David P. Fyhrie

Influences of mechanical stress on prenatal and postnatal skeletal development.

A new theory is introduced to describe some of the influences of mechanical stresses on chondroosseoubiology. It is proposed that degeneration and ossification is a normal process for all cartilage in the appendicular skeleton, which is (1) accelerated by intermittently applied shear stresses (or strain energy), and (2) inhibited or prevented by intermittently applied hydrostatic pressure. These concepts were applied using finite element computer models in an effort to predict the ossification pattern of the prenatal and postnatal femoral anlage. The theoretical calculations successfully predicted the key features of skeletal morphogenesis including the development of (1) the primary ossification site, (2) a tubular diaphysis and marrow cavity, (3) meta-phys-al and epiphyseal trabecular bone, (4) the location and geometry of the growth plate, (5) the appearance and location of the secondary ossific nucleus, and (6) the existence and thickness distribution of articular cartilage. The results suggest that degenerative joint disease in immobilized or non-load-bearing mature joints may be a manifestation of the final stage in the ossification of the anlage. In nonfunctional joints, the absence or reduction of intermittent hydrostatic pressure in the articular cartilage permits cartilage degeneration and the progressive advance of the ossification front toward the joint surface until the articular cartilage has been ossified.

Hypoxia Decreases Sclerostin Expression and Increases Wnt Signaling in Osteoblasts

Mutations in sclerostin function or expression cause sclerosing bone dysplasias, involving decreased antagonism of Wnt/Lrp5 signaling. Conversely, deletion of the VHL tumor suppressor in osteoblasts, which stabilize HIF‐α isoforms and thereby enables HIF‐α/β‐driven gene transcription, increases bone mineral content and cross‐sectional area compared to wild‐type controls. We examined the influence of cellular hypoxia (1% oxygen) upon sclerostin expression and canonical Wnt signaling. Osteoblasts and osteocytes cultured under hypoxia revealed decreased sclerostin transcript and protein, and increased expression and nuclear localization of activated β‐catenin. Similarly, both hypoxia and the hypoxia mimetic DFO increased β‐catenin gene reporter activity. Hypoxia and its mimetics increased expression of the BMP antagonists gremlin and noggin and decreased Smad‐1/5/8 phosphorylation. As a partial explanation for the mechanism of regulation of sclerostin by oxygen, MEF2 reporter assays revealed decreased activity. Modulation of VEGF signaling under normoxia or hypoxia revealed no influence upon Sost transcription. These data suggest that hypoxia inhibits sclerostin expression, through enhanced antagonism of BMP signaling independent of VEGF. J. Cell. Biochem. 110: 457–467, 2010.

Comparison of the linear finite element prediction of deformation and strain of human cancellous bone to 3D digital volume correlation measurements.

The mechanical properties of cancellous bone and the biological response of the tissue to mechanical loading are related to deformation and strain in the trabeculae during function. Due to the small size of trabeculae, their motion is difficult to measure. To avoid the need to measure trabecular motions during loading the finite element method has been used to estimate trabecular level mechanical deformation. This analytical approach has been empirically successful in that the analytical models are solvable and their results correlate with the macroscopically measured stiffness and strength of bones. The present work is a direct comparison of finite element predictions to measurements of the deformation and strain at near trabecular level. Using the method of digital volume correlation, we measured the deformation and calculated the strain at a resolution approaching the trabecular level for cancellous bone specimens loaded in uniaxial compression. Smoothed results from linearly elastic finite element models of the same mechanical tests were correlated to the empirical three-dimensional (3D) deformation in the direction of loading with a coefficient of determination as high as 97% and a slope of the prediction near one. However, real deformations in the directions perpendicular to the loading direction were not as well predicted by the analytical models. Our results show, that the finite element modeling of the internal deformation and strain in cancellous bone can be accurate in one direction but that this does not ensure accuracy for all deformations and strains.

Mechanical property and tissue mineral density differences among severely suppressed bone turnover (SSBT) patients, osteoporotic patients, and normal subjects

Pathogenesis of atypical fractures in patients on long term bisphosphonate therapy is poorly understood, and the type, the manner in which they occur and the fracture sites are quite different from the usual osteoporotic fractures. We hypothesized that the tissue-level mechanical properties and mean degree of mineralization of the iliac bone would differ among 1) patients with atypical fractures and severely suppressed bone turnover (SSBT) associated with long-term bisphosphonate therapy, 2) age-matched, treatment-naïve osteoporotic patients with vertebral fracture, 3) age-matched normals and 4) young normals. Large differences in tissue-level mechanical properties and/or mineralization among these groups could help explain the underlying mechanism(s) for the occurrence of typical osteoporotic and the atypical femoral shaft fractures. Elastic modulus, contact hardness, plastic deformation resistance, and tissue mineral densities of cortical and trabecular bone regions of 55 iliac bone biopsies--12 SSBT patients (SSBT; aged 49-77), 11 age-matched untreated osteoporotic patients with vertebral fracture (Osteoporotic), 12 age-matched subjects without bone fracture (Age-Matched Normal), and 20 younger subjects without bone fracture (Young Normal)--were measured using nanoindentation and quantitative backscattered electron microscopy. For cortical bone nanoindentation properties, only plastic deformation resistance was different among the groups (p<0.05), with greater resistance to plastic deformation in the SSBT group compared to all other groups. For trabecular bone, all nanoindentation properties and mineral density of the trabecular bone were different among the groups (p<0.05). The SSBT group had greater plastic deformation resistance and harder trabecular bone compared to the other three groups, stiffer bone compared to the Osteoporotic and Young Normal groups, and a trend of higher mineral density compared to the Age-Matched Normal and Osteoporotic groups. Lower heterogeneity of modulus and contact hardness for cortical bone of the SSBT and trabecular bone of the Osteoporotic fracture groups, respectively, compared to the non-fractured groups, may contribute to fracture susceptibility due to lowered ability to prevent crack propagation. We tentatively conclude that, in addition to extremely low bone formation rate, atypical fractures in SSBT and/or long-term bisphosphonate treatment may be associated with greater mean plastic deformation resistance properties and less heterogeneous elastic properties of the bone.

Modulation of sclerostin expression by mechanical loading and bone morphogenetic proteins in osteogenic cells

The anabolic effect of dynamic mechanical loading on skeletal architecture has been repeatedly demonstrated, but the cellular and molecular events occurring between load and ultimate bone formation remain obscure. The discovery of sclerostin, an antagonist of Wnt/Lrp5 signaling, and the sclerosing bone dysplasias that result from its mutation suggest its pivotal role in modulating bone formation. We examined expression of Sost mRNA across a variety of clonal cell lines spanning the osteogenic phenotype from immature osteoblast to mature osteocyte. No sclerostin expression was detected in immature MC3T3-E1 osteoblasts and, surprisingly, mature MLO-Y4 osteocytes, whereas immature MLO-A5 osteocytic cells expressed very low levels of Sost. Highest expression was observed in mature UMR 106.01 osteoblasts. We examined the influence of bone morphogenetic proteins on Sost expression. Treatment with BMP-2, -4 or -6 was without effect on Sost in mature MLO-Y4 osteocytes but elicited a robust increase in Sost expression in immature MLO-A5 osteocytes. Oscillatory fluid flow applied to mature UMR 106.01 osteoblasts transiently decreased expression of sclerostin at both the mRNA and protein level. Overall, our results indicate that BMP treatment and in vitro mechanical loading demonstrate opposite effects upon sclerostin expression.

The Effect of Regional Variations of the Trabecular Bone Properties on the Compressive Strength of Human Vertebral Bodies

Cancellous centrum is a major component of the vertebral body and significantly contributes to its structural strength and fracture risk. We hypothesized that the variability of cancellous bone properties in the centrum is associated with vertebral strength. Microcomputed tomography (micro-CT)-based gray level density (GLD), bone volume fraction (BV/TV), and finite element modulus (E) were examined for different regions of the trabecular centrum and correlated with vertebral body strength determined experimentally. Two sets of images in the cancellous centrum were digitally prepared from micro-CT images of eight human vertebral bodies (T10–L5). One set included a cubic volume (1 per vertebral centrum, nxa0=xa08) in which the largest amount of cancellous material from the centrum was included but all the shell materials were excluded. The other set included cylindrical volumes (6 per vertebral centrum, nxa0=xa048) from the anterior (4 regions: front, center, left, and right of the midline of vertebra) and the posterior (2 regions: left and right) regions of the centrum. Significant positive correlations of vertebral strength with GLD (r2xa0=xa00.57, pxa0=xa00.03) and E (r2xa0=xa00.63, pxa0=xa00.02) of the whole centrum and with GLD (r2xa0=xa00.65, pxa0=xa00.02), BV/TV (r2xa0=xa00.72, pxa0=xa00.01) and E (r2xa0=xa00.85, pxa0=xa00.001) of the central region of the vertebral centrum were found. Vertebral strength decreased with increasing coefficient of variation of GLD, BV/TV, and E calculated from subregions of the vertebral centrum. The values of GLD, BV/TV, and E in centrum were significantly smaller for the anterior region than for the posterior region. Overall, these findings supported the significant role of regional variability of centrum properties in determining the whole vertebral strength.

Relating micromechanical properties and mineral densities in severely suppressed bone turnover patients, osteoporotic patients, and normal subjects

Mineralization of bone, from the tissue level to whole bones, is associated with mechanical properties. The relationship between bone tissue mineralization and micromechanical properties may be affected by age, disease, and drug treatment. Patients with severely suppressed bone turnover (SSBT) suffered atypical fractures while on bisphosphonate treatment. The role of tissue level mineralization in predicting material level properties of SSBT bone may be different from that of other osteoporotic patients and of normal subjects. The aim of this study was to compare the relationships between mineralization and micromechanical properties of bone biopsies from patients with SSBT, bisphosphonate-naive osteoporotic patients with typical vertebral fracture, and normal young and age-matched subjects. We used nanoindentation and quantitative backscattered electron microscopy to characterize the elastic modulus, contact hardness, plastic deformation resistance, and tissue mineralization of the biopsies at site-matched locations within each biopsy. The linear mineralization-mechanical property relationships were different among the groups with respect to the intercepts for only cortical bone tissue but not the slopes for cortical and trabecular bone tissues. For a given mineral density, there was a trend of greater plastic deformation resistance in SSBT cortical bone compared to young normal bone. Similarly, there was a trend of greater plastic deformation resistance in osteoporotic trabecular bone compared to young normal bone for a given mineral density. The age-matched normal group had higher elastic modulus and a trend of higher contact hardness compared to the young normal group for a given mineral density. However, the mechanical property-mineralization relationships within an individual were weak, and only 21 of 53 biopsies that were analyzed had at least one significant association between mineralization and a mechanical property measurement for either cortical or trabecular bone tissues. The average properties of microstructural regions (deep and superficial remodeling packets in trabecular bone; osteonal and interstitial regions in cortical bone) were consistent with mineral accumulation with tissue age, with the exception of the SSBT group. SSBT trabecular bone deep packets had higher hardness and plastic deformation resistance than superficial packets, but mineralization levels and tissue modulus were not different between packet types. We conclude that relationships between mineral and mechanical properties were different between fracture and normal groups and between young and old normal groups, and that atypical fracture may be associated with changed microstructural material properties and tissue level mineralization compared to osteoporotic patients with vertebral fracture and normal subjects. We hypothesize that tissue level bone quality may be an important determinant in fracture risk, such that tissue mineral density may predict different material properties in different patient groups.

Do sacrificial bonds affect the viscoelastic and fracture properties of bone

Sacrificial bonds have been suggested as a toughening mechanism for bone tissue. Ionic bridges formed by divalent calcium ions between collagen molecules have been proposed as candidates for sacrificial bonds. If this mechanism is active at the macroscopic level, we should observe changes in mechanical properties of bone when calcium ions are maintained or removed from the tissue. To test this hypothesis, we measured viscoelastic and monotonic mechanical properties of cortical bone subjected to differing ionic environments. Storage modulus of bone could be changed up to 3.8% by the presence or absence of Na+ or Ca++ in the environment in a reversible fashion when bones were monitored continuously during treatments. A long-term one-time treatment increased the viscoelastic properties of bone soaked in Na+ solutions whereas the viscoelastic properties of bones soaked in Ca++ solutions were maintained. However, the strength and toughness of bone specimens soaked and fractured in treatment solutions were not improved. The presence of Ca++ affected the mechanical behavior of mineralized bone tissue at the macro scale. These effects were reversible, consistent with the original proposal. However, these effects may not necessarily indicate an increase in strength or toughness of the tissue at the macro scale.

Mechanical Properties of Ligament and Tendon

In Chap. 2 we explored the architecture and composition of ligaments and tendons, non-calcified but nonetheless extremely tough skeletal tissues responsible for binding bones together, transmitting forces from muscles and constraining motion within normal limits. To briefly review, ligaments and tendons are composed of linearly arranged collagen molecules assembled in a hierarchal fashion into subfibrils, fibrils and fibers. Collagen fibers within ligaments are architecturally orientated to effectively control and constrain joint motion; in tendon they are grouped into distinct but parallel fascicles.

Crutch Weightbearing on Comminuted Humeral Shaft Fractures: A Biomechanical Comparison of Large versus Small Fragment Fixation for Humeral Shaft Fractures

Purpose: This study evaluated the failure properties of length unstable humerii secured with small or large fragment plates. Methods: Two nonlocking plate constructs were examined, a nine-hole 4.5-mm limited contact dynamic compression plate (large fragment group) and a 12-hole 3.5-mm limited contact dynamic compression plate (small fragment group), both on composite humerii with a 1-cm defect to simulate comminution (n = 12 for each group). Each plate construct had similar working lengths and number of fixation points. Mechanical testing was first randomized for stiffness measurements in axial and torsional loads. All constructs were then tested in cyclic axial loads to failure. Results: For axial testing, the large fragment group had a mean stiffness of 1020 ± 195 N/mm compared with 268 ± 67 N/mm in the small fragment group (P < 0.0001). For torsional testing, the large fragment group had a mean stiffness of 1.5 ± 0.05 Nm/degree compared with 0.9 ± 0.04 Nm/degree in the small fragment group (P < 0.0001). Plastic deformation in the large fragment and small fragment groups were 0.09 ± 0.07 mm and 0.20 ± 0.24 mm, respectively (P = 0.1) assessed during cyclic testing up to 300 N. The postcyclic yield force in the large fragment group was 227 ± 30 N and in the small fragment group was 153 ± 5 N (P < 0.0001). The ultimate load in the large fragment and small fragment groups were 800 ± 87 N and 307 ± 15 N, respectively. Conclusion: The results corroborate anticipated plate mechanical behavior with plate stiffness increasing as both plate width and thickness increase. The calculated yield force data suggest that both small and large fragment constructs would experience plastic deformation during bilateral crutch ambulation in a patient weighing 50 kg or more. The large fragment construct is not expected to catastrophically fail when subjected to loads in a patient 90 kg or less. The small fragment construct is predicted to catastrophically fail in patients weighing 70 kg or more.