Alessandra Carriero

Vitamin D Deficiency Induces Early Signs of Aging in Human Bone, Increasing the Risk of Fracture

In addition to decreasing bone mass, vitamin D deficiency causes early aging of the remaining mineralized bone and leads to severe losses in fracture resistance. Vitamin D–Deficient Bone Showing Its Age Vitamin D, which is sometimes called the “sunshine vitamin” because humans can synthesize it in the presence of sunlight, has long been associated with prevention of bone disease. Vitamin D is required for proper absorption of calcium and its uptake into bone, and a lack of vitamin D is known to cause rickets and osteomalacia—diseases in which bone is too soft because of excessive collagenous matrix and its inadequate mineralization. Now, Busse and co-authors provide some evidence that the reverse is also partially true, and vitamin D deficiency can result in areas of overly dense mineralization in the bone. To study the effects of vitamin D deficiency, Busse and colleagues used samples of bone from 30 apparently healthy people. Half of these subjects were deficient in vitamin D, defined by low concentration of vitamin D in the blood and altered macroscopic characteristics of the bone. Through detailed analysis of bone structure and functional tests measuring the bones’ resistance to cracking, the authors characterized the ways in which vitamin D–deficient bone differs from normal. As expected, they found that bones from vitamin D–deficient subjects had a much thicker layer of unmineralized osteoid coating the surface of mineralized bone. However, they also demonstrated that the bone underneath this osteoid layer was more heavily mineralized than normal and had structural characteristics of older and more brittle bone. They explained this phenomenon by noting that osteoclasts, cells that normally remodel the bone, cannot get through the thick osteoid layer. As a result, the areas of bone hidden underneath the osteoid continue to age and mineralize even as the overall bone mineral content progressively decreases. These interesting and unexpected findings about human bone emphasize the negative consequences of vitamin D deficiency, which is all too common, especially at northern latitudes. Additional work will be needed to translate this knowledge into clinical practice, but the detailed understanding of human bone structure may provide some insight into more effective ways to prevent or treat fractures in patients with vitamin D deficiency. Vitamin D deficiency is a widespread medical condition that plays a major role in human bone health. Fracture susceptibility in the context of low vitamin D has been primarily associated with defective mineralization of collagenous matrix (osteoid). However, bone’s fracture resistance is due to toughening mechanisms at various hierarchical levels ranging from the nano- to the microstructure. Thus, we hypothesize that the increase in fracture risk with vitamin D deficiency may be triggered by numerous pathological changes and may not solely derive from the absence of mineralized bone. We found that the characteristic increase in osteoid-covered surfaces in vitamin D–deficient bone hampers remodeling of the remaining mineralized bone tissue. Using spatially resolved synchrotron bone mineral density distribution analyses and spectroscopic techniques, we observed that the bone tissue within the osteoid frame has a higher mineral content with mature collagen and mineral constituents, which are characteristic of aged tissue. In situ fracture mechanics measurements and synchrotron radiation micro–computed tomography of the crack path indicated that vitamin D deficiency increases both the initiation and propagation of cracks by 22 to 31%. Thus, vitamin D deficiency is not simply associated with diminished bone mass. Our analyses reveal the aged nature of the remaining mineralized bone and its greatly decreased fracture resistance. Through a combination of characterization techniques spanning multiple size scales, our study expands the current clinical understanding of the pathophysiology of vitamin D deficiency and helps explain why well-balanced vitamin D levels are essential to maintain bone’s structural integrity.

How tough is brittle bone? Investigating osteogenesis imperfecta in mouse bone.

The multiscale hierarchical structure of bone is naturally optimized to resist fractures. In osteogenesis imperfecta, or brittle bone disease, genetic mutations affect the quality and/or quantity of collagen, dramatically increasing bone fracture risk. Here we reveal how the collagen defect results in bone fragility in a mouse model of osteogenesis imperfecta (oim), which has homotrimeric α1(I) collagen. At the molecular level, we attribute the loss in toughness to a decrease in the stabilizing enzymatic cross‐links and an increase in nonenzymatic cross‐links, which may break prematurely, inhibiting plasticity. At the tissue level, high vascular canal density reduces the stable crack growth, and extensive woven bone limits the crack‐deflection toughening during crack growth. This demonstrates how modifications at the bone molecular level have ramifications at larger length scales affecting the overall mechanical integrity of the bone; thus, treatment strategies have to address multiscale properties in order to regain bone toughness. In this regard, findings from the heterozygous oim bone, where defective as well as normal collagen are present, suggest that increasing the quantity of healthy collagen in these bones helps to recover toughness at the multiple length scales.

Determination of gait patterns in children with spastic diplegic cerebral palsy using principal components

This study developed an objective graphical classification method of spastic diplegic cerebral palsy (CP) gait patterns based on principal component analysis (PCA). Gait analyses of 20 healthy and 20 spastic diplegic CP children were examined to define gait characteristics. PCA was used to reduce the dimensionality of 27 parameters (26 selected kinematics variables and age of the children) for the 40 subjects in order to identify the dominant variability in the data. Fuzzy C-mean cluster analysis was performed plotting the first three principal components, which accounted for 61% of the total variability. Results indicated that only the healthy children formed a distinct cluster; however it was possible to recognise gait patterns in overlapping clusters in children with spastic diplegia. This study demonstrates that it is possible to quantitatively classify gait types in CP using PCA. Graphical classification of gait types could assist in clinical evaluation of the children and serve as a validation of clinical reports as well as aid treatment planning.

Correlation between lower limb bone morphology and gait characteristics in children with spastic diplegic cerebral palsy.

Background: Children with spastic diplegic cerebral palsy (CP) exhibit abnormal walking patterns and frequently develop lower limb, long bone deformities. It is important to determine if any relationship exists between bone morphology and movement of the lower limbs in children with CP. This is necessary to explain and possibly prevent the development of these deformities. Methods: This study investigated the relationship between bone morphology and gait characteristics in 10 healthy children (age range, 6-13 years; mean, 8 years 7 months; SD, ±2 years 7 months) and 9 children with spastic diplegic CP (age range, 6-12 years; mean, 9 years 2.5 months; SD, ±1 year 10.5 months) with no previous surgery. Three-dimensional magnetic resonance images were analyzed to define bone morphology. Morphological characteristics, such as the bicondylar angle, neck-shaft angle, anteversion angle, and tibial torsion, were measured. Gait analyses were performed to obtain kinematic characteristics of CP and normal childrens gait. Principal component analysis was used to reduce the dimensionality of 27 parameters (26 kinematics variables and age of the children) to 8 independent variables. Correlations between gait and bone morphology were determined for both groups of children. Results: Results indicated that in healthy children, hip adduction was correlated with neck-shaft and bicondylar angles. In CP children, pelvic obliquity correlated with neck-shaft angle, and foot rotation with bicondylar angle. In the transverse plane, hip and pelvic rotational kinematics were related to femoral anteversion in healthy children and to tibial torsion in CP children. Conclusion: Different development was observed in femoral and tibial morphology between CP and healthy children. The relationship between bone shape and dynamic gait patterns also varied between these populations. This needs to be taken into account, particularly when surgical treatment is planned. Clinical Relevance: Understanding the relationship between gait abnormality and bone deformity could eventually help in developing treatment regimens that will address gait deviations at the correct level and promote normal bone growth in children with CP.

Altered lacunar and vascular porosity in osteogenesis imperfecta mouse bone as revealed by synchrotron tomography contributes to bone fragility

Osteogenesis imperfecta (brittle bone disease) is caused by mutations in the collagen genes and results in skeletal fragility. Changes in bone porosity at the tissue level indicate changes in bone metabolism and alter bone mechanical integrity. We investigated the cortical bone tissue porosity of a mouse model of the disease, oim, in comparison to a wild type (WT-C57BL/6), and examined the influence of canal architecture on bone mechanical performance. High-resolution 3D representations of the posterior tibial and the lateral humeral mid-diaphysis of the bones were acquired for both mouse groups using synchrotron radiation-based computed tomography at a nominal resolution of 700nm. Volumetric morphometric indices were determined for cortical bone, canal network and osteocyte lacunae. The influence of canal porosity architecture on bone mechanics was investigated using microarchitectural finite element (μFE) models of the cortical bone. Bright-field microscopy of stained sections was used to determine if canals were vascular. Although total cortical porosity was comparable between oim and WT bone, oim bone had more numerous and more branched canals (p<0.001), and more osteocyte lacunae per unit volume compared to WT (p<0.001). Lacunae in oim were more spherical in shape compared to the ellipsoidal WT lacunae (p<0.001). Histology revealed blood vessels in all WT and oim canals. μFE models of cortical bone revealed that small and branched canals, typical of oim bone, increase the risk of bone failure. These results portray a state of compromised bone quality in oim bone at the tissue level, which contributes to its deficient mechanical properties.

Reference point indentation is not indicative of whole mouse bone measures of stress intensity fracture toughness

Bone fragility is a concern for aged and diseased bone. Measuring bone toughness and understanding fracture properties of the bone are critical for predicting fracture risk associated with age and disease and for preclinical testing of therapies. A reference point indentation technique (BioDent) has recently been developed to determine bones resistance to fracture in a minimally invasive way by measuring the indentation distance increase (IDI) between the first and last indentations over cyclic indentations in the same position. In this study, we investigate the relationship between fracture toughness KC and reference point indentation parameters (i.e. IDI, total indentation distance (TID) and creep indentation distance (CID)) in bones from 38 mice from six types (C57Bl/6, Balb, oim/oim, oim/+, Phospho1−/− and Phospho1 wild type counterpart). These mice bone are models of healthy and diseased bone spanning a range of fracture toughness from very brittle (oim/oim) to ductile (Phospho1−/−). Left femora were dissected, notched and tested in 3-point bending until complete failure. Contralateral femora were dissected and indented in 10 sites of their anterior and posterior shaft surface over 10 indentation cycles. IDI, TID and CID were measured. Results from this study suggest that reference point indentation parameters are not indicative of stress intensity fracture toughness in mouse bone. In particular, the IDI values at the anterior mid-diaphysis across mouse types overlapped, making it difficult to discern differences between mouse types, despite having extreme differences in stress intensity based toughness measures. When more locations of indentation were considered, the normalised IDIs could distinguish between mouse types. Future studies should investigate the relationship of the reference point indentation parameters for mouse bone in other material properties of the bone tissue in order to determine their use for measuring bone quality.

Ex vivo determination of bone tissue strains for an in vivo mouse tibial loading model

Previous studies introduced the digital image correlation (DIC) as a viable technique for measuring bone strain during loading. In this study, we investigated the sensitivity of a DIC system in determining surface strains in a mouse tibia while loaded in compression through the knee joint. Specifically, we examined the effect of speckle distribution, facet size and overlap, initial vertical alignment of the bone into the loading cups, rotation with respect to cameras, and ex vivo loading configurations on the strain contour maps measured with a DIC system. We loaded tibiae of C57BL/6 mice (12 and 18 weeks old male) up to 12 N at 8 N/min. Images of speckles on the bone surface were recorded at 1 N intervals and DIC was used to compute strains. Results showed that speckles must have the correct size and density with respect to the facet size of choice for the strain distribution to be computed and reproducible. Initial alignment of the bone within the loading cups does not influence the strain distribution measured during peak loading, but bones must be placed in front of the camera with the same orientation in order for strains to be comparable. Finally, the ex vivo loading configurations with the tibia attached to the entire mouse, or to the femur and foot, or only to the foot, showed different strain contour maps. This work provides a better understanding of parameters affecting full field strain measurements from DIC in ex vivo murine tibial loading tests.

Mechanobiological simulations of prenatal joint morphogenesis

Joint morphogenesis is the process in which prenatal joints acquire their reciprocal and interlocking shapes. Despite the clinical importance of the process, it remains unclear how joints acquire their shapes. In this study, we simulate 3D mechanobiological joint morphogenesis for which the effects of a range of movements (or lack of movement) and different initial joint shapes are explored. We propose that static hydrostatic compression inhibits cartilage growth while dynamic hydrostatic compression promotes cartilage growth. Both pre-cavitational (no muscle contractions) and post-cavitational (with muscle contractions) phases of joint development were simulated. Our results showed that for hinge type motion (planar motion from 45° to 120°) the proximal joint surface developed a convex profile in the posterior region and the distal joint surface developed a slightly concave profile. When 3D movements from 40° to -40° in two planes were applied, simulating a rotational movement, the proximal joint surface developed a concave profile whereas the distal joint surface rudiment acquire a rounded convex profile, showing an interlocking shape typical of a ball and socket joint. The significance of this research is that it provides new and important insights into normal and abnormal joint development, and contributes to our understanding of the mechanical factors driving very early joint morphogenesis. An enhanced understanding of how prenatal joints form is critical for developing strategies for early diagnosis and preventative treatments for congenital musculoskeletal abnormalities such as developmental dysplasia of the hip.

Effects of normal and abnormal loading conditions on morphogenesis of the prenatal hip joint: application to hip dysplasia

Joint morphogenesis is an important phase of prenatal joint development during which the opposing cartilaginous rudiments acquire their reciprocal and interlocking shapes. At an early stage of development, the prenatal hip joint is formed of a deep acetabular cavity that almost totally encloses the head. By the time of birth, the acetabulum has become shallower and the femoral head has lost substantial sphericity, reducing joint coverage and stability. In this study, we use a dynamic mechanobiological simulation to explore the effects of normal (symmetric), reduced and abnormal (asymmetric) prenatal movements on hip joint shape, to understand their importance for postnatal skeletal malformations such as developmental dysplasia of the hip (DDH). We successfully predict the physiological trends of decreasing sphericity and acetabular coverage of the femoral head during fetal development. We show that a full range of symmetric movements helps to maintain some of the acetabular depth and femoral head sphericity, while reduced or absent movements can lead to decreased sphericity and acetabular coverage of the femoral head. When an abnormal movement pattern was applied, a deformed joint shape was predicted, with an opened asymmetric acetabulum and the onset of a malformed femoral head. This study provides evidence for the importance of fetal movements in the prevention and manifestation of congenital musculoskeletal disorders such as DDH.

Phospho1 deficiency transiently modifies bone architecture yet produces consistent modification in osteocyte differentiation and vascular porosity with ageing

PHOSPHO1 is one of principal proteins involved in initiating bone matrix mineralisation. Recent studies have found that Phospho1 KO mice (Phospho1-R74X) display multiple skeletal abnormalities with spontaneous fractures, bowed long bones, osteomalacia and scoliosis. These analyses have however been limited to young mice and it remains unclear whether the role of PHOSPHO1 is conserved in the mature murine skeleton where bone turnover is limited. In this study, we have used ex-vivo computerised tomography to examine the effect of Phospho1 deletion on tibial bone architecture in mice at a range of ages (5, 7, 16 and 34 weeks of age) to establish whether its role is conserved during skeletal growth and maturation. Matrix mineralisation has also been reported to influence terminal osteoblast differentiation into osteocytes and we have also explored whether hypomineralised bones in Phospho1 KO mice exhibit modified osteocyte lacunar and vascular porosity. Our data reveal that Phospho1 deficiency generates age-related defects in trabecular architecture and compromised cortical microarchitecture with greater porosity accompanied by marked alterations in osteocyte shape, significant increases in osteocytic lacuna and vessel number. Our in vitro studies examining the behaviour of osteoblast derived from Phospho1 KO and wild-type mice reveal reduced levels of matrix mineralisation and modified osteocytogenic programming in cells deficient in PHOSPHO1. Together our data suggest that deficiency in PHOSPHO1 exerts modifications in bone architecture that are transient and depend upon age, yet produces consistent modification in lacunar and vascular porosity. It is possible that the inhibitory role of PHOSPHO1 on osteocyte differentiation leads to these age-related changes in bone architecture. It is also intriguing to note that this apparent acceleration in osteocyte differentiation evident in the hypomineralised bones of Phospho1 KO mice suggests an uncoupling of the interplay between osteocytogenesis and biomineralisation. Further studies are required to dissect the molecular processes underlying the regulatory influences exerted by PHOSPHO1 on the skeleton with ageing.