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 bone's 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.

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 microarchitecture 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 microns. 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.

A global relationship between trabecular bone morphology and homogenized elastic properties.

An alternative concept of the relationship between morphological and elastic properties of trabecular bone is presented and applied to human tissue from several anatomical locations using a digital approach. The three-dimensional morphology of trabecular bone was assessed with a microcomputed tomography system and the method of directed secants as well as the star volume procedure were used to compute mean intercept length (MIL) and average bone length (ABL) of 4 mm cubic specimens. Assuming isotropic elastic properties for the trabecular tissue, the general elastic tensors of the bone specimens were determined using the homogenization method and the closest orthotropic tensors were calculated with an optimization algorithm. The assumption of orthotropy for trabecular bone was found to improve with specimen size and hold within 6.1 percent for a 4 mm cube size. A strong global relationship (r2 = 0.95) was obtained between fabric and the orthotropic elastic tensor with a minimal set of five constants. Mean intercept length and average bone length provided an equivalent power of prediction. These results support the hypothesis that the elastic properties of human trabecular bone from an arbitrary anatomical location can be estimated from an approximation of the anisotropic morphology and a prior knowledge of tissue properties.

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 mustrain 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.

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.

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.

A longitudinal study of subchondral plate and trabecular bone in cruciate-deficient dogs with osteoarthritis followed up for 54 months.

OBJECTIVE: To evaluate the sequence of changes in articular cartilage, trabecular bone, and subchondral plate in dogs with osteoarthritis (OA), 3 months, 18 months, and 54 months after anterior cruciate ligament transection (ACLT). METHODS: Specimens of the medial tibial plateau were analyzed with microscopic computed tomography (micro-CT) at a resolution of 60 microns, and biochemical and morphologic changes in the femoral articular cartilage were assessed. RESULTS: At 3 months and 18 months after ACLT, the articular cartilage in the unstable knee showed histologic changes typical of early OA and increased water content and uronic acid concentration; by 54 months, full-thickness ulceration had developed. Micro-CT analysis showed a loss of trabecular bone in the unstable knee, compared with the contralateral knee, at all time points. At both 18 and 54 months, the differences in trabecular thickness and surface-to-volume ratio were greater than at 3 months. Although the mean subchondral plate thickness, especially in the medial aspect of the medial tibial plateau, was greater in the OA knee than in the contralateral knee 18 months and 54 months after ACLT, these differences were not statistically significant; however, the difference was significantly greater at 54 months than at 3 months. CONCLUSION: Thickening of the subchondral bone is not required for the development of cartilage changes of OA in this model. The bony changes that develop after ACLT, however, could result in abnormal transmission of stress to the overlying cartilage and thereby contribute to the progression of cartilage degeneration.

Evaluation of a microcomputed tomography system to study trabecular bone structure.

A new microcomputed tomography (micro-CT) system and thresholding procedure was evaluated as a tool for nondestructive analysis of trabecular bone. Images of 6-mm trabecular bone cubes acquired from the micro-CT system were compared with optical images of corresponding histologic sections to determine the accuracy of representation. The stereologic measures of bone volume fraction (PP) and trabecular plate density (PL) were used to quantify the comparisons. The results showed that the micro-CT measures of PP were not significantly different from those measured from histologic sections and therefore were very accurate. Measures of PL were different by approximately 14%, which translated into discrepancies in trabecular plate thicknesses of about 19 microns. This difference was significantly correlated to the microstructural characteristics of the specific specimen scanned. The precision of both measurements was excellent.

Morphometric and anisotropic symmetries of the canine distal femur.

The purpose of this study was to investigate the homotypical variation in morphologic and anisotropic properties of trabecular bone from paired canine distal femurs. A microcomputed tomography system was used to produce three-dimensional digital reconstructions of the trabecular bone structures. The results showed that the mean bilateral differences in all bone morphology variables were less than 5%. Differences in measures of degree of anisotropy were also less than 5% between matched sites from left and right sides. In general, the bilateral difference in angle of orientation was less than 15 degrees. These data are valuable for calculating the appropriate sample size to use in experiments using the contralateral canine femur as a control. The results and general approach of the study may have significance for the future use of other contralateral trabecular bone sites as controls.

Examination of subchondral bone architecture in experimental osteoarthritis by microscopic computed axial tomography.

Subchondral bone changes have been proposed as an early event in the pathogenesis of osteoarthritis. In this study, microscopic computed axial tomography was used to evaluate the subchondral bone structure in femoral heads from a guinea pig model of osteoarthritis. Examination of trabecular bone within the femoral head showed a highly significant increase in bone fraction in the experimental animals. This was due to the development of trabeculae that were thicker and closer together. We conclude that trabecular remodeling may be an early event in this model of osteoarthritis.