Christopher J. Hernandez

A Biomechanical Analysis of the Effects of Resorption Cavities on Cancellous Bone Strength

We evaluated the effects of resorption cavities on cancellous bone strength using computational methods. Adding cavities to cancellous bone caused reductions in strength and stiffness that were greater than expected from the associated changes in bone volume and more pronounced when cavities were targeted to regions of high tissue strain.

A theoretical analysis of the changes in basic multicellular unit activity at menopause

Bone loss at menopause is an important contributor to the development of osteoporosis in women. Although alterations in bone remodeling are the implied process through which bone is lost at menopause, how menopause influences basic multicellular units (BMUs), the teams of cells that perform bone remodeling, is not completely clear. In this analysis we utilize a computer simulation of BMU activity to evaluate the changes that occur at menopause. Transient and maintained changes in both the rate of bone turnover (expressed as the BMU birthrate or origination frequency) and the focal bone balance (differences between the amount of bone formed and resorbed at each remodeling site) are considered. The magnitude of the change in BMU activity is determined parametrically through comparison to lumbar spine bone mineral density data present in the literature. We find that a change in bone turnover that is maintained after menopause, a transient change in focal bone balance at menopause, or a combination of the two is consistent with bone loss patterns seen clinically. Understanding the changes in BMU activity that occur at menopause could lead to improved strategies to treat and prevent postmenopausal osteoporosis.

Microdamage caused by fatigue loading in human cancellous bone: relationship to reductions in bone biomechanical performance.

Vertebral fractures associated with osteoporosis are often the result of tissue damage accumulated over time. Microscopic tissue damage (microdamage) generated in vivo is believed to be a mechanically relevant aspect of bone quality that may contribute to fracture risk. Although the presence of microdamage in bone tissue has been documented, the relationship between loading, microdamage accumulation and mechanical failure is not well understood. The aim of the current study was to determine how microdamage accumulates in human vertebral cancellous bone subjected to cyclic fatigue loading. Cancellous bone cores (n = 32) from the third lumbar vertebra of 16 donors (10 male, 6 female, age 76±8.8, mean ± SD) were subjected to compressive cyclic loading at σ/E0 = 0.0035 (where σ is stress and E0 is the initial Young’s modulus). Cyclic loading was suspended before failure at one of seven different amounts of loading and specimens were stained for microdamage using lead uranyl acetate. Damage volume fraction (DV/BV) varied from 0.8±0.5% (no loading) to 3.4±2.1% (fatigue-loaded to complete failure) and was linearly related to the reductions in Young’s modulus caused by fatigue loading (r2 = 0.60, p<0.01). The relationship between reductions in Young’s modulus and proportion of fatigue life was nonlinear and suggests that most microdamage generation occurs late in fatigue loading, during the tertiary phase. Our results indicate that human vertebral cancellous bone tissue with a DV/BV of 1.5% is expected to have, on average, a Young’s modulus 31% lower than the same tissue without microdamage and is able to withstand 92% fewer cycles before failure than the same tissue without microdamage. Hence, even small amounts of microscopic tissue damage in human vertebral cancellous bone may have large effects on subsequent biomechanical performance.

Three-Dimensional Dynamic Bone Histomorphometry

Dynamic bone histomorphometry is the standard method for measuring bone remodeling at the level of individual events. Although dynamic bone histomorphometry is an invaluable tool for understanding osteoporosis and other metabolic bone diseases, the techniques two‐dimensional nature requires the use of stereology and prevents measures of individual remodeling event number and size. Here, we used a novel three‐dimensional fluorescence imaging technique to achieve measures of individual resorption cavities and formation events. We performed this three‐dimensional histomorphometry approach using a common model of postmenopausal osteoporosis, the ovariectomized rat. The three‐dimensional images demonstrated the spatial relationship between resorption cavities and formation events consistent with the hemiosteonal model of cancellous bone remodeling. Established ovariectomy was associated with significant increases in the number of resorption cavities per unit bone surface (2.38 ± 0.24 mm−2 sham surgery versus 3.86 ± 0.35 mm−2 bilateral ovariectomy [OVX], mean ± SD, p < 0.05) and total volume occupied by cavities per unit bone volume (0.38% ± 0.06% sham versus 1.12% ± 0.18% OVX, p < 0.001), but there was no difference in surface area per resorption cavity, maximum cavity depth, or cavity volume. In addition, we found that established ovariectomy is associated with increased size of bone formation events because of the merging of formation events (23,700 ± 6,890 µm2 sham verusus 33,300 ± 7,950 µm2 OVX). No differences in mineral apposition rate (determined in 3D) were associated with established ovariectomy. That established estrogen depletion is associated with increased number of remodeling events with only subtle changes in remodeling event size suggests that circulating estrogens may have their primary effect on the origination of new basic multicellular units with relatively little effect on the progression and termination of active remodeling events.

Mechanical Failure Begins Preferentially Near Resorption Cavities in Human Vertebral Cancellous Bone Under Compression

The amount of bone turnover in the body has been implicated as a factor that can influence fracture risk and bone strength. Here we test the idea that remodeling cavities promote local tissue failure by determining if microscopic tissue damage (microdamage) caused by controlled loading in vitro is more likely to form near resorption cavities. Specimens of human vertebral cancellous bone (L4, 7 male and 2 female, age 70±10, mean±SD) were loaded in compression to the yield point, stained for microscopic tissue damage and submitted to three-dimensional fluorescent imaging using serial milling (image voxel size 0.7×0.7×5.0 μm). We found the resulting damage volume per bone volume (DV/BV) was correlated with percent eroded surface (p<0.01, r(2)=0.65), demonstrating that whole specimen measures of resorption cavities and microdamage are related. Locations of microdamage were more than two times as likely to have a neighboring resorption cavity than randomly selected sites without microdamage (relative risk 2.39, 95% confidence interval of relative risk: 2.09-2.73), indicating a spatial association between resorption cavities and microdamage at the local level. Individual microdamage sites were 48,700 (40,100; 62,700) μm(3) in size (median, 25th and 75th percentiles). That microdamage was associated with resorption cavities when measured at the whole specimen level as well as at the local level provides strong evidence that resorption cavities play a role in mechanical failure processes of cancellous bone and therefore have the potential to influence resistance to clinical fracture.

Links Between the Microbiome and Bone.

The human microbiome has been shown to influence a number of chronic conditions associated with impaired bone mass and bone quality, including obesity, diabetes, and inflammatory bowel disease. The connection between the microbiome and bone health, however, has not been well studied. The few studies available demonstrate that the microbiome can have a large effect on bone remodeling and bone mass. The gut microbiome is the largest reservoir of microbial organisms in the body and consists of more than a thousand different species interacting with one another in a stable, dynamic equilibrium. How the microbiome can affect organs distant from the gut is not well understood but is believed to occur through regulation of nutrition, regulation of the immune system, and/or translocation of bacterial products across the gut endothelial barrier. Here we review each of these mechanisms and discuss their potential effect on bone remodeling and bone mass. We discuss how preclinical studies of bone‐microbiome interactions are challenging because the microbiome is sensitive to genetic background, housing environment, and vendor source. Additionally, although the microbiome exhibits a robust response to external stimuli, it rapidly returns to its original steady state after a disturbance, making it difficult to sustain controlled changes in the microbiome over time periods required to detect alterations in bone remodeling, mass, or structure. Despite these challenges, an understanding of the mechanisms by which the gut microbiome affects bone has the potential to provide insights into the dissociation between fracture risk and bone mineral density in patients including those with obesity, diabetes, or inflammatory bowel disease. In addition, alteration of the gut microbiome has the potential to serve as a biomarker of bone metabolic activity as well as a target for therapies to improve bone structure and quality using pharmaceutical agents or pre‐ or probiotics.

Quantitative relationships between microdamage and cancellous bone strength and stiffness.

Microscopic tissue damage (microdamage) is an aspect of bone quality associated with impaired bone mechanical performance. While it is clear that bone tissue submitted to more severe loading has greater amounts of microdamage (as measured through staining), how microdamage influences future mechanical performance of the bone has not been well studied, yet is necessary for understanding the mechanical consequences of the presence of microdamage. Here we determine how stained microdamage generated by a single compressive overload affects subsequent biomechanical performance of cancellous bone. Human vertebral cancellous bone specimens (n=47) from 23 donors (14 males, 9 females, 64-92years of age) were submitted to a compressive overload, stained for microdamage, then reloaded in compression to determine the relationship between the amount of microdamage caused by the initial load and reductions in mechanical performance during the reload. Damage volume fraction (DV/BV) caused by the initial overload was related to reductions in Youngs modulus, yield strength, ultimate strength, and yield strain upon reloading (p<0.05, R(2)=0.18-0.34). The regression models suggest that, on average, relatively small amounts of microdamage are associated with large reductions in reload mechanical properties: a 1.50% DV/BV caused by a compressive overload was associated with an average reduction in Youngs modulus of 41.0±3.2% (mean±SE), an average reduction in yield strength of 63.1±4.5% and an average reduction in ultimate strength of 52.7±4.0%. Specimens loaded beyond 1.2% (1.2-4.0% apparent strain) demonstrated a single relationship between reload mechanical properties (Youngs modulus, yield strength, and ultimate strength) and bone volume fraction despite a large range in amounts of microdamage. Hence, estimates of future mechanical performance of cancellous bone can be achieved using the bone volume fraction and whether or not a specimen was previously loaded beyond ultimate strain. The empirical relationships provided in this study make it possible to estimate the degree of impaired mechanical performance resulting from an observed amount of stained microdamage.

Human Evolution and Osteoporosis-Related Spinal Fractures

The field of evolutionary medicine examines the possibility that some diseases are the result of trade-offs made in human evolution. Spinal fractures are the most common osteoporosis-related fracture in humans, but are not observed in apes, even in cases of severe osteopenia. In humans, the development of osteoporosis is influenced by peak bone mass and strength in early adulthood as well as age-related bone loss. Here, we examine the structural differences in the vertebral bodies (the portion of the vertebra most commonly involved in osteoporosis-related fractures) between humans and apes before age-related bone loss occurs. Vertebrae from young adult humans and chimpanzees, gorillas, orangutans, and gibbons (T8 vertebrae, n = 8–14 per species, male and female, humans: 20–40 years of age) were examined to determine bone strength (using finite element models), bone morphology (external shape), and trabecular microarchitecture (micro-computed tomography). The vertebrae of young adult humans are not as strong as those from apes after accounting for body mass (p<0.01). Human vertebrae are larger in size (volume, cross-sectional area, height) than in apes with a similar body mass. Young adult human vertebrae have significantly lower trabecular bone volume fraction (0.26±0.04 in humans and 0.37±0.07 in apes, mean ± SD, p<0.01) and thinner vertebral shells than apes (after accounting for body mass, p<0.01). Since human vertebrae are more porous and weaker than those in apes in young adulthood (after accounting for bone mass), even modest amounts of age-related bone loss may lead to vertebral fracture in humans, while in apes, larger amounts of bone loss would be required before a vertebral fracture becomes likely. We present arguments that differences in vertebral bone size and shape associated with reduced bone strength in humans is linked to evolutionary adaptations associated with bipedalism.

Three-dimensional characterization of resorption cavity size and location in human vertebral trabecular bone

The number and size of resorption cavities in cancellous bone are believed to influence rates of bone loss, local tissue stress and strain and potentially whole bone strength. Traditional two-dimensional approaches to measuring resorption cavities in cancellous bone report the percent of the bone surface covered by cavities or osteoclasts, but cannot measure cavity number or size. Here we use three-dimensional imaging (voxel size 0.7×0.7×5.0 μm) to characterize resorption cavity location, number and size in human vertebral cancellous bone from nine elderly donors (7 male, 2 female, ages 47-80 years). Cavities were 30.10 ± 8.56 μm in maximum depth, 80.60 ± 22.23∗10(3) μm(2) in surface area and 614.16 ± 311.93∗10(3) μm(3) in volume (mean ± SD). The average number of cavities per unit tissue volume (N.Cv/TV) was 1.25 ± 0.77 mm(-3). The ratio of maximum cavity depth to local trabecular thickness was 30.46 ± 7.03% and maximum cavity depth was greater on thicker trabeculae (p<0.05, r(2)=0.14). Half of the resorption cavities were located entirely on nodes (the intersection of two or more trabeculae) within the trabecular structure. Cavities that were not entirely on nodes were predominately on plate-like trabeculae oriented in the cranial-caudal (longitudinal) direction. Cavities on plate-like trabeculae were larger in maximum cavity depth, cavity surface area and cavity volume than cavities on rod-like trabeculae (p<0.05). We conclude from these findings that cavity size and location are related to local trabecular microarchitecture.

Alterations to the Gut Microbiome Impair Bone Strength and Tissue Material Properties

Alterations in the gut microbiome have been associated with changes in bone mass and microstructure, but the effects of the microbiome on bone biomechanical properties are not known. Here we examined bone strength under two conditions of altered microbiota: (1) an inbred mouse strain known to develop an altered gut microbiome due to deficits in the immune system (the Toll‐like receptor 5–deficient mouse [TLR5KO]); and (2) disruption of the gut microbiota (ΔMicrobiota) through chronic treatment with selected antibiotics (ampicillin and neomycin). The bone phenotypes of TLR5KO and WT (C57Bl/6) mice were examined after disruption of the microbiota from 4 weeks to 16 weeks of age as well as without treatment (n = 7 to 16/group, 39 animals total). Femur bending strength was less in ΔMicrobiota mice than in untreated animals and the reduction in strength was not fully explained by differences in bone cross‐sectional geometry, implicating impaired bone tissue material properties. Small differences in whole‐bone bending strength were observed between WT and TLR5KO mice after accounting for differences in bone morphology. No differences in trabecular bone volume fraction were associated with genotype or disruption of gut microbiota. Treatment altered the gut microbiota by depleting organisms from the phyla Bacteroidetes and enriching for Proteobacteria, as determined from sequencing of fecal 16S rRNA genes. Differences in splenic immune cell populations were also observed; B and T cell populations were depleted in TLR5KO mice and in ΔMicrobiota mice (p < 0.001), suggesting an association between alterations in bone tissue material properties and immune cell populations. We conclude that alterations in the gut microbiota for extended periods during growth may lead to impaired whole‐bone mechanical properties in ways that are not explained by bone geometry.