Lumbar Vertebral Cancellous Bone Research Articles

Protective effect of low-dose risedronate against osteocyte apoptosis and bone loss in ovariectomized rats

Osteocyte apoptosis is the first reaction to estrogen depletion, thereby stimulating osteoclastic bone resorption resulting in bone loss. We investigated the effects of two different risedronate (RIS) doses (high and low) on osteocyte apoptosis, osteoclast activity and bone loss in ovariectomized rats. Forty rats with ovariectomy (OVX) and sham ovariectomy (SHAM) were divided into 4 groups: 1) SHAM rats treated with saline (SHAM); 2) OVX rats treated with saline (OVX); 3) OVX rats treated with low-dose RIS (OVX-LR, 0.08 μg/kg/day); 4) OVX rats treated with high-dose RIS (OVX-HR, 0.8 μg/kg/day). All animals were sacrificed 90 days after surgery for the examinations of osteocyte apoptosis by caspase-3 staining, osteoclast activity by TRAP staining and bone volume by micro-CT scanning in lumbar vertebral cancellous bone. Both low and high dose RIS significantly reduced caspase-3 positive osteocytes, empty lacunae and TRAP positive osteoclasts in OVX rats. Although the difference in caspase-3 positive osteocytes was not significant between the OVX-LR and OVX-HR groups, numerically these cells were significantly more prevalent in OVX-HR (not OVX-LR) group than in SHAM group. TRAP positive osteoclasts were significantly higher in OVX-LR group than in SHAM or OVX-HR group. There was no significant difference in bone volume among the OVX-LR, OVX-HR and SHAM groups, but lower in OVX group alone. However, significant increase in trabecular thickness only occurred in OVX-LR group. We conclude that both low and high dose RIS significantly inhibit osteocyte apoptosis and osteoclast activity in OVX rats, but the low-dose RIS has weaker effect on osteoclast activity. However, low-dose RIS preserves cancellous bone mass and microarchitecture as well as high-dose RIS after estrogen depletion.

Voxel size and measures of individual resorption cavities in three-dimensional images of cancellous bone

Cavities formed by osteoclasts on the surface of cancellous bone during bone remodeling (resorption cavities) are believed to act as stress risers and impair cancellous bone strength and stiffness. Although resorption cavities are readily detected as eroded surfaces in histology sections, identification of resorption cavities in three-dimensional images of cancellous bone has been rare. Here we use sub-micrometer resolution images of rat lumbar vertebral cancellous bone obtained through serial milling ( n = 5) to determine how measures of the number and surface area of resorption cavities are influenced by image resolution. Three-dimensional images of a 1 mm cube of cancellous bone were collected at 0.7 × 0.7 × 5.0 μm/voxel using fluorescence based serial milling and uniformly coarsened to four other resolutions ranging from 1.4 × 1.4 × 5.0 to 11.2 × 11.2 × 10 μm/voxel. Cavities were identified in the three-dimensional image as an indentation on the cancellous bone surface and were confirmed as eroded surfaces by viewing two-dimensional cross-sections (mimicking histology techniques). The number of cavities observed in the 0.7 × 0.7 × 5.0 μm/voxel images (22.0 ± 1.43, mean ± SD) was not significantly different from that in the 1.4 × 1.4 × 5.0 μm/voxel images (19.2 ± 2.59) and an average of 79% of the cavities observed at both of these resolutions were coincident. However, at lower resolutions, cavity detection was confounded by low sensitivity (< 20%) and high false positive rates (> 40%). Our results demonstrate that when image voxel size exceeds 1.4 × 1.4 × 5.0 μm/voxel identification of resorption cavities by bone surface morphology is highly inaccurate. Experimental and computational studies of resorption cavities in three-dimensional images of cancellous bone may therefore require images to be collected at resolutions of 1.4 μm/pixel in-plane or better to ensure consistent identification of resorption cavities.

Microscopic Multi-Directional Permeability Coefficient of Human Lumbar Vertebral Cancellous Bone Tissue

The mechanical performance of cancellous bone is characterized using experimental measure of parameters (elastic moduli, permeability, etc.) which apply poroelasticity theory. Poroelastic investigations of bone are being extended to investigations of micro-mechanisms of remodeling processes and transport of metabolic product in bone. To understand physiological and pathological behavior of human skeletal system, the accurate measurement of biomechanical properties for bone is the one of important works. Particularly, the microscopic measurement is very important since the biomechanical behavior at the small scale of bone could be closely related to the remodeling of bone. But microscopic measurement of permeability coefficient which is one of important parameter of poroelasticity theory was never measured. In this study, a small scale permeability testing machine was developed to measure accurate coefficient of microscopic specimen permeability. Total of twenty one cylindrical cancellous specimens with a diameter of 500 from seven fresh male and female lumbar vertebrae (14, 39, 59, 61, 69, 75, and 77 years) were fabricated using a micro-milling machine having a resolution of 10 . To determine the directional permeability, bone coordinate was set to be x1 (longitudinal axis) and others were set as x2 (posteroanterior axis) and x3 (latero-medial axis). The measured mean permeability (± SD) values of each direction from x1 to x3 were 9 5.56 10− × (S.D. ± 10 7.10 10− × ), 9 6.66 10− × (S.D. ± 10 6.56 10− × ), and 9 7.04 10− × (S.D. ± 10 8.47 10− × ) m2/Pa/sec, respectively. Mean value in the x1 direction of specimens were the smallest (p&lt;0.01) in comparison to x2 and x3 directions.

Lipids isolated from bone induce the migration of human breast cancer cells

Bone is the most common site to which breast cancer cells metastasize. We found that osteoblast-like MG63 cells and human bone tissue contain the bile acid salt sodium deoxycholate (DC). MG63 cells take up and accumulate DC from the medium, suggesting that the bone-derived DC originates from serum. DC released from MG63 cells or bone tissue promotes cell survival and induces the migration of metastatic human breast cancer MDA-MB-231 cells. The bile acid receptor farnesoid X receptor (FXR) antagonist Z-guggulsterone prevents the migration of these cells and induces apoptosis. DC increases the gene expression of FXR and induces its translocation to the nucleus of MDA-MB-231 cells. Nuclear translocation of FXR is concurrent with the increase of urokinase-type plasminogen activator (uPA) and the formation of F-actin, two factors critical for the migration of breast cancer cells. Our results suggest a novel mechanism by which DC-induced increase of uPA and binding to the uPA receptor of the same breast cancer cell self-propel its migration and metastasis to the bone.

Weaning Initiates a Rapid and Powerful Anabolic Phase in the Rat Maternal Skeleton1

Maternal skeletal mineral lost during lactation is rapidly restored after weaning. The purposes of this study were to determine when increases of bone formation occur after weaning, whether the expanding osteoblast population is derived from proliferating progenitors, and to relate these skeletal changes to known endocrine events at weaning. Female rats were allowed to complete one reproductive cycle. Half of these rats were mated a second time and allowed to lactate for 20 days. The other half served as an age-matched, normal estrus cycling comparison group. One day after weaning, the dams and their comparison group were given four injections of bromodeoxyuridine (BrdU) at 8-h intervals. Indices of bone formation and the kinetics of BrdU-labeled cells were measured in lumbar vertebral cancellous bone. At 2 days after weaning, cancellous bone formation rates were substantially greater than those in the nonmated rats. Indices of bone formation more than doubled from the second to seventh day after weaning. At 25 h after the first BrdU injection in the postweaned rats, considerable numbers of labeled cells were observed on or near the bone surface, with about 17% of the osteoblast population labeled. Labeled osteoblasts peaked at 20%-24% compared with 4% in the normal estrus cycling group. Immediately following weaning, there is a profound increase in the osteoblast population in maternal cancellous bone. Many, if not most of these newly formed osteoblasts were derived from proliferating progenitors. It is possible that the endocrine milieu of lactation expands or primes the osteoprogenitor pool for this rapid anabolic phase.

Assessment of bone feature parameters from lumbar trabecular skeletal patterns using mathematical morphology image processing

In this study, bone feature parameters were obtained from digital lumbar vertebral images to determine their potential usefulness in assessing binary trabecular skeletal patterns. The system consisted of a magnification radiographic technique, computed radiography (CR), microfocused X-ray computed tomography (mu-CT), and mathematical morphology image processing. A digital CR image was produced, using a ten-times magnified lumbar vertebral cancellous bone block (1.5 x 1.5 x 1.5 cm(3)). The information was then subjected to mathematical morphology image processing, to extract eight binary trabecular skeletal patterns having different continuities as the operation number ( n) increased. These skeletal patterns were used as test patterns and analyzed quantitatively by calculation of the skeletal pixel percentage (SKP) and star volume analysis (Vsk, volume of skeletal trabecular elements; Vsp, volume of nonskeletal elements). Then the binary skeletal pattern data were converted into the mu-CT format, using a general personal computer, and the images were quantitatively analyzed with mu-CT built-in image analysis software for microstructural indices, fractal dimension analysis, and node-strut analysis. The SKP and Vsk showed an expected decrease as n increased. Concurrently, the Vsp showed an expected increase as n increased. The skeletal thickness (Sk.Th) showed a constant value at n = 1 to 4 and n = 5 to 7, but decreased in a stepwise fashion at n = 1 and n = 5. The skeletal number (Sk.N), bone area fraction (B.Ar/T.Ar) and bone perimeter fraction (B.Pm/T.Ar), decreased as n increased. Skeletal space (Sk.Sp) and perimeter-to-area ratio (B.Pm/B.Ar) increased with increasing n. In the fractal dimension analysis, the values decreased with increasing n and showed changes similar to the image observations. In fact, the SKP, star volume analysis, Sk.N, B.Ar/T.Ar, B.Pm/T.Ar, Sk.Sp, and B.Pm/B.Ar all closely mirrored the observational analysis of the images. Linkage of the skeletal structure as functions of the following parameters could be quantitatively demonstrated. The parameters used were node (Nd) and terminus (Tm). Variations in the total strut length (TSL) and Tm could demonstrate quantitative changes in skeletal structure. These results indicate that the system consisting of a combination of a magnification radiographic technique, CR, mu-CT, and mathematical morphology image processing may be a useful tool for quantitative skeletal structure analysis and the structural assessment of lumbar vertebrae, for the assessment of skeletal structural changes. It is important to choose suitable parameters for the desired structural changes.

Fractal analysis of lumbar vertebral cancellous bone architecture.

Osteoporosis is characterized by bone mineral density (BMD) decreasing and spongy bone rearrangement with consequent loss of elasticity and increased bone fragility. Quantitative computed tomography (QCT) quantifies bone mineral content but does not describe spongy architecture. Analysis of trabecular pattern may provide additional information to evaluate osteoporosis. The aim of this study was to determine whether the fractal analysis of the microradiography of lumbar vertebrae provides a reliable assessment of bone texture, which correlates with the BMD. The lumbar segment of the spine was removed from 22 cadavers with no history of back pain and examined with standard x-ray, traditional tomography, and quantitative computed tomography to measure BMD. The fractal dimension, which quantifies the image fractal complexity, was calculated on microradiographs of axial sections of the fourth lumbar vertebra to determine its characteristic spongy network. The relationship between the values of the BMD and those of the fractal dimension was evaluated by linear regression and a statistically significant correlation (R = 0.96) was found. These findings suggest that the application of fractal analysis to radiological analyses can provide valuable information on the trabecular pattern of vertebrae. Thus, fractal dimensions of trabecular bone structure should be considered as a supplement to BMD evaluation in the assessment of osteoporosis.

Lasofoxifene (CP-336,156) protects against the age-related changes in bone mass, bone strength, and total serum cholesterol in intact aged male rats.

The purpose of this study was to evaluate if long-term (6 months) treatment with lasofoxifene (LAS), a new selective estrogen receptor modulator (SERM), can protect against age-related changes in bone mass and bone strength in intact aged male rats. Sprague-Dawley male rats at 15 months of age were treated (daily oral gavage) with either vehicle (n = 12) or LAS at 0.01 mg/kg per day (n = 12) or 0.1 mg/kg per day (n = 11) for 6 months. A group of 15 rats was necropsied at 15 months of age and served as basal controls. No significant change was found in body weight between basal and vehicle controls. However, an age-related increase in fat body mass (+42%) and decrease in lean body mass (-8.5%) was observed in controls. Compared with vehicle controls, LAS at both doses significantly decreased body weight and fat body mass but did not affect lean body mass. No significant difference was found in prostate wet weight among all groups. Total serum cholesterol was significantly decreased in all LAS-treated rats compared with both the basal and the vehicle controls. Both doses of LAS treatment completely prevented the age-related increase in serum osteocalcin. Peripheral quantitative computerized tomography (pQCT) analysis at the distal femoral metaphysis indicated that the age-related decrease in total density, trabecular density, and cortical thickness was completely prevented by treatment with LAS at 0.01 mg/kg per day or 0.1 mg/kg per day. Histomorphometric analysis of proximal tibial cancellous bone showed an age-related decrease in trabecular bone volume (TBV; -46%), trabecular number (Tb.N), wall thickness (W.Th), mineral apposition rate, and bone formation rate-tissue area referent. Moreover, an age-related increase in trabecular separation (Tb.Sp) and eroded surface was observed. LAS at 0.01 mg/kg per day or 0.1 mg/kg per day completely prevented these age-related changes in bone mass, bone structure, and bone turnover. Similarly, the age-related decrease in TBV and trabecular thickness (Tb.Th) and the age-related increase in osteoclast number (Oc.N) and osteoclast surface (Oc.S) in the third lumbar vertebral cancellous bone were completely prevented by treatment with LAS at both doses. Further, LAS at both doses completely prevented the age-related decrease in ultimate strength (-47%) and stiffness (-37%) of the fifth lumbar vertebral body. These results show that treatment with LAS for 6 months in male rats completely prevents the age-related decreases in bone mass and bone strength by inhibiting the increased bone resorption and bone turnover associated with aging. Further, LAS reduced total serum cholesterol and did not affect the prostate weight in these rats. Our data support the potential use of a SERM for protecting against the age-related changes in bone and serum cholesterol in elderly men.

Lumbar vertebral cancellous bone is capable of responding to PGE2 treatment by stimulating both modeling and remodeling-dependent bone gain in aged male rats.

Previously we found that PGE2 3 mg/kg in 20-month-old male rats induced massive bone formation mainly by modeling dependent bone gain in cortical bone. It is not known whether cancellous bone will respond similarly; thus, we evaluated the effect of PGE2 on cancellous bone of the same aged rats. Thirty-four 20-month-old Wistar male rats were given PGE2 (3 mg/kg/day) or vehicle subcutaneously for 10 and 30 days. Double fluorescent labels were injected 9 and 2 days prior to the sacrifice. Histomorphometry was performed on 1% toluidine blue stained and unstained sagittal sections of lumbar vertebral bodies. The results demonstrated that 10-day PGE2 treatment increased osteoprogenitor cells, osteoblasts (x 2-fold), osteoid (x 4.5-fold), woven bone formation (0.04%), and 40% more trabecular area; it stimulated modeling (x 2-fold) and remodeling-dependent (x 1.5-fold) bone formation with increase of mineralization lag time (MLT, x 7.5-fold). Thirty-day treatment sustained increases in osteoblast numbers, modeling and remodeling-dependent bone formation and further stimulated woven bone formation (6.6%), turnover (x 3-fold), and trabecular area and number (x 2-fold). Osteoprogenitor cells were undetectable along with 70% less osteoid area compared with 10-day treatment but still was 1.5-fold higher than aging controls. MLT returned to aging control level. It was concluded that the aged cancellous bone of 20-month-old male Wistar rat retains a capability of responding to the anabolic effect of PGE2. Osteoblastogenesis and enhanced modeling and remodeling-dependent woven or lamellar formation contribute to this anabolic action. Bone formation differed in that the endocortical surface of cortical bone was stimulated mainly by modeling whereas both modeling and remodeling-dependent bone gain were equally stimulated at the trabecular surface of the lumbar vertebral body.

A new method of comprehensive static histomorphometry applied on human lumbar vertebral cancellous bone

The aim of the present study was to assess age-related changes in the human spine by use of established static histomorphometry, and to determine how these static histomorphometric measures are interrelated in human cancellous bone tissue. The material comprised normal human lumbar vertebral bodies (L-2) from 12 women (19–96 years) and 12 men (23–91 years) selected from a larger autopsy material to give an even age and gender distribution. In addition, L-2 from three female subjects (80, 88, and 90 years) with a known vertebral fracture of L-2 were considered. Approximately 9-mm-thick frontal (mediolateral) slices were embedded in methylmetacrylate, stained with aniline blue, and scanned into a computer with a flatbed image scanner at a high resolution (2400 dpi). With a custom-made computer program the following static histomorphometric measures were determined: trabecular bone volume; marrow space star volume; bone space star volume; anisotropy of bone and marrow phase (star length distribution method); node-strut analysis (node:terminus ratio); trabecular thickness; trabecular number; trabecular separation; and trabecular bone pattern factor. In addition, connectivity density was determined (by the ConnEulor method). All 11 histomorphometric measures, except bone space star volume and the two measures of anisotropy, showed a significant correlation with age. Marrow space star volume ( r = 0.82) and trabecular bone volume ( r = −0.81) showed the highest correlation with age. Furthermore, it was found that all of the histomorphometric measures were correlated, to different degrees. Trabecular bone volume correlated significantly with all ten histomorphometric measures, whereas the two anisotropy measures were poorly correlated to the other measures. Finally, we found the histomorphometric values in this study to be in excellent accordance with various previously published results from studies of human trabecular vertebral bone, the sole exception being marrow space star volume, which was probably due to the small (artificial) region of interest (ROI) that was used in the earlier studies. In conclusion, the new method applied herein allows for easy assessment of age-related changes and also for assessment of relationships between histomorphometric measures in human vertebral cancellous bone.

Skeletal effects of low-dose cyclosporin A in aged male rats: lack of relationship to serum testosterone levels.

The aim of this study was to investigate the skeletal effects of cyclosporin A (CsA) in a dose range relevant to clinical medicine in lumbar vertebral cancellous bone of aged male rats and to correlate these effects with possible changes in serum testosterone levels. Thirty-one 18-month-old male Wistar rats were divided into four weight-matched groups and subcutaneously injected with either 0, 1, 3, or 5 mg of CsA/kg of body weight three times per week After 4 weeks of treatment, all rats were killed after in vivo fluorochrome labeling and the first lumbar vertebrae analyzed by quantitative histomorphometry. Serum was analyzed for total calcium, creatinine, alkaline phosphatase, osteocalcin, parathyroid hormone, total testosterone, and CsA levels. CsA administration resulted in a dose-dependent increase in serum osteocalcin levels and in histomorphometric indices of cancellous bone turnover in the axial skeleton. Furthermore, CsA-treated rats showed a deterioration of vertebral cancellous bone structure with increased discontinuity of the trabecular bone network due to trabecular plate perforations. Serum testosterone levels were not significantly changed by CsA treatment and were uncorrelated to all biochemical or histomorphometric indices of bone turnover. We conclude that the 4-week administration of CsA at doses that are close to those used in transplantation patients induced high turnover osteopenia in the axial skeleton of aged, 18-month-old male rats, and that these effects were likely not mediated by changes in serum testosterone levels.

Effects of droloxifene on prevention of cancellous bone loss and bone turnover in the axial skeleton of aged, ovariectomized rats

The purpose of this study was to determine the efficacy of droloxifene (DRO), an estrogen antagonist/agonist, in preventing ovariectomy (OVX)-induced lumbar vertebral cancellous bone loss and bone turnover in aged female rats. Fifty-three Sprague-Dawley female rats were OVX or sham-operated at 19 months of age, and divided into 6 groups: (I) sham-operated controls; (II) OVX vehicle controls; (III) OVX rats treated with E2 at 30 micrograms/kg/day; (IV)-(VI) OVX rats treated with DRO at either 2.5, 5, or 10 mg/kg p.o. daily. The treatment period was 8 weeks. Static and dynamic cancellous bone histomorphometric parameters were determined on 4 and 10 microns thick, undecalcified, double-fluorescent labeled sections of the fourth lumbar vertebral body. Changes in body weight, uterine weight, and total serum cholesterol were also determined. OVX for 8 weeks in 19-month-old female rats resulted in reduced trabecular bone volume (-18%) and trabecular width (-10%) and increased labeling perimeter (+52%), bone formation rate/bone surface referent (+60%), bone formation rate/bone volume referent (+77%), osteoclast number (+41%), and osteoclast perimeter (+41%). E2 treatment at 30 micrograms/kg/day for 8 weeks prevented OVX-induced cancellous bone loss and decreased bone resorption, bone formation, and bone turnover to the values of sham controls. DRO at 2.5-10 mg/kg/day completely prevented bone loss and bone turnover associated with estrogen deficiency. Osteoclast number and perimeter were significantly decreased in DRO-treated-OVX rats compared to both sham and OVX controls. Trabecular bone volume, trabecular width, labeling perimeter, bone formation rate/bone surface referent, and bone formation rate/bone volume referent showed no differences in DRO-treated OVX rats compared to those of E2-treated OVX rats and sham controls. These histomorphometric results indicated that DRO is an estrogen agonist on cancellous bone of lumbar vertebral bodies of aged, OVX rats. Further, E2 treatment prevented the OVX-induced increase in body weight gain and nonsignificantly reduced total serum cholesterol compared to OVX controls. Body weight gain and total serum cholesterol did not differ between OVX rats treated with E2 and sham controls. In OVX rats treated with DRO, body weight decreased significantly in a dose-response manner, and total serum cholesterol was significantly reduced by 65% to 70% compared to both sham and OVX controls. In addition, treatment with E2 increased uterine weight to the value of sham controls in OVX rats. However, DRO had no effect on uterine weight at either 2.5 or 10 mg/kg/day, while it only slightly but significantly increased uterine weight over OVX controls at 5 mg/kg/day. We conclude that DRO was efficacious in the prevention of lumbar vertebral cancellous bone loss and in the decline of total serum cholesterol but had no effect on uterine weight in the aged, OVX female rats. Our data suggest that DRO is a potentially useful agent for the prevention of vertebral bone loss leading to spinal fractures in postmenopausal women.

Multiplanar variations in the structural characteristics of cancellous bone

A quantitative serial sectioning technique and a video-imaging procedure were used to obtain precise (20-μm voxels, in a 5 mm × 6 mm × 7 mm test volume) digital images of lumbar vertebral cancellous bone specimens. Conventional stereological image analyses were performed on this data to determine multiplanar (bulk) and surface planar variations in cancellous structural properties. Based upon an error analysis of subgroups of the complete serial planar images, our findings suggest that, for a plane resolution of 20 μm, sectioning at an increment ⩽100 μm along an axis is necessary to obtain accurate data of bulk structural properties of cancellous bone. Planar structural variations obtained from the serial sections of cancellous bone revealed a high degree of complexity and heterogeneity of the bone architecture. Bone area centers deviated from the section centroid and showed a helical variation along the primary or superior-inferior loading axis of samples. In comparison with the base planes (perpendicular to the superior-inferior axis), the lateral planes (parallel to the superior-inferior axis) of spine samples showed smaller mean values of structural indices and a more oriented structure. This structural anisotropy may be related to the functional mechanical anisotropy of the samples.

Increased cancellous bone remodeling during lactation in beagles

Changes in bone mass and cancellous bone remodeling parameters were determined during lactation in adult beagles. Bone mineral content was determined by photon absorptiometry, fraction of mineralized cancellous bone area and perimeter to area ratios by image analysis of microradiographs, and bone remodeling parameters by fluorochrome-based histomorphometry. Bone mineral content of the intact proximal humerus and fraction of mineralized tissue areas of lumbar vertebral cancellous bone were decreased during lactation when compared with controls. There were significant increases during lactation in osteoid volumes and osteoid surface, although osteoid seam thickness was not changed. There were significant increases in the fraction of fluorochrome-labeled cancellous bone surfaces in the lactating dogs when compared with controls. The mineralizing surface, bone formation rate and activation frequency were significantly increased in the lactating dogs when compared with controls. There were also significant increases in resorption parameters during lactation. These data indicate that during lactation, bone remodeling and bone turnover is increased. Increased bone remodeling during lactation in a longer-lived mammal may represent an example of a “reversible mineral deficit” which may be an important physiological mechanism in nursing mothers to ensure adequate calcium for milk production. Increased bone remodeling may also provide a physiological mechanism to enhance the capacity of the skeleton to better accommodate the greatly increased and episodic changes in mineral homeostasis during lactation.