Henry P. Schwarcz

Dark-field transmission electron microscopy of cortical bone reveals details of extrafibrillar crystals.

In a previous study we showed that most of the mineral in bone is present in the form of mineral structures, 5-6nm-thick, elongated plates which surround and are oriented parallel to collagen fibrils. Using dark-field transmission electron microscopy, we viewed mineral structures in ion-milled sections of cortical human bone cut parallel to the collagen fibrils. Within the mineral structures we observe single crystals of apatite averaging 5.8±2.7nm in width and 28±19nm in length, their long axes oriented parallel to the fibril axis. Some appear to be composite, co-aligned crystals as thin as 2nm. From their similarity to TEM images of crystals liberated from deproteinated bone we infer that we are viewing sections through platy crystals of apatite that are assembled together to form the mineral structures.

You are not what you eat during physiological stress: Isotopic evaluation of human hair

Variation in δ(13) C and δ(15) N values can be assessed to understand not only diet, but also the influence of physiological factors on an individual. The metabolic balance of an individual can impact isotopic signals in tissues that are forming during the periods of metabolic stress. Fluctuating δ(15) N values are associated with physiological stressors that alter an individuals metabolism such as infection, injury, or pregnancy. This study examines variation in δ(13) C and δ(15) N values along sequentially segmented hair in both modern and archaeological individuals. Subjects with an observable skeletal pathology, known chronic illness, or evidence of pregnancy were compared with controls exhibiting no evidence of physiological stress. The results on hair samples from individuals from 19(th) century Belleville, Ontario, four modern cadavers (two with cancer and two sudden deaths), and two living pregnant women indicate that δ(15) N values are approximately 1‰ higher in individuals with a pathological condition (e.g., infection, fracture, or cancer) and are 1‰ lower during pregnancy, whereas δ(13) C values show less variability. Higher nitrogen values may represent the recycling of nitrogen derived from the breakdown of existing proteins in the body (catabolism), whereas lower δ(15) N values are related to increased utilization of dietary and urea nitrogen for tissue synthesis during pregnancy. These findings suggest that short-term fluctuations of δ(15) N values may be the result of changes in an individuals metabolic balance, and that metabolic imbalance poses a confounding factor to ancient dietary studies when using rapidly growing tissues such as hair.

Scanning transmission electron microscopic tomography of cortical bone using Z-contrast imaging.

Previously we presented (McNally et al., 2012) a model for the ultrastructure of bone showing that the mineral resides principally outside collagen fibrils in the form of 5 nm thick mineral structures hundreds of nanometers long oriented parallel to the fibrils. Here we use high-angle annular dark-field electron tomography in the scanning transmission electron microscope to confirm this model and further elucidate the composite structure. Views of a section cut parallel to the fibril axes show bundles of mineral structures extending parallel to the fibrils and encircling them. The mineral density inside the fibrils is too low to be visualized in these tomographic images. A section cut perpendicular to the fibril axes, shows quasi-circular walls composed of mineral structures, wrapping around apparently empty holes marking the sites of fibrils. These images confirm our original model that the majority of mineral in bone resides outside the collagen fibrils.

The ultrastructure of bone as revealed in electron microscopy of ion-milled sections

Mineral makes up more than half the volume of bone, but its spatial and structural relationship to collagen and other proteins is still a matter of debate. Due to the nanometer-size of bone crystals this matter can be resolved only with transmission electron microscope (TEM) images. Using sections cut with an ultramicrotome, previous investigators determined most mineral lies in the 40nm wide gap zone in collagen fibrils. Using less invasive sectioning methods (ion milling and focused ion beam [FIB]) reveals that most mineral is extrafibrillar, occurring in the form of mineral lamellae, polycrystalline plates 300nm or more long, packed around collagen fibrils in stacks of four or more lamellae <1nm apart. While Ca and P also occur in the gap zone, they do not appear to be in the form of well-crystallized apatite. This new model for bone ultrastructure resolves outstanding problems presented by the previous model.

Bone mineralization is elevated and less heterogeneous in adults with type 2 diabetes and osteoarthritis compared to controls with osteoarthritis alone

PURPOSEnThe purpose of this study was to determine whether trabecular bone mineralization differed in adults with type 2 diabetes compared to adults without type 2 diabetes.nnnMETHODSnProximal femur specimens were obtained following a total hip replacement procedure from men and women ≥65 years of age with and without type 2 diabetes. A scanning electron microscope was used for quantitative backscattered electron imaging (qBEI) analysis of trabecular bone samples from the femoral neck. Gray scale images (pixel size=5.6 μm(2)) were uploaded to ImageJ software and gray level (GL) values were converted to calcium concentrations (weight [wt] % calcium [Ca]) using data obtained with energy dispersive X-ray spectrometry. The following bone mineralization density distribution (BMDD) outcomes were collected: the weighted mean bone calcium concentration (CaMEAN), the most frequently occurring bone calcium concentration (CaPEAK) and mineralization heterogeneity (CaWIDTH). Differences between groups were assessed using the Students t-test for normally distributed data and Mann-Whitney U-test for non-normally distributed data. An alpha value of <0.05 was considered significant.nnnRESULTSnThirty-five Caucasian participants were recruited (mean [standard deviation, SD] age, 75.5 [6.5]years): 14 adults with type 2 diabetes (years since type 2 diabetes diagnosis, 13.5 [7.4]years) and 21 adults without type 2 diabetes. In the adults with type 2 diabetes, bone CaMEAN was 4.9% greater (20.36 [0.98]wt.% Ca versus 19.40 [1.07]wt.% Ca, p=0.015) and CaWIDTH was 9.4% lower (median [interquartile range] 3.55 [2.99-4.12]wt.% Ca versus 3.95 [0.71]wt.% Ca, p<0.001) compared to controls. There was no between-group difference in CaPEAK (21.12 [0.97]wt.% Ca for type 2 diabetes versus 20.44 [1.30]wt.% Ca for controls, p=0.121).nnnCONCLUSIONnThe combination of elevated mean calcium concentration in bone and lower mineralization heterogeneity in adults with type 2 diabetes may have deleterious effects on the biomechanical properties of bone. These microscopic alterations in bone mineralization, which may be mediated by suppressed bone remodeling, further elucidate higher fracture risk in adults with type 2 diabetes.

Caves in caves: evolution of post-depositional macroholes in stalagmites

In a previous paper (Shtober-Zisu et al., 2012) we described millimeter to centime-sized fluid-free holes within the interiors of stalagmites of widely varying origin. We present here further observations of this phenomenon, using X-ray tomography, macroscopic and microscopic observation of sections of twenty-six stalagmites from various sites in North America and the Caribbean region. We can distinguish three types of cavities in speleothems: primary µm-sized fluid inclusions; mm to cm sized holes, aligned along the stalagmite growth axis which are clearly syngenetic; and µm to cm-sized holes away from the growth axis (“off-axis holes or OAHs”) deeply buried inside their host stalagmites, and cutting primary growth layers. Neither axial nor off axis holes contain fluid today. Off-axis holes appear to have been formed by internal corrosion of the calcite host, possibly enhanced by the action of bacteria which were sustained by permeation of through the body of the stalagmite of water containing dissolved organic species. A modern stalagmite from Israel is shown to contain bacteria associated with active hole formation.

The ultrastructure of bone and its relevance to mechanical properties

Bone is a biologically generated composite material comprised of two major structural components: crystals of apatite and collagen fibrils. Computational analysis of the mechanical properties of bone must make assumptions about the geometric and topological relationships between these components. Recent transmission electron microscope (TEM) studies of samples of bone prepared using ion milling methods have revealed important previously unrecognized features in the ultrastructure of bone. These studies show that most of the mineral in bone lies outside the fibrils and is organized into elongated plates 5 nanometers (nm) thick, ~ 80 nm wide and hundreds of nm long. These so-called mineral lamellae (MLs) are mosaics of single 5 nm-thick, 20 – 50 nm wide crystals bonded at their edges. MLs occur either stacked around the 50 nm-diameter collagen fibrils, or in parallel stacks of 5 or more MLs situated between fibrils. About 20% of mineral is in gap zones within the fibrils. MLs are apparently glued together into mechanically coherent stacks which break across the stack rather than delaminating. ML stacks should behave as cohesive units during bone deformation. Finite element computations of mechanical properties of bone show that the model including such features generates greater stiffness and strength than are obtained using conventional models in which most of the mineral, in the form of isolated crystals, is situated inside collagen fibrils.