Inhibition of apoptosis exacerbates fatigue-damage tendon injuries in an in vivo rat model.

Tendinopathy is a common and progressive musculoskeletal disease. Increased apoptosis is an end-stage tendinopathy manifestation, but its contribution to the pathology of the disease is unknown. A previously established in vivo model of fatigue damage accumulation shows that increased apoptosis is correlated with the severity of induced tendon damage, even in early onset of the disease, supporting its implication in the pathogenesis of the disease. Consequently, this study aimed to determine: (1) whether apoptosis could be inhibited after fatigue damage and (2) whether its inhibition could lead to remodeling of the extracellular matrix (ECM) and pericellular matrix (PCM), to ultimately improve the mechanical properties of fatigue-damaged tendons. The working hypothesis was that, despite the low vascular nature of the tendon, apoptosis would be inhibited, prompting increased production of matrix proteins and restoring tendon mechanical properties. Rats received 2 or 5 d of systemic pan-caspase inhibitor (Q-VD-OPh) or dimethyl sulfoxide (DMSO) carrier control injections starting immediately prior to fatigue loading and were sacrificed at days 7 and 14 post-fatigue-loading. Systemic pan-caspase inhibition for 2 d led to a surprising increase in apoptosis, but inhibition for 5 d increased the population of live cells that could repair the fatigue damage. Further analysis of the 5 d group showed that effective inhibition led to an increased population of cells producing ECM and PCM proteins, although typically in conjunction with oxidative stress markers. Ultimately, inhibition of apoptosis led to further deterioration in mechanical properties of fatigue-damaged tendons.

High resolution micro arthrography of hard and soft tissues in a murine model.

OBJECTIVE: Recent developments on high resolution micro computed tomography (µCT) allow imaging of soft tissues in small animal joints. Nevertheless, µCT images cannot distinguish soft tissues from synovial fluid due to their similar mass density, limiting the 3D assessment of soft tissues volume and thickness. This study aimed to evaluate a lead chromate contrast agent for µCΤ arthrography of rat knee joints ex vivo. DESIGN: Intact tibiofemoral rat joints were injected with the contrast agent at different concentrations and imaged using a µCT at 2.7 µm isotropic voxel size. Cartilage thickness was measured using an automated procedure, validated against histological measurements, and analyzed as a function of µCT image resolution. Changes in hard and soft tissues were also analyzed in tibiofemoral joints 4 weeks after surgical destabilization of the medial meniscus (DMM). RESULTS: The contrast agent diffused well throughout the whole knee cavity without penetrating the tissues, therefore providing high contrast at the boundaries between soft tissues and synovial fluid space. Thickness analysis of cartilage demonstrated a high similarity between histology and µ-arthrography approaches (R(2) = 0.90). Four weeks after surgical DMM, the development of osteophytes (Oph) and cartilage ulcerations was recognizable with µCT, as well as a slight increase in trabecular bone porosity, and decrease in trabecular thickness. CONCLUSIONS: A lead chromate-based contrast agent allowed discriminating the synovial fluid from soft tissues of intact knee joints, and thus made possible both qualitative and quantitative assessment of hard and soft tissues in both intact and DMM tibiofemoral joints using high resolution µCT.

Ovariectomy increases vascular calcification via the OPG/RANKL cytokine signalling pathway.

BACKGROUND: Observational studies suggest a strong relationship between menopause and vascular calcification. Receptor activator of nuclear factor-kappaBeta ligand (RANKL) and osteoprotegerin (OPG) are critical regulators of bone remodelling and modulate vascular calcification. We assessed the hypothesis that ovariectomy increases vascular calcification via the OPG/RANKL axis. MATERIALS AND METHODS: Age-matched sexually mature rabbits were randomized to ovariectomy (OVX, n = 12) or sham procedure (SHAM, n = 12). One month post-procedure, atherosclerosis was induced by 15 months 0.2%-cholesterol diet and endothelial balloon denudations (at months 1 and 3). Aortic atherosclerosis was assessed in vivo by magnetic resonance imaging (MRI) at months 9 and 15. At sacrifice, aortas were harvested for ex vivo microcomputed tomography (microCT) and molecular analysis of the vascular tissue. RESULTS: Vascular calcification density and calcific particle number were significantly greater in OVX than SHAM (8.4 +/- 2.8 vs. 1.9 +/- 0.6 mg cm(-3), P = 0.042, and 94 +/- 26 vs. 33 +/- 7 particles cm(-3), P = 0.046, respectively). Calcification morphology, as assessed by the arc angle subtended by the largest calcific particle, showed no difference between groups (OVX 33 +/- 7 degrees vs. SHAM 33 +/- 5 degrees , P = 0.99). By Western blot analysis, OVX increased the vascular OPG:RANKL ratio by 66%, P = 0.029, primarily by decreasing RANKL (P = 0.019). At month 9, MRI demonstrated no difference in atheroma volume between OVX and SHAM, and no significant change was seen by the end of the study. CONCLUSIONS: In contrast to bone, vascular OPG:RANKL ratio increased in response to ovariectomy with a corresponding fourfold increase in arterial calcification. This diametrical organ-specific response may explain the comorbid association of osteoporosis with calcifying atherosclerosis in post-menopausal women.

Strain amplification and integrin based signaling in osteocytes.

Recent morphological studies have suggested that osteocyte processes are directly attached at discrete locations along the canalicular wall by beta3 integrins at the apex of infrequent, previously unrecognized, canalicular projections. This discovery has led to a new paradigm for the initiation of intracellular signaling, which provides a possible long sought after molecular mechanism for the initiation of intracellular signaling in bone cells. The quantitative feasibility of this hypothesis is explored with a detailed theoretical model, which predicts that axial strains due to the sliding of actin microfilaments about the fixed integrin attachments are in order of magnitude larger than the radial strains in the previously proposed strain amplification theory and two orders of magnitude greater than whole tissue strains.

Strength of cancellous bone trabecular tissue from normal, ovariectomized and drug-treated rats over the course of ageing.

Hormone therapy (HT) drugs and bisphosphonates prevent osteoporosis by inhibiting osteoclastic bone resorption. However, the effects of osteoporosis and anti-resorptive drugs on the mechanical behavior of the bone tissue constituting individual trabeculae have not yet been quantified. In this study, we test the hypothesis that the mechanical properties of bone trabecular tissue will differ for normal, ovariectomized and drug-treated rat bones over the course of ageing. Microtensile testing is carried on individual trabeculae from tibial bone of ovariectomized (OVX) rats, OVX rats treated with tibolone and placebo-treated controls. The method developed minimizes errors due to misalignment and stress concentrations at the grips. The local mineralization of single trabeculae is compared using micro-CT images calibrated for bone mineral content assessment. Our results indicate that ovariectomy in rats increases the stiffness, yield strength, yield strain and ultimate stress of the mineralized tissue constituting trabecular bone relative to normal; we found significant differences (P < 0.05) at 14, 34 and 54 weeks of treatment. These increases are complemented by a significant increase in the mineral content at the tissue level, although overall bone mineral density and mass are reduced. With drug treatment, the properties remain at, or slightly below, the placebo-treated controls levels for 54 weeks. The higher bone strength in the OVX group may cause the trabecular architecture to adapt as seen during osteopenia/osteoporosis, or alternately it may compensate for loss of trabecular architecture. These findings suggest that, in addition to the effects of osteoporosis and subsequent treatment on bone architecture, there are also more subtle processes ongoing to alter bone strength at the tissue level.

Loss of osteocyte integrity in association with microdamage and bone remodeling after fatigue in vivo.

As a result of fatigue, bone sustains microdamage, which is then repaired by bone-remodeling processes. How osteoclastic activity is targeted at the removal of microdamaged regions of bone matrix is unknown. In the current studies, we tested the hypothesis that changes in osteocyte integrity, through the initiation of regulated cell death (apoptosis), are associated with fatigue-related microdamage and bone resorption. Ulnae of adult rats were fatigue-loaded to produce a known degree of matrix damage. Osteocyte integrity was then assessed histomorphometrically from terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-nick end labeling (TUNEL)-stained sections to detect cells undergoing DNA fragmentation associated with apoptosis; toluidine blue-stained sections were used for secondary morphological confirmation. Ten days after loading, large numbers of TUNEL-positive osteocytes were found in bone surrounding microcracks and in bone surrounding intracortical resorption spaces (approximately 300% increases over controls, p < 0.005). TUNEL labeling in loaded ulnae at sites distant from microcracks or resorption foci did not differ from that in control bone. Osteocytes in toluidine blue-stained sections showed equivalent trends to TUNEL-stained sections, with significant increases in pyknotic nuclei and empty lacunae associated with microcracks and intracortical resorption spaces. TUNEL-positive osteocytes were observed around bone microdamage by 1 day after loading (p < 0.01 relative to baseline), and their number remained elevated throughout the entire experimental period. Increases in empty lacunae and decreases in normal osteocyte numbers were observed over time as well. These studies show that (1) osteocyte apoptosis is induced by bone fatigue, (2) this apoptosis is localized to regions of bone that contain microcracks, and (3) osteoclastic resorption after fatigue also coincides with regions of osteocyte apoptosis. The strong associations between microdamage, osteocyte apoptosis, and subsequent bone remodeling support the hypothesis that osteocyte apoptosis provides a key part of the activation or signaling mechanisms by which osteoclasts target bone for removal after fatigue-induced matrix injury.

Temporal and spatial characterization of regenerate bone in the lengthened rabbit tibia.

A rabbit model of bilateral tibial lengthening was used to investigate temporal and spatial changes in new bone volume and architecture during regenerate bone formation. Tibiae were lengthened 9.0 mm at 0.75 mm/day after a 6-day latency period. Animals were euthanized at four time points, and new bone volume and architecture within the distraction gap were assessed by microcomputed tomography and histomorphometry. New bone formation began before day 18 postsurgery and increased markedly between day 18 (completion of distraction) and day 24. This period of high bone formation activity might therefore be optimal for biologic and mechanical interventions aimed at enhancing bone regeneration. Regions of both endochondral and intramembranous bone formation were observed throughout the consolidation period. Significant increases in bone volume fraction were observed early in the consolidation period and were attributed to significant increases in trabecular thickness. This suggested that increased mineral density in the gap tissue with time was a consequence of increased osteoblast activity and associated trabecular thickening. New bone formation was shown to be highly oriented toward the distraction axis throughout lengthening. More bone formed consistently in lateral and proximal regions of the distraction gap, perhaps due to improved blood supply or progenitor cell availability in these areas. No differences in trabecular architecture were detected between regions having more or less bone volume, suggesting that bony tissue differentiation in all regions of the distraction gap was similar. Homotypical variations in measures of bone architecture were small; thus, these outcome variables seem appropriate for determining the effects of biological and mechanical interventions on bone regeneration in this animal model.

Variations in three-dimensional cancellous bone architecture of the proximal femur in female hip fractures and in controls.

Cubes of cancellous bone were obtained from proximal femora of women with hip fractures (n = 26) and from female cadaveric controls (n = 32) to compare architecture and mechanics between groups. Specimens were scanned on a microcomputed tomography system. Stereologic algorithms and model-based estimates were applied to the data to characterize the three-dimensional cancellous microstructure. Cubes were mechanically tested to failure to obtain mechanical properties. Specimens from control subjects had significantly higher bone volume fraction, trabecular number, and connectivity than specimens from patients with hip fractures; no difference in trabecular thickness was observed between groups. Both maximum modulus and ultimate stress were significantly higher in the control than in the fracture group, consistent with the higher bone volume found in the control group. No statistical differences in any of these architectural or mechanical variables were found when groups were matched for bone volume. Specimens from both patients with hip fractures and controls demonstrated strong relationships between trabecular number and bone volume fraction that were statistically equivalent, suggesting that for a given bone mass, both groups have the same overall number of trabeculae. However, there was an architectural difference between fracture and control groups in terms of the three-dimensional spatial arrangement of trabeculae. Fracture specimens had a significantly more anisotropic (oriented) structure than control specimens, with proportionately fewer trabecular elements transverse to the primary load axis, even when matched for bone volume. Relationships between mechanical and architectural parameters were significantly different between groups, suggesting that fracture and control groups have different structure-mechanics relationships, which we hypothesize may be a consequence of the altered three-dimensional structure between groups.

The role of interstitial fluid flow in the remodeling response to fatigue loading.

Load-induced fluid flow enhances molecular transport through bone tissue and relates to areas of bone resorption and apposition. Remodeling activity is highly coordinated and necessitates a means for cellular communication via intracellular and extracellular means. Osteocytes, osteoblasts, and osteoclasts, which reside in disparate locations within the tissue, communicate intracellularly via the cellular syncytium and extracellularly via the pericellular fluid space of the lacunocanalicular system. Both of these communications systems are physically disrupted by microdamage incurred during fatigue loading of bone. The purpose of this study was to develop an analytical model to understand the role of interstitial fluid flow in the remodeling response to fatigue loading. Adequate transport was assumed a prerequisite for maintenance of cell viability in bone. Diffusive and convective transport were simulated through the lacunocanalicular network in a healthy undamaged state as well as in a damaged state after fatigue loading. The model predicts that fatigue damage impedes transport from the blood supply, depleting the concentration of molecular entities in and downstream from areas of damage. Furthermore, the presence of microcracks alters the distribution of molecular entities between individual lacunae. These effects were confirmed by the results of an in vivo pilot study in which fluorescent, flow-visualizing agents pooled within microcracks and were absent from areas surrounding microcracks, corresponding to areas deprived of fluid flow. Loss of osteocyte viability is coupled to targeting and initiation of new remodeling activity. Taken as a whole, these data suggest a link between interstitial fluid flow, mass transport, maintenance of osteocyte viability, and modulation of remodeling activity.

Bone microdamage and skeletal fragility in osteoporotic and stress fractures.

The accumulation of bone microdamage has been proposed as one factor that contributes to increased skeletal fragility with age and that may increase the risk for fracture in older women. This paper reviews the current status and understanding of microdamage physiology and its importance to skeletal fragility. Several questions are addressed: Does microdamage exist in vivo in bone? If it does, does it impair bone quality? Does microdamage accumulate with age, and is the accumulation of damage with age sufficient to cause a fracture? The nature of the damage repair mechanism is reviewed, and it is proposed that osteoporotic fracture may be a consequence of a positive feedback between damage accumulation and the increased remodeling space associated with repair.

Failure mechanisms in human vertebral cancellous bone.

Specimens of human vertebral cancellous bone were compressed to well past mechanical failure (15% strain) in the infero-superior direction. The mechanisms of failure were examined microscopically and histologically. The primary mechanism of failure was shown to be microscopic cracking rather than overt failure of the trabecular elements. The morphology of the cracks was consistent with an hypothesis that they were the result of shear stress (or strain) in the tissue. Complete fracture of trabeculae was confined to elements oriented transversely to the direction of loading. The tissue's ultimate strength and residual strength after compressive failure were both strongly correlated to tissue stiffness (R2 = 0.88 and R2 = 0.71, respectively). It is proposed that cancellous bone strength may be a consequence of the adaptation of bone stiffness to applied stresses. With removal of the load, all specimens recovered at least 94% of their original height. Implications of energy dissipation by microcracking for recovery and maintenance of overall trabecular architecture are discussed.

Aging and matrix microdamage accumulation in human compact bone.

Bone matrix microdamage in bone matrix, evidenced as microcracks, occurs consequent to cyclic loading. Microdamage caused by in vivo loading has been described in human rib cortex; however, the existence and extent of microcracks in human long bone cortices are largely unknown. Using histomorphometric methods to examine the incidence and localization of microcracks in human femoral compact bone specimens, we found that the amount of microdamage present in femoral compact bone increases dramatically with increasing age. Least squares regression analysis showed that in males, microcrack density (Cr.De., #/mm2) increases exponentially with age (r2 = 0.70). In females, Cr.De. also increases as an exponential function of increasing age (r2 = 0.79), at a significantly higher rate than in male specimens (p < 0.001). The current studies indicate that with increasing age, bone microdamage accumulates more rapidly than intrinsic processes can effect its repair. A combination of cumulative loading history, focal changes in material properties and alteration in the ability of the tissue to perceive and/or react to microcracks may all play role in this accumulation of bone microdamage with aging. This accumulation of microdamage in bone will contribute to decreased strength and stiffness. In addition, and perhaps most significantly for understanding aging and increased bone fragility, matrix microdamage in composite materials like bone will result in a profoundly reduced resistance to fracture. The importance of this accumulation of matrix microdamage in human bone with increasing age in contributing to the increased fragility of the aging skeleton is discussed.

Osteocyte density in woven bone.

Woven bone forms rapidly during tissue growth, following injury and in response to certain anabolic stimuli. Functional differences between woven and lamellar bone may be due, in part, to differences in osteocyte density (cells per unit tissue). Woven bone has been estimated to contain four to eight times more osteocytes than lamellar bone, although primary data to support this assertion are limited. Given recent findings implicating osteocytes as regulators of bone remodeling, bone formation and bone volume, such large differences in osteocyte density between woven and lamellar bone may have important consequences. In this study, we compared the density of osteocyte lacunae (lacunae/mm(2) tissue) in rat lamellar bone with that in woven bone formed under several different circumstances. We found that the lacunar density of lamellar cortical bone in the rat (834+/-83 cells/mm2, mean+/-SD) did not differ significantly from that of periosteal woven bone formed via intramembranous osteogenesis, either in response to mechanical loading (921+/-204 cells/mm2) or in the periosteal buttressing region of the fracture callus (1138+/-168 cells/mm2). In contrast, lacunar density of endochondrally derived woven bone in the center (gap) region of fracture callus was nearly 100% greater (1875+/-270 cells/mm2) than in lamellar cortical bone while lacunar density of primary spongiosa of the growth plate was 40% greater (1674+/-228 cells/mm2) than that in lamellar cancellous bone (1189+/-164). These findings demonstrate that lacunar density in woven bone varies depending on skeletal site and developmental history and appears to be elevated in endochondrally derived woven bone adjacent to marrow space. Given the considerable evidence supporting osteocytes as local initiators of bone remodeling, we suggest that woven bone with increased lacunar density may undergo remodeling at an accelerated rate.

In vivo trabecular microcracks in human vertebral bone.

Human vertebral cancellous bone from white males (N = 19), black males (N = 16), white females (N = 12), and black females (N = 17) was examined histologically for the presence, numerical density, and morphology of in vivo microscopic cracking (microdamage). Two patterns of microcracks, linear and cross-hatched, were observed. Linear microcracks were observed in both the central portion and near surfaces of trabeculae. Those inside trabeculae were usually single microcracks approximately 50 microns in length and were found in both cement lines and in interstitial bone matrix. Linear microcracks near the trabecular surface were usually multiple parallel cracks approximately 80 microns in length. Microcracks with a cross-hatched appearance were less prevalent. They were observed primarily in vertically oriented trabeculae and were often surrounded by an area of diffuse staining. Two-way ANOVA revealed no differences in microcrack density (Cr.Dn; #/mm2) between males and females [mean (SD) 5.13 (5.02) vs. 5.41 (6.26), respectively], but whites had significantly higher microcrack density than blacks [7.00 (5.71) vs. 3.63 (4.98), respectively, p < 0.05]. White males had a significantly higher microcrack density than black males [7.60 (5.56) vs. 2.21 (1.78), respectively, p < 0.05]. Although not statistically significant, white females also had higher microcrack density than black females. In contrast to what has been reported in the femur, regression analysis found no statistically significant relationship between microcrack density in the spine and age for any of the four race-gender groups. However, significant power relationships were found between microcrack density and bone area fraction for all groups except for black females. The difference between axial and appendicular bone remodeling rates, and their implications for microdamage accumulation, are discussed.

Examination of compact bone microdamage using back-scattered electron microscopy.

A new staining technique using heavy metals (lead-uranyl acetate) has been developed to allow visualization of bone microdamage using both light microscopy and scanning electron microscopy operated in its back-scattered mode (BSE). At the light microscopic level, the number of microcracks counted in sequential sections of human ribs is the same for both the traditional basic fuchsin method of differentially staining microcracks and the new lead-uranyl acetate procedure. With BSE study, however, the number of microcracks observed is significantly reduced in all samples, because of the reduction of projection effect error associated with surface based imaging techniques. Application of the lead-uranyl acetate staining technique to ex vivo-loaded crack propagation specimens showed an extensive ultrastructurally disrupted region associated with the crack path through bone, consistent with the damage process zone around cracks in toughened composite materials.

Mechanical and morphological effects of strain rate on fatigue of compact bone.

Compact bone specimens were cyclically loaded in uniaxial tension for one million cycles; loading was performed at either of two physiological strain rates (0.01 s-1 or 0.03 s-1) and a physiological strain range (0-1200 microstrain). Microdamage in loaded and nonloaded control specimens was then assessed histomorphometrically. Fatigue, evidence by stiffness loss, was observed at both strain rates and was significantly greater in specimens loaded at the high experimental strain rate than in specimens loaded at the low strain rate. Morphologically, this fatigue corresponded to increased numbers of microcracks in the bone. These data show that fatigue and resultant microdamage are realistic expectations of cyclic loading within the physiological strain range. The rate at which strains are developed influences the fatigue behavior of compact bone, suggesting that cyclic loading at high physiological strain rates, characteristic of vigorous activities, is more damaging to compact bone than loading at lower physiological strain rates.

Long-term fatigue behavior of compact bone at low strain magnitude and rate.

Fatigue behavior of compact bone at physiological strain ranges was examined in vitro. Standardized specimens of bovine compact bone were cyclically loaded in uniaxial tension of 0-1200 or 0-1500 microstrain for up to 13-37 million cycles to study the long-term fatigue properties. All specimens exhibited fatigue during the first several million cycles of loading, evidenced by a gradual decrease of specimen modulus during this initial loading period; mean modulus loss for all specimens was approximately 6%. After this initial stiffness loss, specimen modulus stabilized and did not change again for the duration of the loading. Osteonal bone specimens lost significantly more stiffness than primary bone specimens during the early loading history, but neither microstructural type progressed to fatigue failure. These data suggest that some fatigue of compact bone is a realistic expectation of the normal loading environment, but this fatigue does not progress to fatigue failure within a physiologically reasonable number of cycles when tested in vitro at strain magnitudes like those measured in living animals. Implications for fatigue/stress fractures in vivo are discussed.

Prevention of fracture healing in rats by an inhibitor of angiogenesis.

Angiogenesis is considered essential to fracture healing, but its role in the healing process remains poorly understood. Angiogenesis inhibitors, which block new blood vessel formation by specifically targeting vascular cells, are currently under development for use in cancer chemotherapy, and are potentially powerful tools for defining the consequences of angiogenic impairment on fracture healing. In this study, we directly tested the effects of the angiogenesis inhibitor TNP-470 on the healing of closed femoral fractures in an established rat model system. Beginning 1 day after fracture, animals received either angiogenesis inhibitor at a therapeutically effective antitumor dose, or a weight-adjusted amount of carrier vehicle. The progress of fracture healing was assessed at weekly intervals for 21 days by radiography and histology; functional assessment was carried out at day 24 by biomechanical testing. By all three criteria, treatment with the angiogenesis inhibitor completely prevented fracture healing. Formation of both callus and periosteal woven bone were suppressed, indicating that both the intramembranous and endochondral pathways of osteogenesis were affected. The resulting tissue resembled "atrophic nonunions" often seen clinically in cases of failed fracture healing, but rarely achieved in animal models. These results show that angiogenesis is essential to very early stages of fracture healing, and suggest this model system may be useful for understanding the mechanisms underlying fracture nonunions due to vascular impairment. Finally, the data raise the possibility that impairment of fracture healing may be an adverse effect of clinical treatments with antiangiogenic drugs.

Influence of nonenzymatic glycation on biomechanical properties of cortical bone.

In this study, the influence of nonenzymatic glycation (NEG) on the mechanical properties of bone and bone collagen were investigated. Bovine cortical bone specimens were incubated in ribose to cause collagen cross-links in vitro, and nondestructive mechanical testing was used to determine tensile and compressive elastic modulus as a function of incubation time. Mechanical properties associated with yield, postyield, and final fracture of bone were determined at the end of the incubation period. The stiffness of the collagen network was measured using stress relaxation tests of demineralized bone cylinders extracted periodically throughout the incubation period. It was found that accumulation of nonenzymatic glycation end-products in cortical bone caused stiffening of the type I collagen network in bone (r2 = 0.92; p < 0.001) but did not significantly affect the overall stiffness of the mineralized bone (p = 0.98). The ribosylated group had significantly more NEG products and higher yield stress and strain than the control group (p < 0.05). Postyield properties including postyield strain and strain energy were lower in the ribosylated group but were not significantly different from the control group (p = 0.24). Compared with the control group, the ribosylated group was characterized by significantly higher secant modulus and lower damage fraction (p < 0.05). Taken together, the results of this study suggest that collagen in bone is susceptible to the same NEG-mediated changes as collagen in other connective tissues and that an increased stiffness of the collagen network in bone due to NEG may explain some of the age-related increase in skeletal fragility and fracture risk.

Human vertebral cancellous bone surface distribution.

The three-dimensional distribution of bone surface and the bone volume fraction (BV/TV) of 110 human vertebral cancellous bone specimens from seven individuals were measured using a three-dimensional radiographic method (microcomputed tomography). The ratios of the three principal projections of bone surface per total volume were found to be relatively constant for specimens examined in this study. The constancy of the projected surface ratios means that the fraction of the total bone surface oriented in any direction does not change markedly with BV/TV. Bone volume fraction was a good predictor of bone surface per total volume (BS/TV) for a one-parameter nonlinear model (r2 = 0.92). The results of this pilot study suggest that the changes in surface distribution which occur during age-related bone loss are largely predetermined rather than adaptive. The results are also consistent with the idea that cancellous bone tends to maintain a constant ratio of trabecular number for the principal directions. If these inferences from the data are correct, the morphogenetic processes which create the initial adult trabecular pattern become of primary interest. A model was developed which explained the strong relationship between BS/TV and BV/TV. The model was used to demonstrate the importance of morphogenetic processes.