.05, all regions) independently of age, height and bone mineral content. CONCLUSION: The age-related shift medially of the centre of mass of the femoral neck and trochanter may have adverse effects on fracture resistance in the event of a fall, so compromising the beneficial effects of walking on fitness, strength and risk of falling. The role of more diverse physical activity patterns in old age that impose loading on the supero-lateral cortex of the femur, involving for example hip flexion and stretching, needs investigation for their ability to correct this medial shifting of the centre of mass.

Sex hormone status may modulate rate of expansion of proximal femur diameter in older women alongside other skeletal regulators.

CONTEXT: Little is known of associations between hip geometry and skeletal regulators. This is important because geometry is a determinant of both hip function and resistance to fracture. OBJECTIVE: We aimed to determine the effects of sex hormone status and other candidate regulators on hip geometry and strength. SUBJECTS AND METHODS: A random sample of 351 women aged 67-79 had two to four hip dual-energy x-ray absorptiometry scans performed over 8 yr of follow-up. Hip structural analysis software was used to measure subperiosteal diameter (PD) and the distance from the center of mass to the lateral cortical margin (d-lat) on three 5-mm-thick cross-sectional regions: narrow neck, intertrochanter, and shaft. Section modulus (Z), bone mineral density (grams per centimeter squared), and an index of bone mineral content (cross-sectional area) were calculated as estimators of bone strength. Serum analytes measured at baseline included SHBG, estradiol, PTH, creatinine, albumin, vitamin D metabolites, and glutamate- and gamma-carboxyglutamate-osteocalcin (OC). A linear mixed model was used to model associations with predictor variables, including testing whether the predictors significantly modified the effect of aging. RESULTS: Aging was associated with increasing PD and d-lat, and higher baseline SHBG significantly modified this effect, in the case of PD, increasing the rates of change at the narrow neck region by 19% for SHBG level 2 sd higher than population mean (P = 0.026). Higher baseline creatinine was independently associated with faster increases in PD and d-lat with aging (P < 0.041). Z declined faster with aging if baseline PTH was higher, and higher albumin had a contrary effect. Z was positively associated with free estradiol and inversely associated with SHBG and glutamate-OC. CONCLUSION: These results show large effects of SHBG on the regulation of proximal femur expansion and bending resistance, probably acting as a surrogate for low bioavailable estrogen. Potentially important effects for fracture resistance in old age were also revealed for PTH, markers related to renal function and the nutritional markers albumin and undercarboxylated OC.

Osteoclastic cortical erosion as a determinant of subperiosteal osteoblastic bone formation in the femoral neck's response to BMU imbalance. Effects of stance-related loading and hip fracture.

Femoral neck fractures have previously been shown to be associated with increased cortical and endocortical remodeling, reduced wall thickness of endocortical packets and cortical porosity. Femoral neck width is associated positively with history of lifetime physical activity; so we hypothesized that exposure to mechanical loading may influence the subperiosteal osteoblastic response to the weakening effect of intracortical bone resorption. In 21 femoral neck biopsies from female subjects (13 with hip fracture), there was a positive association between osteoblastic periosteal alkaline phosphatase expression shown in frozen sections and the percentage of cortical canals internal to the subperiosteal surface showing evidence of osteoclastic erosion (Goldner's stain; p =0.03). This was stronger in the plane of locomotor loading and particularly strong in the inferior (compression) cortex ( p =0.002). In 35 cases and 23 age/gender-matched postmortem controls, osteoid-bearing cortical canals (%) were significantly elevated in the fracture cases compared with the controls within the anterior region. There was also a significant correlation between cortical and endocortical %OS/BS (percentage osteoid surface to bone surface) (fracture, n =12; control, n =12) over the whole biopsy ( p =0.041). Generally, these associations of intracortical with endocortical remodeling were consistent with both envelopes being regulated by common processes. These results support the concept that the slow growth of femoral neck width by subperiosteal apposition of bone occurs directly or, otherwise, in response to the weakening of the cortex as it is "trabecularized" by imbalance of bone multicellular units (BMU). This process, in turn, depends on cortical thinning and enlargement of canals with the formation of giant, composite osteons, the whole being more marked in cases of future hip fracture.

Femoral neck fragility: genes or environment?

As with other diseases that show exponentially increasing rates, hip fracture probably requires multiple prior events to become manifest. It is common ground that there are genetic, environmental, lifestyle and perhaps dietary determinants of risk of osteoporotic fracture as well as interactions between them. The key to secondary prevention is the understanding of how these components can be integrated into an effective assessment of the major risks of hip fracture after a previous fracture has occurred. The key to primary prevention is to understand both the pathological and physiological basis of hip fragility. It is not unreasonable to suppose that in a western lifestyle our limited and stereotypic patterns of locomotion from middle age onwards may offer considerably less protection than, for example, the more physically demanding activity of subsistence farming.

NOS isoforms in adult human osteocytes: multiple pathways of NO regulation?

Until now, eNOS has been considered to be the predominant osteocytic nitric oxide synthase (NOS) isoform in bone. We previously studied the distribution of eNOS protein expression in the human femoral neck because of its possible involvement in the response to load. Studies in rat and human fracture callus have shown that nNOS mRNA is expressed sometime after fracture, but no study has yet immunolocalized NOS isoforms in mature adult human bone. In this study, we have examined the distribution of NOS isoforms in iliac osteocytes. Frozen sections (10 microm) were cut from transiliac biopsies from 8 female osteoporotic patients (range, 56-80 years) and from 7 female postmortem femoral neck biopsies (range, 65-90 years). Sections were incubated overnight in antiserum for eNOS, nNOS, or iNOS followed by peroxidase/VIP substrate detection. We used eNOS and iNOS antisera directed against the C-terminus. For nNOS, three different antisera were used, two binding to different C-terminal epitopes and one binding to N-terminal epitope. Sections were then incubated in propidium iodide or methyl green to detect all osteocytes. eNOS antibody was able to detect eNOS epitopes in osteocytes. All three nNOS antibodies detected nNOS epitopes in osteocytes, but those directed against the C-terminus had higher detection rates. iNOS was rarely seen. In the iliac crest, the percentage of osteocytes positive for nNOS was higher than that for eNOS (cortical: nNOS 84.04%, eNOS 61.78%, P < 0.05; cancellous: nNOS 82.33%, eNOS 65.21%, P < 0.05). In the femoral neck, the percentage of osteocytes positive for nNOS (60.98%) was also higher than that for eNOS (40.41%), although this difference was not statistically significant. In conclusion, both eNOS and nNOS isoforms are present in osteocytes in the iliac crest and femoral neck.

Discrimination between cases of hip fracture and controls is improved by hip structural analysis compared to areal bone mineral density. An ex vivo study of the femoral neck.

In vivo bone densitometry is affected by measurement inaccuracies arising from the assumptions made about soft tissue and marrow composition. This study tested the hypothesis that section modulus (SM, a measure of bending resistance) when measured ex vivo, would discriminate cases of hip fracture from controls better than areal bone mineral density (aBMD). The biopsies were from (n = 22, female) subjects that had suffered an intracapsular hip fracture. The control material (n = 24, female) was from post-mortem subjects. Serial peripheral quantitative computed tomography (pQCT) 1-mm thick cross-sectional images of femoral neck previously embedded in methacrylate were obtained with the Densiscan 1000 pQCT densitometer and matched for lateral location. The image voxels were converted to units of bone mass, which were then used to derive the section modulus. The data were used to derive means from which receiver operating characteristic (ROC) curves could be generated. The area under the curves (AUC) showed that discrimination between the fracture cases and controls was better for SM than aBMD [SM: AUC = 0.83 (95% confidence interval: 0.71, 0.96), aBMD: AUC = 0.70 (0.54, 0.85); P = 0.034]. To simulate the forces experienced during a sideways fall, the model's neutral axis was rotated by 210 degrees. The results for section modulus were predictable from those at 0 degrees (r(2) = 0.97). We conclude that biomechanical analysis of the distribution of bone within the femoral neck may offer a marked improvement in the ability to discriminate patients with an increased risk of intracapsular fracture. Progress towards implementing this form of analysis in clinical densitometry should improve its diagnostic value, but may depend in part on better image resolution and more accurate corrections for the variability between subjects in regional soft tissue composition.

Bone remodeling at the endocortical surface of the human femoral neck: a mechanism for regional cortical thinning in cases of hip fracture.

UNLABELLED: Endocortical remodeling and wall thickness (W.Th.) were measured in femoral neck bone from 12 female fracture cases (81.3 +/- 1.5 years) and 12 sex-matched controls (81.9 +/- 1.9 years). Regionally, osteoid and eroded surface were increased, whereas W.Th. was reduced. These processes likely contribute to cortical bone loss seen in hip fracture. INTRODUCTION: Because periosteal expression of alkaline phosphatase was similar between cases and controls, we hypothesized that the mechanism causing the marked femoral neck cortical thinning associated with hip fracture may be net endocortical bone loss. METHODS: Twelve female cases of femoral neck fracture (mean age = 81.3 +/- 1.5 years) and 12 age- and sex-matched postmortem controls (mean age = 81.9 +/- 1.9 years) were included in the study. Samples of their femoral neck bone were embedded in methyl methacrylate, sectioned at 10 microm, and stained with Solochrome cyanine R and Goldner's trichrome for the detection of osteoid (%OS/BS) and resorption surfaces (%ES/BS) respectively. In addition, wall thickness (W.Th.) and lamellar thickness (Lm.Th.) data were also collected from identifiable endocortical bone packets as a measure of formative potential. RESULTS AND CONCLUSIONS: %OS/BS was significantly elevated in the anterior (control = 3.4 +/- 0.7: fracture = 11.0 +/- 2.3; p = 0.0001), inferior (3.4 +/- 1.0: 9.9 +/- 3.0; p = 0.0009), and posterior quadrants (3.2 +/- 0.8: 9.1 +/- 2.3; p = 0.0021). Only for anterior region was increased %ES/BS demonstrated in the fracture group (2.8 +/- 0.6: 5.3 +/- 0.7; p = 0.055). W.Th. (mm) was reduced only in the inferior region of the fracture cases (control = 33.7 +/- 1.2: fracture = 30.6 +/- 0.9; p = 0.013), whereas Lm.Th. was also reduced inferiorly (control = 2.7 +/- 0.08: fracture = 2.5 +/- 0.08; p = 0.042). These data suggest that an endocortical remodeling imbalance involving reduced bone formation within inferior region coupled with elevated anterior resorption may make an important contribution to the cortical thinning observed in cases of femoral neck fracture.

The effects of hormone replacement therapy on cortical bone in postmenopausal women. A histomorphometric study.

Investigations of the actions of estrogen on the skeleton have mainly focused on cancellous bone and there are no reported histomorphometric studies of the effects of oestrogen on cortical bone in humans. The aim of this study was to investigate the effects of both conventional hormone replacement therapy (HRT) and high-dose oestradiol on cortical bone in postmenopausal women. Transiliac biopsies were obtained from nine postmenopausal women aged 54-71 yr before and after 2 yr (mean, 23.5 months) of conventional HRT and in seven postmenopausal women aged 52-67 yr after long-term, high-dose oestradiol implant therapy (at least 14 yr). Indices of bone turnover, remodeling, and cortical structure were assessed by image analysis. Cortical width was highest in the women treated with high-dose oestrogen therapy (2.29 +/- 0.78 mm; mean +/- SD) and lowest in untreated women (1.36 +/- 0.60 mm; P=0.014). The proportion of canals with an eroded surface was significantly lower in the high-dose oestrogen group than in women before or after conventional HRT (3.03 +/- 3.7% vs. 11.1 +/- 7.1% and 10.5 +/- 8.6%; P=0.017 and 0.05, respectively). Bone formation rate (microm2/microm/day) in untreated women was significantly higher than in the high-dose oestrogen group (0.121 +/- 0.072 vs. 0.066 +/- 0.045, respectively; P=0.05), values in women treated with conventional HRT being intermediate. Our results provide the first histomorphometric evidence in postmenopausal women of dose-dependent oestrogen-induced suppression of bone turnover in iliac crest cortical bone. There was also a trend toward higher wall width with increasing dose of oestrogen, consistent with the previously reported anabolic effect in cancellous bone.

Hip section modulus, a measure of bending resistance, is more strongly related to reported physical activity than BMD.

We hypothesized that measures of physical activity would have a closer relationship with section modulus (SM), an indicator of bending resistance, than with bone mineral density (BMD) because physical activity might expand the bony envelope, which tends to reduce BMD for a constant bone mineral content. Four hundred twenty-three men and 436 women (mean age 72 years, SD =3) were recruited from a prospective population-based cohort study to a study of hip bone loss. Hip BMD was measured on two occasions 2-5 years apart (mean 2.7, DXA-Hologic 1,000 W). Hip structural analysis (HSA) software was used to calculate SM and BMD from the DXA scans on three narrow regions: the narrow neck (NN), intertrochanter (IT) and shaft (S). A physical activity and lifestyle questionnaire was administered at baseline. Multivariate repeated measures analysis of variance was used to model the associations between personal attributes (weight, height, age), physical activity and lifestyle variables with SM, cross-sectional area (CSA), sub-periosteal diameter (PD) and BMD. Men and women were analysed together after tests for interactions with gender, which were found not to be significant. In all regions female gender was associated with having lower values of all outcomes, and body weight was positively associated with all outcomes, i.e., SM, CSA, PD and BMD ( P<0.0001). Sub-periosteal diameter was positively associated with reported lifetime physical activity (IT and S, P<0.0001). There was a significant decline of BMD with age at the NN and S regions ( P<0.026), and the PD increased with age (NN and S, P<0.019). Previous fracture history was associated with having lower values of BMD, SM and CSA (except for S; P<0.022). Both section modulus and CSA were positively associated with heavy physical activity after age 50 years in all regions ( P<0.019), whereas NN BMD was the only BMD associate of heavy physical activity after 50 ( P=0.036). Time spent per week on recreational activities classified as no impact activity was positively associated with BMD, CSA and SM (multivariate P<0.016). In conclusion, proximal femur diameter is associated positively with reported life-long physical activity. If this is mediated through a loading related effect on sub-periosteal expansion, BMD would be an unsatisfactory outcome measure in physical activity studies since it is inversely related to projected bone area. SM in contrast was associated with several measures of recent physical activity and relates more directly to the bending experienced by the proximal femur in response to a given load. These data are consistent with an effect of mechanical loading to regulate bone strength through an anabolic effect maximal in the subperiosteal cortex, where the highest loading-related strains are experienced.

Evidence for bone formation on the external "periosteal" surface of the femoral neck: a comparison of intracapsular hip fracture cases and controls.

Age-related expansion of the external surface of the femoral neck in order to offset generalized bone loss is potentially an important mechanism whereby hip strength and hence resistance to hip fracture is maintained. However, it has been widely assumed that bone formation is precluded from this external interface due to the presence of a synovial membrane associated with the hip joint. In this study we have demonstrated histologically that bone formation does indeed occur on the outer "periosteal" surface of the proximal femoral neck. It was therefore hypothesized that an impairment or reduction in periosteal bone formation might be seen in cases of femoral neck fracture compared with age-matched controls. Qualitative analysis of whole femoral neck samples from female subjects and age- and sex-matched post-mortem controls demonstrated that these groups expressed similar distributions of the bone formation marker, alkaline phosphatase (AP), at the periosteal surface [whole biopsy mean % periosteal AP-positive surface: control=16.0 (range=0.5-43.0), fracture=13.4 (range=1.0-34.6), p=0.44]. In conclusion, despite a wide intersubject variation, bone formation at the femoral neck periosteum is a feature of elderly women even if they have had a hip fracture.

Increased cancellous bone in the femoral neck of patients with coxarthrosis (hip osteoarthritis): a positive remodeling imbalance favoring bone formation.

Osteoporosis is caused by an imbalance between bone resorption and formation which results in an absolute reduction in bone mass. In a previous study we highlighted a condition, osteoarthritis of the hip (coxarthrosis, cOA), where an imbalance between resorption and formation provided beneficial effects in the form of an absolute increase in bone mass. We demonstrated that the femoral neck in patients with cOA had increased cancellous bone area, connectivity and trabecular thickness which might contribute to the protection against fracture associated with the condition. The aim of the present study was to analyze forming and resorbing surfaces in coxarthritic cancellous bone to assess whether increased formation or reduced resorption could be responsible for these structural changes. Whole cross-sectional femoral neck biopsies were obtained from 11 patients with cOA and histomorphometric parameters compared with 14 age- and sex-matched cadaveric controls. The ratio of osteoid surface to bone surface was 121% ( p<0.001) higher in the cases but there was no significant difference in resorptive surface. The percentage osteoid volume to bone volume (%OV/BV; +270%, p<0.001) and osteoid width (O.Wi; +127%, p<0.001) were also higher in the cases. This study suggests that the increased cancellous bone mass seen in cases of cOA is due to increased bone formation rather than decreased bone resorption. Investigation of the cellular and biochemical basis for these changes might provide new insights into the pathogenesis of osteoarthritis and highlight novel biological mechanisms regulating bone multicellular unit (BMU) balance that could be relevant to developing new interventions against hip and other osteoporotic fractures.

Increased femoral neck cancellous bone and connectivity in coxarthrosis (hip osteoarthritis).

Patients with coxarthrosis (cOA) have a reduced incidence of intracapsular femoral neck fracture, suggesting that cOA offers protection. The distribution of bone in the femoral neck was compared in cases of coxarthrosis and postmortem controls to assess the possibility that disease-associated changes might contribute to reduced fragility. Whole cross-section femoral neck biopsies were obtained from 17 patients with cOA and 22 age- and sex-matched cadaveric controls. Densitometry was performed using peripheral quantitated computed tomography (pQCT) and histomorphometry on 10-microm plastic-embedded sections. Cortical bone mass was not different between cases and controls (P > 0.23), but cancellous bone mass was increased by 75% in cOA (P = 0.014) and histomorphometric cancellous bone area by 71% (P < 0.0001). This was principally the result of an increase of apparent density (mass/vol) of cancellous bone (+45%, P = 0.001). Whereas cortical porosity was increased in the cases (P < 0.0001), trabecular width was also increased overall in the cases by 52% (P < 0.001), as was cancellous connectivity measured by strut analysis (P < 0.01). Where osteophytic bone was present (n = 9) there was a positive relationship between the amount of osteophyte and the percentage of cancellous area (P < 0.05). Since cancellous bone buttresses and stiffens the cortex so reducing the risk of buckling, the increased cancellous bone mass and connectivity seen in cases of cOA probably explain, at least in part, the ability of patients with cOA to resist intracapsular fracture of the femoral neck during a fall.

The ratio of osteocytic incorporation to bone matrix formation in femoral neck cancellous bone: an enhanced osteoblast work rate in the vicinity of hip osteoarthritis.

Recently it has been shown that an inactivating mutation in the TGFb-SMAD3 signaling pathway, which increases the conversion of osteoblasts to osteocytes, is accompanied by bone loss combined with increased osteocyte density. We hypothesized that increased matrix TGFb, known to occur in osteoarthritis, might cause the reverse of these effects in man. Because coxarthrosis (cOA) is associated with a reduced risk of femoral neck fracture, whole cross-section femoral neck biopsies were obtained from 11 patients with femoral neck fracture, 14 patients with cOA, and 22 age-and sex-matched controls. Lacunar density (Lc x mm2), osteocyte density (Ot x mm2), and cancellous wall width (Cn x W x Wi), were compared between cases of coxarthrosis, femoral neck fracture (FNF) and controls. In cOA, Lc.mm2 was reduced by 24% (P <0.001) while in FNF it was increased by 20% (P <0.001). Cn x W x Wi was increased in cOA by 22% (P <0.05) and in FNF was reduced by 27% (P <0.001). Lc x mm2 was inversely related to percentage cancellous bone area (adj. r2 = 0.373; P <0.01) and wall widths, r2 = 0.382, P <0.001. The reduction in osteocyte lacunar density coupled with increased wall width is consistent with a model of cOA effects on bone in which increased levels of matrix TGFb might prolong the effective lifespan or work rate of the osteoblast and delay its incorporation into the matrix as an osteocyte. One possible approach to strengthening bone in osteoporosis might be to enhance the effective lifespan of the osteoblast by modulating TGFb-related pathway activity in its local environment.

Osteocyte density in aging subjects is enhanced in bone adjacent to remodeling haversian systems.

The osteocyte is a candidate regulatory cell for bone remodeling. Previously, we demonstrated that there is a substantial (approximately 50%) loss of osteocytes from their lacunae in the cortex of the elderly femoral neck. Higher occupancy was evident in tissue exhibiting high remodeling and high porosity. The present study examines the distribution of osteocytes within individual osteonal systems at differing stages of the remodeling cycle. In 22 subjects, lacunar density, osteocyte density, and their quotient, the percent lacunar occupancy, was assessed up to a distance of 65 microm from the canal surface in six quiescent, resorbing, and forming osteons. In both forming (p = 0.024) and resorbing (p = 0.034) osteons, osteocyte densities were significantly higher in cases of hip fracture than controls. However, there were no significant between-group differences in lacunar occupancy. In both cases and controls, osteocyte density (p < 0.0001; mean difference +/-SEM: 157 +/- 34/mm2) and lacunar occupancy (p = 0.025; mean difference: 8.1 +/- 3.4%) were shown to be significantly higher in forming compared with quiescent osteons. Interestingly, resorbing systems also exhibited significantly elevated osteocyte density in both the fracture and the control group combined (mean difference 76 +/- 23/mm2; p = 0.003). Lacunar occupancy was also greater in resorbing compared with quiescent osteons (both groups combined: p = 0.022; mean difference: 5.7 +/- 2.3%). Elevated osteocyte density and lacunar occupancy in forming compared with quiescent systems was expected because of the likely effects of aging on quiescent osteons. However, the higher levels of these parameters in resorbing compared with quiescent systems was the opposite of what we expected and suggests that, in addition to their postulated mechanosensory role in the suppression of remodeling and bone loss, osteocytes might also contribute to processes initiating or maintaining bone resorption.

Patterns of osteocytic endothelial nitric oxide synthase expression in the femoral neck cortex: differences between cases of intracapsular hip fracture and controls.

Evidence indicates that extensive amalgamation of adjacent resorbing osteons is responsible for destroying the microstructural integrity of the femoral neck's inferior cortex in osteoporotic hip fracture. Such osteonal amalgamation is likely to involve a failure to limit excessive resorption, but its mechanistic basis remains enigmatic. Nitric oxide (NO) inhibits osteoclastic bone destruction, and in normal bone cells its generation by endothelial nitric oxide synthase (eNOS, the predominant bone isoform) is enhanced by mechanical stimuli and estrogen, which both protect against fracture. To determine whether eNOS expression in osteocytes reflects their proposed role in regulating remodeling, we have examined patterns of osteocyte eNOS immunolabeling in the femoral neck cortex of seven cases of hip fracture and seven controls (females aged 68-96 years). The density of eNOS+ cells (mm(-2)) was 53% lower in the inferior cortex of the fracture cases (p < 0.0004), but was similar in the superior cortex. eNOS+ osteocytes were, on average, 22% further from their nearest blood supply, than osteocytes in general (p < 0.0001) and the nearest eNOS+ osteocyte was 57% further from its nearest canal surface (p < 0.0001). This differential distribution of eNOS+ osteocytes was significantly more pronounced in the cortices of fracture cases (p < 0.0001). We conclude that the normal regional and osteonal pattern of eNOS expression by osteocytes is disrupted in hip fracture, particularly at sites that are loaded most by physical activity. These results suggest that eNOS+ osteocytes may normally act as sentinels confining resorption within single osteons. A reduction in their number, coupled to an increase in their remoteness from canal surfaces, may thus permit the irreversible merging of resorbing osteons, and thus contribute to the marked increase in the fragility of osteoporotic bone.

Super-osteons (remodeling clusters) in the cortex of the femoral shaft: influence of age and gender.

Previous studies of cortical remodeling in the fractured femoral neck indicated that the merging of spatially clustered remodeling osteons could result in the formation of deleteriously large cavities associated with femoral neck fracture. This study aimed to identify whether remodeling osteons in the femoral shaft were also clustered and to assess the influence of age and gender. Microradiographic images of femoral mid-shaft cross-sections from 66 subjects over 21 years of age were analyzed to determine the number, size and location of all Haversian canals. Those most recently remodeled were identified using an edge-detection algorithm highlighting the most marked differential gradients in grey levels. Cluster analysis (JMP software) of these osteons identified the proportion of recently remodeled osteons that were within 0.75 mm clusters. As in the femoral neck, remodeling osteons were significantly more clustered than could occur by chance (real, 59.4%; random, 39.4%; P < 0.0001). The density of these clusters (number/mm(2)) was not significantly associated with subject age or gender but was greatest near the periosteum and decreased toward the marrow cavity (periosteal 0.043 +/- 0.004; mid-cortex 0.028 +/- 0.003; endosteal 0.017 +/- 0.002). Cortical porosity increased with age. The presence of giant canals (diameter >385 microm) was inversely related to the presence of clusters (R(2) = 0.237, P < 0.0001). This data suggest that remodeling osteons tend to be spatially colocalized in the shaft as they are in the neck of the femur and their presence is independent of age or gender. We propose that these remodeling clusters be termed super-osteons. The negative relationship between super-osteons and giant canals raises the intriguing possibility that loss of the control of remodeling depth results in the merging of osteonal systems to form deleteriously large cortical cavities with a marked reduction in bone strength.

Osteocyte lacunar occupancy in the femoral neck cortex: an association with cortical remodeling in hip fracture cases and controls.

In adult humans, osteocytes die and disappear from their lacunae in the cortex of bones which remodel slowly, such as the proximal femur, and osteocyte death is particularly prevalent in the elderly. We have investigated the statistical determinants of osteocyte density in microscopic fields (0.71 mm2) within thin, complete femoral neck cross-sections cut from biopsies embedded in methyl methacrylate and stained with solochrome cyanine R. Lacunae were counted under phase contrast and osteocytes within lacunae were counted in the same fields under epifluorescence. The percentage of lacunae containing an osteocyte varied between 12.4% and 99.2%, according to subject and quadrantic region of the cortex examined. The microscopic determinants of field-specific osteocyte density included the porosity measured in the field itself and the regional measurement of the proportion of cortical canals bearing osteoid. There was significant variation between subjects and, within subjects, between cortical regions. Also the inferior region showed a significantly higher density of lacunae than the superior region (+8.2%; P = 0.013). However, cases of fracture were not significantly different from controls with respect to osteocyte lacunar occupancy after adjusting for osteoid-bearing canals and porosity. It is concluded that in subjects in their 7th-9th decades of age, osteocyte lacunar occupancy is statistically associated with bone turnover, implying that high turnover (locally young bone age) might favor lacunar occupancy (ln% osteoid; P = 0.021). Alternative explanations of the association are that porosity reflects a better nutritional supply via the vasculature or that porosity of the cortex is associated with osteocyte density through an effect of osteocytes on bone remodeling.

Intracapsular hip fracture and the region-specific loss of cortical bone: analysis by peripheral quantitative computed tomography.

Generalized bone loss within the femoral neck accounts for only 15% of the increase in intracapsular hip fracture risk between the ages of 60 and 80 years. Conventional histology has shown that there is no difference in cancellous bone area between cases of intracapsular fracture and age and sex-matched controls. Rather, a loss of cortical bone thickness and increased porosity is the key feature with the greatest change occurring in those regions maximally loaded during a fall (the inferoanterior [IA] to superoposterior [SP] axis). We have now reexamined this finding using peripheral quantitative computed tomography (pQCT) to analyze cortical and cancellous bone areas, density, and mass in a different set of ex vivo biopsy specimens from cases of intracapsular hip fracture (female, n = 16, aged 69-92 years) and postmortem specimens (female, n = 15, aged 58-95 years; male, n = 11, aged 56-86 years). Within-neck location was standardized by using locations at which the ratio of maximum to minimum external diameters was 1.4 and at more proximal locations. Cortical widths were analyzed using 72 radial profiles from the center of area of each of the gray level images using a full-width/half-maximum algorithm. In both male and female controls, cancellous bone mass increased toward the femoral head and the rate of change was gender independent. Cancellous bone mass was similar in cases and controls at all locations. Overall, cortical bone mass was significantly lower in the fracture cases (by 25%; p < 0.001) because of significant reductions in both estimated cortical area and density. These differences persisted at locations that are more proximal. The mean cortical width in the cases was significantly lower in the IA (22.2%;p = 0.002) and inferior regions (19%;p < 0.001). The SP region was the thinnest in both cases and controls. These data confirm that a key feature in the etiology of intracapsular hip fracture is the site-specific loss of cortical bone, which is concentrated in those regions maximally loaded during a fall on the greater trochanter. An important implication of this work is that the pathogenesis of bone loss leading to hip fracture must be by a mechanism that varies in its effect according to location within the femoral neck Key candidate mechanisms would include those involving locally reduced mechanical loading. This study also suggests that the development of noninvasive methodologies for analyzing the thickness and estimated densities of critical cortical regions of the femoral neck could improve detection of those at risk of hip fracture.