Erin M.R. Bigelow

The use of nano-computed tomography to enhance musculoskeletal research.

Abstract Advances in computed tomography (CT) imaging are opening new avenues toward more precise characterization and quantification of connective tissue microarchitecture. In the last two decades, micro-computed tomography (microCT) has significantly augmented destructive methods for the 3D micro-analysis of tissue structure, primarily in the bone research field. Recently, microCT has been employed in combination with contrast agents to generate contrast-enhanced images of soft tissues that are otherwise difficult to visualize due to their native radiodensity. More recent advances in CT technology have enabled ultra-high resolution imaging by utilizing a more powerful nano-focused X-ray source, such as that found in nano-computed tomography (nanoCT) systems. NanoCT imaging has facilitated the expansion of musculoskeletal research by reducing acquisition time and significantly expanding the range of samples that can be imaged in terms of size, age and tissue-type (bone, muscle, tendon, cartilage, vessels and adipose tissue). We present the application and early results of nanoCT imaging in various tissue types and how this ultra-high resolution imaging modality is capable of characterizing microstructures at levels of details previously not possible. Contrast-enhanced imaging techniques to enable soft-tissue visualization and characterization are also outlined.

Genetic perturbations that impair functional trait interactions lead to reduced bone strength and increased fragility in mice

Functional adaptation may complicate the choice of phenotype used in genetic studies that seek to identify genes contributing to fracture susceptibility. Often, genetic variants affecting one trait are compensated by coordinated changes in other traits. Bone fracture is a prototypic example because mechanical function of long bones (stiffness and strength) depends on how the system coordinately adjusts the amount (cortical area) and quality (tissue-mineral density, TMD) of bone tissue to mechanically offset the natural variation in bone robustness (total area/length). We propose that efforts aimed at identifying genes regulating fracture resistance will benefit from better understanding how functional adaptation contributes to the genotype-phenotype relationship. We analyzed the femurs of C57BL/6J-Chr(A/J)/NaJ Chromosome Substitution Strains (CSSs) to systemically interrogate the mouse genome for chromosomes harboring genes that regulate mechanical function. These CSSs (CSS-i, i=the substituted chromosome) showed changes in mechanical function on the order of -26.6 to +11.5% relative to the B6 reference strain after adjusting for body size. Seven substitutions showed altered robustness, cortical area, or TMD, but no effect on mechanical function (CSS-4, 5, 8, 9, 17, 18, 19); six substitutions showed altered robustness, cortical area, or TMD, and reduced mechanical function (CSS-1, 2, 6, 10, 12, 15); and one substitution also showed reduced mechanical function but exhibited no significant changes in the three physical traits analyzed in this study (CSS-3). A key feature that distinguished CSSs that maintained function from those with reduced function was whether the system adjusted cortical area and TMD to the levels needed to compensate for the natural variation in bone robustness. These results provide a novel biomechanical mechanism linking genotype with phenotype, indicating that genes control function not only by regulating individual traits, but also by regulating how the system coordinately adjusts multiple traits to establish function.

Systematic evaluation of skeletal mechanical function.

Many genetic and environmental perturbations lead to measurable changes in bone morphology, matrix composition, and matrix organization. Here, straightforward biomechanical methods are described that can be used to determine whether a genetic or environmental perturbation affects bone strength. A systematic method is described for evaluating how bone strength is altered in the context of morphology and tissue‐level mechanical properties, which are determined in large part from matrix composition, matrix organization, and porosity. The methods described include computed tomography, whole‐bone mechanical tests (bending and compression), tissue‐level mechanical tests, and determination of ash content, water content, and bone density. This strategy is intended as a first step toward screening mice for phenotypic effects on bone and establishing the associated biomechanical mechanism by which function has been altered, and can be conducted without a background in engineering. The outcome of these analyses generally provides insight into the next set of experiments required to further connect cellular perturbation with functional change. Curr. Protoc. Mouse Biol. 3:39‐67

Femoral Neck External Size but not aBMD Predicts Structural and Mass Changes for Women Transitioning Through Menopause

The impact of adult bone traits on changes in bone structure and mass during aging is not well understood. Having shown that intracortical remodeling correlates with external size of adult long bones led us to hypothesize that age‐related changes in bone traits also depend on external bone size. We analyzed hip dual‐energy X‐ray absorptiometry images acquired longitudinally over 14 years for 198 midlife women transitioning through menopause. The 14‐year change in bone mineral content (BMC, R2 = 0.03, p = 0.015) and bone area (R2 = 0.13, p = 0.001), but not areal bone mineral density (aBMD, R2 = 0.00, p = 0.931) correlated negatively with baseline femoral neck external size, adjusted for body size using the residuals from a linear regression between baseline bone area and height. The dependence of the 14‐year changes in BMC and bone area on baseline bone area remained significant after adjusting for race/ethnicity, postmenopausal hormone use, the 14‐year change in weight, and baseline aBMD, weight, height, and age. Women were sorted into tertiles using the baseline bone area‐height residuals. The 14‐year change in BMC (p = 0.009) and bone area (p = 0.001) but not aBMD (p = 0.788) differed across the tertiles. This suggested that women showed similar changes in aBMD for different structural and biological reasons: women with narrow femoral necks showed smaller changes in BMC but greater increases in bone area compared to women with wide femoral necks who showed greater losses in BMC but without large compensatory increases in bone area. This finding is opposite to expectations that periosteal expansion acts to mechanically offset bone loss. Thus, changes in femoral neck structure and mass during menopause vary widely among women and are predicted by baseline external bone size but not aBMD. How these different structural and mass changes affect individual strength‐decline trajectories remains to be determined.

Moving toward a prevention strategy for osteoporosis by giving a voice to a silent disease

A major unmet challenge in developing preventative treatment programs for osteoporosis is that the optimal timing of treatment remains unknown. In this commentary we make the argument that the menopausal transition (MT) is a critical period in a woman’s life for bone health, and that efforts aimed at reducing fracture risk later in life may benefit greatly from strategies that treat women earlier with the intent of keeping bones strong as long as possible. Bone strength is an important parameter to monitor during the MT because engineering principles can be applied to differentiate those women that maintain bone strength from those women that lose bone strength and are in need of early treatment. It is critical to understand the underlying mechanistic causes for reduced strength to inform treatment strategies. Combining measures of strength with data on how bone structure changes during the MT may help differentiate whether a woman is losing strength because of excessive bone resorption, insufficient compensatory bone formation, trabeculae loss, or some combination of these factors. Each of these biomechanical mechanisms may require a different treatment strategy to keep bones strong. The technologies that enable physicians to differentially diagnose and treat women in a preventive manner, however, have lagged behind the development of prophylactic treatments for osteoporosis. To take advantage of these treatment options, advances in preventive treatment strategies for osteoporosis may require developing new technologies with imaging resolutions that match the pace by which bone changes during the MT and supplementing a womans bone mineral density (BMD)-status with information from engineering-based analyses that reveal the structural and material changes responsible for the decline in bone strength during the menopausal transition.

Canalization Leads to Similar Whole Bone Mechanical Function at Maturity in Two Inbred Strains of Mice

Previously, we showed that cortical mineralization is coordinately adjusted to mechanically offset external bone size differences between A/J (narrow) and C57BL/6J (wide) mouse femora to achieve whole bone strength equivalence at adulthood. The identity of the genes and their interactions that are responsible for establishing this homeostatic state (ie, canalization) remain unknown. We hypothesize that these inbred strains, whose interindividual differences in bone structure and material properties mimic that observed among humans, achieve functional homeostasis by differentially adjusting key molecular pathways regulating external bone size and mineralization throughout growth. The cortices of A/J and C57BL/6J male mouse femora were phenotyped and gene expression levels were assessed across growth (ie, ages 2, 4, 6, 8, 12, 16 weeks). A difference in total cross‐sectional area (p < 0.01) and cortical tissue mineral density were apparent between mouse strains by age 2 weeks and maintained at adulthood (p < 0.01). These phenotypic dissimilarities corresponded to gene expression level differences among key regulatory pathways throughout growth. A/J mice had a 1.55‐ to 7.65‐fold greater expression among genes inhibitory to Wnt pathway induction, whereas genes involved in cortical mineralization were largely upregulated 1.50‐ to 3.77‐fold to compensate for their narrow diaphysis. Additionally, both mouse strains showed an upregulation among Wnt pathway antagonists corresponding to the onset of adult ambulation (ie, increased physiological loads). This contrasts with other studies showing an increase in Wnt pathway activation after functionally isolated, experimental in vivo loading regimens. A/J and C57BL/6J long bones provide a model to develop a systems‐based approach to identify individual genes and the gene‐gene interactions that contribute to trait differences between the strains while being involved in the process by which these traits are coordinately adjusted to establish similar levels of mechanical function, thus providing insight into the process of canalization.

Differential Adaptive Response of Growing Bones From Two Female Inbred Mouse Strains to Voluntary Cage-Wheel Running: ADAPTIVE RESPONSE OF BONE TO VOLUNTARY CAGE-WHEEL RUNNING

The phenotypic response of bones differing in morphological, compositional, and mechanical traits to an increase in loading during growth is not well understood. We tested whether bones of two inbred mouse strains that assemble differing sets of traits to achieve mechanical homeostasis at adulthood would show divergent responses to voluntary cage‐wheel running. Female A/J and C57BL6/J (B6) 4‐week‐old mice were provided unrestricted access to a standard cage‐wheel for 4 weeks. A/J mice have narrow and highly mineralized femora and B6 mice have wide and less mineralized femora. Both strains averaged 2 to 9.5 km of running per day, with the average‐distance run between strains not significantly different (p = 0.133). Exercised A/J femora showed an anabolic response to exercise with the diaphyses showing a 2.8% greater total area (Tt.Ar, p = 0.06) and 4.7% greater cortical area (Ct.Ar, p = 0.012) compared to controls. In contrast, exercised B6 femora showed a 6.2% (p < 0.001) decrease in Tt.Ar (p < 0.001) and a 6.7% decrease in Ct.Ar (p = 0.133) compared to controls, with the femora showing significant marrow infilling (p = 0.002). These divergent morphological responses to exercise, which did not depend on the daily distance run, translated to a 7.9% (p = 0.001) higher maximum load (ML) for exercised A/J femora but no change in ML for exercised B6 femora compared to controls. A consistent response was observed for the humeri but not the vertebral bodies. This differential outcome to exercise has not been previously observed in isolated loading or forced treadmill running regimes. Our findings suggest there are critical factors involved in the metabolic response to exercise during growth that require further consideration to understand how genotype, exercise, bone morphology, and whole‐bone strength interact during growth.

The Effect of Capsulectomy on Hip Joint Biomechanics

Objectives: Hip arthroscopy is performed in young adult patients with hip pain. During arthroscopy, capsulectomy is performed to improve intraoperative visualization. The stability of the hip joint after capsulectomy is relatively unknown. Our study evaluated anterior hip stability in capsular sectioned states with labral injury. We theorized the load required for anterior translation would decrease with greater capsular injury. Methods: Ten human cadaveric pelvises (20 hips, mean 54.25 years) without radiographic evidence of degeneration or dysplasia were harvested. The hips were prepared and mounted onto a custom built testing fixture. Two pelvises were excluded due to fixation issues for one or both hips. The 16 hips were tested in five states (table 1). Each hip was tested in neutral position with a 20N compressive force. The capsular incision was made in-line with the femoral neck. The labrectomy was made at 2:30 position. The sutured capsule was created using #2 Ethibond suture 1cm apart along the incision. The partial capsulectomy was made by resecting the iliofemoral ligament, leaving the pubofemoral and ischiofemoral ligaments intact. Total capsulectomy state was created by removing the remaining capsule. The load at 12mm of anterior translation was recorded for each state after two preconditioning trials. Results: A repeated measures ANOVA with a Bonferroni adjustment showed no difference between all-intact vs. sutured-intact. It also demonstrated no significant difference between the sutured-intact and the sutured labrectomy states. A repeated measures ANOVA with a Bonferroni adjustment was run to compare the capsular states with a 1cm partial labrectomy (table 2). There was a significant difference between the sutured-labrectomy and the partial capsulectomy (p=0.0146) and between the sutured-labrectomy and the total capsulectomy (p=0.0005). There was also a significant difference when comparing the partial capsulectomy and the total capsulectomy (p=0.0377). Conclusion: The findings demonstrate that both the capsule and labrum play a role in providing anterior hip stability. These results indicate that the iliofemoral ligament is crucial for preventing anterior translation in labral injured states. Intraoperative capsulectomy should be avoided in patients with large irreparable labral tears to prevent postoperative anterior hip instability