Robert W. Goulet

The relationship between the structural and orthogonal compressive properties of trabecular bone

In this study, cubes of trabecular bone with a wide range of structural properties were scanned on a micro-computed tomography system to produce complete three-dimensional digitizations from which morphological and architectural parameters could be measured in a nondestructive manner. The cubes were then mechanically tested in uniaxial compression in three orthogonal directions and to failure in one direction to find the orthogonal tangent elastic moduli and ultimate strengths. After testing, the cubes were weighed and ashed to determine the apparent and ash densities. A high correlation between the basic stereologic measurements was found, indicating that there is a relationship between the amount of bone and number of trabeculae in cancellous bone. Regression analysis was used to estimate the modulus and ultimate strength; these regressions accounted for 68-90% of the variance in these measures. These relationships were dependent on the metaphyseal type and donor, with the modulus also dependent on the direction of testing. This indicates that the properties of the individual trabeculae, as well as their amount and organization, may be important in predicting the mechanical properties of cancellous bone.

Measurement and significance of three-dimensional architecture to the mechanical integrity of trabecular bone

SummaryThe mechanical properties of trabecular bone have been shown to vary significantly with age, anatomic location, and metabolic condition. Efforts towards predicting its behavior have been extensive, and significant relationship between measures of density and mechanical integrity have been reported. Unfortunately, the significant heterogeneity in trabecular bone anisotropy contributes to significant unexplained variance in its strength and modulus when predicted using scalar measures of mass or density. As a result, numerous investigators have attempted to include measures of architecture in an effort to more rigorously investigate potential physiologic optimization strategies, as well as account for the increased fragility associated with advancing age. In our laboratories we have utilized a unique three-dimensional, microcomputed tomography system to measure trabecular plate thickness, trabecular plate separation, trabecular plate number, surface to volume ratio, bone volume fraction, anisotropy, and connectivity in isolated specimens of trabecular bone. The results of these studies demonstrate that in normal bone, more than 80% of the variance in its mechanical behavior can be explained by measures of density and orientation. The independent measures of connectivity and trabecular plate number were found to be significantly correlated with bone volume fraction, suggesting a potential strategy in the formation of trabecular bone. It might be hypothesized, however, that the relationship between bone volume fraction and connectivity may be substantially altered under conditions associated with aging, fragility, or metabolic bone disease. This hypothesis would be consistent with the histologic, evidence of reduced connectivity in osteopenic patients.

Type I collagen mutation alters the strength and fatigue behavior of Mov13 cortical tissue

Despite advances in understanding the molecular basis of Osteogenesis Imperfecta, the mechanisms by which type I collagen mutations compromise whole bone function are not well understood. Previously, we have shown that a heterozygous type I collagen mutation is associated with increased brittleness of long bones from Mov13 transgenic mice, a model of the mild form of Osteogenesis Imperfecta. In the current study, we investigated tissue-level damage processes by testing the hypothesis that the fatigue properties of Mov13 tissue were significantly compromised relative to littermate controls. We also quantified tissue structure and mineral content to explain variations in the fatigue behavior. Micro-beam specimens were machined from the anterior and posterior quadrants of Mov13 and control femurs and subjected to cyclic bending at one of four stress levels. Mov13 tissue exhibited a 22-25% reduction in tissue bending strength and a similar reductions in fatigue life and the stress level at which damage was apparent. These results provided tissue-level evidence that damage accumulation mechanisms were significantly compromised in Mov13 cortical tissue. Given that significant alterations in tissue structure were observed in Mov13 femurs, the results of this study support the idea that Mov13 femurs were brittle because alterations in tissue structure associated with the mutation interfered with normal damage processes. These results provide new insight into the pathogenesis of Osteogenesis Imperfecta and are consistent with bone behaving as a damaging composite material, where damage accumulation is central to bone fracture.

Mechanical Stimulation of Tissue Repair in the Hydraulic Bone Chamber

A hydraulically activated bone chamber model was utilized to investigate cellular and microstructural mechanisms of mechanical adaptation during bone repair. Woven trabecular bone and fibrotic granulation tissue filled the initially empty chambers by 8 weeks postimplantation into canine tibial and femoral metaphyses. Without mechanical stimulation, active bone remodeling to lamellar trabecular bone and reconstitution of marrow elements were observed between 8 and 24 weeks. In subsequent loading studies, the hydraulic mechanism was activated on one randomly chosen side of 10 dogs following 8 weeks of undisturbed bone repair. The loading treatment applied an intermittent compressive force (18 N, 1.0 Hz, 1800 cycles/day) for durations of a few days up to 12 weeks. Stereological analysis of three‐dimensional microcomputed tomography images revealed an increase in trabecular plate thickness and connectivity associated with the loaded repair tissue microstructure relative to unloaded contralateral controls. These microstructural alterations corresponded to an over 600% increase in the apparent modulus of the loaded bone tissue. A significant increase in the percentage of trabecular surfaces lined by osteoblasts immunopositive for type I procollagen after a few days of loading provided further evidence for mechanical stimulation of bone matrix synthesis. The local principal tissue strains associated with these adaptive changes were estimated to range from approximately −2000 to +3000 μstrain using digital image‐based finite element methods. This study demonstrates the sensitivity of bone tissue and cells to a controlled in vivo mechanical stimulus and identifies microstructural mechanisms of mechanical adaptation during bone repair. The hydraulic bone chamber is introduced as an efficient experimental model to study the effects of mechanical and biological factors on bone repair and regeneration.

Use of microcomputed tomography scanning as a new technique for the evaluation of membranous bone.

Previous basic bone studies in cranial bone biology and bone grafting have used calipers, volume displacement, and cephalometric tracings to measure membranous bone and to infer fundamental properties of cranial bone. These tools have limited accuracy and reproducibility. Histomorphometry has also been used in the quantitative analysis of cranial bone; however, two-dimensional histology is unable to capture a precise representation of the three-dimensional structure of bone. For the first time, we have used the advanced technology of three-dimensional microcomputed tomographic (micro-CT) scanning as a highly accurate and automated tool to precisely measure changes in bone stereology, volume and projection, and microarchi-tecture in the evaluation of membranous bone. The advantages of this technology are numerous and include the rapid and nondestructive three-dimensional analysis of bone microstructure at resolutions between 10 and 75 μm. Measures of “connectivity” in three dimensions and the architectural parameter of “anisotropy” are available through micro-CT imaging but can only be inferred through two-dimensional histological series. We successfully imaged two full-thickness cranial bone specimens and one cancellous iliac bone graft. The images demonstrate a similarity between the two membranous specimens and a marked difference in comparison with the endochondral graft. These differences are borne out by mathematical analysis, and their significance is discussed. The utility of micro-CT in the evaluation of membranous bone was displayed by its ability to rapidly calculate differences in bone stereology and to quantitatively measure morphological changes at an ultrastructural level. We believe the benefits of this system will prove to be extremely useful for investigations into the basic biology of membranous bone, bone grafts, and craniofacial interfaces, and we encourage its use by other scientific investigators in the field of craniofacial surgery as they strive for more scientifically rigorous tools to understand the basic biology of membranous bone.

Direct calculation of the surface-to-volume ratio for human cancellous bone.

There are many diseases which cause detrimental changes in the trabecular structure of cancellous bone, leading to mechanical failure of the tissue. One approach to understanding the mechanisms of these diseases is to create idealized models that recreate the morphology of the tissue. This paper presents a partial development of such a model. Further histological methods must be developed before a complete definition of morphologically valid models is possible. In a histological section of cancellous bone, the orientation and length of the trabecular surfaces determine how a line drawn across the bone section will intersect the bone-marrow interface. The distribution of the average length between intersections for a set of parallel lines is defined as the mean intercept length distribution. In this paper, the average surface morphology and volume of the average structure of cancellous bone is determined from an examination of the mean intercept length. The average structure of cancellous bone contains a repeated structural element (SE). As a result, the basic bone structure is analogous to a brick wall made from many similar bricks. For a group of 107 specimens, a strong relationship between structural element volume (SE.V) and bone volume fraction (BV/TV) is demonstrated, SE.V = 0.017 kappa (BV/TV)-2.05 mm3, R2 = 0.93, with kappa a model-dependent constant. For the same specimens, the structural element surface (SE.S) showed the relationship, SE.S = 0.144 kappa (BV/TV)-1.35, R2 = 0.92. As a result of the inverse square dependence of structural element volume on bone volume fraction, it is predicted that cancellous bone strength is inversely proportional to structural element volume.

Hierarchical Structure of Bone and Micro-Computed Tomography

Bone is highly complex, with multiple hierarchical levels of structure. Micro-CT has been able to provide much information about the properties of bone at several of these levels at the mid-range of bones hierarchical structure, and it will continue to provide a valuable tool for further characterizing bone in various conditions and explaining mechanisms of bone failure.

A new technique for the quantitative analysis of cranial suture biology

OBJECTIVE Our objective was to assess the ability of the microcomputed tomography scanner to correctly image normal and synostosed cranial sutures at the ultrastructural level. DESIGN AND METHODS Two specimens of coronal sutures were collected from operative specimens. After appropriate preparation, histological sections were obtained and stained with toluene blue for evaluation. Representative histological sections were compared to microcomputed tomography slices. RESULTS AND CONCLUSIONS With microcomputed tomography, we successfully imaged one normal and one synostosed human coronal suture and performed a quantitative analysis of these specimens. Microcomputed tomography scanning was found to be a highly accurate imaging device for the evaluation of cranial suture development. Microcomputed tomography offers three-dimensional imaging at the microscopic level and allows for rapid quantitative analysis of bone architecture, including several measurements unavailable through histologic analysis. We believe that microcomputed tomography can play an important role in imaging and in the quantitative analysis of the stereology of bone microarchitecture. Among its advantages, microcomputed tomography is able to image many more slices than are obtainable through histology, and the method is not prone to human error. Microcomputed tomography slices are generated without destruction of the specimen and without loss or corruption of reproducible data. Structure-oriented slices from microcomputed tomography together with cellular-oriented sections from histology are complementary in the overall quantitative analysis of cranial sutures.

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.