Characterization of complex, co-adapted skeletal biomechanics phenotypes: a needed paradigm shift in the genetics of bone structure and function.

The genetic architecture of skeletal biomechanical performance has tremendous potential to advance our knowledge of the biological mechanisms that drive variation in skeletal fragility and osteoporosis risk. Research using traditional approaches that focus on specific gene pathways is increasing our understanding of how and to what degree those pathways may affect population-level variation in fracture susceptibility, and shows that known pathways may affect bone fragility through unsuspected mechanisms. Non-traditional approaches that incorporate a new appreciation for the degree to which bone traits co-adapt to functional loading environments, using a wide variety of redundant compensatory mechanisms to meet both physiological and mechanical demands, represent a radical departure from the dominant reductionist paradigm and have the potential to rapidly advance our understanding of bone fragility and identification of new targets for therapeutic intervention.

Intracortical bone remodeling variation shows strong genetic effects.

Intracortical microstructure influences crack propagation and arrest within bone cortex. Genetic variation in intracortical remodeling may contribute to mechanical integrity and, therefore, fracture risk. Our aim was to determine the degree to which normal population-level variation in intracortical microstructure is due to genetic variation. We examined right femurs from 101 baboons (74 females, 27 males; aged 7-33 years) from a single, extended pedigree to determine osteon number, osteon area (On.Ar), haversian canal area, osteon population density, percent osteonal bone (%On.B), wall thickness (W.Th), and cortical porosity (Ct.Po). Through evaluation of the covariance in intracortical properties between pairs of relatives, we quantified the contribution of additive genetic effects (heritability [h (2)]) to variation in these traits using a variance decomposition approach. Significant age and sex effects account for 9 % (Ct.Po) to 21 % (W.Th) of intracortical microstructural variation. After accounting for age and sex, significant genetic effects are evident for On.Ar (h (2) = 0.79, p = 0.002), %On.B (h (2) = 0.82, p = 0.003), and W.Th (h (2) = 0.61, p = 0.013), indicating that 61-82 % of the residual variation (after accounting for age and sex effects) is due to additive genetic effects. This corresponds to 48-75 % of the total phenotypic variance. Our results demonstrate that normal, population-level variation in cortical microstructure is significantly influenced by genes. As a critical mediator of crack behavior in bone cortex, intracortical microstructural variation provides another mechanism through which genetic variation may affect fracture risk.

Reproductive status and sex show strong effects on knee OA in a baboon model.

OBJECTIVE: We aimed to characterize severity and occurrence of knee osteoarthritis (OA), and effects of age, sex, body mass, and reproductive status on population-level normal variation in this condition in the baboon, a natural model of human knee OA. METHODS: We visually inspected articular cartilage of distal right femora of 464 baboons (309 females, 155 males) and assigned an OA severity score (comparable to a modified Outerbridge score) from 1 = unaffected to 4 = advanced OA (eburnation). Presence/absence of osteophytes was recorded. We tested for significant effects of age, sex, body mass, and, in females, reproductive status (pre-, peri-, or post-menopausal) on OA. When appropriate, analyses were repeated on an age-matched subset (153 of each sex). RESULTS: Knee OA was more frequent and severe in older animals (P < 0.0001), but significant age variation was apparent in each severity grade. Sex differences within the younger and older age groups suggest that males develop knee OA earlier, but females progress more quickly to advanced disease. There is a strong relationship between reproductive status and OA severity grade in females (P = 0.0005) with more severe OA in peri- and post-menopausal female baboons, as in humans. CONCLUSIONS: Idiopathic knee OA is common in adult baboons. Occurrence and severity are influenced strongly by reproductive status in females, and by sex with regard to patterns of disease progression - providing an animal model to investigate sex-specific variation in OA susceptibility in which the environmental heterogeneity inherent in human populations is vastly reduced.

A multiscale modeling approach to scaffold design and property prediction.

The optimum scaffold architecture for bone tissue regeneration is a porous structure with a narrow range of pore sizes, pore density, and a high degree of interconnectivity among pores. To achieve such a design, the microstructure of the scaffold material must be optimized in order to satisfy both biological and mechanical function requirements. In this paper, we present a multiscale modeling approach for designing a scaffold with an optimized porosity and mechanical properties made from a two-phase composite of spherical hydroxyapatite (HAp) particles embedded in a collagen matrix. In particular, first-principles computation is used to calculate the elastic properties and theoretical strengths of nanoscaled HAp particles. The constitutive properties of the HAp/collagen composites are subsequently computed as a function of HAp content via FEM-based micromechanical modeling. The constitutive relations of the composite are then utilized to optimize the mechanical properties of a three-dimensional scaffold for either cortical or cancellous bone by varying the pore size, pore density and volume fractions of HAp in the composite. For the pore size, pore density, volume fractions of HAp considered, the scaffold can be designed to match the mechanical properties of cancellous bone, but not those of cortical bone. The optimized scaffold is one with a pore diameter of 1000 microm, a channel diameter of 100 microm, 27% pore density and at least 20% HAp by volume.

Heritability of lumbar trabecular bone mechanical properties in baboons.

Genetic effects on mechanical properties have been demonstrated in rodents, but not confirmed in primates. Our aim was to quantify the proportion of variation in vertebral trabecular bone mechanical properties that is due to the effects of genes. L3 vertebrae were collected from 110 females and 46 male baboons (6-32 years old) from a single extended pedigree. Cranio-caudally oriented trabecular bone cores were scanned with microCT then tested in monotonic compression to determine apparent ultimate stress, modulus, and toughness. Age and sex effects and heritability (h(2)) were assessed using maximum likelihood-based variance components methods. Additive effects of genes on residual trait variance were significant for ultimate stress (h(2)=0.58), toughness (h(2)=0.64), and BV/TV (h(2)=0.55). When BV/TV was accounted for, the residual variance in ultimate stress accounted for by the additive effects of genes was no longer significant. Toughness, however, showed evidence of a non-BV/TV-related genetic effect. Overall, maximum stress and modulus show strong genetic effects that are nearly entirely due to bone volume. Toughness shows strong genetic effects related to bone volume and shows additional genetic effects (accounting for 10% of the total trait variance) that are independent of bone volume. These results support continued use of bone volume as a focal trait to identify genes related to skeletal fragility, but also show that other focal traits related to toughness and variation in the organic component of bone matrix will enhance our ability to find additional genes that are particularly relevant to fatigue-related fractures.

Improving fracture toughness of dental nanocomposites by interface engineering and micromechanics.

The fracture toughness of dental nanocomposites fabricated by various methods of mixing, silanization, and loadings of nanoparticles had been characterized using fatigue-precracked compact-tension specimens. The fracture mechanisms near the crack tip were characterized using atomic force microscopy (AFM), transmission electron microscopy (TEM), and scanning electron microscopy (SEM). The near-tip fracture processes in the nanocomposties were identified to involve several sequences of fracture events, including: (1) particle bridging, (2) debonding at the poles of particle/matrix interface, and (3) crack deflection around the particles. Analytical and finite-element methods were utilized to model the observed sequences of fracture events to identify the source of fracture toughness in the dental nanocomposites. Theoretical results indicated that silanization and nanoparticle loadings improved the fracture toughness of dental nanocomposites by a factor of 2 to 3 through a combination of enhanced interface toughness by silanization, crack deflection, as well as crack bridging. A further increase in the fracture toughness of the nanocomposites can be achieved by increasing the fracture toughness of the matrix, nano-filled particles, or the interface.

Microstrain fields for cortical bone in uniaxial tension: optical analysis method.

This study employed an optical strain measurement method, called microdisplacements by machine vision photogrammetry (DISMAP), to measure both the global and local strain fields in microtensile specimens of cortical bone subjected to controlled uniaxial tension. The variation of local maximum principal strains was measured within the gauge region of samples as a function of applied tensile stress during testing. High gradients of local strain appeared around microstructural features in stressed bone even while the global strain for the entire gauge region showed a strong linear correlation with increasing tensile stress (r2 = 0.98, p < 0.0001). The highest local strain around microstructural features in bone was 11.5-79.5 times higher than the global strain.

Microstructural strain near osteocyte lacuna in cortical bone in vitro.

Mechanical factors affect bone remodeling such that increased mechanical demand results in net bone formation, whereas decreased demand results in net bone resorption. Two proposed mechanical signals are stress-generated fluid flow forces acting on cells and bone matrix deformation itself. A prominent current theory is that bone cells are more responsive to fluid flow than to mechanical strain. Recent experiments support this conclusion: bone cells increase their production of osteopontin (OPN) mRNA, prostaglandin (PGE(2)), and nitric oxide (NO) in response to fluid flow in contrast to cells stimulated by mechanical strain levels similar to those measured in vivo. However, when cells are subjected to substrate strains levels many times greater than those measured in vivo, increased biological activity again results. We assert that it is neither fluid flow nor matrix deformation per se, but rather the resulting cell deformation that causes cell biological response. Machined specimens of undamaged bovine cortical bone were subjected to increasing levels of macroscopic strain while observed under an optical microscope at 220X. Continuum level strain was measured using a standard foil strain gauge attached to the back of the specimen and ranged from 500 to 6,000 microstrain. Images of the specimen surface at each strain level were captured. To determine the level of osteocyte deformation that results from fluid flow in vitro, MLO-Y4 cells were cultured on collagen coated 190 cm2 plastic sheets and subjected to steady fluid flow at 16 dynes/cm(2). Images representing the initial undisturbed cell configuration and the configuration of the cells after ten minutes of fluid flow were acquired from a videotape of the flow experiment. The captured unloaded vs. loaded image pairs were analyzed to determine the local deformation and strain fields using a digital stereoimaging system. When subjected to a nominal continuum strain level approximately equal to that measured in humans in vivo during rigorous activity (2,000 microstrain), the local, osteocyte level strains can be as high as 12,000 to 15,000 microstrain (1.2% to 1.5%). Average osteocyte strains due to fluid flow in vitro increase from 7,972 microstrains after 16 seconds of flow to 22,856 microstrains after 64 seconds of flow. In contrast, maximum strains measured in vivo are approximately 1,800 microstrain in humans and up to 3,000 microstrain in other species. These data may help to explain why bone cells are more sensitive to fluid flow than substrate strain; fluid forces result in cell deformations much higher than those considered to be "physiological".

Primary adult human bone cells do not respond to tissue (continuum) level strains.

Bone adapts to its mechanical environment, and, since the late 1800s, investigators have presumed that this adaptation relates to strain magnitude. Indeed, overwhelming evidence supports the view that either strain or some strain-related quantity stimulates bone adaptation or remodeling. Virtually all investigators, implicitly or explicitly, assume that the level of strain magnitude responsible for bone adaptation is that measured by strain gauges in vivo (i.e., 100-2500 microstrain) and that bone cells are directly deformed by strained matrix. We present evidence that bone cell deformation in this range does not cause bone adaptation. First, bone cells in vitro typically do not respond to average (continuum) levels of strain magnitude. Second, bone cells in vitro do respond to fluid flow-induced shear stresses in these ostensible physiological ranges. Third, in vivo strain magnitudes presumed to stimulate remodeling reflect only averages, and not local peaks, which are 2-15 times higher. Thus, we hypothesize that sensing cells do not respond to levels of strain presumed to be physiological.

Machine vision photogrammetry: a technique for measurement of microstructural strain in cortical bone.

Understanding local microstructural deformations and strains in cortical bone may lead to a better understanding of cortical bone damage development, fracture, and remodeling. Traditional experimental techniques for measuring deformation and strain do not allow characterization of these quantities at the microstructural level in cortical bone. This study describes a technique based on digital stereoimaging used to measure the microstructural strain fields in cortical bone. The technique allows the measurement of material surface displacements and strains by comparing images acquired from a specimen at two distinct stress states. The accuracy of the system is investigated by analyzing an undeformed image set; the test image is identical to the reference image but translated by a known pixel amount. An increase in the correlation sub-image train parameter results in an increase in displacement measurement accuracy from 0.049 to 0.012 pixels. Errors in strain calculated from the measured displacement field were between 39 and 564 microstrain depending upon the sub-image train size and applied image displacement. The presence of a microcrack in cortical bone results in local strain at the crack tip reaching 0.030 (30,000 microstrain) and 0.010 (10,000 microstrain) near osteocyte lacunae. It is expected that the use of this technique will allow a greater understanding of bone strength and fracture as well as bone mechanotransduction.