Structural features of subchondral bone cysts and adjacent tissues in hip osteoarthritis.

OBJECTIVE: Focal lesions within the subchondral bone, termed subchondral bone cysts (SBCs), are clinically accepted radiographic markers of advanced osteoarthritis (OA), but their etiology in the hip is not well understood. DESIGN: This study used micro-computed tomography (µCT), and histological and immunocytological analysis to examine the prevalence, size, location, and morphological and cellular features of SBCs found within 34 femoral heads (14 male, 20 female; age range = 43-80 years) obtained from total hip arthroplasty procedures. RESULTS: SBCs were common-present in 91% of the femoral heads examined-and frequently commuted with the surface of the femoral head, but otherwise showed no preferred anatomical location. Few associations were found between SBC features and patient characteristics such as BMI, age and sex. SBCs were also heterogenous in composition, ranging from fibrous (most common) to predominantly fatty (least common) and often containing vasculature, nerve fibers, cartilage islands, and bony spicules. Despite this heterogeneity, focal abnormalities in bone density and cartilage thickness were consistently observed. Bone adjacent to SBCs was denser than that in the primary compressive group, and cartilage thickness in regions overlying SBCs was lower than in non-overlying regions. In contrast to these local bony changes, µCT-based finite element analyses indicated that the stiffness of the primary compressive group was only mildly affected by SBCs. CONCLUSIONS: These findings indicate that SBCs in the femoral head involve extensive perturbations in cellular activity, culminating in myriad skeletal tissue types and spatially heterogenous changes in bone and cartilage morphology that are likely to affect OA progression.

A comprehensive mapping of the structure and gene organisation in the sheep MHC class I region.

BACKGROUND: The major histocompatibility complex (MHC) is a chromosomal region that regulates immune responsiveness in vertebrates. This region is one of the most important for disease resistance because it has been associated with resistance or susceptibility to a wide variety of diseases and because the MHC often accounts for more of the variance than other loci. Selective breeding for disease resistance is becoming increasingly common in livestock industries, and it is important to determine how this will influence MHC polymorphism and resistance to diseases that are not targeted for selection. However, in sheep the order and sequence of the protein coding genes is controversial. Yet this information is needed to determine precisely how the MHC influences resistance and susceptibility to disease. METHODS: CHORI bacterial artificial chromosomes (BACs) known to contain sequences from the sheep MHC class I region were sub-cloned, and the clones partially sequenced. The resulting sequences were analysed and re-assembled to identify gene content and organisation within each BAC. The low resolution MHC class I physical map was then compared to the cattle reference genome, the Chinese Merino sheep MHC map published by Gao, et al. (2010) and the recently available sheep reference genome. RESULTS: Immune related class I genes are clustered into 3 blocks; beta, kappa and a novel block not previously identified in other organisms. The revised map is more similar to Bovidae maps than the previous sheep maps and also includes several genes previously not annotated in the Chinese Merino BAC assembly and others not currently annotated in the sheep reference chromosome 20. In particular, the organisation of nonclassical MHC class I genes is similar to that present in the cattle MHC. Sequence analysis and prediction of amino acid sequences of MHC class I classical and nonclassical genes was performed and it was observed that the map contained one classical and eight nonclassical genes together with three possible pseudogenes. CONCLUSIONS: The comprehensive physical map of the sheep MHC class I region enhances our understanding of the genetic architecture of the class I MHC region in sheep and will facilitate future studies of MHC function.

Imaging techniques for the assessment of fracture repair.

Imaging of a healing fracture provides a non-invasive and often instructive reproduction of the fracture repair progress and the healing status of bone. However, the interpretation of this reproduction is often qualitative and provides only an indirect and surrogate measure of the mechanical stability of the healing fracture. Refinements of the available imaging techniques have been suggested to more accurately determine the healing status of bone. Plain radiographs provide the ability to determine the degree of bridging of the fracture gap and to quantify the amount of periosteal callus formation. Absorptiometric measures including dual X-ray absorptiometry and computed tomography provide quantitative information on the amount and the density of newly formed bone around the site of the fracture. To include the effect of spatial distribution of newly formed bone, finite element models of healing fracture can be employed to estimate its load bearing capacity. Ultrasound technology not only avoids radiation doses to the patients but also provides the ability to additionally measure vascularity in the surrounding soft tissue of the fracture and in the fracture itself.

The intravertebral distribution of bone density: correspondence to intervertebral disc health and implications for vertebral strength.

UNLABELLED: This study's goal was to determine associations among the intravertebral heterogeneity in bone density, bone strength, and intervertebral disc (IVD) health. Results indicated that predictions of vertebral strength can benefit from considering the magnitude of the density heterogeneity and the congruence between the spatial distribution of density and IVD health. INTRODUCTION: This study aims to determine associations among the intravertebral heterogeneity in bone density, bone strength, and IVD health METHODS: Regional measurements of bone density were performed throughout 30 L1 vertebral bodies using micro-computed tomography (µCT) and quantitative computed tomography (QCT). The magnitude of the intravertebral heterogeneity in density was defined as the interquartile range and quartile coefficient of variation in regional densities. The spatial distribution of density was quantified using ratios of regional densities representing different anatomical zones (e.g., anterior to posterior regional densities). Cluster analysis was used to identify groups of vertebrae with similar spatial distributions of density. Vertebral strength was measured in compression. IVD health was assessed using two scoring systems. RESULTS: QCT- and µCT-based measures of the magnitude of the intravertebral heterogeneity in density were strongly correlated with each other (p < 0.005). Accounting for the interquartile range in regional densities improved predictions of vertebral strength as compared to predictions based only on mean density (R (2) = 0.59 vs. 0.43; F-test p-value = 0.018). Specifically, after adjustment for mean density, vertebral bodies with greater heterogeneity in density exhibited higher strength. No single spatial distribution of density was associated with high vertebral strength. Analyses of IVD scores suggested that the health of the adjacent IVDs may modulate the effect of a particular spatial distribution of density on vertebral strength. CONCLUSIONS: Noninvasive measurements of the intravertebral distribution of bone density, in conjunction with assessments of IVD health, can aid in predictions of bone strength and in elucidating biomechanical mechanisms of vertebral fracture.

The effect of intravertebral heterogeneity in microstructure on vertebral strength and failure patterns.

UNLABELLED: The goal of this study was to determine the influence of intravertebral heterogeneity in microstructure on vertebral failure. Results show that noninvasive assessments of the intravertebral heterogeneity in density improve predictions of vertebral strength and that local variations in microstructure are associated with locations of failure in the vertebral body. INTRODUCTION: The overall goal of this study was to determine the influence of intravertebral heterogeneity in microstructure on vertebral failure. METHODS: Trabecular density and microarchitecture were quantified for 32 thoracic vertebrae using micro-computed tomography (µCT)-based analyses of 4.81 mm, contiguous cubes throughout the centrum. Intravertebral heterogeneity in density was defined as the interquartile range and quartile coefficient of variation of the cube densities. The vertebrae were compressed to failure to measure stiffness, strength, and toughness. Pre- and post-compression µCT images were analyzed using digital volume correlation to quantify failure patterns in the vertebrae, as defined by the distributions of residual strain. RESULTS: Failure patterns consisted of large deformations in the midtransverse plane with concomitant endplate biconcavity and were linked to the intravertebral distribution of bone tissue. Low values of connectivity density and trabecular number, and high values of trabecular separation, were associated with high strains. However, local microstructural properties were not the sole determinants of failure. For instance, the midtransverse plane experienced the highest strain (p < 0.008) yet had the highest density, lowest structure model index, and lowest anisotropy (p < 0.013). Accounting for the intravertebral heterogeneity in density improved predictions of strength and stiffness as compared to predictions based only on mean density (strength: R(2) = 0.75 vs. 0.61, p < 0.001; stiffness: R(2) = 0.44 vs. 0.26, p = 0.001). CONCLUSIONS: Local variations in microstructure are associated with failure patterns in the vertebra. Noninvasive assessments of the intravertebral heterogeneity in density--which are feasible in clinical settings--can improve predictions of vertebral strength and stiffness.

Smaller, weaker, and less stiff bones evolve from changes in subsistence strategy.

UNLABELLED: We propose a computational model with which to examine the evolution of bone. Our results indicate that changes in subsistence strategy have influenced the evolution of bone growth and mechanoregulation, and predict that bone size, stiffness, and structural strength may decrease in future generations, bringing increased risk of fracture and prevalence of osteoporosis. INTRODUCTION: Archeological data suggest that bone size and strength have decreased over evolution. We hypothesize that changing evolutionary pressures and levels of physical activity, both arising from changes in subsistence strategy, have affected the evolution of bone. We propose a computational model with which to examine the evolution of bone growth and mechanoregulation due to the transitions from hunter-gatherer to agricultural to modern lifestyles. METHODS: The evolution of genes governing growth and mechanoregulation in a population of bones is simulated, where each individual is represented by a 2-D bone cross-section. Genetic variability is assumed to modulate growth through mechanoregulatory factors that direct periosteal expansion, endosteal expansion/infilling, and ash content accretion in response to strains incurred during walking. RESULTS: The model predicts decreases in cortical area and section modulus (a measure of structural strength) and increases in maximum compressive strain over the course of the simulation, meaning evolution of smaller, less strong, and less stiff bones is predicted for the population average. The model predicts small but continued decreases in size, strength, and stiffness in modern populations, despite the absence of a strong evolutionary advantage to efficient bones during this phase. CONCLUSION: In conclusion, our results show that changing loading regimes and evolutionary pressures may have influenced the evolution of bone growth and mechanoregulation, and predict that bone size and strength may continue to decrease in future generations, bringing increased risk of fracture and prevalence of osteoporosis.

Polymorphism of sheep MHC Class IIb gene TAPASIN.

The Major Histocompatibility Complex (MHC) is one of the most gene dense regions in the genome and studies in several species have shown significant associations between the MHC and disease. The endoplasmic reticular glycoprotein, tapasin, is involved in the MHC class I antigen presentation pathway. Sheep TAPASIN is located in the class IIb region of the MHC. Sheep TAPASIN was subcloned from BAC and cosmid genomic clones and DNA sequenced. TAPASIN is 9549bp in length and encodes a protein of 447 amino acids. The structure of sheep TAPASIN was similar to other mammals and consisted of eight exons with a distinctively larger intron between exon three and four. Sheep TAPASIN gene had high sequence identity with other mammalian TAPASINs. The TAPASIN gene sequence is conserved across many mammalian species and is possibly maintained through purifying selection with the average ratio of ds/dn of 3.9. Twenty-six SNPs in sheep TAPASIN were identified.

Use of microindentation to characterize the mechanical properties of articular cartilage: comparison of biphasic material properties across length scales.

OBJECTIVE: Small scale mechanical testing techniques offer new possibilities for defining changes in mechanical properties that accompany the morphological, histological, and biochemical abnormalities of osteoarthritis (OA). The goal of this study was to investigate the use of microindentation in characterizing the biphasic material properties of articular cartilage. Direct comparisons of the biphasic properties (E, k and nu) determined using microindentation were made to those determined on the same specimens using standard macroscale testing techniques. METHODS: Deep-zone bovine articular cartilage specimens (n=10) were tested in macroscale confined and unconfined compression. For microindentation testing, the biphasic properties were determined by conducting finite element simulations of the microindentation experiments for different combinations of values of biphasic properties and identifying the combination yielding the best match to each microindentation curve. Paired t-tests were performed to compare each of E, k and nu between the macro- and microscale. RESULTS: The microscale values for E, k and nu were 0.74 (0.53, 0.95)MPa, 0.66 (0.022, 0.110)x10(-16)m(4)/Ns, and 0.16 (0.08, 0.24), respectively. A significant difference between the macro- and microscale measurements was observed for k (P<0.0001), but not for E or nu (P=0.88, 0.16). CONCLUSIONS: The agreement in Young's modulus and Poisson's ratio between the results of the microindentation and macroscale tests supports the use of microindentation for characterization of some of the biphasic material properties of articular cartilage. The observed differences in permeability between macro- and microscales are consistent with evidence in the literature of a length-scale dependence to this property.

BMP2 is essential for post natal osteogenesis but not for recruitment of osteogenic stem cells.

The effects of BMP2 on bone marrow stromal cell differentiation and bone formation after bone marrow ablation were determined using C57 BL/6J (B6) mice. Inhibition of BMP2 expression with lentiviral BMP2 shRNA prevented both mineralized nodule formation in vitro and bone formation in vivo, and blocked the expression of Runx2 and osterix, transcriptional determinants of terminal osteogenic differentiation. No effect was observed on the expression of Sox9, a transcription factor, which is the one of the first transcriptional determinant to be expressed in committed chondroprogenitor and osteoprogenitor cells. In vitro studies showed that exogenously added BMP7 rescued the expression of osterix and enhanced the expression of Sox9, but had no effect on the expression of Runx2, while it only partially recovered the development of mineral deposition in the cultures. On the other hand, the exogenous addition of BMP2 rescued both Runx2 and osterix expression, did not enhance the expression of Sox9, but fully recovered the inhibition of mineral deposition in the cultures. Using antibodies against CD146 and Sox9, immunohistological examination of the cell populations found in the medullary space three days after bone marrow ablation, showed qualitatively equal numbers of cells expressing these skeletal progenitor and stem cell markers in control and BMP2 shRNA treated animals. Fluorescence Activated Cell Sorting (FACS) analysis of the cells found with the marrow cavities at three days after marrow ablation using CD146 antibody showed near equal numbers of immunopositive cells in both control and shRNA treated animals. In summary, the differences observed in vitro for BMP2 and BMP7 on osteogenic gene expression and mineralization suggest that they have differing effects on bone cell differentiation. These results further demonstrate that in vivo BMP2 is a central morphogenetic regulator of post natal osteoprogenitor differentiation, but does not affect recruitment of progenitors to the osteoblastic lineage.

Measurement of fracture callus material properties via nanoindentation.

In bone fracture healing, the extent to which the injured bone regains stability and strength depends on the mechanical properties of the tissues that are formed during healing. While many techniques have been used to quantify the overall mechanical behavior of fracture calluses, few data exist on the material properties of individual callus tissues. The overall goal of this study was to quantify these material properties. Nanoindentation was performed at multiple locations across thin (200mum), longitudinal sections of rat fracture callus at 35 days post fracture. Following indentation, sections were stained with alizarin red S and alcian blue to obtain semi-quantitative estimates of tissue mineral content and proteoglycan content, respectively. Indentation moduli varied over three orders of magnitude (0.61-1010MPa) throughout the callus. Much of this variation was due to the presence of multiple tissue types: the indentation moduli of granulation tissue, chondroid tissue and woven bone ranged 0.61-1.27MPa (median=0.99MPa), 1.39-4.42MPa (median=2.89MPa) and 26.92-1010.00MPa (median=132.00MPa), respectively. In regions of alizarin red staining, the indentation modulus was correlated (r=0.62, P=0.04) with stain intensity, suggesting a positive correlation between modulus and mineral content in woven bone. In addition, the indentation modulus of woven bone along the periosteal aspect of the cortex increased with distance from the fracture gap (P=0.004). These results demonstrate the usefulness of nanoindentation in characterizing the elastic properties of the heterogeneous mixture of tissues present in bone fracture callus.

Biomechanics of trabecular bone.

Trabecular bone is a complex material with substantial heterogeneity. Its elastic and strength properties vary widely across anatomic sites, and with aging and disease. Although these properties depend very much on density, the role of architecture and tissue material properties remain uncertain. It is interesting that the strains at which the bone fails are almost independent of density. Current work addresses the underlying structure-function relations for such behavior, as well as more complex mechanical behavior, such as multiaxial loading, time-dependent failure, and damage accumulation. A unique tool for studying such behavior is the microstructural class of finite element models, particularly the "high-resolution" models. It is expected that with continued progress in this field, substantial insight will be gained into such important problems as osteoporosis, bone fracture, bone remodeling, and design/analysis of bone-implant systems. This article reviews the state of the art in trabecular bone biomechanics, focusing on the mechanical aspects, and attempts to identify important areas of current and future research.

Dependence of yield strain of human trabecular bone on anatomic site.

Understanding the dependence of human trabecular bone strength behavior on anatomic site provides insight into structure-function relationships and is essential to the increased success of site-specific finite element models of whole bones. To investigate the hypothesis that the yield strains of human trabecular bone depend on anatomic site, the uniaxial tensile and compressive yield properties were compared for cylindrical specimens from the vertebra (n=61), proximal tibia (n=31), femoral greater trochanter (n=23), and femoral neck (n=27) taken from 61 donors (67+/-15years). Test protocols were used that minimized end artifacts and loaded specimens along the main trabecular orientation. Yield strains by site (mean+/-S.D.) ranged from 0.70+/-0.05% for the trochanter to 0.85+/-0.10% for the femoral neck in compression, from 0.61+/-0.05% for the trochanter to 0.70+/-0.05% for the vertebra in tension, and were always higher in compression than tension (p<0.001). The compressive yield strain was higher for the femoral neck than for all other sites (p<0.001), as was the tensile yield strain for the vertebra (p<0.007). Analysis of covariance, with apparent density as the covariate, indicated that inter-site differences existed in yield stress even after adjusting statistically for density (p<0.035). Coefficients of variation in yield strain within each site ranged from only 5-12%, consistent with the strong linear correlations (r(2)=0.94-0.98) found between yield stress and modulus. These results establish that the yield strains of human trabecular bone can differ across sites, but that yield strain may be considered uniform within a given site despite substantial variation in elastic modulus and yield stress.

Nonlinear behavior of trabecular bone at small strains.

Study of the behavior of trabecular bone at strains below 0.40 percent is of clinical and biomechanical importance. The goal of this work was to characterize, with respect to anatomic site, loading mode, and apparent density, the subtle concave downward stress-strain nonlinearity, that has been observed recently for trabecular bone at these strains. Using protocols designed to minimize end-artifacts, 155 cylindrical cores from human vertebrae, proximal tibiae, proximal femora, and bovine proximal tibiae were mechanically tested to yield at 0.50 percent strain per second in tension or compression. The nonlinearity was quantified by the reduction in tangent modulus at 0.20 percent and 0.40 percent strain as compared to the initial modulus. For the pooled data, the mean +/- SD percentage reduction in tangent modulus at 0.20 percent strain was 9.07+/- 3.24 percent in compression and 13.8 +/- 4.79 percent in tension. At 0.40 percent strain, these values were 23.5 +/- 5.71 and 35.7+/- 7.10 percent, respectively. The magnitude of the nonlineari't depended on both anatomic site (p < 0.001) and loading mode (p < 0.001), and in tension was positively correlated with density. Calculated values of elastic modulus and yield properties depended on the strain range chosen to define modulus via a linear curve fit (p < 0.005). Mean percent differences in 0.20 percent offset yield strains were as large as 10.65 percent for some human sites. These results establish that trabecular bone exhibits nonlinearity at low strains, and that this behavior can confound intersite comparisons of mechanical properties. A nonlinear characterization of the small strain behavior of trabecular bone was introduced to characterize the initial stress-strain behavior more thoroughly.

Mechanical properties of carbonated apatite bone mineral substitute: strength, fracture and fatigue behaviour.

The synthesis and properties of carbonated apatite materials have received considerable attention due to their importance for medical and dental applications. Such apatites closely resemble the mineral phase of bone, exhibiting superior osteoconductive and osteogenic properties. When formed at physiological temperature they present significant potential for bone repair and fracture fixation. The present study investigates the mechanical properties of a carbonated apatite cancellous bone cement. Flexural strength was measured in three and four point bending, and the fracture toughness and fatigue crack-growth behaviour was measured using chevron and disc-shaped compact tension specimens. The average flexural strength was found to be approximately 0.468 MPa, and the fracture toughness was approximately 0.14 MPa radical m. Fatigue crack-growth rates exhibited a power law dependence on the applied stress intensity range with a crack growth exponent m=17. The fatigue threshold value was found to be approximately 0.085 MPa radical m. The mechanical properties exhibited by the carbonated apatite were found to be similar to those of other brittle cellular foams. Toughness values and fatigue crack-growth thresholds were compared to other brittle foams, bone and ceramic materials. Implications for structural integrity and longer term reliability are discussed.