The importance of intrinsic damage properties to bone fragility: a finite element study.

As the average age of the population has increased, the incidence of age-related bone fracture has also increased. While some of the increase of fracture incidence with age is related to loss of bone mass, a significant part of the risk is unexplained and may be caused by changes in intrinsic material properties of the hard tissue. This investigation focused on understanding how changes to the intrinsic damage properties affect bone fragility. We hypothesized that the intrinsic (µm) damage properties of bone tissue strongly and nonlinearly affect mechanical behavior at the apparent (whole tissue, cm) level. The importance of intrinsic properties on the apparent level behavior of trabecular bone tissue was investigated using voxel based finite element analysis. Trabecular bone cores from human T12 vertebrae were scanned using microcomputed tomography (µCT) and the images used to build nonlinear finite element models. Isotropic and initially homogenous material properties were used for all elements. The elastic modulus (E(i)) of individual elements was reduced with a secant damage rule relating only principal tensile tissue strain to modulus damage. Apparent level resistance to fracture as a function of changes in the intrinsic damage properties was measured using the mechanical energy to failure per unit volume (apparent toughness modulus, W(a)) and the apparent yield strength (σ(ay), calculated using the 0.2% offset). Intrinsic damage properties had a profound nonlinear effect on the apparent tissue level mechanical response. Intrinsic level failure occurs prior to apparent yield strength (σ(ay)). Apparent yield strength (σ(ay)) and toughness vary strongly (1200% and 400%, respectively) with relatively small changes in the intrinsic damage behavior. The range of apparent maximum stresses predicted by the models was consistent with those measured experimentally for these trabecular bone cores from the experimental axial compressive loading (experimental: σ(max) = 3.0-4.3 MPa; modeling: σ(max) = 2-16 MPa). This finding differs significantly from previous studies based on nondamaging intrinsic material models. Further observations were that this intrinsic damage model reproduced important experimental apparent level behaviors including softening after peak load, microdamage accumulation before apparent yield (0.2% offset), unload softening, and sensitivity of the apparent level mechanical properties to variability of the intrinsic properties.

Shear ground reaction force variation among equine arena surfaces.

Shear forces at the surface-hoof interface affect hoof slide, surface grip, forces transferred to the limb, and injury risk. However, the variation in shear forces among surfaces with different compositions have not been quantified. Shear ground reaction forces were measured on five dirt and seven synthetic arena surfaces. Cohesion/adhesion and angle of internal friction/coefficient of friction were calculated. Surface composition, surface temperature, cushion depth, and moisture content were also measured. The effects of surface material (dirt/synthetic) on shear properties were assessed using analysis of variance (ANOVA; P < 0.05). The relationships between surface composition or management properties and shear properties were analyzed using linear correlation. Shear properties were not different between dirt and synthetic surface categories; however, surface fiber content was correlated with adhesion and coefficient of friction. These correlations predict that more fiber will decrease soil adhesion (r = -0.75; P < 0.01) and increase the coefficient of friction (r = 0.81; P < 0.01). Furthermore, maximum shear force was significantly correlated with cushion depth (r = 0.61; P < 0.01) and moisture content (r = 0.57; P < 0.01), where shear force was greater on surfaces with thicker cushion layers or higher moisture content. The findings suggest that shear mechanical behavior is more dependent on surface composition than surface material categories (dirt/synthetic) and also indicate that arena owners can influence shear forces by adjusting either surface composition or management.

Arena surface vertical impact forces vary with surface compaction.

Mechanical properties of arena surfaces are extrinsic factors for musculoskeletal injury. Vertical impact forces of harrowed and compacted cushion were measured at five locations on 12 arena surfaces (five dirt, seven synthetic [dirt and fiber]). Eight variables related to impact force, displacement, and acceleration were calculated. Surface temperature, cushion depth and moisture content were also measured. The effects of surface material type (dirt/synthetic) and cushion compaction (harrowed/compacted) on vertical impact properties were assessed using an analysis of variance. Relationships of manageable surface properties with vertical impact forces were examined through correlations. Compacted cushion exhibited markedly higher vertical impact force and deceleration with lower vertical displacement than harrowed cushion (P < 0.001), and the effect was greater on dirt than synthetic surfaces (P = 0.039). Vertical displacement (P = 0.021) and soil rebound (P = 0.005) were the only variables affected by surface type. Surface compaction (harrowed, compacted) had a significantly greater effect on vertical impact forces than surface type (dirt, synthetic). By reducing surface compaction through harrowing, extrinsic factors related to musculoskeletal injury risk are reduced. These benefits were more pronounced on dirt than synthetic surfaces. These results indicate that arena owners should regularly harrow surfaces, particularly dirt surfaces.

Cartilage stress-relaxation is affected by both the charge concentration and valence of solution cations.

OBJECTIVE: Understanding the mechanical functions of specific cartilage molecules such as aggrecan is important for understanding both healthy cartilage and disease progression. Cartilage is primarily composed of chondrocytes and an extracellular matrix consisting of multiple biopolymers, ions, and water. Aggrecan is one matrix biopolymer which consists of a core protein and multiple anionic glycosaminoglycans. Previous research has demonstrated that the stiffness of extracted aggrecan decreases under increased solution cation concentration, and the purpose of this study was to determine whether changes in solution ion concentration resulted in changes in tissue-level viscoelastic properties. METHODS: Middle-zone explants of bovine calf patellofemoral cartilage were harvested and cultured overnight before mechanical testing. Repeated stress-relaxation and cyclical loading tests were performed after equilibration in solutions of 0.15 M and 1 M NaCl and 0.075 M and 0.5 M CaCl(2). A stretched exponential model was fit to the stress-relaxation data. Storage and loss moduli were determined from the cyclical loading data. RESULTS: Changes in ionic strength and species affected both stress-relaxation and cyclical loading of cartilage. Stress-relaxation was faster under higher ionic strength. CaCl(2) concentration increases resulted in decreased peak stress, while NaCl increases resulted in decreased equilibrium stress. Storage and loss moduli were affected differently by NaCl and CaCl(2). CONCLUSIONS: These results show that cartilage stress-relaxation proceeds faster under higher concentrations of solution cations, consistent with the theory of polymer dynamics. These data demonstrate the complexity of cartilage mechanical properties and suggest that aggrecan stiffness may be important in tissue-level cartilage viscoelastic properties.

On sampling bones for microcracks.

This paper addresses the problem of designing experiments to measure microcrack density in cortical bone. Microcracks are relatively scarce in bone cross-sections, and their size requires microscope settings having small fields of view. Thus, substantial time is required to count cracks in each cross-section. Consequently, most studies evaluate a relatively small cross-sectional area from each specimen, the chance of finding a crack in any given field is small, and there is a significant chance of not finding even one crack in the specimens representing a particular subject. Therefore, a statistical model for microcrack counting was created to develop guidelines for sampling bones for microcracks. Three questions were addressed. 1) What are the relationships of sample size to variability in microcrack density results and the probability of crackless specimens? 2) How can sample size be chosen a priori so as to reduce the probability of crackless specimens and the associated variability in the data to an acceptable level? 3) What are the confidence intervals for the mean density of microcracks measured using microscopic counting? Using a Poisson model for the distribution of microcracks within microscope fields the total area (mm(2)) that should be examined for each specimen is given by A(s)=-ln(F)/Cr.Dn, where Cr.Dn is the expected microcrack density for an individual sample and F is the desired probability (expressed as a fraction) that the individual sample will contain no microcracks. This equation is validated against 8 results from three different experiments.

Volume effects on fatigue life of equine cortical bone.

Materials, including bone, often fail due to loading in the presence of critical flaws. The relative amount, location, and interaction of these flaws within a stressed volume of material play a role in determining the failure properties of the structure. As materials are generally imperfect, larger volumes of material have higher probabilities of containing a flaw of critical size than do smaller volumes. Thus, larger volumes tend to fail at fewer cycles compared with smaller volumes when fatigue loaded to similar stress levels. A material is said to exhibit a volume effect if its failure properties are dependent on the specimen volume. Volume effects are well documented in brittle ceramics and composites and have been proposed for bone. We hypothesized that (1) smaller volumes of cortical bone have longer fatigue lives than similarly loaded larger volumes and (2) that compared with microstructural features, specimen volume was able to explain comparable amounts of variability in fatigue life. In this investigation, waisted rectangular specimens (n=18) with nominal cross-sections of 3x4 mm and gage lengths of 10.5, 21, or 42 mm, were isolated from the mid-diaphysis of the dorsal region of equine third metacarpal bones. These specimens were subjected to uniaxial load controlled fatigue tests, with an initial strain range of 4000 microstrain. The group having the smallest volume exhibited a trend of greater log fatigue life than the larger volume groups. Each volume group exhibited a significant positive correlation between the logarithm of fatigue life and the cumulative failure probability, indicating that the data follow the two-parameter Weibull distribution. Additionally, log fatigue life was negatively correlated with log volume, supporting the hypothesis that smaller stressed volumes of cortical bone possess longer fatigue lives than similarly tested larger stressed volumes.

Modelling the effect of race surface and racehorse limb parameters on in silico fetlock motion and propensity for injury.

BACKGROUND: The metacarpophalangeal joint (fetlock) is the most commonly affected site of racehorse injury, with multiple observed pathologies consistent with extreme fetlock dorsiflexion. Race surface mechanics affect musculoskeletal structure loading and injury risk because surface forces applied to the hoof affect limb motions. Race surface mechanics are a function of controllable factors. Thus, race surface design has the potential to reduce the incidence of musculoskeletal injury through modulation of limb motions. However, the relationship between race surface mechanics and racehorse limb motions is unknown. OBJECTIVE: To determine the effect of changing race surface and racehorse limb model parameters on distal limb motions. STUDY DESIGN: Sensitivity analysis of in silico fetlock motion to changes in race surface and racehorse limb parameters using a validated, integrated racehorse and race surface computational model. METHODS: Fetlock motions were determined during gallop stance from simulations on virtual surfaces with differing average vertical stiffness, upper layer (e.g. cushion) depth and linear stiffness, horizontal friction, tendon and ligament mechanics, as well as fetlock position at heel strike. RESULTS: Upper layer depth produced the greatest change in fetlock motion, with lesser depths yielding greater fetlock dorsiflexion. Lesser fetlock changes were observed for changes in lower layer (e.g. base or pad) mechanics (nonlinear), as well as palmar ligament and tendon stiffness. Horizontal friction and fetlock position contributed less than 1° change in fetlock motion. MAIN LIMITATIONS: Simulated fetlock motions are specific to one horse's anatomy reflected in the computational model. Anatomical differences among horses may affect the magnitude of limb flexion, but will likely have similar limb motion responses to varied surface mechanics. CONCLUSIONS: Race surface parameters affected by maintenance produced greater changes in fetlock motion than other parameters studied. Simulations can provide evidence to inform race surface design and management to reduce the incidence of injury.

Long-term ovariectomy decreases ovine compact bone viscoelasticity.

Changes in bone mineral density associated with estrogen depletion in humans do not account for all of the associated change in fracture risk, and it is possible that some of this variation may lie in changes of other aspects of bone quality. The purpose of this study was to investigate changes in viscoelastic behavior of compact bone that may be associated with estrogen depletion. Changes in compact bone viscoelastic properties associated with three years of ovariectomy were investigated with dynamic mechanical analysis (low-amplitude 3-point bending at frequencies of 1-20 Hz) using beams milled from the diaphysis of the ovine radius. The viscoelastic storage modulus was significantly (5.2%) lower at the higher frequencies for the ovariectomized animals. The general anatomic variation in storage modulus, in which cranial sectors had higher values than caudal sectors, did not change with ovariectomy. The loss tangent (tandelta, a measure of damping) was also greatly decreased (up to 83%) at high frequencies in the ovariectomized animals. Anatomic variation in tandelta at low (6-12 Hz) frequencies (cranial and caudal sectors having higher values than lateral or medial sectors) was enhanced with ovariectomy. Changes in viscoelastic properties associated with long-term estrogen depletion could be responsible for a significant reduction in the toughness or strength of a bone without concomitant changes in screening modalities used to evaluate bone quality (e.g., DXA, QCT, QUA).

Summary--Measuring "bone quality".

The idea of bone quality is well-established in the literature and represents a real conundrum in the treatment of osteoporosis. On the one hand, there are measurements for patients that predict fracture risk for the population as a whole, but between individual patients, one will fracture but another will not, despite the fact that all of the technical measurements we use to predict fracture risk are the same. There are, of course, many aspects of bone mechanical properties that cannot yet be measured in patients. The session began with a discussion of what bone quality is, then the speakers presented work on novel aspects of bone properties that could help explain why fracture prediction in vivo is inexact.

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.

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.

Gompertzian growth curves in parathyroid tumours: further evidence for the set-point hypothesis.

BACKGROUND: Clinical and cell kinetic data in parathyroid tumours show that their rate of growth slows down progressively and that tumour size approaches an asymptotic value. The Gompertz equation has been widely used in oncology to model growth retardation in malignant tumours; we describe its first application to a benign tumour. METHODS: In 41 patients with radiation associated hyperparathyroidism, individual solutions were derived for the Gompertz equation: Nt = Exp[A/ a(1 - Exp - at)], where A is the rate constant (years-1) for initial exponential growth and a is the rate constant (years-1) for exponential decline in A. Input data comprised three estimates of tumour age at surgery, 100%, 75% and 50% of the time since irradiation, cell number estimated from tumour weight, and current tumour growth rate, representing the difference between current cell birth rate, estimated from the prevalence of mitotic figures, and an assumed mean rate of cell loss of 5%. RESULTS: With 100% tumour age, geometric mean values were 2.76 for A, 0.134 for a, and 0.87 g for the growth asymptote. As assumed tumour age decreased, the rate constants increased and the growth asymptotes declined from 22% to 9% greater than the geometric mean tumour weight. Depending on assumed tumour age, the rate constants were about 15-45 times smaller than in myeloma and in testicular tumours, and the growth asymptotes about 2500 and about 60 times smaller, respectively. A and a were highly correlated (r2 = 0.993), with a slope of 20.9 and no significant intercept. Depending on assumed tumour age, the geometric mean time from the initial mutation to the first cell division ranged from 39 to 92 days, much longer than in malignant tumours. CONCLUSIONS: (1) The Gompertz modelling demonstrates that both the nonprogressive clinical course and the slow growth of parathyroid tumours can be accounted for by a single mutation. (2) The extremely low values for A and a, and consequent very long delay before the first cell division, support the notion that the initial mutation does not affect a growth regulatory gene, but increases growth indirectly via an increase in secretory set-point, the clone of mutant cells behaving as if they were in a hypocalcaemic environment until the plasma calcium rises to the new set-point. (3) The clinical characteristics of radiation-induced parathyroid tumours are modelled more closely if there is a substantial delay between time of irradiation and onset of tumour growth. (4) The rate constants A and a are highly correlated because the variability of tumour weight on a logarithmic scale is much lower than the variability of the rate constants.

Fatigue damage-fracture mechanics interaction in cortical bone.

Fatigue loading causes accumulation of damage that may lead to the initiation of a macrocrack and result in a catastrophic failure of bone. The objective of this study was to examine the influence of fatigue damage on crack growth parameters in bovine cortical bone. Nineteen rectangular beam specimens (4 x 4 x 48 mm) were machined from bovine tibiae. The long axis of the beams was aligned with the long axis of bones. Using a four-point bending fatigue setup, ten specimens were fatigue-damaged to different levels as indicated by stiffness loss. A through-thickness notch was machined at the center of each damaged and undamaged beam. The notched specimens were then monotonically loaded beyond failure using a three-point bending protocol. Critical stress intensity factor, K(I), and work to critical load, W(Q), were significantly lower in the damaged group than in the undamaged group (p < 0.03). When the undamaged specimens were assigned a percent stiffness loss of zero and pooled with the damaged group, significant negative correlations of percent stiffness loss with K(I) (R = 0.58, p < 0.01), W(Q) (R = 0.54, p < 0.02), maximum load, P(max) (R = 0.59, p < 0.008), deflection at maximum load, Delta(max) (R = 0.48, p < 0.04), structural stiffness, S(max) (R = 0.53, p < 0.02), W(max) (R = 0.55, p < 0.02), and load at 1.4 mm deflection (a value beyond failure but without complete fracture), P(1.4) (R = 0.47, p < 0.05), were found. Post hoc analysis revealed that the average load-deflection curve from the damaged group was transformable into that from the undamaged group through a special shift on the load-deflection plane. Fatigue damage reduces bone stiffness and resistance to crack initiation, maximum load-carrying capacity, and deflection before and after failure in cortical bone. The data suggest there is a single rule that governs the overall effect of fatigue damage on the fracture behavior of cortical bone.

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.

Bone stiffness predicts strength similarly for human vertebral cancellous bone in compression and for cortical bone in tension.

The yield strength and ultimate strength of cortical and cancellous bone tissue are very highly correlated to bone stiffness. For samples of human vertebral cancellous bone in compression and for bovine cortical bone in tension, the coefficient of determination (r2) for regression between ultimate strength and stiffness was 0.89 and 0.92, and between yield strength and stiffness it was 0.94 and 0.93, respectively. The slope of the regression for human vertebral cancellous bone ultimate strength predicted by stiffness was not statistically different from similar regressions for cortical bone in tension in either a bovine sample or in published data from multiple species. We believe that the observed correlation results from the evolutionary need to build sufficiently strong bones using cells that are sensitive to deformation and that directly control bone stiffness, but not strength. The practical significance of this work is that an in vivo estimate of bone stiffness (e.g., from ultrasound measurement) may be a surrogate for bone strength.

Effects of vertebral bone fragility and bone formation rate on the mineralization levels of cancellous bone from white females.

Back-scattered electron microscopy was used to study mineralization levels of human iliac cancellous bone of white females (N = 49). Mineralization levels were assessed by converting bone pixel grayscale levels to atomic number (Z) using known calibration standards. The data set consisted of bone biopsies from normal and vertebral fracture subjects that had either high or low values for bone formation rate (BFR(s)) within their respective groups (fracture/low BFR(s), N = 12; fracture/high BFR(s), N = 10; normal/low BFR(s), N = 12; normal/high BFR(s), N = 15). The following three measures of mineralization were quantitatively determined for each specimen: an overall mean mineralization (Z(mean)), the mineralization of trabecular packets deep within the interior of trabeculae (Z(deep)), and the mineralization of superficial exterior packets (Z(superficial)). Two-way analysis of variance revealed that the high BFR(s) group had a significantly lower Z(superficial) than the low BFR(s) group [mean (SD) 10.383 (0.270) vs. 10.563 (0.289)], and there was no significant interaction. BFR(s) had no effect on Z(mean) or Z(deep). For the pooled data, Z(deep) was significantly higher than Z(superficial) [10.866 (0.242) vs. 10.471 (0.291)]. There was no significant difference in Z(mean), Z(deep), or Z(superficial) between normals and those with vertebral fracture, but the standard deviations of the mineralization measures in the fracture group were at least double that of the normal group. Frequency histograms show that the two groups have fundamentally different mineralization distributions. The normal group demonstrates typical Gaussian distributions centered around the mean, and the distributions of the fracture group are bimodal, with peaks occurring at either the high or low tails of the distributions of the normal group. We hypothesize that both low and high patterns of mineralization might detrimentally affect bone material properties, with low mineralization levels causing reduced stiffness and strength and high mineralization resulting in reduced fracture toughness. The degree to which the mineralization differences may affect strength and stiffness of individual elements is estimated. The higher standard deviations of mineralization measures in the fracture group may reflect an inability to properly regulate trabecular level stress and strain. Forward stepwise regression analysis showed significant relationships between Ob.S/OS and both Z(superficial) and Z(mean), suggesting that the osteoblast may play an important role in regulating mineralization.

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.

Decline in osteocyte lacunar density in human cortical bone is associated with accumulation of microcracks with age.

Despite osteocytes' ideal position to sense the local environment and thereby influence bone remodeling, the function of osteocytes in bone remains controversial. In this study, histomorphometric examination of male and female femoral middiaphyseal cortical bone was conducted to determine if bone's remodeling response, indicated by tissue porosity and accumulation of damage, is associated with osteocyte lacunar density (number of osteocyte lacunae per bone area). The results support the sensory role of the osteocyte network as the decline in osteocyte lacunar density in human cortical bone is associated with the accumulation of microcracks and increase in porosity with age. Porosity and microcrack density increased exponentially with a decline in osteocyte lacunar density indicating that a certain minimum number of osteocytes is essential for an "operational" network. No gender-related differences were found in the relationship of osteocyte lacunar density to age, porosity, or microcrack density. The coefficient of variation of osteocyte lacunar density increased linearly with age, indicating that aging bone tissue is characterized by increased heterogeneity in the spatial organization of osteocytes. Osteocyte lacunar density, porosity, and microcrack density exhibited the same exponential probability density distribution in the donor population, indicating their regulation by similar biological phenomena.

Intracortical remodeling in adult rat long bones after fatigue loading.

Intracortical remodeling in the adult skeleton removes and replaces areas of compact bone that have sustained microdamage. Although studies have been performed in animal species in which there is an existing baseline of remodeling activity, laboratory rodents have been considered to have limited suitability as models for cortical bone turnover processes because of a lack of haversian remodeling activity. Supraphysiological cyclic axial loading of the ulna in vivo was used to induce bending with consequent fatigue and microdamage. Right ulnae of adult Sprague-Dawley rats were fatigue-loaded to a prefailure stopping point of 30% decrease in ulnae whole bone stiffness. Ten days after the first loading, left ulnae were fatigued in the same way. Ulnae were harvested immediately to allow comparison of the immediate response of the left ulna to the fatigue loads, and the biological response of the right leg to the fatigue challenge. Histomorphometry and confocal microscopy of basic fuchsin-stained bone sections were used to assess intracortical remodeling activity, microdamage, and osteocyte integrity. Bone microdamage (linear microcracks, as well as patches of diffuse basic fuchsin staining within the cortex) occurred in fatigue-loaded ulnar diaphyses. Ten days after fatigue loading, intracortical resorption was activated in ulnar cortices. Intracortical resorption occurred in preferential association with linear-type microcracks, with microcrack number density reduced almost 40% by 10 days after fatigue. Resorption spaces were also consistently observed within areas of the cortex in which no bone matrix damage could be detected. Confocal microscopy studies showed alterations of osteocyte and canalicular integrity around these resorption spaces. These studies reveal that: (1) rat bone undergoes intracortical remodeling in response to high levels of cyclic strain, which induce microdamage in the cortex; and (2) intracortical resorption is associated both with bone microdamage and with regions of altered osteocyte integrity. From these studies, we conclude that rats can initiate haversian remodeling in long bones in response to fatigue, and that osteocyte death or damage may provide one of the stimuli for this process.

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.