Osteopontin regulates type I collagen fibril formation in bone tissue.

Osteopontin (OPN) is a non-collagenous protein involved in biomineralization of bone tissue. Beyond its role in biomineralization, we show that osteopontin is essential to the quality of collagen fibrils in bone. Transmission electron microscopy revealed that, in Opn-/- tissue, the organization of the collagen fibrils was highly heterogeneous, more disorganized than WT bone and comprised of regions of both organized and disorganized matrix with a reduced density. The Opn-/- bone tissue also exhibited regions in which the collagen had lost its characteristic fibrillar structure, and the crystals were disorganized. Using nanobeam electron diffraction, we show that damage to structural integrity of collagen fibrils in Opn-/- bone tissue and their organization causes mineral disorganization, which could ultimately affect its mechanical integrity. STATEMENT OF SIGNIFICANCE: This study presents new evidence about the role of osteopontin (OPN) - a non-collagenous protein - on the structure and organization of the organic and mineral matrix in bone. In previous work, osteopontin has been suggested to regulate the nucleation and growth of bone mineral crystals and to form sacrificial bonds between mineralized collagen fibrils to enhance bone's toughness. Our findings show that OPN plays a crucial role before mineralization, during the formation of the collagen fibrils. OPN-deficient bones present a lower collagen content compared to wild type bone and, at the tissue level, collagen fibrils organization can be significantly altered in the absence of OPN. Our results suggest that OPN is critical for the formation and/or remodeling of bone collagen matrix. Our findings could lead to the development of new therapeutic strategies of bone diseases affecting collagen formation and remodeling.

On the Relation of Bone Mineral Density and the Elastic Modulus in Healthy and Pathologic Bone.

PURPOSE OF REVIEW: Osteoporosis could lead to the bone mechanical failure. To examine the bone health, mechanical properties are often estimated from the images of the bone density. Here, we review the relationships that have been experimentally determined between mineral density and the elastic modulus and factors that affect these relationships. RECENT FINDINGS: Studies, which have investigated the relation between the elastic modulus and bone mineral at the bulk scale, have shown that approximately 70% of variations in the elastic modulus can be explained based on the amount of mineral in bone. At the tissue level, however, higher resolution techniques are used to characterize the density and modulus more locally, and this leads to the correlation of mineral with modulus to be not as strong as that of the bulk level and often times, insignificant. This observation indicates the importance of structural hierarchy and mineral crystal organization in determining the local stiffness of the bone tissue. At the bulk level in bone (cm scale), modulus (E) is related to density (ρ) through a power law relationship (E ∝ ρα). At the tissue level (µm-mm scale), the relationship between the modulus and density is weak, likely due to the effect of microstructural features at small length scales.

Mutant cartilage oligomeric matrix protein (COMP) compromises bone integrity, joint function and the balance between adipogenesis and osteogenesis.

Mutations in COMP (cartilage oligomeric matrix protein) cause severe long bone shortening in mice and humans. Previously, we showed that massive accumulation of misfolded COMP in the ER of growth plate chondrocytes in our MT-COMP mouse model of pseudoachondroplasia (PSACH) causes premature chondrocyte death and loss of linear growth. Premature chondrocyte death results from activation of oxidative stress and inflammation through the CHOP-ER pathway and is reduced by removing CHOP or by anti-inflammatory or antioxidant therapies. Although the mutant COMP chondrocyte pathologic mechanism is now recognized, the effect of mutant COMP on bone quality and joint health (laxity) is largely unknown. Applying multiple analytic approaches, we describe a novel mechanism by which the deleterious consequences of mutant COMP retention results in upregulation of miR-223 disturbing the adipogenesis - osteogenesis balance. This results in reduction in bone mineral density, bone quality, mechanical strength and subchondral bone thickness. These, in addition to abnormal patterns of ossification at the ends of the femoral bones likely contribute to precocious osteoarthritis (OA) of the hips and knees in the MT-COMP mouse and PSACH. Moreover, joint laxity is compromised by abnormally thin ligaments. Altogether, these novel findings align with the PSACH phenotype of delayed ossification and bone age, extreme joint laxity and joint erosion, and extend our understanding of the underlying processes that affect bone in PSACH. These results introduce a novel finding that miR-223 is involved in the ossification defect in MT-COMP mice making it a therapeutic target.

Elastic modulus varies along the bovine femur.

Bone is a heterogeneous material and its mechanical properties vary within the body. Variations in the mechanical response of different bone samples taken from the body cannot be fully explained by only looking at local compositional information at the tissue level. Due to different states of the stress within bones, one might expect that mechanical properties change over the length of a bone; this has not been a matter of systematic research in previous studies. In this study, the distribution of the tissue elastic modulus along the bovine femur is investigated using three-point bending tests. Two bovine femora were split to seven and eight blocks from proximal to distal metaphysis, respectively and twenty beam shaped bone samples were extracted and tested from each block. Based on our findings, the longitudinal elastic modulus follows a gradient pattern along the bovine femur as it increases along the bone from the proximal metaphysis to mid-diaphysis and then decreases toward the distal metaphysis again. Considering long bones to be subjected to bending loads, this mechanism alters the bone structure to support load in the regions where it is needed; similar as outlined by Wolff's law. In another part of this study, microfocus X-ray computed tomography (µCT) was found unable to predict the same trend of changes for the elastic modulus via image-based or density-based elastic moduli calculations. This is insofar important as conventional finite element models of bone are often directly shaped from µCT data. Based on our findings, it seems that current computed tomography based finite element models generated in this manner may not adequately capture the local variation of material behavior of bone tissue, but this may be improved by considering the changes of the elastic modulus along the femur.

Characterization of the MPS I-H knock-in mouse reveals increased femoral biomechanical integrity with compromised material strength and altered bone geometry.

Mucopolysaccharidosis type I (MPS I), is an autosomal recessive lysosomal storage disorder caused by a deficiency in the α-L-iduronidase enzyme, resulting in decreased enzymatic activity and accumulation of glycosaminoglycans. The disorder phenotypically manifests with increased urine glycosaminoglycan excretion, facial dysmorphology, neuropathology, cardiac manifestations, and bone deformities. While the development of new treatment strategies have shown promise in attenuating many symptoms associated with the disorder, the bone phenotype remains unresponsive. The aim of this study was to investigate and further characterize the skeletal manifestations of the Idua-W392X knock-in mouse model, which carries a nonsense mutation corresponding to the IDUA-W402X mutation found in Hurler syndrome (MPS I-H) patients. µCT analysis of the microarchitecture demonstrated increased cortical thickness, trabecular number, and trabecular connectivity along with decreased trabecular separation in the tibiae of female homozygous Idua-W392X knock-in (IDUA-/-) mice, and increased cortical thickness in male IDUA-/- tibiae. Cortical density, as determined by µCT, and bone mineral density distribution, as determined by quantitative backscattered microscopy, were equivalent in IDUA-/- and wildtype (Wt) bone. However, tibial porosity was increased in IDUA-/- cortical bone. Raman spectroscopy results indicated that tibiae from female IDUA-/- had decreased phosphate to matrix ratios and increased carbonate to phosphate ratios compared to Wt female tibiae, whereas these ratios remained equivalent in male IDUA-/- and Wt tibiae. Femora demonstrated altered geometry and upon torsional loading to failure analysis, female IDUA-/- mouse femora exhibited increased torsional ultimate strength, with a decrease in material strength relative to Wt littermates. Taken together, these findings suggest that the IDUA-/- mutation results in increased bone torsional strength by altering the overall bone geometry and the microarchitecture which may be a compensatory response to increased porosity, reduced bone tensile strength and altered physiochemical composition.

Cement lines and interlamellar areas in compact bone as strain amplifiers - contributors to elasticity, fracture toughness and mechanotransduction.

Bone is multi-scale hierarchical composite material making the prediction of fragility, as well as pinning it to a certain cause, complicated. For proper mechanical simulation and reflection of bone properties in models, microscopic structural features of bone tissue need to be included. This study sets out to gain a mechanistic insight into the role of various microstructural features of bone tissue in particular cement lines and interlamellar areas. Further the hypothesis that compliant interlamellar areas and cement lines within osteonal bone act as strain amplifiers was explored. To this end, a series of experimentally-based micromechanical finite element models of bovine osteonal bone were developed. Different levels of detail for the bone microstructure were considered and combined with the results of physical three-point bending tests and an analytical composite model of a single osteon. The objective was to examine local and global effects of interface structures. The geometrical and microstructural characteristics of the bone samples were derived from microscopy imaging. Parametric finite element studies were conducted to determine optimal values of the elastic modulus of interstitial bone and interlamellar areas. The average isotropic elastic modulus of interfaces suggested in this study is 88.5MPa. Based on the modelling results, it is shown that interfaces are areas of accumulated strain in bone and are likely to act as potential paths for crack propagation. The strain amplification capability of interface structures in the order of 10 predicted by the models suggests a new explanation for the levels of strain required in bone homoeostasis for maintenance and adaptation.