Karl J. Jepsen

Guidelines for assessment of bone microstructure in rodents using micro-computed tomography

Use of high‐resolution micro–computed tomography (µCT) imaging to assess trabecular and cortical bone morphology has grown immensely. There are several commercially available µCT systems, each with different approaches to image acquisition, evaluation, and reporting of outcomes. This lack of consistency makes it difficult to interpret reported results and to compare findings across different studies. This article addresses this critical need for standardized terminology and consistent reporting of parameters related to image acquisition and analysis, and key outcome assessments, particularly with respect to ex vivo analysis of rodent specimens. Thus the guidelines herein provide recommendations regarding (1) standardized terminology and units, (2) information to be included in describing the methods for a given experiment, and (3) a minimal set of outcome variables that should be reported. Whereas the specific research objective will determine the experimental design, these guidelines are intended to ensure accurate and consistent reporting of µCT‐derived bone morphometry and density measurements. In particular, the methods section for papers that present µCT‐based outcomes must include details of the following scan aspects: (1) image acquisition, including the scanning medium, X‐ray tube potential, and voxel size, as well as clear descriptions of the size and location of the volume of interest and the method used to delineate trabecular and cortical bone regions, and (2) image processing, including the algorithms used for image filtration and the approach used for image segmentation. Morphometric analyses should be based on 3D algorithms that do not rely on assumptions about the underlying structure whenever possible. When reporting µCT results, the minimal set of variables that should be used to describe trabecular bone morphometry includes bone volume fraction and trabecular number, thickness, and separation. The minimal set of variables that should be used to describe cortical bone morphometry includes total cross‐sectional area, cortical bone area, cortical bone area fraction, and cortical thickness. Other variables also may be appropriate depending on the research question and technical quality of the scan. Standard nomenclature, outlined in this article, should be followed for reporting of results.

Cyclic hydrostatic pressure enhances the chondrogenic phenotype of human mesenchymal progenitor cells differentiated in vitro

Much attention has been given to the influences of bioactive factors on mesenchymal progenitor cell differentiation and proliferation, but few studies have examined the effect of mechanical factors on these cells. This study examined the effects of cyclic hydrostatic pressure on human bone marrow‐derived mesenchymal progenitor cells undergoing chondrogenic differentiation. Aggregates of bone marrow‐derived mesenchymal progenitor cells were cultured in a defined chondrogenic medium and were subjected to cyclic hydrostatic pressure. Aggregates were loaded at various time points: single (day 1 or 3) or multiple (days 1–7). At 14 and 28 days, aggregates were harvested for histology, immunohistochemistry, and quantitative DNA and matrix macromolecule analysis. The aggregates loaded for a single day did not demonstrate significant changes in proteoglycan and collagen contents compared with the non‐loaded controls. In contrast, for the multi‐day loaded aggregates, statistically significant increases in proteoglycan and collagen contents were found on both day 14 and day 28. Aggregates loaded for seven days were larger and histological staining indicated a greater matrix/cell ratio. This study indicates that hydrostatic pressure enhances the cartilaginous matrix formation of mesenchymal progenitor cells differentiated in vitro, and suggests that mechanical forces may play an important role in cartilage repair and regeneration in vivo.

Abnormal collagen fibrils in tendons of biglycan/fibromodulin-deficient mice lead to gait impairment, ectopic ossification, and osteoarthritis

Small leucine‐rich proteoglycans (SLRPs) regulate extracellular matrix organization, a process essential in development, tissue repair, and metastasis. In vivo interactions of biglycan and fibromodulin, two SLRPs highly expressed in tendons and bones, were investigated by generating biglycan/fibromodulin double‐deficient mice. Here we show that collagen fibrils in tendons from mice deficient in biglycan and/or fibromodulin are structurally and mechanically altered resulting in unstable joints. As a result, the mice develop successively and progressively 1) gait impairment, 2) ectopic tendon ossification, and 3) severe premature osteoarthritis. Forced use of the joints increases ectopic ossification and osteoarthritis in the double‐deficient mice, further indicating that structurally weak tendons cause the phenotype. The study shows that mutations in SLRPs may predispose to osteoarthritis and offers a valuable and unique animal model for spontaneous osteoarthritis characterized by early onset and a rapid progression of the disease

Understanding bone strength: size isn’t everything

In vivo models, particularly mouse mutations, are increasingly being used to investigate the impact of the absence or overexpression of a gene product on musculoskeletal load-bearing capacity. Skeletal functional integrity can be assessed by structural strength tests that measure how well the whole bone can bear loads. Although the importance of performing these tests is well-recognized, care must be taken in designing the experiments and interpreting the data. The aim of this report is to clarify the relationship of whole bone structural strength to material and geometric properties and the interpretation of these data in the context of in vivo models, especially mice. In particular, we emphasize that there is no alternative to testing whole bone strength and that conclusions regarding bone mechanical function based solely on geometry or bone mineral content are inappropriate and likely misleading. What is a whole bone structural test and what does it measure? Different types of loads, such as bending or torsion, can be applied to whole bones in vitro to determine the structure’s stiffness and failure load (structural strength). The structural stiffness is a measure of the resistance to deformation under the applied load, and the structural strength is the load required to fail the whole bone. These two whole bone measurements are structural properties and are influenced by both the material from which the structure is composed (the tissue material properties) as well as how and where that material is distributed (the geometric form of the tissue) (Figure 1). Therefore, both material and geometric properties are required to assess the structural integrity of a long bone, and neither material nor geometry alone is sufficient to predict the structural failure load. Currently, there is no substitute for a mechanical test to measure whole bone structural behavior; no alternative parameter has been identified that is fully indicative of strength and can serve as a surrogate measure. Bone material properties are the tissue level mechanical properties that describe the constituent material and are independent of the size and shape of the bone. Material properties include the tissue ultimate stress and modulus of elasticity. These tissue properties are determined by machining precise samples from the bone of interest and testing them in a particular loading mode. The material properties are influenced by compositional measures such as mineral density, collagen content, and ash fraction. In addition to composition, factors such as collagen cross-linking, collagen fiber orientation, mineral crystal size, and the microstructural organization (e.g., lamellae, osteons) also influence material behavior. From a mechanical perspective, the composition and organization of the material clearly influence the tissue’s ability to bear loads, but most measures, except for mineral density, have not yet been related directly to the tissue properties derived from mechanical tests. When designing a structural test, the relevant material and geometric measures are determined by the loading mode applied to the whole bone to measure strength (e.g., torsion, bending, or compression) as well as the outcome parameter of interest (e.g., stiffness or failure load) (Figure 1). For example, if we test a bone to failure in torsion, then we will measure the torsional load to failure (a structural parameter). The appropriate geometric and material properties are the torsional section modulus (a geometric parameter) and the ultimate shear stress of the bone (a material parameter). The section modulus represents the geometric resistance to torsion and increases as the material lies further from the axis of rotation (Figure 2). The ultimate shear stress is the strength of the bone tissue when loaded in torsion. A biomechanics tutorial by Turner and Burr has provided a more complete presentation of mechanical assessment of whole bone and bone tissue. The contribution of structural, geometric and material analyses can be illustrated with a hypothetical example (Figure 3). Consider the case of a mutant and wild-type comparison in which animal age, gender, and weight are matched, but the bone material and geometry may be affected by the mutation. A whole bone torsion test to failure showed that both the control and mutant failed at the same torque of 8.7 Nzmm. Based on this analysis alone, we would conclude that the mutation had no effect. Additional analyses, either geometric or material, would be necessary to reveal the true effect of the mutation. On the other hand, if we only measure the geometry, we find the mutant bone to have a 21% lower section modulus than wild-type. Therefore, based on geometry alone, we might conclude that the mutant is structurally weaker than the control. However, combined with the structural information, we would know that the smaller mutant bones must have increased material properties to achieve the same structural failure load. Conversely, a tissue level material test would determine that the ultimate shear stress is 26% higher in the mutant than the wild-type. Therefore, based on material differences alone, we might conclude that the mutant is structurally stronger than the control. In each case, global conclusions based on a single analysis (structural, geometric, or material) are different, contradictory, and potentially incorrect. Ideally, all three tests would be required, but, at a minimum, combining two analyses is sufficient to understand the effect of the mutation on the structural properties of the whole bone. Address for correspondence and reprints: Marjolein C. H. van der Meulen, Ph.D., Sibley School of Mechanical and Aerospace Engineering, Cornell University, 219A Upson Hall, Ithaca, NY 14853. E-mail: mcv3@cornell.edu Bone Vol. 29, No. 2 August 2001:101–104

Genetic architecture of complex traits: Large phenotypic effects and pervasive epistasis

The genetic architecture of complex traits underlying physiology and disease in most organisms remains elusive. We still know little about the number of genes that underlie these traits, the magnitude of their effects, or the extent to which they interact. Chromosome substitution strains (CSSs) enable statistically powerful studies based on testing engineered inbred strains that have single, unique, and nonoverlapping genetic differences, thereby providing measures of phenotypic effects that are attributable to individual chromosomes. Here, we report a study of phenotypic effects and gene interactions for 90 blood, bone, and metabolic traits in a mouse CSS panel and 54 traits in a rat CSS panel. Two key observations emerge about the genetic architecture of these traits. First, the traits tend to be highly polygenic: across the genome, many individual chromosome substitutions each had significant phenotypic effects and, within each of the chromosomes studied, multiple distinct loci were found. Second, strong epistasis was found among the individual chromosomes. Specifically, individual chromosome substitutions often conferred surprisingly large effects (often a substantial fraction of the entire phenotypic difference between the parental strains), with the result that the sum of these individual effects often dramatically exceeded the difference between the parental strains. We suggest that strong, pervasive epistasis may reflect the presence of several phenotypically-buffered physiological states. These results have implications for identification of complex trait genes, developmental and physiological studies of phenotypic variation, and opportunities to engineer phenotypic outcomes in complex biological systems.

A syndrome of joint laxity and impaired tendon integrity in lumican- and fibromodulin-deficient mice

Lumican and fibromodulin regulate the assembly of collagens into higher order fibrils in connective tissues. Here, we show that mice deficient in both of these proteoglycans manifest several clinical features of Ehlers-Danlos syndrome. TheLum −/− Fmod −/− mice are smaller than their wild type littermates and display gait abnormality, joint laxity, and age-dependent osteoarthritis. Misaligned knee patella, severe knee dysmorphogenesis, and extreme tendon weakness are the likely causes for joint laxity in the double-nulls. Fibromodulin deficiency alone leads to significant reduction in tendon stiffness in theLum +/+ Fmod −/− mice, with further loss in stiffness in a Lum gene dose-dependent way. At the protein level, we show marked increase of lumican in Fmod −/− tendons, which may partially rescue the tendon phenotype in this genotype. These results establish fibromodulin as a key regulator and lumican as a modulator of tendon strength. A disproportionate increase in small diameter immature collagen fibrils and a lack of progression to mature, large diameter fibrils in the Fmod −/−background may constitute the underlying cause of tendon weakness and suggest that fibromodulin aids fibril maturation. This study demonstrates that the collagen fibril-modifying proteoglycans, lumican and fibromodulin, are candidate genes and key players in the pathogenesis of certain types of Ehlers-Danlos syndrome and other connective tissue disorders.

APPLICATION OF HOMOGENIZATION THEORY TO THE STUDY OF TRABECULAR BONE MECHANICS

It is generally accepted that the strength and stiffness of trabecular bone is strongly affected by trabecular microstructure. It has also been hypothesized that stress induced adaptation of trabecular bone is affected by trabecular tissue level stress and/or strain. At this time, however, there is no generally accepted (or easily accomplished) technique for predicting the effect of microstructure on trabecular bone apparent stiffness and strength or estimating tissue level stress or strain. In this paper, a recently developed mechanics theory specifically designed to analyze microstructured materials, called the homogenization theory, is presented and applied to analyze trabecular bone mechanics. Using the homogenization theory it is possible to perform microstructural and continuum analyses separately and then combine them in a systematic manner. Stiffness predictions from two different microstructural models of trabecular bone show reasonable agreement with experimental results, depending on metaphyseal region, (R2 greater than 0.5 for proximal humerus specimens, R2 less than 0.5 for distal femur and proximal tibia specimens). Estimates of both microstructural strain energy density (SED) and apparent SED show that there are large differences (up to 30 times) between apparent SED (as calculated by standard continuum finite element analyses) and the maximum microstructural or tissue SED. Furthermore, a strut and spherical void microstructure gave very different estimates of maximum tissue SED for the same bone volume fraction (BV/TV). The estimates from the spherical void microstructure are between 2 and 20 times greater than the strut microstructure at 10-20% BV/TV.

Three-dimensional reconstruction of fracture callus morphogenesis

Rat and mouse femur and tibia fracture calluses were collected over various time increments of healing. Serial sections were produced at spatial segments across the fracture callus. Standard histological methods and in situ hybridization to col1a1 and col2a1 mRNAs were used to define areas of cartilage and bone formation as well as tissue areas undergoing remodeling. Computer-assisted reconstructions of histological sections were used to generate three-dimensional images of the spatial morphogenesis of the fracture calluses. Endochondral bone formation occurred in an asymmetrical manner in both the femur and tibia, with cartilage tissues seen primarily proximal or distal to the fractures in the respective calluses of these bones. Remodeling of the calcified cartilage proceeded from the edges of the callus inward toward the fracture producing an inner-supporting trabecular structure over which a thin outer cortical shell forms. These data suggest that the specific developmental mechanisms that control the asymmetrical pattern of endochondral bone formation in fracture healing recapitulated the original asymmetry of development of a given bone because femur and tibia grow predominantly from their respective distal and proximal physis. These data further show that remodeling of the calcified cartilage produces a trabecular bone structure unique to fracture healing that provides the rapid regain in weight-bearing capacity to the injured bone. (J Histochem Cytochem 54:1215-1228, 2006)

Osteocyte apoptosis and control of bone resorption following ovariectomy in mice

INTRODUCTION Osteocyte apoptosis has been linked to bone resorption resulting from estrogen depletion and other resorptive stimuli; however, precise spatial and temporal relationships between the two events have not been clearly established. The purpose of this study was to characterize the patterns of osteocyte apoptosis in relation to bone resorption following ovariectomy to test whether osteocyte apoptosis occurs preferentially in areas known to activate resorption. Moreover, we report that osteocyte apoptosis is necessary to initiate endocortical remodeling in response to estrogen withdrawal. MATERIALS AND METHODS Adult female C57BL/6J mice (17 weeks old) underwent either bilateral ovariectomy (OVX), or sham surgery (SHAM) and were euthanized on days 3, 7, 14, or 21 days after OVX. Diaphyseal cross-sections were stained by immunohistochemistry for activated caspase-3 as a marker of apoptosis. The percentages of caspase-positive stained osteocytes (Casp+Ot.) were measured along major and minor anatomical axes around the femoral diaphysis to evaluate the distribution of osteocyte apoptosis after estrogen loss; resorption surface was measured at the adjacent endocortical regions. In a second study to test whether osteocyte apoptosis plays a regulatory role in the initiation of bone resorption, a group of OVX mice received the pan-caspase inhibitor, QVDOPh, to inhibit osteocyte apoptosis. Remaining experimental and sham groups received either QVD or Vehicle. RESULTS OVX increased osteocyte apoptosis in a non-uniform distribution throughout the femoral diaphyses. Increases in Casp+osteocytes were predominantly located in the posterior diaphyseal cortex. Here, the number of apoptotic osteocytes 4- to 7-fold higher than sham controls (p<0.005) by day 3 post-OVX and remained elevated. Increases in resorption post-OVX also occurred along the posterior endocortical surface overlying the region of osteocyte apoptosis, but these increases occurred only at 14 and 21 days post-OVX (p<0.002) well after the increases in osteocyte apoptosis. Treatment with QVD in OVX animals suppressed osteocyte apoptosis, with levels in QVD-treated samples equivalent to baseline. Moreover, the increases in osteoclastic resorption normally observed after estrogen loss did not occur in OVX mice treated with QVD. CONCLUSIONS The results of this study demonstrate that osteocyte apoptosis following estrogen loss occur regionally, rather than uniformly throughout the cortex. We also showed that estrogen loss increased osteocyte apoptosis. Apoptotic osteocytes were overwhelmingly localized within the posterior cortical region, the location where endocortical resorption was subsequently activated in ovariectomized mice. Finally, the increases in osteoclastic resorption normally observed after estrogen withdrawal did not occur in the absence of osteocyte apoptosis indicating that this apoptosis is necessary to activate endocortical remodeling following estrogen loss.

Relationship Between Bone Morphology and Bone Quality in Male Tibias: Implications for Stress Fracture Risk†

Biomechanical properties were assessed from the tibias of 17 adult males 17‐46 years of age. Tissue‐level mechanical properties varied with bone size. Narrower tibias were comprised of tissue that was more brittle and more prone to accumulating damage compared with tissue from wider tibias.